Heparan sulfates (HSs) are extraordinarily complex extracellular sugar molecules that are critical components of multiple signaling systems controlling neuronal development. The molecular complexity of HSs arises through a series of specific modifications, including sulfations of sugar residues and epimerizations of their glucuronic acid moieties. The modifications are introduced nonuniformly along protein-attached HS polysaccharide chains by specific enzymes. Genetic analysis has demonstrated the importance of specific HS-modification patterns for correct neuronal development. However, it remains unclear whether HS modifications provide a merely permissive substrate or whether they provide instructive patterning information during development. This study shown with single-cell resolution that highly stereotyped motor axon projections in C. elegans depend on specific HS-modification patterns. By manipulating extracellular HS-modification patterns, axons can be cell specifically rerouted, indicating that HS modifications are instructive. This axonal rerouting is dependent on the HS core protein lon-2/glypican and both the axon guidance cue slt-1/Slit and its receptor eva-1. These observations suggest that a changed sugar environment instructs slt-1/Slit-dependent signaling via eva-1 to redirect axons. These experiments provide genetic in vivo evidence for the 'HS code' hypothesis which posits that specific combinations of HS modifications provide specific and instructive information to mediate the specificity of ligand/receptor interactions (Bülow, 2008).

Heparan sulfate is ubiquitous at the cell surface, where it is expressed predominantly as covalent modifications of proteoglycans of either the transmembrane syndecan family or the glycosylphosphatidylinositol (GPI)-anchored glypican family. Heparan sulfate has been proposed to function as a "coreceptor" for a number of "heparin-binding" growth factors. Although little is known about functional differences between individual members of the glypican gene family, mutations in both the Drosophila gene dally and the human gene for glypican-3 strongly suggest that at least some glypicans do function in cellular growth control and morphogenesis. In particular, deletion of the human glypican-3 gene is responsible for Simpson-Golabi-Behmel syndrome, and its associated pre- and postnatal tissue overgrowth, increased risk of embryonal tumors during early childhood, and numerous visceral and skeletal anomalies. An mRNA has been identified and characterized that encodes a 572-amino-acid member of the glypican gene family (glypican-5) that is most related (50% amino acid similarity, 39% identity) to glypican-3. Glypican-5 mRNA is detected as a 3.9- and 4.4-kb transcript in adult and neonatal mouse brain total RNA, and in situ hybridization results localize transcript primarily to restricted regions of the developing central nervous system, limb, and kidney in patterns consistent with a role in the control of cell growth or differentiation. Interestingly, glypican-5 localizes to 13q31-32 of the human genome, deletions of which are associated with human 13q- syndrome, a developmental disorder with a pattern of defects that shows significant overlap with the pattern of glypican-5 expression (Saunders, 1997).

The glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. The pattern of glypican-1 mRNA and protein expression was examined in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of glypican-1 expression are found throughout the brain and skeletal system. In the brain, glypican-1 mRNA is widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localizes strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, glypican-1 is found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 is also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression is observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development (Litwack, 1998).

A study was carried out of the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, it was found that a significant portion of the glypican and biglycan immunoreactivity colocalizes with nuclear staining by propidium iodide and is also seen in isolated nuclei. In certain regions, staining is selective, insofar as glypican and biglycan immunoreactivity in the nucleus is seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals; these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells is greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining is seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrates transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Therefore these data suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes (Liang, 1997).

