InteractiveFly: GeneBrief

Syndecan: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - Syndecan

Synonyms -

Cytological map position - 57E1--6

Function - receptor

Keywords - axon guidance, Robo/Slit interaction

Symbol - Sdc

FlyBase ID: FBgn0010415

Genetic map position -

Classification - heparan sulfate proteoglycan, syndecan domain

Cellular location - surface transmembrane

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Nguyen, M. U., Kwong, J., Chang, J., Gillet, V. G., Lee, R. M. and Johnson, K. G. (2016). The extracellular and cytoplasmic domains of Syndecan cooperate postsynaptically to promote synapse growth at the Drosophila neuromuscular junction. PLoS One 11: e0151621. PubMed ID: 26987116
The heparan sulfate proteoglycan (HSPG) Syndecan (Sdc) is a crucial regulator of synapse development and growth in both vertebrates and invertebrates. In Drosophila, Sdc binds via its extracellular heparan sulfate (HS) sidechains to the receptor protein tyrosine phosphatase LAR to promote the morphological growth of the neuromuscular junction (NMJ). To date, however, little else is known about the molecular mechanisms by which Sdc functions to promote synapse growth. This study shows that all detectable Sdc found at the NMJ is provided by the muscle, strongly suggesting a post-synaptic role for Sdc. Both the cytoplasmic and extracellular domains of Sdc are required to promote synapse growth or to rescue Sdc loss of function. The results are reported of a yeast two-hybrid screen using the cytoplasmic domains of Sdc as bait, and several novel candidate binding partners were identified for the cytoplasmic domains of Sdc. Together, these studies provide new insight into the mechanism of Sdc function at the NMJ, and provide enticing future directions for further exploring how Sdc promotes synapse growth.
Warren, J. L., Jr., Hoxha, E., Jumbo-Lucioni, P. and De Luca, M. (2017). Reduction of Syndecan transcript levels in the insulin-producing cells affects glucose homeostasis in adult Drosophila melanogaster. DNA Cell Biol [Epub ahead of print]. PubMed ID: 28945109
Signaling by direct cell-matrix interactions has been shown to impact the transcription, secretion, and storage of insulin in mammalian beta cells. However, more research is still needed in this area. Syndecans are transmembrane heparan sulfate proteoglycans that function independently and in synergy with integrin-mediated signaling to mediate cell adhesion to the extracellular matrix. This study used the model organism Drosophila melanogaster to determine whether knockdown of the Syndecan (Sdc) gene expression specifically in the insulin-producing cells (IPCs) might affect insulin-like peptide (ILP) production and secretion. IPCs of adult flies produce three ILPs (ILP2, ILP3, and ILP5), which have significant homology to mammalian insulin. Flies with reduced Sdc expression in the IPCs did not show any difference in the expression of ilp genes compared to controls. However, they had significantly reduced levels of the circulating ILP2 protein, higher circulating carbohydrates, and were less glucose tolerant than control flies. Finally, IPCs-specific Sdc knockdown led to reduced levels of head Glucose transporter1 gene expression, extracellular signal-regulated kinase phosphorylation, and reactive oxygen species. Taken together, these findings suggest a cell autonomous role for Sdc in insulin release in D. melanogaster.

Biochemical studies suggest that axon guidance activity requires cell-surface heparan sulfate to promote binding of mammalian Slit/Robo homologs (Liang, 1999; Hu, 2001). The Drosophila homolog of Syndecan (reviewed in Couchman, 2003), a heparan sulfate proteoglycan (HSPG), is required for proper Slit signaling. Slit, the ligand for the Roundabout (Robo) receptors, is secreted from midline cells of the Drosophila central nervous system (CNS). It acts as a short-range repellent that controls midline crossing of axons and allows growth cones to select specific pathways along each side of the midline. In addition, Slit directs the migration of muscle precursors and ventral branches of the tracheal system, showing that it provides long-range activity beyond the limit of the developing CNS. Syndecan (Sdc) mutations have been generated; they affect all aspects of Slit activity and cause robo-like phenotypes. sdc interacts genetically with robo and slit, and double mutations cause a synergistic strengthening of the single-mutant phenotypes. The results suggest that Syndecan is a necessary component of Slit/Robo signaling and is required in the Slit target cells (Steigemann, 2003).

Genetic assays provide a sensitive means of detecting an in vivo interaction between different components in a pathway, but they do not show that the association is direct. Thus, a biochemical assay was developed to determine whether Sdc binds to Slit and/or Robo in cellular extracts in which all three proteins are endogenously expressed. Immunoprecipitation of either Slit or Robo and subsequent detection with anti-Sdc antibodies reveals that Sdc associates with both Slit and its receptor, suggesting the possibility of a ternary complex. This association is specific because no Sdc is trapped by nonspecific IgG or N-Cadherin antibodies that successfully IP other signaling molecules. Thus, Sdc participates directly in a complex with Slit and Robo (Johnson, 2004).

Mammalian Slit proteins are capable of binding to the HSPG Glypican, and this in vitro interaction is sensitive to heparinase III treatment (Liang, 1999). Furthermore, heparinase III treatment significantly reduced the binding of human Slit2 to Robo1-transfected cells and reduced its biological activity (Hu, 2001). In contrast to mammals, the Drosophila genome contains only a single Slit-coding gene, which acts through three receptors: Robo, Robo2, and Robo3. These receptors are expressed in an overlapping set of axons of the longitudinal tracts that run parallel to the midline of the developing CNS. In order to identify an endogenous HSPG that might participate in Slit signaling, the expression of HSPG-encoding genes was examined by whole-mount in situ hybridization, looking for HSPG encoding genes that are expressed in patterns that include the expression domains of Slit and/or Robo. syndecan (sdc), which encodes a conserved transmembrane protein containing a heparan-sulfate-modified extracellular domain (Spring, 1994), shows zygotic patterns overlapping all tissues affected in slit mutants and in domains directly adjacent to regions of slit expression (Steigemann, 2003).

If Sdc participates in the control of Slit/Robo signaling and is not expressed in Slit-expressing cells, it is likely to be coexpressed with the Robo receptors. Robo2 and Robo3 are expressed in a subset of the Robo-expressing axons. Thus, the anti-Robo antibody labels all axons in which the three Drosophila Slit receptors are active. In order to localize the Sdc-expressing cells, Sdc-specific antibodies were generated to immunostain embryos in combination with anti-Robo antibodies. Specificity of the antibody staining is indicated by the absence of staining in homozygous sdc mutant embryos and by panneural overexpression using the GAL4/UAS system . Panneural Sdc expression was achieved with a UAS-dependent transgene containing an sdc cDNA (composed of the translated exons; sdc-RA in response to the elav-GAL4-driver). Sdc and Robo are coexpressed in the longitudinal axons from stage 13 onward. Sdc protein is also expressed across the midline in positions corresponding to the commissural axons that lack Robo. The heparinase sensitivity of the mammalian Slit/Robo interaction, the molecular nature of Sdc, and its coexpression with Robo (and thus also with Robo2 and Robo3) are consistent with a role of Sdc in Slit signaling (Steigemann, 2003).

In order to test this proposal, sdc mutants were generated by imprecise excision of a P element inserted in the first intron of the sdc gene. Two sdc mutations were generated, the alleles sdc97 and sdc23, both lacking the first exon, the transcription start site, and parts of the first intron. In contrast to sdc97, the translation start site as well as parts of the signal peptide encoded by the second exon are removed in the sdc23 allele. Both deletion mutants fail to express the sdc transcript in all regions of the embryo where transcripts are detected in wild-type embryos. Furthermore, Sdc could not be detected by anti-Sdc antibodies in the mutant embryos. These findings indicate that the newly generated sdc mutants are either strong hypomorphic or amorphic alleles (Steigemann, 2003).

Homozygous sdc mutants are semilethal and show identical phenotypes in the CNS and in the muscle pattern. In order to unambiguously demonstrate that the lack of sdc activity is responsible for the mutant phenotype observed, Sdc-RA was panneurally expressed using the GAL4/UAS system in sdc mutant individuals. The neural phenotype of the mutants was rescued, indicating that the mutant phenotype was caused by the lack of Sdc and that the transgene provides functional Sdc activity (Steigemann, 2003).

In order to examine the possible defects in axonal guidance and muscle patterning, sdc mutant embryos were stained with both Fasciclin II (FasII; mAB 1D4) antibodies, which label three longitudinal axon tracts at each side of the midline, and anti-Mhc antibodies, which visualize the muscle pattern. The results show that the lack of sdc activity causes phenocopies of robo and robo2 mutants; i.e., it affects both midline guidance of axons and the establishment of the muscle pattern. The defects in CNS axon guidance were strikingly similar to robo2 mutants but less pronounced than in robo mutants. The muscle and CNS phenotypes were also weaker than in slit mutants, in which signaling through all Robo receptors is impaired (Steigemann, 2003).

It was next asked whether sdc activity participates in the control of robo and/or slit expression, or vice versa, by examining the strength and patterns of expression of each gene in sdc, robo, and slit mutant embryos. The results showed that there was no crossregulatory effect on gene expression and localization of the proteins. In addition, it was asked whether panneural overexpression of sdc interferes with axonal guidance and the establishment of the muscle patterns. Panneural sdc expression was strongly induced in transgene-bearing wild-type embryos, but no effect on the FasII-expressing axons, the commissures, and the mytotube pattern could be observed. Collectively, the results suggest that in the absence of sdc activity, both slit and robo expression as well as the production and localization of the proteins were not affected, but the effectiveness of the Slit signal is strongly reduced in sdc mutants. In addition, panneural sdc overexpression does not interfere with Slit signaling (Steigemann, 2003).

In order to link embryonic Sdc requirement genetically to Slit/Robo signaling, it was next asked whether sdc mutations can enhance loss-of-function slit and robo phenotypes. It was found that the number of ventral muscles, which cross the midline dorsal of the CNS in homozygous sdc and robo2 single mutants, is significantly increased in double mutant combinations of sdc and robo2, resulting in a muscle phenotype indistinguishable from slit and homozygous robo, robo2 double mutants. In the CNS, the FasII-expressing longitudinal fascicles of robo2, sdc double mutants converged into a single thick axon bundle at the ventral midline, resembling the effects seen with slit mutants. Similar observations were obtained with the mAb BP102 against all CNS axons, showing strongly condensed fascicles in robo2, sdc double mutant embryos. The synergistic strengthening of both the muscle and the CNS phenotypes in robo2, sdc double mutants, which are similar to a weak slit mutant phenotype, indicates that only some Slit-derived repellent activity is received along the midline. In contrast to robo2, the robo mutant phenotype was not significantly enhanced by the simultaneous lack of sdc. The data suggest that Robo can, in part, compensate for the lack of Robo2 and vice versa and that Robo is more sensitive to reduced Sdc-dependent Slit activity than Robo2 (Steigemann, 2003).

The results imply that sdc, slit, and robo are components of the same genetic circuitry. This proposal was tested by genetic means, asking whether the gene activities interact in vivo. Loss of only one copy of sdc led to the development of a normal muscle pattern, whereas the simultaneous absence of one copy of both slit and sdc in slit/+, sdc/+ double heterozygous embryos caused an increase in the number of longitudinal transverse muscles. Furthermore, the number of FasII-expressing inner fascicles that cross the midline is increased (3.3%) as compared to slit heterozygous embryos (0.6%;). More clearly, homozygous sdc mutant embryos, which also lack one copy of either robo, robo2, or slit, show an enhanced axonal guidance defect with multiple midline crossings of the fascicles (72%, 47%, and 95%, respectively), a phenotype very similar to the robo mutant. These results establish that Sdc acts in the same genetic circuitry as Slit and the Robo receptor family and represents a critical component of the Slit/Robo signaling pathway (Steigemann, 2003).

Axonal expression of Sdc suggests that its activity is required in the Robo-expressing Slit target cells. In order to test this inference, cell-specific rescue experiments were performed by expressing Sdc from either an elav-GAL4 or a sim-GAL4 driven UAS transgene in Robo-expressing neurons and Slit-expressing midline cells, respectively. The axon guidance defects of the sdc mutant were entirely rescued by Sdc expression in neurons, whereas no rescuing activity was seen in response to Sdc expression in midline cells. These findings indicate that sdc activity is not required for the production and/or secretion of Slit, but in the reception and/or the transmission of the signal in the target cells (Steigemann, 2003).

The results present evidence that axonal and myotube guidance in Drosophila require the transmembrane HSPG Sdc and that Sdc is an essential component of the Slit/Robo signaling pathway. These findings are consistent with earlier results showing that heparinase III treatment both weakens the Slit/Robo interaction and the biological effect of Slit signaling in mammalian cell culture, but is surprising in the light of the demonstration that mammalian Slit associates with the HSPG Glypican (Liang, 1999). Therefore dally and perlecan mutations were examined for potential interaction with Slit/Robo signaling and no effect was found. In addition, no defects were detected in sdc mutants reminiscent of impaired Hedgehog, Wingless, or FGF signaling, pathways previously shown to be sensitive to the levels of other HSPGs. Furthermore, Slit signaling in sdc mutants is also impaired in other tissues including the tracheal system. Thus, Slit/Robo signaling seems to primarily or even specifically require Sdc among the known Drosophila HSPGs. The difference in the nature of the HSPG that interacts with Slit in Drosophila and mammals may either reflect a species-dependent difference or a difference between the in vivo situation shown in this study and the previously published in vitro analysis. Other remarkable results are the spatially restricted requirement for Sdc as suggested by its expression domains adjacent to Slit-expressing cells and the finding that no defects, other than those related to Slit/Robo signaling, could be observed. Furthermore, overexpression of Sdc in the Slit target tissue did not interfere with proper Slit signaling. This result and the finding that overexpression of Sdc in the Slit-secreting midline glia cells also has no effect on axonal pathfinding and myotube guidance make it unlikely that Sdc functions as a simple ligand gatherer of soluble Slit. In addition, the rescue and protein localization studies indicate that Sdc functions in the target tissue and not in the Slit-secreting cells and acts in a Robo receptor-dependent manner. Sdc could, therefore, function to stabilize and/or transduce the Slit signal. Sdc may act as a coreceptor that presents Slit to Robo receptors or stabilizes the ligand receptor complex. Alternatively, signaling across the cell membrane might involve the Sdc-dependent organization of microdomains within the target cell membrane as has been proposed for vertebrate Sdc proteins (Steigemann, 2003).


Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila

Fragile X syndrome (FXS), the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1) gene product (FMRP), an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1) null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs): GPI-anchored glypican Dally-like protein (Dlp) and transmembrane Syndecan (Sdc). Earlier work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg) ligand abundance and downstream Frizzled-2 (Fz2) receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb), and downstream ERK phosphorylation (dpERK) are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb) and downstream signaling via phosphorylation of the transcription factor MAD (pMAD) seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1) Wg and Jeb trans-synaptic signaling, and (2) synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state (Friedman, 2013).

FXS is widely considered a disease state arising from synaptic dysfunction, with pre- and postsynaptic defects well characterized in the Drosophila disease model. There has been much work documenting FXS phenotypes in humans as well as in animal models, but there has been less progress on mechanistic underpinnings. This study focuses on the extracellular synaptomatrix in FXS owing to identification of pharmacological and genetic interactions between FMRP and secreted MMPs, a mechanism that is conserved in mammals. Other studies have also highlighted the importance of the synaptomatrix in synaptogenesis, particularly the roles of membrane-anchored HSPGs as co-receptors regulating trans-synaptic signaling. Importantly, it has been shown that FMRP binds HSPG mRNAs, thereby presumably repressing translation. Based on these multiple lines of evidence, this study hypothesized that the FMRP-MMP-HSPG intersection provides a coordinate mechanism for the pre- and postsynaptic defects characterizing the FXS disease state, with trans-synaptic signaling orchestrating synapse maturation across the synaptic cleft (Friedman, 2013).

In testing this hypothesis, a dramatic upregulation of GPI-anchored glypican Dlp and transmembrane Sdc HSPGs was discovered at dfmr1 null NMJ synapses. Indeed, these are among the largest synaptic molecular changes reported in the Drosophila FXS disease model. Importantly, HSPGs have been shown to play key roles in synaptic development. For example, the mammalian HSPG Agrin has long been known to regulate acetylcholine receptors, interconnected with a glycan network modulating trans-synaptic signaling. In Drosophila, Dlp, Sdc and Perlecan HSPGs mediate axon guidance, synapse formation and trans-synaptic signaling. Previous work on dlp mutants reports elevated neurotransmission, paradoxically similar to the Dlp overexpression phenotype shown in this study. However, the previous study does not show Dlp overexpression electrophysiological data, although it does show increased active zone areas consistent with strengthened neurotransmission. The same study reports that Dlp overexpression decreases bouton number on muscle 6/7, which differs from finding in this study of increased bouton number on muscle 4. Because HSPG co-receptors regulate trans-synaptic signaling, dfmr1 mutants were tested for changes in three established pathways at the Drosophila NMJ. Strong alterations in both Wg and Jeb pathways were found, with anterograde signaling being downregulated in both cases. In contrast, no change was found in the retrograde BMP Gbb pathway, suggesting that FMRP plays specific roles in modulating anterograde trans-synaptic signaling during synaptogenesis (Friedman, 2013).

The defect in Jeb signaling seems to be simple to understand, with decreased synaptomatrix ligand abundance coupled to decreased dpERK nuclear localization. However, there is no known link to HSPG co-receptor regulation. It has been shown earlier that Jeb signaling is regulated by another synaptomatrix glycan mechanism, providing a clear precedent for this level of regulation. In contrast, the Wnt pathway exhibits an inverse relationship between Wg ligand abundance (elevated) and Fz2-C nuclear signaling (reduced). This apparent contradiction is explained by the dual activity of the Dlp co-receptor, which stabilizes extracellular Wg to retain it at the membrane, but also competes with the Fz2 receptor. This ‘exchange-factor mechanism’ is competitively dependent on the ratio of Dlp co-receptor to Fz2 receptor, with a higher ratio causing more Wg to be competed away from Fz2. Indeed, it has been demonstrated that the same elevated Wg surface retention couples to decreased downstream Fz2-C signaling in an independent HSPG regulative mechanism at the Drosophila NMJ. This study suggests that in the dfmr1 null synapse, highly elevated Dlp traps Wg, thereby preventing it from binding Fz2 to initiate signaling (Friedman, 2013).

Dysregulation of the Wg nuclear import pathway (FNI) provides a plausible mechanism to explain synapse development defects underlying the FXS disease state, with established roles in activity-dependent modulation of synaptic morphogenesis and neurotransmission. FXS has long been associated with defects in activity-dependent architectural modulation, including postsynaptic spine formation, synapse pruning and functional plasticity. Although it is surely not the only player, aberrant Wg signaling could play a part in these deficiencies. Importantly, it has been shown that the FNI pathway is involved in shuttling large RNA granules out of the postsynaptic nucleus, providing a potential intersection with the FMRP RNA transport mechanism. However, the Wg FNI pathway is not the only Wnt signaling at the Drosophila NMJ, with other outputs including the canonical, divergent canonical and planar cell polarity pathways, which could be dysregulated in dfmr1 nulls. For example, a divergent canonical retrograde pathway proceeds through GSK3β (Shaggy) to alter microtubule assembly, and the FXS disease state is linked to dysregulated GSK3β and microtubule stability misregulation via Drosophila Futsch/mammalian MAP1B. Moreover, it has been shown that the secreted HSPG Perlecan (Drosophila Trol) regulates bidirectional Wnt signaling to affect Drosophila NMJ structure and/or function, via anterograde FNI and retrograde divergent canonical pathways. It is also important to note that previous studies show that a reduction in the FNI pathway, due to decreased Fz2-C trafficking to the nucleus, leads to decreased NMJ bouton number. Future work is needed to fully understand connections between FMRP, HSPGs, the multiple Wnt signaling pathways and the established defects in the synaptic microtubule cytoskeleton in the FXS disease state (Friedman, 2013).

Adding to the complications of FXS trans-synaptic signaling regulation, it was shown that two trans-synaptic signaling pathways are suppressed in parallel: the Wg and Jeb pathways. Possibly even more promising for clinical relevance, it has been established that the Jeb signaling functions as a repressor of neurotransmission strength at the Drosophila NMJ, with jeb and alk mutants presenting increased evoked synaptic transmission. Consistently, loss of FMRP leads to increased EJC amplitudes, which could be due, at least partially, to misregulated Jeb-Alk signaling. Importantly, it has been shown that dfmr1 null neurotransmission defects are due to a combination of pre- and postsynaptic changes, and that there is a non-cell-autonomous requirement for FMRP in the regulation of functional changes in the synaptic vesicle (SV) cycle underlying neurotransmission strength. Additionally, jeb and alk mutants exhibit synaptic structural changes consistent with this FMRP interaction, including a larger NMJ area and synaptic bouton maturation defects, which are markedly similar to the structural overelaboration phenotypes of the FXS disease state. These data together suggest that altered Jeb-Alk trans-synaptic signaling plays a role in the synaptic dysfunction characterizing the dfmr1 null. The study proposes that Wg and Jeb signaling defects likely interact, in synergistic and/or antagonistic ways, to influence the combined pre- and postsynaptic alterations characterizing the FXS disease state (Friedman, 2013).

Although trans-synaptic signaling pathways, and in particular both Wnt and Jeb-Alk pathways, have been proposed to be involved in the manifestation of a number of neurological disorders, this study provides the first evidence that aberrant trans-synaptic signaling is causally involved in an FXS disease model. The study proposes a mechanism in which FMRP acts to regulate trans-synaptic ligands by depressing expression of membrane-anchored HSPG co-receptors. HSPG overexpression alone is sufficient to cause both synaptic structure and function defects characterizing the FXS disease state. Increasing HSPG abundance in the postsynaptic cell is enough to increase the number of presynaptic branches and synaptic boutons, as well as elevate neurotransmission. Correlation with these well-established dfmr1 null synaptic phenotypes suggests that HSPG elevation could be a causal mechanism. Conclusively, reversing HSPG overexpression in the dfmr1 null is sufficient to correct Wnt and Jeb signaling, and to restore normal synaptic structure and function. Because there is no dosage compensation, HSPG heterozygosity offsets the elevation caused by loss of dfmr1. Correcting both Dlp and Sdc HSPGs in the dfmr1 background restores Wg and Jeb signaling to control levels. Correcting Dlp levels by itself restores synaptic architecture, but both Dlp and Sdc have to be corrected to restore normal neurotransmission in dfmr1 null synapses. Taken together, these results from the Drosophila FXS disease model provide exciting new insights into the mechanisms of synaptic phenotypes caused by the loss of FMRP, and promising avenues for new therapeutic treatment strategies (Friedman, 2013).

Protein Interactions

The LG4 (laminin G-like) domain of the laminin alpha4 chain is responsible for the significantly higher affinity of the alpha4 chain to heparin than found for other alpha chains; four basic residues were identified to be essential for this activity. By creating GST (glutathione S-transferase)-fused LG1, LG2, LG4 and LG5 proteins, it was found that only LG4 is active for the adhesion of human HT1080 cells, human umbilical vein endothelial cells and Drosophila haemocytes Kc167 with a half-saturating concentration of 20 microg/ml. Adhesion was counteracted by treatment of the cells with heparin, heparan sulphate and heparitinase I. Upon mutating the four basic residues essential for heparin binding within LG4, the adhesion activity was abolished. Pull-down experiments using glutathione beads/GST-fusion proteins indicate a direct interaction of LG4 with syndecan-4, which might be the major receptor for cell adhesion. Neither the release of glypican-1 by treating human cells with phosphatidylinositol-specific phospholipase C nor targeted knockdown of dally or dally-like protein impaired the cell-adhesion activity. Since the LG4-LG5 domain of the alpha4 chain is cleaved in vivo from the main body of laminin-8 (alpha4beta1gamma1), it is suggested that the heparan sulphate proteoglycan-binding activity of LG4 is significant in modulating the signalling of Wnt, Decapentaplegic and fibroblast growth factors (Yamashita, 2004).

Heparin-column chromatography and elastase-digestion of medium from hemocyte Kc167 gave Drosophila laminin alpha3/5betagamma trimer, alpha3/5LG2-3 and alpha3/5LG4-5 modules with eluting NaCl concentrations of 450, 280 and 450 mM, respectively. Kc167 cells bound dish surface with alpha3/5betagamma trimer or alpha3/5LG4-5, but not with alpha3/5LG2-3 modules. Cell binding was counteracted by treating with heparin or heparan sulfate. RNA interference of Syndecan in Kc167 cells impaired the binding, but that of dally or dally-like did not. Green fluorescent protein-expressing hemocytes also bound surface with alpha3/5betagamma trimer or alpha3/5LG4-5 module. Thus, Syndecan-dependent binding of hemocytes to laminin may have a potential role in sessile hemocytes islets formation in T2-A8 segments of Drosophila (Narita, 2004).

The heparan sulfate proteoglycan syndecan is an in vivo ligand for the Drosophila LAR receptor tyrosine phosphatase

Receptor tyrosine phosphatases (RPTPs) are essential for axon guidance and synaptogenesis in Drosophila. Each guidance decision made by embryonic motor axons during outgrowth to their muscle targets requires a specific subset of the five neural RPTPs. The logic underlying these requirements, however, is still unclear, partially because the ligands recognized by RPTPs at growth cone choice points have not been identified. RPTPs in general are still 'orphan receptors' because, while they have been found to interact in vitro with many different proteins, their in vivo ligands are unknown. This study uses a new type of deficiency screen to identify the transmembrane heparan sulfate proteoglycan Syndecan (Sdc) as a ligand for the neuronal RPTP LAR. LAR interacts with the glycosaminoglycan chains of Syndecan in vitro with nanomolar affinity. Genetic interaction studies using Sdc and Lar LOF mutations demonstrate that Sdc contributes to LAR's function in motor axon guidance. Overexpression of Sdc on muscles is shown to generate the same phenotype as overexpression of LAR in neurons, and genetic removal of LAR suppresses the phenotype produced by ectopic muscle Sdc. Finally, it is shown that there is at least one additional, nonproteoglycan, ligand for LAR encoded in the genome. Taken together, these results demonstrate that Sdc on muscles can interact with neuronal LAR in vivo and that binding to Sdc increases LAR's signaling efficacy. Thus, Sdc is a ligand that can act in trans to positively regulate signal transduction through LAR within neuronal growth cones (Fox, 2005).

Genetic removal of Sdc from embryos bearing Lar mutations increases the penetrance of the characteristic Lar ISNb bypass phenotype. This effect on penetrance is as large as those usually observed when a second RPTP is genetically removed from a single Rptp mutant (e.g., removal of DPTP69D from a Lar mutant). Removal of Sdc increases penetrance for both hypomorphic and (zygotic) null Lar mutations. The effect on the null penetrance is likely due to reduction of maternally contributed LAR function. However, it was found that Sdc mutations alone do not produce ISNb bypass at a significant frequency, even when both maternal and zygotic Sdc are removed. One explanation for this finding might be that Sdc is partially redundant with Dally and/or Dlp, since these are cell-surface HSPGs expressed in a similar pattern to Sdc (Fox, 2005).

To test this model, Dally and Dlp expression were reduced in an Sdc mutant background. It is difficult to assess the appropriate extent of reduction for this experiment. Glypicans cannot be completely removed, since this would produce embryos with severe early phenotypes due to loss of Hedgehog and Wingless signaling. Zygotic triple mutants (Sdc dally dlp) were generated, and, also, dally and dlp dsRNAs were injected into Sdc maternal/zygotic mutant embryos (dally/dlp RNAi would affect both maternal and zygotically contributed mRNAs). The genetic triple mutants had CNS phenotypes that are stronger than the Sdc phenotype but displayed few motor axon guidance errors. Dally/Dlp-injected Sdc mutant embryos had more severe phenotypes, but ISNb guidance was not selectively affected. Overall, the data suggest that Sdc is not redundant with Dally or Dlp and that its absence is likely to be compensated for by non-HSPG proteins. Perhaps the second LAR ligand detected by embryo staining is redundant with Sdc. Like Sdc, this ligand is expressed both on CNS axons and in lines in the periphery (Fox, 2005).