Cell-surface proteoglycans have been implicated in cell responses to growth factors, extracellular matrix, and cell adhesion molecules. M12, one of the most abundant membrane-associated proteoglycans in the adult rat brain, is an approximately 65 kDa glycosylphosphatidylinositol-linked protein that bears heparan sulfate chains. To assess its identity, M12 was purified and internal peptide sequences obtained. Comparison of the results with the protein sequence predicted by a cDNA cloned from PC12 cells indicates that M12 is rat glypican, a proteoglycan first cloned from human fibroblasts. In addition, antibodies raised against a rat glypican fusion protein specifically detect the 65 kDa brain proteoglycan core protein, both by immunoprecipitation and by Western blotting. Northern blot analysis using a rat glypican probe also detects glypican message in the adult, as well as the developing rat brain. In situ hybridization with glypican RNA probes shows that glypican is expressed in a subset of structures in the adult rat nervous system. These include the hippocampus, dorsal thalamus, amygdala, cerebral cortex, piriform cortex, olfactory tubercle, several cranial nerve nuclei, the ventral horn of the spinal cord, and the dorsal root ganglia. Several other brain regions exhibit little or no hybridization over background. In most cases where glypican hybridization is observed, the signal can be localized specifically to the cell bodies of identifiable neurons, for example, spinal motoneurons and hippocampal pyramidal cells. In the cerebral cortex, glypican hybridization is found in layers 2/3, 5, and 6, but is missing from 1 and 4. The data suggest that glypican is expressed primarily by subpopulations of projection neurons in the adult rat nervous system (Litwack, 1994).

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. Reported here are the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain. The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican, it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons (Stipp, 1994).

The expression of cell surface heparan sulfate proteoglycans in rat heart was investigated by Northern blot analysis with specific cDNA probes. In adult heart mRNAs for both syndecan-3 and glypican are abundantly expressed. Lower levels of syndecan-2 mRNA and very low levels of syndecan-1 mRNA are also detected. Analysis of RNA isolated from hearts of rats of various ages reveals that the levels of both syndecan-3 and glypican mRNA increase dramatically at birth, and continued to be expressed at high levels in adult animals. To determine which of these proteoglycans is expressed in cardiomyocytes, primary cultures of cardiomyocytes and nonmyocytes isolated from neonatal rat hearts were analyzed for proteoglycan expression. Glypican mRNA is localized almost exclusively to cardiomyocytes. Syndecan-3 mRNA is not detected in myocytes, but is detected in the nonmyocyte cells. Biochemical characterization of cardiomyocyte glypican reveals that it is a phosphatidylinositol-anchored heparan sulfate proteoglycan. Results of immunofluorescent staining of rat hearts with anti-glypican antibodies are consistent with the Northern blot data, and localize glypican to the lateral regions of myocyte plasma membrane that contact the basement membrane, as well as sites of myocyte adhesion junctions. At the latter site glypican colocalizes with vinculin. Visualization of basic fibroblast growth factor binding sites by means of a tissue slice overlay assay also reveals colocalization with glypican. These results demonstrate developmental and cell-type-specific expression of membrane heparan sulfate proteoglycans in the heart. They also show that glypican is a major heparan sulfate proteoglycan expressed on the cardiomyocyte plasma membrane (Asundi, 1997).

An avian cDNA homolog of human and rat glypicans has been cloned from a stage 17 chicken heart cDNA library and used to analyze the distribution of this proteoglycan during development by Northern analysis and whole mount in situ hybridization. At stages 7-12, strong signals are detected in the cephalic region of the neural folds, rostral portion of paraxial mesoderm, and newly formed epithelial somites. At stages 20-25, strong expression is observed in the mantle zone of the telencephalon, the apical epidermal ridge and proximal region of developing limb. Transcripts also are found in the truncus arteriosus and arteriovenous-canal region of the heart, but not in the myocardium. This distribution pattern suggests that the avian glypican may be involved in the morphogenesis of limb, somite, heart, and brain. The expression of glypican also overlaps FGFs in limb bud, FGF receptors in heart and somite, and NGF receptors in forebrain. The affinity of heparan sulfate proteoglycans for growth factors and the distribution of the avian glypican are consistent with a role for this molecule in growth factor-mediated signals (Niu, 1996).