A genetic epistasis experiment demonstrates that Sdc acts in trans (as a ligand) to regulate LAR function. However, the data allow this conclusion to be reached only for SNa bifurcation, which is affected by LAR overexpression but not by loss of LAR. In its regulation of the decision of ISNb growth cones to enter the muscle field, Sdc could also act as a ligand, since it is expressed at the appropriate time on patches of cells near the muscle field entry site that could be contacted by LAR-expressing ISNb growth cones at this choice point. Alternatively, Sdc could act as a coreceptor at this choice point since it is expressed on the motor nerves together with LAR. Finally, it is not known if the Sdc that interacts with LAR during ISNb axon guidance is attached to the cell surface or has been shed by proteolytic cleavage. If released then Sdc is the essential ligand; Sdc could be expressed by either muscles or neurons and transported to the choice point (Fox, 2005).

These results show that the cell-surface HSPG Sdc is an in vivo ligand for LAR and indicate that it positively regulates LAR signaling during motor axon guidance. Sdc’s GAG chains bind directly to LAR with high affinity, and this binding requires basic sequences in the first Ig domain of LAR. Further work will be required to determine whether binding to Sdc directly stimulates LAR’s phosphatase activity, relocalizes LAR within the growth cone,or facilitates LAR signaling by another mechanism (Fox, 2005).

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. The Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function (Johnson, 2006).

Converging lines of evidence suggest that membrane-associated HSPGs serve an important purpose in the assembly, function and plasticity of excitatory synapses. The ancient HSPG families of syndecans and glypicans are necessary for Drosophila to regulate distinct aspects of synaptic morphogenesis. Genetic and biochemical data indicate that Sdc and Dlp interact with LAR to control presynaptic properties. Because LAR-family RPTPs have been shown to control the formation of excitatory synapses in Drosophila, C. elegans, and mammals, these findings may represent a more general mechanism for regulating synaptic morphogenesis and function. Despite the importance of LAR-family RPTPs during cellular morphogenesis inside and outside of the nervous system, the lack of physiologically relevant extracellular binding partners has made it challenging to study this well-conserved group of receptors (Johnson, 2006).

The data show that Sdc promotes the formation of presynaptic boutons. This Sdc function appears to be mediated by LAR, as supported by parallel phenotypes, direct binding, in vivo colocalization, and three types of double mutant analysis between Sdc and LAR. Despite the fact that Sdc exhibits gain-of-function activity and endogenous expression on both sides of the synapse, neuronal and muscle-specific rescue experiments show that Sdc function is mainly presynaptic. Because LAR is required only in neurons to promote synapse growth, these findings support a model in which Sdc acts as a neuronal cell-autonomous agonist of LAR. This is somewhat surprising, given the fact that soluble forms of Sdc bind to LAR and that endogenous Sdc appears to be released from the presynaptic membrane to fill the subsynaptic reticulum. One way for Sdc to act presynaptically would be to bind to LAR even before the two proteins are presented on the neuronal surface. Because Dlp has a competitive advantage over Sdc for binding to LAR, a prebound complex of Sdc and LAR would have the ability to stimulate synapse growth before the phosphatase could be inhibited by Dlp. Such a mechanism could provide a time- and/or HSPG concentration-dependent switch from bouton addition to active zone assembly (Johnson, 2006).

Sdc could promote LAR activity in collaboration with an additional cell-type-specific membrane protein. Data from a parallel study has also identified Sdc-LAR interactions during embryonic motor axon guidance. However, in contrast to CNS pathfinding, complete loss of Sdc alone has no significant effect on motor pathfinding, suggesting that additional LAR ligands exist in the early embryo. Recent experiments with the vertebrate LAR ortholog PTP-s suggest that non-HSPG ligands may regulate its ability to promote retinal axon outgrowth. Although it remains a formal possibility, an additional ligand may not be needed to account for the NMJ growth-promoting activity of LAR because the larval synaptic phenotype in Sdc mutants is nearly as strong as the growth defect in LAR mutants (Johnson, 2006).

Active zone assembly is vital for neurotransmission at the synapse and has been proposed as a means to modulate synaptic function over time. Analysis of Dlp reveals that synaptic glypicans are required to regulate active zone morphology and function. Moreover, Dlp is limiting for active zone morphogenesis, consistent with an instructive role. Because the activities of Dlp appear opposite to those of LAR, it is proposed that the high affinity binding of Dlp to LAR induces an inhibition of receptor function. This hypothesis is supported by the double RNAi experiments showing that the LAR effect on Ena phosphorylation is epistatic to the effect of Dlp, indicating that Dlp acts upstream of LAR. Because loss of Dlp at the NMJ did not induce a significant change in the number of presynaptic boutons, the results lead to a model in which Dlp is specialized for control of active zone properties. Such a function might provide a means to independently regulate and spatially distinguish LAR inhibition from LAR activation. In any case, the presence of active zone phenotypes in dlp but not in Sdc reveals specialization among synaptic HSPGs (Johnson, 2006).

LAR regulates both NMJ growth and active zone morphogenesis. Thus, LAR appears to provide a link between two important synaptic properties that are regulated by different extracellular factors. A mechanism to couple bouton growth and active zone formation would make sense because active zones appear early in the nascent bouton. Because LAR catalytic activity is necessary for bouton addition, and yet LAR inhibition by Dlp appears necessary for proper active zone formation, LAR's role at the active zone may be primarily structural. For example, LAR may simply provide an anchorage point for synaptic components like the scaffolding protein Liprin-alpha that regulates active zone formation. Alternatively, LAR may exist in distinct yet active signaling states, one of which is dependent on PTP activity (promoting synapse growth), and one of which is dependent on recruitment of signaling molecules (controlling active zone assembly). Because loss of Dlp or LAR has opposite effects on quantal content at the NMJ, it is attractive to speculate that the Dlp-LAR pathway normally provides a means to modulate the strength of neurotransmission, either during NMJ growth or during synaptic plasticity. LAR PTPs are required for normal physiology and plasticity at mammalian hippocampal synapses (Johnson, 2006).

Sdc and Dlp are both HSPGs that bind to LAR and thus might be expected to act similarly, but the results show that their functions are different. One way to account for the specificity might be a difference in the effect of soluble versus cell-surface HSPGs on LAR. Some ligand molecules such as Ephrins function when clustered at high density (e.g., on a membrane surface) but fail to activate their receptors when presented in a soluble, monomeric form. Another possibility could be that LAR binding or signaling is differentially influenced by direct protein-protein interactions with the Sdc versus Dlp core proteins. The two HSPGs have very different core structures; Sdc is a transmembrane molecule with HS modification sites near the N terminus, whereas Dlp is a GPI-anchored protein with HS sites proximal to the membrane and a large disulphide bonded globular domain located more distally. It may also be relevant that Dlp consistently binds more effectively to LAR than Sdc, with KD measurements in solution showing an affinity approximately 2-fold higher. These results suggest a competition model in which Dlp displaces Sdc, possibly to favor the stabilization of active zones after new growth at the synapse. In this model, presynaptic growth would be initially promoted by Sdc and would then be limited or halted by Dlp binding after formation of close membrane contact between the nerve and muscle. Such a mechanism could insure a transition from growth to synapse stabilization and could participate in subsequent maintenance or plasticity of the synapse (Johnson, 2006).

Of course other molecules influence synapse size, and these might include coligands or coreceptors that may bind to Sdc, Dlp, and/or LAR. Potential candidates might include bone morphogenic protein (BMP), the type II BMP receptor Wishful thinking (Wit), or the Wnt ortholog Wingless (Wg), which have all been shown to regulate NMJ morphology in Drosophila. However, in addition to significant phenotypic differences compared to the HSPGs, overexpression studies indicate that neither BMP nor Wg are limiting for NMJ morphogenesis. In contrast, Sdc and Dlp are both limiting for different aspects of synapse development, consistent with an instructive role in this context. Consistent with this notion, Syndecan-2 is sufficient to promote dendritic spine maturation during hippocampal synaptogenesis in culture. Although vertebrate Syndecan-2 has yet to be tested at the synapse by loss of function, the colocalization and parallel biology of Synecan-2 and vertebrate LAR-family receptors strongly suggest conservation in the regulation of synaptic LAR (Johnson, 2006).

The genetic and biochemical studies described in this study have identified a partnership between HSPGs and LAR in Drosophila NMJ development that sets precedents for (1) the in vivo requirement for members of the syndecan and glypican families in synapse growth and electrophysiological function, (2) the specificity of HSPG function at the synapse, with distinct actions of Sdc and Dlp, and (3) biochemical identification of Sdc and Dlp as LAR binding partners, plus genetic evidence to place them in a pathway regulating biological function at the synapse (Johnson, 2006).

A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling

A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product,functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation, were characterized. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects (Dani, 2012).

It is well known that synaptic interfaces harbor heavily-glycosylated membrane proteins, glycolipids and ECM molecules, but understanding of glycan-mediated mechanisms within this synaptomatrix is limited. A genomic screen aimed to systematically interrogate glycan roles in both structural and functional development in the genetically-tractable Drosophila NMJ synapse. 130 candidate genes were screened, classified into 8 functional families: N-glycan biosynthesis, O-glycan biosynthesis, GAG biosynthesis, glycoprotein/proteoglycan core proteins, glycan modifying/degrading enzymes, glycosyltransferases, sugar transporters and glycan-binding lectins. From this screen, 103 RNAi knockdown conditions were larval viable, whereas 27 others produced early developmental lethality. 35 genes had statistically significant effects on different measures of morphological development: 27 RNAi-mediated knockdowns increased synaptic bouton number, 9 affected synapse area (2 increased, 7 decreased) and 2 genes increased synaptic branch number. These data suggest that overall glycan mechanisms predominantly serve to limit synaptic morphogenesis. 13 genes had significant effects on the functional differentiation of the synapse, with 12 increasing transmission strength and only 1 decreasing function upon RNAi knockdown. Thus, glycan-mediated mechanisms also predominantly limit synaptic functional development. A very small fraction of tested genes (CG1597; pgant35A, CG7480; veg, CG6657; hs6st, CG4451; sulf1, CG6725 and CG11874) had effects on both morphology and function. A large percentage of genes (~30%) showed morphological defects with no corresponding effect on function, while only 7% of genes showed functional alterations without morphological defects, and <5% of all genes affect both. These results suggest that glycans have clearly separable roles in modulating morphological and functional development of the NMJ synapse (Dani, 2012).

A growing list of neurological disorders linked to the synapse are attributed to dysfunctional glycan mechanisms, including muscular dystrophies, cognitive impairment and autism spectrum disorders. Drosophila homologs of glycosylation genes implicated in neural disease states include ALG3 (CG4084), ALG6 (CG5091), DPM1 (CG10166), FUCT1 (CG9620), GCS1 (CG1597), MGAT2 (CG7921), MPDU1 (CG3792), PMI (CG33718) and PPM2 (CG12151). Two of these genes, Gfr (CG9620) and CG1597, showed synaptic morphology phenotypes in the RNAi screen. Given that connectivity defects are clearly implicated in cognitive impairment and autism spectrum disorders, it would be of interest to explore the glycan mechanism affecting synapse morphology in Drosophila models of these disease states. Glycans are well known to modulate extracellular signaling, including ligands of integrin receptors, to regulate intercellular communication. In the genetic screen, several O-glycosyltransferases mediating this mechanism were identified to show morphological (GalNAc-T2, CG6394; pgant35A, CG7480, O-fut2, CG14789; rumi, CG31152) and functional (pgant5, CG31651; pgant35A, CG7480) synaptic defects upon RNAi knockdown. These findings suggest that known integrin-mediated signaling pathways controlling NMJ synaptic structural and functional development are modulated by glycan mechanisms. The screen showed CG6657 RNAi knockdown affects functional differentiation, consistent with reports that this gene regulates peripheral nervous system development. The corroboration of the screen results with published reports underscores the utility of RNAi-mediated screening to identify glycan mechanisms, and supports use of the screen results for bioinformatic/meta-analysis to link observed phenotypes to neurophysiological/pathological disease states and to direct future glycan mechanism studies at the synapse (Dani, 2012).

From this screen, the two functionally-paired genes sulf1 and hs6st were selected for further characterization. As in the RNAi screen, null alleles of these two genes had opposite effects on synaptic functional differentiation but similar effects on synapse morphogenesis, validating the corresponding screen results. The two gene products have functionally-paired roles; Hs6st is a heparan sulfate (HS) 6-O-sulfotransferase, and Sulf1 is a HS 6-O-endosulfatase. These activities control sulfation of the same C6 on the repeated glucosamine moiety in HS GAG chains found on heparan sulfate proteoglycans (HSPGs). At the Drosophila NMJ, two HSPGs are known to regulate synapse assembly; the GPI-anchored glypican Dally-like protein (Dlp), and the transmembrane Syndecan (Sdc). In contrast, the secreted HSPG Perlecan (Trol) is not detectably enriched at the NMJ, and indeed appears to be selectively excluded from the perisynaptic domain. In other developmental contexts, the membrane HSPGs Dlp and Sdc are known to act as co-receptors for WNT and BMP ligands, regulating ligand abundance, presentation to cognate receptors and therefore signaling. Importantly, the regulation of HSPG co-receptor abundance has been shown to be dependent on sulfation state mediated by extracellular sulfatases. Consistently, upregulation of Dlp and Sdc was observed in sulf1 null synapses, whereas Dlp was reduced in hs6st null synapses. In the developing Drosophila wing disc, HSPG co-receptors increase levels of the Wg ligand due to extracellular stabilization, and the primary function of Dlp in this developmental context is to retain Wg at the cell surface. Likewise, in developing Drosophila embryos, a significant fraction of Wg ligand is retained on the cell surfaces in a HSPG-dependent manner, with the HSPG acting as an extracellular co-receptor. Syndecan also modulates ligand-dependent activation of cell-surface receptors by acting as a co-receptor. At the NMJ, regulation of both these HSPG co-receptors occurs in the closely juxtaposed region between presynaptic bouton and muscle subsynaptic reticulum, in the exact same extracellular space traversed by the secreted trans-synaptic Wg and Gbb signals. It is therefore proposed that altered Dlp and Sdc HSPG co-receptors in sulf1 and hs6st mutants differentially trap/stabilize Wg and Gbb trans-synaptic signals at the interface between motor neuron and muscle, to modulate the extent and efficacy of intercellular signaling driving synaptic development (Dani, 2012).

HS sulfation modification is linked to modulating the intercellular signaling driving neuronal differentiation . In particular, WNT and BMP ligands are both regulated via HS sulfation of their extracellular co-receptors, and both signals have multiple functions directing neuronal differentiation, including synaptogenesis. In the Drosophila wing disc, extracellular WNT (Wg) ligand abundance and distribution was recently shown to be strongly elevated in sulf1 null mutants. Moreover, sulf1 has also recently been shown to modulate BMP signaling in other cellular contexts. Consistently, this study has shown increased WNT Wg and the BMP Gbb abundance and distribution in sulf1 null NMJ synapses. The hs6st null also exhibits elevated Wg and Gbb at the synaptic interface, albeit the increase is lower and results in differential signaling consequences. In support of this contrasting effect, extracellular signaling ligands are known to bind HSPG HS chains differentially dependent on specific sulfation patterns. It is important to note that the sulf1 and hs6st modulation of trans-synaptic signals is not universal, as Jelly Belly (Jeb) ligand abundance and distribution was not altered in the sulf1 and hs6st null conditions. This indicates that discrete classes of secreted trans-synaptic molecules are modulated by distinct glycan mechanisms to control NMJ structure and function (Dani, 2012).

At the Drosophila NMJ, Wg is very well characterized as an anterograde trans-synaptic signal and Gbb is very well characterized as a retrograde trans-synaptic signal. In Wg signaling, the dFz2 receptor is internalized upon Wg binding and then cleaved so that the dFz2-C fragment is imported into muscle nuclei. In hs6st nulls, increased Wg ligand abundance at the synaptic terminal corresponds to an increase in dFz2C punctae in muscle nuclei as expected. In contrast, the increase in Wg at the sulf1 null synapse did not correspond to an increase in the dFz2C-terminus nuclear internalization, but rather a significant decrease. One explanation for this apparent discrepancy is the 'exchange factor' model based on the biphasic ability of the HSPG co-receptor Dlp to modulate Wg signaling. In the Drosophila wing disc, this model suggests that the transition of Dlp co-receptor from an activator to repressor of signaling depends on Wg cognate receptor dFz2 levels, such that a low ratio of Dlp:dFz2 potentiates Wg-dFz2 interaction, whereas a high ratio of Dlp:dFz2 prevents dFz2 from capturing Wg. In sulf1 null synapses, a very great increase was observed in Dlp abundance (~40% elevated) with no significant change in the dFz2 receptor. In contrast, at hs6st null synapses there is a decrease in Dlp abundance (15% decreased) together with a significant increase in dFz2 receptor abundance (~25% elevated). Thus, the higher Dlp:dFz2 ratio in sulf1 nulls could explain the decrease in Wg signal activation, evidenced by decreased dFz2-C terminus import into the muscle nucleus. In contrast, the Dlp:Fz2 ratio in hs6st is much lower, supporting activation of the dFz2-C terminus nuclear internalization pathway. This previously proposed competitive binding mechanism dependent on Dlp co-receptor and dFz2 receptor ratios predicts the observed synaptic Wg signaling pathway modulation in sulf1 and hs6st dependent manner (Dani, 2012).

At the Drosophila NMJ, Gbb is very well characterized as a retrograde trans-synaptic signal, with muscle-derived Gbb causing the receptor complex Wishful thinking (Wit), Thickveins (Tkv) and Saxaphone (Sax) to induce phosphorylation of the transcription factor mothers against Mothers against decapentaplegic (P-Mad). Mutation of Gbb ligand, receptors or regulators of this pathway have shown that Gbb-mediated retrograde signaling is required for proper synaptic differentiation and functional development. Further, loss of Gbb signaling results in significantly decreased levels of P-Mad in the motor neurons. This study shows that accumulation of Gbb in sulf1 and hs6st null synapses causes elevated P-Mad signaling at the synapse and P-Mad accumulation in motor neuron nuclei. Importantly, sulf1 null synapses show a significantly higher level of P-Mad signaling compared to hs6st null synapses, and this same change is proportionally found in P-Mad accumulation within the motor neuron nuclei. These findings indicate differential activation of Gbb trans-synaptic signaling dependent on the HS sulfation state is controlled by the sulf1 and hs6st mechanism, similar to the differential effect observed on Wg trans-synaptic signaling. Genetic interaction studies show that these differential effects on trans-synaptic signaling have functional consequences, and exert a causative action on the observed bi-directional functional differentiation phenotypes in sulf1 and hs6st nulls. Genetic correction of Wg and Gbb defects in the sulf1 null background restores elevated transmission back to control levels. Similarly, genetic correction of Wg and Gbb in hs6st nulls restores the decreased transmission strength back to control levels. These results demonstrate that the Wg and Gbb trans-synaptic signaling pathways are differentially regulated and, in combination, induce opposite effects on synaptic differentiation (Dani, 2012).

Both wg and gbb pathway mutants display disorganized and mislocalized presynaptic components at the active zone (e.g. Bruchpilot; Brp) and postsynaptic components including glutamate receptors (e.g. Bad reception; Brec/GluRIID). Consistently, the bi-directional effects on neurotransmission strength in sulf1 and hs6st mutants are paralleled by dysregulation of these same synaptic components. Changes in presynaptic Brp and postsynaptic GluR abundance/distribution causally explain the bi-directional effects on synaptic functional strength between sulf1 and hs6st null mutant states. Alterations in active zone Brp and postsynaptic GluRs also agree with assessment of spontaneous synaptic activity. Null sulf1 and hs6st synapses showed opposite effects on miniature evoked junctional current (mEJC) frequency (presynaptic component) and amplitude (postsynaptic component). Further, quantal content measurements also support the observation of bidirectional synaptic function in the two functionally paired nulls. Genetic correction of Wg and Gbb defects in both sulf1 and hs6st nulls restores the molecular composition of the pre- and postsynaptic compartments back to wildtype levels. When both trans-synaptic signaling pathways are considered together, these data suggest that HSPG sulfate modification under the control of functionally-paired sulf1 and hs6st jointly regulates both WNT and BMP trans-synaptic signaling pathways in a differential manner to modulate synaptic functional development on both sides of the cleft (Dani, 2012).

This paper has presented the first systematic investigation of glycan roles in the modulation of synaptic structural and functional development. A host of glycan-related genes were identified that are important for modulating neuromuscular synaptogenesis, and these genes are now available for future investigations, to determine mechanistic requirements at the synapse, and to explore links to neurological disorders. As proof for the utilization of these screen results, this study has identified extracellular heparan sulfate modification as a critical platform of the intersection for two secreted trans-synaptic signals, and differential control of their downstream signaling pathways that drive synaptic development. Other trans-synaptic signaling pathways are independent and unaffected by this mechanism, although it is of course possible that a larger assortment of signals could be modulated by this or similar mechanisms. This study supports the core hypothesis that the extracellular space of the synaptic interface, the heavily-glycosylated synaptomatrix, forms a domain where glycans coordinately mediate regulation of trans-synaptic pathways to modulate synaptogenesis and subsequent functional maturation (Dani, 2012).



Syndecan is prominent in lymph glands (hematopoetic organs), in the PNS and CNS and along the basal surfaces of gut ectoderm (Spring, 1994).

The majority of cell surface HS polymers are carried by two classes of HSPGs: the transmembrane Syndecans and the glysosylphosphatidyl inositol (GPI)-linked Glypicans. While mammalian species express a number of genes in each of these classes, Drosophila has one Syndecan gene (sdc) and two Glypican genes (dally and dallylike). Although some degree of core-protein specificity has been observed in functional comparisons between different HSPGs during early pattern formation and cell fate determination, true loss-of-function mutations have yet to be analyzed for sdc and dallylike (dlp). Although anti-Sdc antibodies had been previously described, this study has developed methods to image Sdc protein at improved resolution and showed that the antibody recognizes Sdc and that Sdc localizes to both longitudinal and commissural axons pathways within the Drosophila embryonic CNS (Johnson, 2004).


The presentation of secreted axon guidance factors plays a major role in shaping central nervous system connectivity. Recent work suggests that heparan sulfate (HS) regulates guidance factor activity; however, the in vivo axon guidance roles of its carrier proteins (heparan sulfate proteoglycans, or HSPGs) are largely unknown. The HSPG Syndecan (Sdc) is critical for the fidelity of Slit repellent signaling at the midline of the Drosophila CNS, consistent with the localization of Sdc to CNS axons. sdc mutants exhibit consistent defects in midline axon guidance, plus potent and specific genetic interactions supporting a model in which HSPGs improve the efficiency of Slit localization and/or signaling. Testing this hypothesis, Slit distribution was shown to be altered in sdc mutants and Slit and its receptor were shown to bind to Sdc. However, when the function of the transmembrane Sdc was compared to a different class of HSPG that localizes to CNS axons (Dallylike), functional redundancy was found, suggesting that these proteoglycans act as spatially specific carriers of common HS structures that enable growth cones to interact with and perceive Slit as it diffuses away from its source at the CNS midline (Johnson, 2004).

The availability of two P-element insertions (P10608 and KG06163) into the sdc locus made it possible to address sdc function by making a small excision-induced deletion [Df(2R)48] removing the first two exons of sdc, including the promotor and 5' untranslated region, the translational start codon, and the signal sequence. To confirm the prediction that this deletion eliminates Sdc protein expression, Df(2R)48 embryos were stained with an antibody that recognizes the extracelluar domain near the transmembrane region of Sdc (Spring, 1994). Only a minute residual signal was found that may represent either nonspecific background or limited perdurance of maternally loaded Sdc. Importantly, simultaneous staining of these mutants with anti-Robo antibodies revealed that although levels of Robo expression appeared normal in Df(2R)48, Robo-positive axons now crossed the CNS midline where Slit concentration is normally at its highest. It was also found that ventral muscles overshoot their insertion sites in strong sdc mutants, reminiscent of defects in slit mutants. To be certain that perturbation of the neighboring genes sara and FKB13 was not responsible for the guidance errors, mutations in each locus were examined and also a sara rescue construct was introduced into the Df(2R)48 background. It was found that these flanking genes do not contribute to the midline phenotype of Df(2R)48. Because HS is known to facilitate cell fate decisions in other contexts, both neuronal and midline glial patterning were examined, but no defects were found. Moreover, levels of Slit expression in midline glia appeared to be comparable to wild-type levels (Johnson, 2004).

To quantify the midline guidance defects in different sdc alleles, mAb 1D4 was used to visualize ipsilateral axon fascicles and scored for the frequency of ectopic midline crossing. An allelic series of phenotypic penetrance was found ranging from 5%-40%, suggesting that the efficiency of axon guidance depends on the amount of Sdc present. However, because Sdc family members are known to be proteolytically processed and released from the cell surface, it was important to determine whether Sdc functions autonomously in axons. A sdc cDNA under the control of the GAL4 upstream activating sequence was used, and its ability to rescue the Df(2R)48 phenotype under the control of either a midline glial-specific GAL4 source (slit-GAL4) or a postmitotic neuron-specific source (elav-GAL4) was examined. Only neuronal expression of Sdc rescues the guidance errors, suggesting that Sdc acts locally to increase growth cone sensitivity to Slit (Johnson, 2004).

Because sdc axon phenotypes suggest the Slit signaling system's failure to restrict midline crossing, it was asked if sdc mutations display specific genetic interactions with mutations in slit or its receptors. Using the same assay that identified Slit as the Robo ligand, embryos transheterozygous for sdc and mutations in several loci were compared. Highly significant (p < 0.005) interactions were found between sdc and both slit and robo in this assay. This interaction appears to be specific because no enhancement is observed when sdc is combined with a mutation in the receptor tyrosine phosphatase gene DPTP69D, which is known to contribute to midline guidance. Although no interaction is seen between sdc and single mutations in robo2 or robo3, crosses between sdc and double mutants removing robo and one of the other robo family genes (e.g., robo, robo2) reveal significant increases in the interaction when they are compared to robo alone. These genetic results suggest that Sdc acts in the Slit-Robo pathway (Johnson, 2004).

A biochemical assay was developed to determine whether Sdc binds to Slit and/or Robo in cellular extracts in which all three proteins are endogenously expressed. Immunoprecipitation of either Slit or Robo and subsequent detection with anti-Sdc antibodies reveals that Sdc associates with both Slit and its receptor, suggesting the possibility of a ternary complex. This association is specific because no Sdc is trapped by nonspecific IgG or N-Cadherin antibodies that successfully immunoprecipitate other signaling molecules. Thus, Sdc participates directly in a complex with Slit and Robo (Johnson, 2004).

Having gathered multiple lines of evidence revealing a role for Sdc in Slit signaling, popular models for the underlying mechanism could be tested. HS has been proposed to support the patterning activity of several secreted ligands by restricting their diffusion from a focal source. If this were the case for Sdc in Slit signaling, a change in the distribution of Slit at sites distant from the midline glia would be predicted. To examine this, an immunohistochemical method was optimized to allow visualization of Slit protein not only on the surface of midline glia but also within the CNS neuropil. No gross qualitative difference was found in the pattern of Slit distribution between Df(2R)48 and wild-type controls. However, rigorous quantitative analysis of confocal images with an independent axon surface marker (LAR) as an internal control to ensure comparable signal strength reveals that sdc mutants show a highly significant change in the pattern of Slit accumulation throughout the neuropil. Slit is still highly expressed on midline glia in sdc mutants, but the Slit signal is significantly reduced in the neuropil. These data are highly reproducible and are consistent with the ligand-trapping model anticipated from previous studies on HS (Johnson, 2004).

The striking difference in protein structure between Syndecans and Glypicans raises two related questions: (1) is there core-protein specificity to HSPG function on the growth cone surface, or are these proteins acting mainly as carriers for a common HS structure? and (2) are the cytoplasmic domains of Sdc, known to interact with signaling proteins inside the cell, essential for Sdc function during axon guidance? Both of these questions were answered by testing the specificity of Sdc relative to anther HSPG. Of course, it was important to compare Sdc to an HSPG normally localized to axons if possible. Using an existing antibody, it was possible to show that Glypican Dallylike (Dlp) is expressed on the surface of embryonic axons in a pattern nearly identical to that of Sdc. This antibody recognizes Dlp ectopically expressed by a UAS-dlp transgene. When UAS-dlp was expressed in a Df(2R)48 background under the control of a Slit-GAL4 driver, no significant rescue was found of the sdc midline phenotype. However, neuron-specific expression of Dlp generated a highly significant degree of functional rescue (p < 0.005). This clearly shows that an increase in Dlp expression can compensate for the loss of Sdc, consistent with the finding that Slit can bind to at least one mammalian Glypican. In addition, this experiment shows that the unique intracellular signaling motifs found in Sdc are not essential for Slit signaling (Johnson, 2004).