Transcripts for avian glypican are found in endocardial cushions, limb buds, somites and forebrain of early chick embryos. Since avian glypican is not well characterized, the cellular localization, regulation of expression, and possible function during cardiac development have been studied. A polyclonal antibody was raised against a 20-amino acid peptide corresponding to an antigenic sequence within avian glypican core protein. The antibody recognizes the expressed core protein in bacterial lysates and the endogenous HSPG in the proteoglycan fraction from chick forebrain. Immunolocalization studies indicate that the core protein is associated with cell membranes. The level of mRNA for avian glypican in MEQC (myc embryonic quail cardiomyocytes) grown in medium containing 10% fetal calf serum was compared to the message levels in cells grown without serum for 3 days. By Northern analysis, glypican transcripts increase markedly after serum starvation. Up-regulation of glypican transcripts by serum withdrawal is partially prevented by addition of TGFbeta-1 and bFGF, suggesting that these growth factors may regulate its expression. MEQC cells deprived of serum migrate into clumps that can be blocked by an antisense OND (oligodeoxynucleotide) to the mRNA encoding the avian glypican. The same antisense OND inhibits the migration of endothelial cells from chick tubular heart explants over the surface of collagen gels. These results indicate that avian glypican may play a role in cell migration during development of endocardial cushions (Niu, 1998).

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, the polarity of the expression of the cell surface heparan sulfate proteoglycans was examined in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identifies glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this is expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicate that sorting mechanisms, rather than differential turnover, account for this bipolar expression of glypican. Chlorate treatment does not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells express wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein result in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms have intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Inverse glycanation-apical sorting relationships in glypican may ensure near constant deliveries of HS to the apical compartment, or, alternatively, "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains (Mertens, 1996).

OCI-5, the rat homolog of human glypican 3 (GPC3), is believed to be involved in morphogenesis and growth control during development. The finding that GPC3 is mutated in patients with the Simpson-Golabi-Behmel overgrowth syndrome is consistent with this idea. In this report, using RNA in situ hybridization, expression of OCI-5 in the developing intestine is shown to be present in both endoderm- and mesenchyme-derived cells in a phased manner related to age and proximal/distal position. To investigate the mechanism of its regulation during intestinal development, OCI-5 expression was studied in the primitive rat intestinal epithelial cell line IEC-18. The expression of the OCI-5 transcript is increased in IEC-18 cells at confluence, in low calcium media, and during spheroid culture, all conditions which result in the cells acquiring a more rounded cell shape. In contrast, cytoskeletal disruption with colchicine causes cells to flatten and spread and abolishes both the confluence- and the low calcium-dependent induction of OCI-5. Treatment with vanadate, a phosphatase inhibitor, causes cells to acquire a spindle-shaped morphology and prevents OCI-5 induction in all situations. Nuclear run-on analysis demonstrates that the rate of OCI-5 transcription is increased at confluence, in low calcium media, and during spheroid culture of IEC-18, and decreased by treatment of cells with colchicine. Together, these data suggest that OCI-5 expression is regulated in IEC-18 by cell shape. The pattern of expression of OCI-5 in the developing intestine is consistent with it playing a role in epithelial-mesenchymal interactions during intestinal morphogenesis, when cell shape changes are likely to occur (Li, 1997).

Glypican is a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan (HSPG) releasable by phosphatidylinositol-specific phospholipase C (PI-PLC) from the surface of differentiated skeletal muscle cells. The processing, location and amount of this HSPG in skeletal muscle cells has been evaluated during differentiation. Immunoprecipitation of incubation medium obtained from differentiated cells incubated with [35S]sulfate by specific antibodies against glypican isolated from Schwann cells demonstrates that the antisera precipitates an intact HSPG. Immunoblot analysis of the proteins released by PI-PLC after heparitinase treatment reveals the presence of a main band of 64 and a faint band of 62 kDa, whereas the sizes of the core proteins for glypican present in the incubation media are 62 and 59 kDa. Pulse-chase experiments indicates that glypican present in the membrane is spontaneously released into the culture medium with a t1/2 of 12 h. The level of expression of glypican was analyzed during in vitro differentiation. The specific amount of the PI-PLC releasable HSPG increases about fourfold during cell differentiation. No changes are detected in the level of the mRNA for glypican. Indirect analysis reveals that in myotubes glypican is present on the cell surface as well as associated with the extracellular matrix (ECM). These results indicate that glypican is present, at least, in two different compartments on the surface of skeletal muscle cells (Brandan, 1996).