In conclusion, it has been found that Sdc localizes to developing axons, is required for accurate growth cone navigation at the CNS midline, and interacts genetically and physically with Slit and Robo. Although a full account of HSPG functional specificity awaits the analysis of mutations in dallylike, the fact that both Syndecan and Glypican can serve interchangeably to improve the efficiency of growth cone repulsion suggests a model in which the total amount of cell surface HS determines the sensitivity of Robo-expressing growth cones to the midline repellent. Of course, the possibility cannot be ruled out that the highly conserved cytoplasmic domains of Sdc play a more subtle modulatory function in this context or an essential function in some distinct context. This might explain the difference in the efficiency of Dlp and Sdc in the rescue of sdc guidance defects. These and other answers will come from future dissection of the Sdc mechanism and the action of HSPGs in neural development (Johnson, 2004).

The heparan sulfate proteoglycans Dally-like and Syndecan have distinct functions in axon guidance and visual-system assembly in Drosophila

Heparan sulfate proteoglycans (HSPGs), a class of glycosaminoglycan-modified proteins, control diverse patterning events via their regulation of growth-factor signaling and morphogen distribution. In C. elegans, zebrafish, and the mouse, heparan sulfate (HS) biosynthesis is required for normal axon guidance, and mutations affecting Syndecan (Sdc), a transmembrane HSPG, disrupt axon guidance in Drosophila embryos. Glypicans, a family of glycosylphosphatidylinositol (GPI)-linked HSPGs, are expressed on axons and growth cones in vertebrates, but their role in axon guidance has not been determined. This study demonstrates that the Drosophila glypican Dally-like protein (Dlp) is required for proper axon guidance and visual-system function. Mosaic studies reveal that Dlp is necessary in both the retina and the brain for different aspects of visual-system assembly. Sdc mutants also show axon guidance and visual-system defects, some that overlap with dlp and others that are unique. dlp+ transgenes are able to rescue some sdc visual-system phenotypes, but sdc+ transgenes are ineffective in rescuing dlp abnormalities. Together, these findings suggest that in some contexts HS chains provide the biologically critical component, whereas in others the structure of the protein core is also essential (Rawson, 2005).

The distribution of Dlp was examined in the developing visual system by using a monoclonal antibody that specifically recognizes Dlp in tissues. In Drosophila, the adult eye is comprised of approximately 800 sensory units, or ommatidia, each with eight distinct photoreceptors, R1-R8. In the eye imaginal disc, Dlp was found on photoreceptor cell bodies and on cells within the morphogenetic furrow. Axons from R1-R6 terminate in the lamina, the first optic ganglion, whereas those from R7 and R8 project to the medulla. In the optic lobe, Dlp is present on photoreceptor axons at the boundary between the lamina and adjacent tissues and along the lamina plexus. Dlp is also observed in the medulla neuropil, medulla glia, medulla neuropil glia, neuroblasts of the proliferative centers, and in the mushroom body neuropil (Rawson, 2005).

To evaluate the function of Dlp in axon guidance, a photoreceptor-specific monoclonal antibody (24B10) was used to visualize photoreceptor projections in dlp mutants. In 50% of dlp mutant hemispheres, the lamina plexus was irregular and thickened. Additionally, 80% of dlp mutant larvae had fibers that aberrantly crossed between ommatidial bundles and/or photoreceptor process expansions outside the normal termination zone of the lamina plexus. Examination of dlp mutant pupae revealed that 80% of optic lobes contain irregularities in the R7 and R8 medulla termini. Crossover of R7 axons to neighboring medulla cartridges was observed (~50%) and misrouting of R7/R8 axons (~20%) (Rawson, 2005).

Visual-system function was assessed in dlp mutants by recording electroretinogram (ERG) profiles in adult flies. A wild-type ERG is composed of the photoreceptor response generated by a light-induced depolarization of the photoreceptor neurons and of the two transient voltage changes, the 'on-transient' and the 'off-transient', resulting from currents related to synaptic transmission during the initiation and termination of the light stimulus. dlp mutants show statistically significant defects in the photoreceptor response and in both on- and off-transients, suggesting that Dlp is required for proper photoreceptor currents and synaptic transmission (Rawson, 2005).

Axon guidance in the visual system depends on photoreceptor specification, as well as on glial-cell migration and lamina-neuron differentiation. Although dlp mutants have reduced and roughened eyes, thin sections of dlp eyes demonstrate that all photoreceptors are present and retain proper polarity in each ommatidium. Staining with several photoreceptor-specific markers confirm that dlp mutant photoreceptors differentiate properly. Likewise, glial cells and lamina neurons were found in the correct number and location in dlp mutants, indicating that patterning defects of these critical cells cannot account for the observed axon-guidance defects (Rawson, 2005).

Because Dlp is expressed in several visual-system elements and cell types, somatic mosaic studies were conducted to determine which cells require dlp for visual-system assembly. Using a method that generates clones encompassing a majority of cells in the retina, crossover of axons between ommatidial bundles and photoreceptor process expansions outside the lamina plexus was observed in 67% of animals with dlp mutant photoreceptors projecting to a heterozygous brain. Conversely, R7/R8 termination defects were absent from the medulla of 40 hr pupae with dlp mutant retinas and dlp/+ optic lobes. These results indicate that Dlp is required in the eye to specify proper axon guidance to the lamina, but not to the medulla (Rawson, 2005).

The photoreceptor-specific requirement for Dlp was further evaluated via the mosaic analysis with a repressible cell marker (MARCM) technique to visualize axons from small dlp mutant clones. dlp mutant photoreceptors display ectopic axon outgrowths directed away from their proper targets in the lamina (37% of dlp/dlp axons). Ectopic axon processes were four times more prevalent on axon bundles near the boundaries of the lamina than on those in more-central regions. This suggests that expression of Dlp on photoreceptors may be important for the detection of repellant cues that prevent aberrant axon outgrowth (Rawson, 2005).

Finally, the tissue-specific requirement was evaluated for Dlp in physiological function of the visual system. Using a mosaic strategy that generated eyes composed solely of dlp mutant photoreceptors, no see statistically significant defects were seen in ERG recordings compared to controls. These results demonstrate that the ERG defects of dlp mutants are produced by loss-of-function in the optic lobe, not in the eye (Rawson, 2005).

Drosophila Syndecan (Sdc) represents another class of HSPG, and it is required for normal axon guidance in the embryo. Antibody specific for Sdc revealed that this proteoglycan is present on photoreceptors in the retina and on photoreceptor projections to the optic lobe. Like Dlp, Sdc is enriched throughout the lamina plexus and at the boundary between the lamina and adjacent tissues. Sdc immunoreactivity was also detectable at a low level on cells just medial to the lamina plexus, the medulla glia, but was absent from some Dlp-expressing cells such as the mushroom body and the medulla neuropil glia (Rawson, 2005).

Analysis of sdc null mutant larvae revealed that 50% of optic lobes had photoreceptor-projection abnormalities and/or lamina-plexus defects including gaps (29%) and gross disorganization (21%, n = 28). Compared to dlp mutants, sdc mutants showed a low penetrance of the lamina-thickening (4%) and lamina-axon-crossover phenotypes. Additionally, some sdc mutants had R7/R8-axon misrouting in the larval stage. sdc mutant pupae showed crossover of R7 axons between medullary cartridges (100%) and defective axon pathfinding to the medulla (86%) but a low penetrance of R7/R8-termini disruption (~10%), a phenotype common in dlp mutants (80%-100%). As is the case for dlp, sdc mutants do not show defects in the specification of photoreceptors, glia, or lamina neurons, confirming that the above phenotypes are not secondary to other overt patterning deficiencies. Overall, dlp and sdc mutants share similar axon-guidance phenotypes, but each has a largely distinct level of penetrance for a given defect (Rawson, 2005).

Electrophysiological analysis revealed that sdc mutants have grossly abnormal ERGs, with defective photoreceptor depolarization and complete absence of on- and off-transients. These ERG abnormalities -- particularly the virtual loss of photoreceptor depolarization -- are distinct from those found in dlp animals. In addition, whereas dlp null mutants have a reduced and roughened eye, sdc mutants do not. These findings are consistent with distinct molecular functions for Dlp and Sdc during visual-system assembly (Rawson, 2005).

Sdc and Dlp are both heparan sulfate-modified proteoglycans, and Dlp expression is capable of limited rescue of sdc axon-guidance abnormalities in the embryo. To determine whether these two cell-surface molecules have overlapping functions in the visual system, a series of rescue experiments was performed. With or without a GAL4 driver, dlp mutant animals bearing a UAS-dlp+ construct show complete rescue of pupal axon-pathfinding defects and ERG abnormalities, suggesting that low levels of Dlp expression are sufficient to provide the function necessary for normal axon guidance and visual-system assembly. Despite this, ubiquitous expression of Sdc was unable to rescue the axon-guidance abnormalities of dlp mutants, demonstrating that Dlp and Sdc are not functionally interchangeable during photoreceptor axon guidance (Rawson, 2005).

The ability of UAS-dlp+ transgenes to rescue axon-guidance phenotypes of sdc mutant larvae and pupae was tested. Pupal sdc mutant phenotypes, including misrouting of R7/R8 photoreceptors to the medulla and crossover of R7 to neighboring medulla cartridges, are largely rescued with neuronal-directed expression of Sdc. Conversely, neuron-specific expression of Sdc in sdc mutants is not sufficient to rescue photoreceptor projection defects to the larval lamina or the ERG abnormalities in adults, suggesting that expression of Sdc in additional cell types is critical for complete restoration of axonal patterning and visual-system function. In contrast to the inability of Sdc expression to rescue dlp mutant phenotypes, neuron-specific expression of Dlp restores proper R7/R8 photoreceptor projection to the medulla of sdc mutant pupae. This rescue demonstrates that a structurally distinct HSPG can provide some of the functions normally served by Sdc (Rawson, 2005).

In vertebrates, glypicans are expressed on axons and growth cones in the developing nervous system. Whereas previous findings have established a function for glypicans in growth-factor signaling, their role in axon guidance has not been reported. Consistent with the expression pattern of Dlp on axons and glial cells of the developing visual system, dlp mutants show defects in photoreceptor projections to the lamina and medulla. Mosaic analysis demonstrates that Dlp is required on photoreceptors for some, but not all, aspects of axon guidance, serving functions in both peripheral and central components of the visual system. dlp is not required in the retina for normal electrophysiological responses to light, indicating that the synaptic-transmission defects in dlp mutant adults are due to loss of Dlp activity in the optic lobes (Rawson, 2005).

Comparison of sdc and dlp mutants as well as transgene rescue experiments demonstrates that Sdc and Dlp have some overlapping functions in visual-system assembly but others that are unique. For example, photoreceptor misrouting to the medulla in sdc mutants can be rescued by neuron-specific expression of dlp+, consistent with the capacity of dlp+ to rescue midline-crossover defects in sdc mutant embryos. In contrast to the ability of dlp+ transgenes to rescue sdc mutants, UAS-sdc+ constructs are unable to rescue any of the dlp mutant phenotypes, even under conditions where very modest levels of Dlp rescues completely. These findings show that Dlp functions cannot be readily provided by another unrelated HSPG, and they argue that the conserved sequence elements of the Dlp core protein are critical for these unique functions (Rawson, 2005).

Effects of HSPGs on axon pathfinding have been considered principally in the context of classical axon-guidance pathways, Slit-Robo signaling in particular. Although there is genetic evidence from mouse, Drosophila, and C. elegans that HSPGs affect Slit-Robo signaling, there are many other possibilities. HSPGs have been extensively characterized as molecules that affect both morphogen signaling and distributions. Recent studies demonstrating that the classical morphogens Wnt, Hh, and BMP also play a bona fide role in axon guidance suggest the possibility that HSPGs govern axon guidance by affecting morphogen function or distributions during this process (Rawson, 2005).

Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels

This study identified a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. It is concluded that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion (Schulz, 2011; Full text of article).


Expression of Syndecans

Nodes of Ranvier correspond to specialized axonal domains where voltage-gated sodium channels are highly concentrated. In the peripheral nervous system, they are covered by Schwann cells microvilli, where three homologous cytoskeletal-associated proteins, ezrin, radixin and moesin (ERM proteins) are enriched. These glial processes are thought to play a crucial role in organizing axonal nodal domains during development. However, little is known about the molecules present in Schwann cell processes that could mediate axoglial interactions. The aim of this study is to identify by immunocytochemistry transmembrane proteins enriched in Schwann cells processes that could interact, directly or indirectly, with axonal proteins. Syndecan-3 (S3) and syndecan-4 (S4), two proteoglycans expressed in Schwann cells, are enriched in perinodal processes in rat sciatic nerves. S3 labeling is localized in close vicinity of sodium channels as early as post-natal day 2, and highly concentrated at nodes of Ranvier in the adult. S4 immunoreactivity accumulates at nodes later, and is also prominent in internodal regions of myelinated fibers. Both S3 and S4 co-localize with ezrin in perinodal processes. These data identify S3 and S4 as transmembrane proteins specifically enriched in Schwann cell perinodal processes, and suggest that S3 may be involved in early axoglial interactions during development (Goutebroze, 2003).

Modifications of Syndecans

A comparative analysis was carried out of heparan sulfate (HS) and chondroitin sulfate (CS) chains of the ectodomains of hybrid type transmembrane proteoglycans, syndecan-1 and -4, synthesized simultaneously by normal murine mammary gland epithelial cells. Although the HS chains were structurally indistinguishable, intriguingly the CS chains were structurally and functionally distinct, probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS. The CS chains of the two syndecans comprised nonsulfated, 4-O-, 6-O-, and 4,6-O-disulfated N-acetylgalactosamine-containing disaccharide units and were significantly different, with a higher degree of sulfation for syndecan-4. Functional analysis using a BIAcore system showed that basic fibroblast growth factor (bFGF) specifically bound only to the HS chains of both syndecans, whereas midkine (MK) and pleiotrophin (PTN) bound not only to the HS but also to the CS chains. Stronger binding of MK and PTN to the CS chains of syndecan-4 than those of syndecan-1 was revealed, supporting the structural and functional differences. Intriguingly, removal of the CS chains decreased the association and dissociation rate constants of MK, PTN, and bFGF for both syndecans, suggesting the simultaneous binding of these growth factors to both types of chains, producing a ternary complex that transfers the growth factors to the corresponding cell surface receptors more efficiently compared with the HS chains alone. The involvement of the core protein was also shown in the binding of MK and PTN to syndecan-1, suggesting the possibility of cooperation with the HS and/or CS chains in the binding of these growth factors and their delivery to the cell surface receptors (Deepa, 2004).

Cleavage of syndecan by metalloproteinase

The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with syndecan-1 in HEK293T cells promotes syndecan-1 shedding, and concentration of cell-associated syndecan-1 is reduced. Treatment of cells with MMP inhibitor BB-94 or tissue inhibitor of MMP (TIMP)-2 but not TIMP-1 interferes with the syndecan-1 shedding promoted by MT1-MMP expression. In contrast, syndecan-1 shedding induced by 12-O-tetradecanoylphorbol-13-acetate treatment is inhibited by BB-94 but not by either TIMP-1 or TIMP-2. Shedding of syndecan-1 is also induced by MT3-MMP but not by other MT-MMPs. Recombinant syndecan-1 core protein is cleaved by recombinant MT1-MMP or MT3-MMP preferentially at the Gly245-Leu246 peptide bond. HT1080 fibrosarcoma cells stably transfected with the syndecan-1 cDNA (HT1080/SDC), that express endogenous MT1-MMP, spontaneously shed syndecan-1. Migration of HT1080/SDC cells on collagen-coated dishes is significantly slower than that of control HT1080 cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induces accumulation of syndecan-1 on the cell surface, concomitant with further retardation of cell migration. Substitution of Gly245 of syndecan-1 with Leu significantly reduces shedding from HT1080/SDC cells and cell migration. These results suggest that the shedding of syndecan-1 promoted by MT1-MMP through the preferential cleavage of Gly245-Leu246 peptide bond stimulates cell migration (Endo, 2003).

Interaction of CASK/LIN-2 and syndecan heparan sulfate proteoglycan

The syndecan family of cell surface heparan sulfate proteoglycans interacts via their cytoplasmic C-terminal tail with the PDZ domain of CASK/LIN-2, a membrane-associated guanylate kinase homolog. The syndecan-CASK interaction may be involved in intercellular signaling and/or cell adhesion. Syndecan-1 to syndecan-4 have distinctive mRNA distributions in adult rat brain by in situ hybridization, with syndecan-2 and -3 being the major syndecans expressed in neurons of the forebrain. At the protein level, syndecan-2 and -3 are differentially localized within neurons; syndecan-3 is concentrated in axons, whereas syndecan-2 is localized in synapses. The synaptic accumulation of syndecan-2 occurs late in synapse development. CASK is a cytoplasmic-binding partner for syndecans, and its subcellular distribution changes strikingly during development, shifting from a primarily axonal distribution in the first 2 postnatal weeks to a somatodendritic distribution in adult brain. This change in CASK distribution correlates temporally and spatially with the expression patterns of syndecan-3 and -2, consistent with the association of both of these syndecans with CASK in vivo. In support of this, a complex of CASK and syndecan-3 was coimmunoprecipitated from brain extracts. These results indicate that specific syndecans are differentially expressed in various cell types of the brain and are targeted to distinct subcellular compartments in neurons, where they may serve specialized functions. Moreover, CASK is appropriately expressed and localized to interact with both syndecan-2 and -3 in different compartments of the neuron throughout postnatal development (Hsueh, 1999).

CASK, the rat homolog of a gene (LIN-2) required for vulval differentiation in Caenorhabditis elegans, is expressed in mammalian brain, but its function in neurons is unknown. CASK is distributed in a punctate somatodendritic pattern in neurons. By immunogold EM, CASK protein is concentrated in synapses, but is also present at nonsynaptic membranes and in intracellular compartments. This immunolocalization is consistent with biochemical studies showing the presence of CASK in soluble and synaptosomal membrane fractions and its enrichment in postsynaptic density fractions of rat brain. By yeast two-hybrid screening, a specific interaction was identified between the PDZ domain of CASK and the COOH terminal tail of syndecan-2, a cell surface heparan sulfate proteoglycan (HSPG). The interaction was confirmed by coimmunoprecipitation from heterologous cells. In brain, syndecan-2 localizes specifically at synaptic junctions where it shows overlapping distribution with CASK, consistent with an interaction between these proteins in synapses. Cell surface HSPGs can bind to extracellular matrix proteins, and are required for the action of various heparin-binding polypeptide growth/differentiation factors. The synaptic localization of CASK and syndecan suggests a potential role for these proteins in adhesion and signaling at neuronal synapses (Hsueh, 1998).

Syndecan defines precise spindle orientation by modulating Wnt signaling in C. elegans

Wnt signals orient mitotic spindles in development, but it remains unclear how Wnt signaling is spatially controlled to achieve precise spindle orientation. This study shows that C. elegans syndecan is required for precise orientation of a mitotic spindle in response to a Wnt cue. SDN-1 is the predominant heparan sulfate (HS) proteoglycan in the early C. elegans embryo, and that loss of HS biosynthesis or of the SDN-1 core protein results in misorientation of the spindle of the ABar blastomere. The ABar and EMS spindles both reorient in response to Wnt signals, but only ABar spindle reorientation is dependent on a new cell contact and on HS and SDN-1. SDN-1 transiently accumulates on the ABar surface as it contacts C, and is required for local concentration of Dishevelled (MIG-5; see Drosophila Dishevelled) in the ABar cortex adjacent to C. These findings establish a new role for syndecan in Wnt-dependent spindle orientation (Dejima, 2014).

Syndecans and FGF signaling

The formation of distinctive basic FGF-heparan sulfate complexes is essential for the binding of bFGF to its cognate receptor. In previous experiments, cell-surface heparan sulfate proteoglycans extracted from human lung fibroblasts could not be shown to promote high affinity binding of bFGF when added to heparan sulfate-deficient cells that express FGF receptor-1 (FGFR1). In alternative tests to establish whether cell-surface proteoglycans can support the formation of the required complexes, K562 cells were first transfected with the IIIc splice variant of FGFR1 and then transfected with constructs coding for either syndecan-1, syndecan-2, syndecan-4 or glypican, or with an antisense syndecan-4 construct. Cells cotransfected with receptor and proteoglycan show a two- to three- fold increase in neutral salt-resistant specific 125I-bFGF binding in comparison to cells transfected with only receptor or cells cotransfected with receptor and anti-syndecan-4. Exogenous heparin enhances the specific binding and affinity cross-linking of 125I-bFGF to FGFR1 in receptor transfectants that are not cotransfected with proteoglycan, but has no effect on this binding and decreases the yield of bFGFR cross-links in cells that are cotransfected with proteoglycan. Receptor-transfectant cells show a decrease in glycophorin A expression when exposed to bFGF. This suppression is dose-dependent and obtained at significantly lower concentrations of bFGF in proteoglycan-cotransfected cells. Complementary cell-free binding assays indicate that the affinity of 125I-bFGF for an immobilized FGFR1 ectodomain is increased threefold when the syndecan-4 ectodomain is coimmobilized with receptor. Equimolar amounts of soluble syndecan-4 ectodomain, in contrast, have no effect on this binding. It is concluded that, at least in K562 cells, syndecans and glypican can support bFGF-FGFR1 interactions and signaling, and that cell-surface association may augment their effectiveness (Steinfeld, 1996).

The outgrowth of the mesoderm of the developing limb bud in response to the apical ectodermal ridge (AER) is mediated at least in part by members of the FGF family. Recent studies have indicated that FGFs need to interact with heparin sulfate proteoglycans in order to bind to and activate their specific cell surface receptors. Syndecan-3 is an integral membrane heparin sulfate proteoglycan that is highly expressed by the distal mesodermal cells of the chick limb bud that are undergoing proliferation and outgrowth in response to the AER. Maintenance of high-level syndecan-3 expression by the subridge mesoderm of the chick limb bud is directly or indirectly dependent on the AER, since its expression is severely impaired in the distal mesoderm of the limb buds of limbless and wingless mutant embryos, which lack functional AERs capable of directing the outgrowth of limb mesoderm. Exogenous FGF-2 maintains a domain of high-level syndecan-3 expression in the outgrowing mesodermal cells of explants of the posterior mesoderm of normal limb buds cultured in the absence of the AER and in the outgrowing subapical mesoderm of explants of limbless mutant limb buds, which lack a functional AER. These results suggest that the domain of high-level syndecan-3 expression in the subridge mesoderm of normal limb buds is maintained by FGFs produced by the AER. Polyclonal antibodies against a syndecan-3 fusion protein inhibit the ability of FGF-2 to promote the proliferation and outgrowth of the posterior subridge mesoderm of limb buds cultured in the absence of the AER. These results suggest that syndecan-3 plays an essential role in limb outgrowth by mediating the interaction of FGFs produced by the AER with the underlying mesoderm of the limb bud (Dealy, 1997).

Syndecan-4 is a heparan sulfate-carrying core protein that has been directly implicated in fibroblast growth factor 2 (FGF2) signaling. Recent studies have suggested that many signaling proteins localize to the raft compartment of the plasma cell membrane. To establish whether syndecan-4 is present in the raft compartment, the distribution has been studied of the core protein and an Fc receptor (FcR)-syndecan-4 chimera prior to and following clustering with FGF2 or antibodies. Whereas unclustered syndecan-4 was present predominantly in the non-raft membrane compartment, clustering induced extensive syndecan-4 redistribution to the rafts, as demonstrated by the sucrose gradient centrifugation and live confocal microscopy. Although syndecan-4 and caveolin-1 moved in tandem, syndecan-4 was not present in caveolae, a major subset of raft compartments. It is concluded that syndecan-4 clustering induces its redistribution to the non-caveolae raft compartment. This process may play an important role in syndecan-4-mediation of FGF2 signaling (Tkachenko, 2002).

Full activity of fibroblast growth factors (FGFs) requires their internalization in addition to the interaction with cell surface receptors. Recent studies have suggested that the transmembrane proteoglycan syndecan-4 functions as a FGF2 receptor. The molecular basis of syndecan endocytosis and its role in FGF2 internalization was investigated in endothelial cells. Syndecan-4 uptake, induced either by treatment with FGF2 or by antibody clustering, requires the integrity of plasma membrane lipid rafts for its initiation, occurs in a non-clathrin-, non-dynamin-dependent manner and involves Rac1, which is activated by syndecan-4 clustering. FGF2 was internalized in a complex with syndecan-4 in 70 kDa dextran-containing endocytic vesicles. FGF2 and syndecan-4 but not dextran endocytosis were blocked by the dominant negative Rac1, while amiloride and the dominant-negative Cdc42 blocked internalization of dextran in addition to FGF2 and syndecan-4. Taken together, these results demonstrate that FGF2 endocytosis requires syndecan-4 clustering-dependent activation of Rac1 and the intact CDC42-dependent macropinocytic pathway (Tkachenko, 2004).

Syndecans and integrin signaling

The alpha(v)beta(3) integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. The alpha(v)beta(3) integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to alpha(v)beta(3) requires the S1 ectodomain (ED); ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances alpha(v)beta(3) recognition of vitronectin, and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt alpha(v)beta(3)-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated alpha(v)beta(3) integrins (Beuvais, 2004).

Syndecans and TGF-beta signaling

TGF-beta has multiple functions including increasing extracellular matrix deposition in fibrosis. It functions through a complex family of cell surface receptors that mediate downstream signaling. A transmembrane heparan sulfate proteoglycan, syndecan-2 (S2), can regulate TGF-beta signaling. S2 protein increases in the renal interstitium in diabetes and regulates TGF-beta-mediated increased matrix deposition in vitro. Transfection of renal papillary fibroblasts with S2 or a S2 construct that has a truncated cytoplasmic domain (S2DeltaS) promotes TGF-beta binding and S2 core protein ectodomain directly binds TGF-beta. Transfection with S2 increased the amounts of type I and type II TGF-beta receptors (TbetaRI and TbetaRII), whereas S2DeltaS was much less effective. In contrast, S2DeltaS dramatically increases the level of type III TGF-beta receptor (TbetaRIII), betaglycan, whereas S2 results in a decrease. Syndecan-2 specifically co-immunoprecipitates with betaglycan but not with TbetaRI or TbetaRII. This is a novel mechanism of control of TGF-beta action that may be important in fibrosis (Chen, 2004).

Syndecan and neurite outgrowth - N-syndecan binds a protein complex containing Src family tyrosine kinases

N-syndecan (syndecan-3) is a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and is suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. N18 neuroblastoma cells were transfected with a rat N-syndecan cDNA and it was shown that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. It is suggested that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway (Kinnunen, 1998).

Syndecan and Slit/robo signaling

Using an affinity matrix in which a recombinant glypican-Fc fusion protein expressed in 293 cells was coupled to protein A-Sepharose, at least two proteins were isolated from rat brain that were detected by SDS-polyacrylamide gel electrophoresis as a single 200-kDa silver-stained band, from which 16 partial peptide sequences were obtained by nano-electrospray tandem mass spectrometry. Mouse expressed sequence tags containing two of these peptides were employed for oligonucleotide design and synthesis of probes by polymerase chain reaction and enabled the isolation from a rat brain cDNA library a 4.1-kilobase clone that encoded two of the peptide sequences and represented the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. Comparisons with recently published sequences also showed that these peptides were derived from proteins that are members of the Slit/MEGF protein family, which share a number of structural features such as N-terminal leucine-rich repeats and C-terminal epidermal growth factor-like motifs, and in Drosophila Slit is necessary for the development of midline glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523-1534 amino acids, and the peptide sequences correspond best to those present in human Slit-1 and Slit-2. Binding of these ligands to the glypican-Fc fusion protein requires the presence of the heparan sulfate chains, but the interaction appears to be relatively specific for glypican-1 insofar as no other identified heparin-binding proteins were isolated using the affinity matrix. Northern analysis demonstrated the presence of two mRNA species of 8.6 and 7.5 kilobase pairs using probes based on both N- and C-terminal sequences, and in situ hybridization histochemistry showed that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where glypican-1 mRNA and immunoreactivity are found. These results therefore indicate that Slit family proteins are functional ligands of glypican-1 in nervous tissue and suggest that their interactions may be critical for certain stages of central nervous system histogenesis (Liang, 1999).