Skeletal muscle has the remarkable capacity to regenerate new muscle fibers in the event of injury or disease. This capacity lies in the satellite cells, which are myogenic stem cells residing in adult muscle. While the signals that activate satellite cells to divide in vivo are not fully understood, satellite cells grown in culture respond to the mitogenic action of fibroblast growth factor (FGF). Satellite cells from the dystrophic mdx mouse are more sensitive to FGF in culture than satellite cells from normal mice. The basis for this heightened sensitivity of mdx satellite cells to FGF was investigated by measuring the number and affinity of protein and heparan sulphate proteoglycan (HSPG) receptors for FGF. HSPG receptors are elevated over four-fold in the mdx cells, as compared with cells from normal animals. This observation was supported by measuring the synthesis of heparan sulphate (HS) and chondroitin sulphate (CS) by satellite cells in culture. Mdx satellite cells synthesized approximately ten times more of these sulphated glycosaminoglycans (GAGs) than did normal cells. For muscle fibroblasts, however, no significant differences were found between normal and mdx cells in the number or affinity of protein or HSPG receptors, or in the amount of sulphated GAGs synthesized. It is proposed that the increase in FGF HSPG receptors is the basis for the heightened response of mdx satellite cells to FGF in culture and may reflect exposure of the cells to growth factors in the degenerating mdx muscle (Crisona, 1998).

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding a Drosophila glypican, and in GPC3, the gene for human glypican-3, implicate glypicans in the control of cell growth and division. So far, five members of the glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, a sixth member of the glypican family has been identified. The glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to glypican-4 (63% identity). Expression of this protein in Namalwa cells shows a core protein of approximately 60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human glypican-6, contains nine exons. Like GPC5, the gene encoding glypican-5, GPC6 maps to chromosome 13q32. Clustering of the GPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, glypican-1, glypican-2, glypican-4, and glypican-6 appear to define a subfamily of glypicans, differing from the subfamily that thus far comprises glypican-3 and glypican-5. Northern blottings indicate that glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might be regulated, in part, by the presence of glypican-6 on the cell surface (Veugelers, 1999).

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre- and postnatal overgrowth with visceral and skeletal anomalies. To identify the causative gene, breakpoints in two female patients with X;autosome translocations were identified. The breakpoints occur near the 5' and 3' ends of a gene, GPC3, that spans more than 500 kilobases in Xq26; in three families, different microdeletions encompassing exons cosegregate with SGBS. GPC3 encodes a putative extracellular proteoglycan, glypican 3, that is inferred to play an important role in growth control in embryonic mesodermal tissues where it is selectively expressed. Initial western- and ligand-blotting experiments suggest that glypican 3 forms a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action (Pilia, 1996).

The Glypican family of heparan sulfate proteoglycans regulates Wnt signaling and convergent extension (CE) in vertebrate embryos. They are predicted to be glycosylphosphatidylinositol (GPI)-tethered membrane-bound proteins, but there is no functional evidence of their regulation by the GPI synthesis complex. Down syndrome critical region protein 5 (Dscr5, also known as Pigp) is a component of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) complex, and is associated with specific features of Down syndrome. This study reports that Dscr5 regulates CE movements through the non-canonical Wnt pathway. Both dscr5 overexpression and knockdown impair convergence and extension movements. Dscr5 functionally interacted with Knypek/Glypican 4 and is required for its localization at the cell surface. Knockdown of dscr5 disrupts Knypek membrane localization and causes an enhanced Frizzled 7 receptor endocytosis in a Caveolin-dependent manner. Furthermore, dscr5 knockdown promotes specific Dishevelled degradation by the ubiquitin-proteosome pathway. These results reveal a functional link between Knypek/Glypican 4 and the GPI synthesis complex in the non-canonical Wnt pathway, and provide the new mechanistic insight that Dscr5 regulates CE in vertebrate embryos by anchoring different Wnt receptors at the cell surface and maintaining Dishevelled stability (Shao, 2009).