Slit proteins are a family of secreted guidance proteins that can repel neuronal migration and axon growth via interaction with their cellular roundabout receptors (Robos). Slit2-Robo-1 interactions were enhanced by cell-surface heparan sulfate. Removal of heparan sulfate decreases the affinity of Slit for Robo by about threefold. In addition, removal of cell-surface heparan sulfate by heparinase III abolishes the chemorepulsive response to Slit2 normally shown by both the migrating neurons and growing axons. These results indicate essential roles for cell-surface heparan sulfate in the repulsive activities of Slit2 (Hu, 2001).

Syndecan interacts with PKC

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation. The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase C alpha (PKC alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha activity, leading to 'superactivation' of the enzyme, apparently through an interaction with its catalytic domain. Yeast two-hybrid and in vitro binding assays have been performed to determine the interaction sites between 4V and PKC alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence [4V(YF): LGKKPIFKK] did not interact with PKC alpha, indicating that tyrosine 192 in the syndecan-4 cytoplasmic domain might be critical for this interaction. Further assays identified a novel interaction site in the C terminus of the catalytic domain of PKC alpha (amino acid sequence 513-672). This encompasses the autophosphorylation sites, which are implicated in activation and stability. Yeast two-hybrid data were confirmed by in vitro binding and coimmunoprecipitation assays. The interaction of syndecan-4 with PKC alpha appears unique since PKC delta and epsilon did not interact with 4V in yeast two-hybrid assays or coimmunoprecipitate with syndecan-4. Finally, overexpression of syndecan-4 in rat embryo fibroblast cells, but not expression of the YF mutant, increased PKC alpha localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKC alpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase (Lim, 2003).

During cell-matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibers. The syndecan-4 cytoplasmic domain directly binds to and activates PKC-alpha (protein kinase C-alpha) in vitro. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, it has been shown that syndecan-4 interacts with PKC-alpha in vivo and that this interaction is mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increases the extent of interaction between syndecan-4 and PKC-alpha. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increases the level of endogenous PKC-alpha and enhances the translocation of PKC-alpha into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibit a slowed down-regulation of PKC-alpha in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-alpha immunocomplex kinase assays also showed that syndecan-4 overexpression increases the activity of membrane PKC-alpha. Taken together, these results suggest that syndecan-4 interacts with PKC-alpha in vivo and regulates its localization, activity and stability (Keum, 2004).

A role for Syndecan-4 in neural induction involving ERK- and PKC-dependent pathways

Syndecan-4 (Syn4) is a heparan sulphate proteoglycan that is able to bind to some growth factors, including FGF, and can control cell migration. This study describes a new role for Syn4 in neural induction in Xenopus. Syn4 is expressed in dorsal ectoderm and becomes restricted to the neural plate. Knockdown with antisense morpholino oligonucleotides reveals that Syn4 is required for the expression of neural markers in the neural plate and in neuralised animal caps. Injection of Syn4 mRNA induces the cell-autonomous expression of neural, but not mesodermal, markers. Two parallel pathways are involved in the neuralising activity of Syn4: FGF/ERK, which is sensitive to dominant-negative FGF receptor and to the inhibitors SU5402 and U0126, and a PKC pathway, which is dependent on the intracellular domain of Syn4. Neural induction by Syn4 through the PKC pathway requires inhibition of PKCdelta and activation of PKCalpha. PKCalpha inhibits Rac GTPase and c-Jun is a target of Rac. These findings might account for previous reports implicating PKC in neural induction and suggest a link between FGF and PKC signalling pathways during neural induction (Kuriyama, 2009).

Syn4 modulates FGF signalling through its extracellular domain (containing the GAG-binding region, which will present heparin sulphates to which FGF is expected to bind) and by an effect on the transduction of intracellular signals. The data support the idea that FGF is required for neural induction and that Syn4 is a likely modulator, by showing that the inhibition of FGF receptor and of MAPK activity impair neural induction by Syn4. Syn4 could act as a co-receptor of the FGF receptor or as a presenter of the FGF ligand, through binding of FGF to the GAG side-chains, to facilitate the activation of FGF receptor (Kuriyama, 2009).

However, Syn4 also plays a separate role in neural induction involving PKC. It is proposed that this involves inhibition of PKC{delta} and activation of PKC{alpha}, and that PKC{alpha} is an inhibitor of the small GTPase Rac. Since the BMP-inhibiting effects of FGF act through MAPK, this pathway could account for the BMP-inhibition-independent role of FGF signalling in neural induction. Rac is a well-known regulator of cell migration that acts by controlling actin polymerisation, but has not previously been implicated in neural induction. Evidence that Rac can control JNK activity suggested the hypothesis that Syn4/PKC{alpha} might inhibit Rac activity by an increase in AP-1 (c-Fos/c-Jun) activity that is mediated through inhibition of JNK (Kuriyama, 2009).

PKC{alpha} has never been connected with the signalling pathways now known to be involved in neural induction. It was originally shown that PKC{alpha} is activated and translocated to the membrane during neural induction, and it was suggested that this is required to confer neural competence on the ectoderm. This study has confirmed and extended these observations by showing that expression of PKC{alpha} in ventral ectoderm or in animal caps can act as a neuralising signal and that PKC{alpha} activity is regulated by interactions with Syn4 and PKC{delta}. PKC{delta} appears to work as a repressor of PKC{alpha}, whereas Syn4 appears to be required for PKC{alpha} activity; however, it was also shown that PKC{alpha} is required for the neuralising activity of Syn4. Thus, this finding allows proposal of a link between the PKC and FGF pathways, both of which have been identified as being involved in neural induction (Kuriyama, 2009).

These observations have parallels in studies of migrating cells. Syn4 interacts with PIP2, and this stabilises the oligomeric structure of Syn4 and promotes the association of PKC{alpha} and Syn4; the catalytic domain of PKC{alpha} binds to the cytoplasmic domain of Syn4, and PKC{alpha} is 'superactivated'. This interaction between PKC{alpha} and Syn4 provides a satisfactory explanation for the observation that neural induction by Syn4 requires PKC{alpha} and vice versa. In addition, during cell migration, PKC{delta} phosphorylates Syn4, decreases its affinity for PIP2 and abolishes its capacity to activate PKC{alpha}. This study has found a similar negative regulation between PKC{alpha} and PKC{delta} during early neural plate development (Kuriyama, 2009).

Syndecan interacts with laminin

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, the human alpha3LG4/5 fragment was recombinantly expressed in mammalian cells, and this fragment was shown to induce adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin-sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule (Okamoto, 2003).

The laminin alpha1 chain G domain has multiple biological activities. Cell binding sequences in the laminin alpha1 chain G domain have been identified by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. A recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides were used to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promotes both cell attachment and heparin binding. Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 and AG75, inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75, inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding (Suzuki, 2003).

Degradation of syndecan by presenilin

The syndecans play critical roles in several signal transduction pathways. The core proteins of these heparan sulfate proteoglycans are characterized by highly conserved transmembrane and intracellular domains which are required for signaling across the membrane and for interaction with cytosolic proteins. However, regulatory mechanisms controlling these functions remain largely unknown. upon ligand-induced primary proteolytic cleavage within the ectodomain, the intracellular domain of syndecan 3 is released by regulated intramembrane proteolysis. The cleavage is mediated by presenilin/gamma-secretase complex and negatively regulates the plasma membrane targeting of the transcriptional cofactor CASK (Schulz, 2004).

Syndecans mediate hedgehog function

Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS-PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS-PGs for function. Because the HS-PG syndecan-3 is preferentially expressed by proliferating chondrocytes, whether it mediates Ihh action was tested. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh-N) in absence or presence of heparinase I or syndecan-3 neutralizing antibodies. While rIhh-N stimulated proliferation in control cultures, it failed to do so in heparinase- or antibody-treated cultures. In reciprocal gain-of-function studies, chondrocytes were made to overexpress syndecan-3 by an RCAS viral vector. Cells became more responsive to rIhh-N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4-5 chick wing buds and effects of syndecan-3 misexpression were monitored over time. Syndecan-3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan-3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan-3 and to identify a specific member of the syndecan family as mediator of hedgehog function (Shimo, 2004).

Intracellular signaling downstream of syndecan as revealed using syndecan mutation

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that co-operates with integrins during cell-matrix interactions for the assembly of focal adhesions and actin stress fibers and in the phosphorylation of focal adhesion kinase (FAK) on Tyr397. These cellular events are regulated by the small GTPase Rho, and in the absence of syndecan-4 ligation, cellular levels of GTP-bound Rho are decreased, implicating syndecan-4 in the regulation of the small GTPases. Compared with wild type cells, fibronectin-adherent syndecan-4-null fibroblasts showed enhanced lamellipodia and increased Rac1 activity that is down-regulated by re-expression of syndecan-4 in the mutant cells. Consistent with the role for Rac1 in activating p38 and JNK signaling, syndecan-4-null cells display higher levels of active p38 MAPK and JNK that are abolished by the expression of a dominant-negative RacN17 mutant. Since p38 and JNK regulate gene expression by phosphorylating and activating transcription factors, both the phosphorylation state and the transcriptional activity of the ATF-2 transcription factor were compared, as a direct p38 and JNK target in syndecan-4-null and wild type cells. In the absence of syndecan-4, both ATF-2 phosphorylation and transcriptional activity are significantly more elevated compared with wild type cells, and both activities are decreased either by the re-expression of syndecan-4 or by the expression of RacN17. These results reveal a novel function for syndecan-4 in modulating nuclear transcriptional activity and indicate an underlying mechanism that acts at the level of Rac1-p38/JNK signaling (Saoncella, 2004).

Migrating cells mediate long-range WNT signaling

In amniotes, it is widely accepted that WNTs secreted by the dorsal neural tube form a concentration gradient that regulates early somite patterning and myotome organization. This study demonstrates in the chicken embryo that WNT protein is not secreted to act at a distance, but rather loaded onto migrating neural crest cells that deliver it to somites. Inhibiting neural crest migration or ablating their population has a profound impact on the WNT response in somites. Furthermore, it was shown that a central player in the efficient delivery of WNT to somites is the heparan sulfate proteoglycan GPC4, expressed by neural crest. Together, these data describe a novel mode of signaling whereby WNT proteins hitch a ride on migratory neural crest cells to pattern the somites at a distance from its source (Serralbo, 2014).

Syndecan in C. elegans

In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in Western blot analysis. In the sdn-1 deletion mutant allele the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment, is seen. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 deletion mutants lack two glycosylation sites. Hence, the missing protein in the Western blot analysis probably corresponds to SDN-1. In addition, SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 deletion mutants. These mutants show a defective egg-laying phenotype. The results show the identification, localization and some functional aspects of syndecan in the nematode C. elegans (Minniti, 2004).

During nervous system development, axons that grow out simultaneously in the same extracellular environment are often sorted to different target destinations. Since there is only a restricted set of guidance cues known, regulatory mechanisms are likely to play a crucial role in controlling cell migration and axonal pathfinding. Heparan sulfate proteoglycans (HSPGs) carry long chains of differentially modified sugar residues that have been proposed to encode specific information for nervous system development. This study shows that the cell surface proteoglycan syndecan SDN-1 functions autonomously in neurons to control the neural migration and guidance choices of outgrowing axons. Epistasis analysis suggests that heparan sulfate (HS) attached to SDN-1 can regulate guidance signaling by the Slit/Robo pathway. Furthermore, SDN-1 acts in parallel with other HSPG core proteins whose HS side chains are modified by the C5-epimerase HSE-5, and/or the 2O-sulfotransferase HST-2, depending on the cellular context. Taken together, these experiments show that distinct HS modification patterns on SDN-1 are involved in regulating axon guidance and cell migration in C. elegans (Rhiner, 2005).

Loss of SDN-1 function interferes with the migration of the HSN, CAN and ALM neurons, which, together with Q neuroblasts, are the only groups of neurons that migrate long distances in C. elegans. Because similar ALM migration defects have been reported for mutations in sax-3/Robo, SDN-1 might also modulate Slit/Robo signaling in cell migration, in addition to its role in the regulation of Slit signaling in midline guidance indicated by the data. However, lack of SDN-1 does not perturb cell migration in general; for example, sex myoblast migration is normal in sdn-1(zh20) animals. Furthermore, sdn-1 null mutants exhibit no circumferential distal tip cell (DTC) migration defects. Mutations in the gene encoding perlecan/UNC-52, a basement membrane HSPG, enhance the DTC migration defects of UNC-6/netrin signaling mutants -- an effect that can be partially suppressed by mutations disrupting growth factor-like signaling. Whether SDN-1 also contributes to signaling by EGL-20/WNT, UNC-129/TGF-ß or EGL-17/FGF still needs to be determined (Rhiner, 2005).

EphB/Syndecan-2 signaling in dendritic spine morphogenesis

The cell surface proteoglycan syndecan-2 can induce dendritic spine formation in hippocampal neurons. The EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and this phosphorylation event is crucial for syndecan-2 clustering and spine formation. Syndecan-2 is tyrosine phosphorylated and forms a complex with EphB2 in mouse brain. Dominant-negative inhibition of endogenous EphB receptor activities blocks clustering of endogenous syndecan-2 and normal spine formation in cultured hippocampal neurons. This is the first evidence that Eph receptors play a physiological role in dendritic spine morphogenesis. These observations suggest that spine morphogenesis is triggered by the activation of Eph receptors: this causes tyrosine phosphorylation of target molecules, such as syndecan-2, in presumptive spines (Ethell, 2001).

Dendritic spines are the principal postsynaptic targets for excitatory synapses. In recent years, these small protrusions on the surface of dendrites have attracted significant interest because changes in their morphology are implicated in synaptic plasticity and long-term memory. Dendritic spines undergo morphological changes in a developmentally regulated and activity-dependent manner. Abnormal spine morphologies have been reported in several neurodevelopmental disorders, including fragile X syndrome. The molecular mechanisms that govern spine morphogenesis are not completely understood, but several different physiological and molecular factors have been shown to affect spine morphology. These factors include synaptic activity and plasticity, actin filament reorganization, calcium dynamics, and protein phosphorylation (Ethell, 2001 and references therein).

Dendritic spine formation occurs during the late stages of development after neuronal connectivity has been established. Before the appearance of mature spines, dendrites exhibit long, thin filopodia-like protrusions without a bulbous head. As the brain matures, these dendritic filopodia disappear, and spines, which typically have mushroom-like and stubby shapes, begin to appear. Primary cultures of rat hippocampal neurons provide an excellent system in which the process of spine formation can be studied in vitro. At 1 week in vitro, these neurons possess predominantly filopodia-like protrusions. Over the next few weeks, these dendritic filopodia gradually decrease in number and are progressively replaced by protrusions that have mushroom-like and stubby shapes. After 3-4 weeks, the majority of the protrusions exhibit mature spine morphologies. The cell surface proteoglycan syndecan-2 has been shown to play a role in spine formation (Ethell, 2001 and references therein).

Syndecan-2 is a member of the syndecan family of transmembrane heparan sulfate proteoglycans. There is increasing evidence that syndecans are involved in transmembrane signaling by interacting with cytoskeletal and signaling molecules. The cytoplasmic domain of syndecans can be subdivided into a highly conserved juxtamembrane segment (C1 region), another conserved segment at the C terminus (C2 region), and a variable segment (V region) located between the C1 and C2 regions. The EFYA (Glu-Phe-Tyr-Ala) sequence at the C terminus serves as the binding site for at least four cytoplasmic proteins, namely syntenin, CASK, synectin, and synbindin. Moreover, the cytoplasmic domain contains 4 tyrosine residues that are conserved among all syndecans. Some of these tyrosine residues are phosphorylated in vitro, and tyrosine phosphorylation has been speculated to play important roles in syndecan-mediated signal transduction (Ethell, 2001 and references therein).

Potential functional roles of syndecan-2 in synapses were first suggested by its interaction with the synaptic PDZ domain protein CASK. Syndecan-2 is clustered at dendritic spines of mature hippocampal neurons in culture and its accumulation occurs concomitant with the morphological maturation of spines. More importantly, transfection of syndecan-2 induces the formation of morphologically mature dendritic spines in immature (8 days in vitro [DIV]) hippocampal neurons. Deletion studies have demonstrated that the C1 and V regions of the syndecan-2 cytoplasmic domain (which contains two potential tyrosine phosphorylation sites) are required for syndecan-2 clustering on dendrites and the induction of mature spines. Based on these data, it is hypothesized that tyrosine phosphorylation of syndecan-2 is the crucial upstream event that leads to dendritic spine formation. This premise then suggests that tyrosine kinase(s) present in dendritic spines play a role in spine formation by phosphorylating syndecan-2 (Ethell, 2001 and references therein).

The Eph family is a large family of receptor tyrosine kinases. Upon stimulation by ephrin ligands, Eph receptors activate signaling cascades in various biological systems. While Eph receptors have been studied primarily in the context of axon guidance during development, there have been suggestions that they may play some roles in synapses in the adult brain. It is speculated that Eph receptors are the kinases involved in the syndecan-2-induced spine formation for several reasons.: (1) syndecan-3, another member of the syndecan family, is tyrosine phosphorylated by recombinant EphB1 in vitro; (2) some Eph receptors interact with syntenin, a syndecan-2 binding PDZ domain protein, and (3) most importantly, some Eph receptors, including EphB2, are present in dendritic spines. These observations have led to this investigation of the possibility that Eph receptors are involved in syndecan-2 phosphorylation during dendritic spine formation (Ethell, 2001 and references therein).

In this paper, it is demonstrated that EphB2 is a crucial tyrosine kinase that phosphorylates syndecan-2 during dendritic spine formation. Furthermore, inhibition of endogenous EphB receptor activities by dominant-negative EphB2 blocks endogenous syndecan-2 clustering and normal spine formation. These results demonstrate a physiological role for EphB2/syndecan-2 signaling in dendritic spine morphogenesis. These findings provide a basis for the role of cell surface ligand-receptor interactions in spine morphogenesis and suggest that the signaling cascade leading to the formation of mature spines is triggered by the activation of Eph receptors by their extracellular ligands (Ethell, 2001).

Syndecan and muscle regeneration

Skeletal muscle regeneration is a highly complex and regulated process that involves muscle precursor proliferation and differentiation and probably requires the participation of heparin binding growth factors such as FGFs, HGF and TGFbeta. Heparan sulfate proteoglycans, key components of cell-surfaces and ECM, modulate growth factor activities and influence cell growth and differentiation. Their expression in forming muscle masses during development and in cell culture, suggest their participation in the regulation of myogenesis. In the present study, heparan sulfate proteoglycan expression in skeletal muscle regeneration induced by barium chloride injection was evaluated. Expression of muscle differentiation markers and neuromuscular junction (NMJ) components was characterized. Immunoblots with anti-Delta-heparan sulfate antibody showed that four major species -- perlecan, glypican, syndecan-3 and syndecan-4 -- are transiently up-regulated. The first three were detected at the surface or basement membranes of newly formed myotubes by specific indirect immunofluorescence. Syndecan-3, a satellite cell marker, showed the earliest and most significant increase. Experiments involving myoblast grafting into regenerating muscle showed that C2C12 cell clones, with inhibited syndecan-3 expression resulting from antisense transfection, presented a normal proliferation rate but an impaired capacity to fuse and form skeletal muscle fibers. These data constitute the first in vivo evidence suggesting the requirement of a specific heparan sulfate proteoglycan for successful skeletal muscle regeneration (Casar, 2004).

Syndecan-3 and syndecan-4 function as coreceptors for tyrosine kinases and in cell adhesion. Syndecan-3-/- mice exhibit a novel form of muscular dystrophy characterized by impaired locomotion, fibrosis, and hyperplasia of myonuclei and satellite cells. Explanted syndecan-3-/- satellite cells mislocalize MyoD, differentiate aberrantly, and exhibit a general increase in overall tyrosine phosphorylation. Following induced regeneration, the hyperplastic phenotype is recapitulated. While there are fewer apparent defects in syndecan-4-/- muscle, explanted satellite cells are deficient in activation, proliferation, MyoD expression, myotube fusion, and differentiation. Further, syndecan-4-/- satellite cells fail to reconstitute damaged muscle, suggesting a unique requirement for syndecan-4 in satellite cell function (Cornelison, 2004).

Syndecans and cell spreading

Raji cells expressing syndecan-1 (Raji-S1) adhere and spread when plated on heparan sulfate-binding extracellular matrix ligands or monoclonal antibody 281.2, an antibody directed against the syndecan-1 extracellular domain. Cells plated on monoclonal antibody 281.2 initially extend a broad lamellipodium, a response accompanied by membrane ruffling at the cell margin. Membrane ruffling then becomes polarized, leading to an elongated cell morphology. The syndecan-1 cytoplasmic domain is not required for these activities, suggesting important roles for the syndecan-1 transmembrane and/or extracellular domains in the assembly of a signaling complex necessary for spreading. Work described here demonstrates that truncation of the syndecan-1 extracellular domain does not affect the initial lamellipodial extension in the Raji-S1 cells but does inhibit the active membrane ruffling that is necessary for cell polarization. Replacement of the entire syndecan-1 transmembrane domain with leucine residues completely blocks the cell spreading. These data demonstrate that the syndecan-1 transmembrane and extracellular domains have important but distinct roles in Raji-S1 cell spreading; the extracellular domain mediates an interaction that is necessary for dynamic cytoskeletal rearrangements whereas an interaction of the transmembrane domain is required for the initial spreading response (McQuade, 2003).

Syndecans and wound repair

Wound repair is a tightly regulated process stimulated by proteases, growth factors, and chemokines, which are modulated by heparan sulfate. To characterize further the role of the heparan sulfate proteoglycan syndecan-1 in wound repair, mice were generated overexpressing syndecan-1 (Snd/Snd) and dermal wound repair was studied. Wound closure, reepithelialization, granulation tissue formation, and remodeling were delayed in Snd/Snd mice. Soluble syndecan-1 was increased, and shedding was prolonged in wounds from Snd/Snd mice. Excess syndecan-1 increased the elastolytic activity of wound fluids. Additionally, cells in the granulation tissue and keratinocytes at wound edges showed markedly reduced proliferation rates in Snd/Snd mice. Skin grafting experiments between Snd/Snd and control mice indicated that the slower growth rate was mainly due to a soluble factor in the Snd/Snd mouse skin. Syndecan-1 immunodepletion and further degradation experiments identified syndecan-1 ectodomain as a dominant negative inhibitor of cell proliferation. These studies indicate that shed syndecan-1 ectodomain may enhance proteolytic activity and inhibit cell proliferation during wound repair (Elenius, 2004).

Syndecan-4 is a ubiquitously expressed heparan sulfate proteoglycan that modulates cell interactions with the extracellular matrix. It is transiently up-regulated during tissue repair by cells that mediate wound healing. Syndecan-4 is essential for optimal fibroblast response to the three-dimensional fibrin-fibronectin provisional matrix that is deposited upon tissue injury. Interference with syndecan-4 function inhibits matrix contraction by preventing cell spreading, actin stress fiber formation, and activation of focal adhesion kinase and RhoA mediated-intracellular signaling pathways. Tenascin-C is an extracellular matrix protein that regulates cell response to fibronectin within the provisional matrix. Syndecan-4 is also required for tenascin-C action. Inhibition of syndecan-4 function suppresses tenascin-C activity and overexpression of syndecan-4 circumvents the effects of tenascin-C. In this way, tenascin-C and syndecan-4 work together to control fibroblast morphology and signaling and regulate events such as matrix contraction that are essential for efficient tissue repair (Midwood, 2004).

Syndecans and cancer

Syndecan-1 was overexpressed in T47D, MCF-7, or Hs578t human breast carcinoma cell lines, mimicking overexpression observed in carcinomas in vivo. Overexpression of syndecan-1, or its ectodomain alone fused to a glycosylphosphatidylinositol anchor (GPI-mS1ED), promotes cell rounding in 2D culture. Deletions within the syndecan-1 ectodomain (S1ED) implicate an active site within the core protein between the glycosaminoglycan attachment region and the transmembrane domain. Polyclonal antibodies directed against the ectodomain, or treatment with the tyrosine kinase inhibitor genistein, block activity and revert GPI-mS1ED overexpressing cells to a normal morphology. Extracellular matrix (ECM)-dependent signaling appears to be targeted, since GPI-mS1ED cells attach and spread similarly to control cells in response to E-cadherin engagement, but fail to spread on integrin-dependent ligands. However, integrin-dependent cell attachment, and integrin activation and subsequent FAK phosphorylation are unaffected, suggesting that the syndecan regulates the integration of signaling following matrix adhesion. In 3D culture, where syndecan-1 may have a more critical role in cell behavior, the disrupted signaling leads to poorly cohesive, invasive colonies. Thus, altered matrix-dependent signaling due to increased levels of cell surface syndecan-1 may lead to epithelial cell invasion during early stages of tumorigenesis (Burbach, 2004).

Among the four members of the syndecan family there exists a high level of divergence in the ectodomain core protein sequence. This has led to speculation that these core proteins bear important functional domains. However, there is little information regarding these functions and thus far the biological activity of syndecans has been attributed largely to their heparan sulfate chains. Cell surface syndecan-1 has been shown to inhibit invasion of tumor cells into three dimensional gels composed of type I collagen. Inhibition of invasion is dependent on syndecan's heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein is also indicated. To more closely examine this possibility and to map the regions of the ectodomain essential for syndecan-1-mediated inhibition of invasion, a panel of syndecan-1 mutational constructs was generated and each construct transfected individually into myeloma tumor cells. The anti-invasive effect of syndecan-1 is dramatically reduced by deletion of an ectodomain region close to the plasma membrane. Further mutational analysis identified a stretch of 5 hydrophobic amino acids, AVAAV (aa 222-226), critical for syndecan-1-mediated inhibition of cell invasion. This invasion regulatory domain is 26 amino acids from the start of the transmembrane domain. Importantly, this domain is functionally specific because its mutation does not affect syndecan-1-mediated cell binding to collagen, syndecan-1-mediated cell spreading or targeting of syndecan-1 to specific domains on the cell surface. This invasion regulatory domain may play an important role in inhibiting tumor cell invasion thus explaining the observed loss of syndecan-1 in some highly invasive cancers (Langford, 2004).

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. This work demonstrates the following: (1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells; (2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS; (3) syndecan-1 does not directly inhibit HPSE-1 enzymatic activity; (4) the presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays; (5) inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggests that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis (Reiland, 2004).


Search PubMed for articles about Drosophila Syndecan

Beauvais, D. M., Burbach, B. J. and Rapraeger, A. C. (2004). The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells. J. Cell Biol. 167(1): 171-81. 15479743

Burbach, B. J., Ji, Y. and Rapraeger, A. C. (2004). Syndecan-1 ectodomain regulates matrix-dependent signaling in human breast carcinoma cells. Exp. Cell Res. 300(1): 234-47. 15383330

Casar, J. C., et al. (2004). Heparan sulfate proteoglycans are increased during skeletal muscle regeneration: requirement of syndecan-3 for successful fiber formation. J. Cell Sci. 117(Pt 1): 73-84. 14627628

Chen, L., Klass, C. and Woods, A. (2004). Syndecan-2 regulates transforming growth factor-beta signaling. J. Biol. Chem. 279(16): 15715-8. 14976204

Cornelison, D. D., et al. (2004). Essential and separable roles for Syndecan-3 and Syndecan-4 in skeletal muscle development and regeneration. Genes Dev. 18(18): 2231-6. 15371336

Couchman, J.R. (2003). Syndecans: proteoglycan regulators of cell-surface microdomains?. Nat. Rev. Mol. Cell Bio. 4: 926-937. 14685171

Dani, N., Nahm, M., Lee, S. and Broadie, K. (2012). A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling. PLoS Genet 8: e1003031. PubMed ID: 23144627

Dealy, C. N., et al. (1997). FGF-stimulated outgrowth and proliferation of limb mesoderm is dependent on Syndecan-3. Dev. Biol. 184: 343-350. PubMed Citation: 9133440

Deepa, S. S., et al. (2004). Chondroitin sulfate chains on syndecan-1 and syndecan-4 from normal murine mammary gland epithelial cells are structurally and functionally distinct and cooperate with heparan sulfate chains to bind growth factors. A novel function to control binding of midkine, pleiotrophin, and basic fibroblast growth factor. J. Biol. Chem. 279(36): 37368-76. 15226297

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Biological Overview

date revised: 2 July 2015

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