The cDNA corresponding to DM-GRASP, a 95 kd cell surface protein expressed on a restricted population of axons has been cloned and sequenced and found to be a new member of the immunoglobulin superfamily of adhesion molecules. Consequently it has been named DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons (Burns, 1991).

A 95- to 100-kDa cell surface glycoprotein, named BEN (for bursal epithelium and neurons), is widely expressed during chicken embryonic development. In the central nervous system, it is restricted to subsets of neurons including the motoneurons and the inferior olivary nucleus neurons (which provide the cerebellum with the climbing fibers) where its expression occurs during the phase of axonogenesis and synaptogenesis. BEN expression extends to a variety of tissues originating from the three embryonic germ layers. BEN immunopurified from neural, epithelial, and hemopoietic tissues is differently glycosylated and may or may not carry the HNK-1 epitope. A full-length cDNA encoding this protein has been cloned. Analysis of its sequence reveals that BEN is a member of the immunoglobulin superfamily. Two proteins with an identical cDNA sequence have recently been reported: DM-GRASP and SC1. Their pattern of expression and structural properties are consistent with those reported for BEN. Therefore BEN, DM-GRASP, and SC1 are likely to be the same protein of the immunoglobulin superfamily (Pourquie, 1992).

The mAb E 21 recognizes a cell surface glycoprotein selectively associated with fish retinal ganglion cell axons that are in a state of growth. All retinal axons and ganglion cells in goldfish embryos stain for E 21. However, in adult fish E 21 immunoreactivity exhibits a patterned distribution in ganglion cells in the marginal growth zone of the continuously enlarging fish retina and the new axons emerging from these cells in the retina, optic nerve, and optic tract. The E 21 antigen is absent from older axons, except the terminal arbor layer in the tectum (the Stratum fibrosum et griseum superficiale) where the antigen is uniformly distributed. Upon optic nerve transection, the previously unlabeled axons reacquire an E 21 immunoreactivity as they regenerate throughout their paths to the tectum. However, several months after ONS, E 21 staining disappears from the regenerated axons over most of their lengths but reappears, as in normal fish, in the terminal arbor layer. The immunoaffinity-purified E 21 antigen, called Neurolin, has an apparent molecular mass of 86 kD and contains the HNK1/L2 carbohydrate moiety, like several members of the class of cell adhesion proteins of the Ig superfamily. The NH2-terminal amino acid sequence has homologies to the cell adhesion proteins DM-Grasp recently described in the chicken. Thus, retinal ganglion cell axons express Neurolin during their development and are able to reexpress this candidate cell adhesion protein during axonal regeneration, suggesting that Neurolin is functionally important for fish retinal axon growth (Paschke, 1992).

A full-length zebrafish cDNA clone and a partial mouse cDNA clone similar to chick DM-GRASP were isolated and analyzed. The nucleotide sequence of the full-length zebrafish clone shares 54% identity, and predicts 39% amino acid identity, with chick DM-GRASP. The partial mouse clone shares 76% nucleotide identity, and predicts 76% amino acid identity, with chick DM-GRASP. The predicted proteins encoded by both of these clones exhibit conserved structural domains that are characteristic of the chick protein. These features may identify them as a distinct subfamily within the immunoglobulin superfamily of cell adhesion proteins. Expression of the zebrafish DM-GRASP protein is similar to chick DM-GRASP and is principally restricted to a small subset of developing sensory and motor neurons during axonogenesis. Zebrafish DM-GRASP expression was temporally regulated and limited to specific axon domains. This regional expression correlates with fasciculated axon domains. These results suggest that the zebrafish and mouse cDNA clones represent the respective fish and mammalian homologs of chick DM-GRASP. The highly selective expression of zebrafish DM-GRASP suggests that it is involved in the selective fasciculation and guidance of axons along their normal pathways (Kanki, 1994).

The polymerase chain reaction was used to isolate cDNAs coding for goldfish and zebrafish neurolin, a previously identified 86 kDa cell surface glycoprotein in the goldfish visual system. Sequence analysis has demonstrated that neurolin belongs to the immunoglobulin superfamily and is 51% similar to the chick cell adhesion protein DM-GRASP/SC-1/BEN. Northern analysis with a riboprobe coding for the C-terminus of neurolin detected two mRNAs of 3.7 kb and 3.3 kb in both embryonic and adult goldfish. Several monoclonal and polyclonal antibodies were generated against immunopurified goldfish neurolin and two have been shown to crossreact with zebrafish proteins. Both antibodies identify a zebrafish protein of the same molecular weight as goldfish neurolin on immunoblots. Immunohistochemical studies with these antibodies in the zebrafish retinotectal system demonstrate labeling on young ganglion cells and growing retinal axons in a pattern similar to that found in goldfish. The similarity of neurolin to a known cell adhesion proteins, its expression on developing retinal ganglion cells and axons in both embryos and adult fish, and its re-expression during retinal axon regeneration in the goldfish suggests that neurolin is important during axonal growth in the fish central nervous system (Laessing, 1994).

The expression of neurolin, the fish homolog of the cell adhesion protein DM-GRASP/BEN/SC-1, is dynamically regulated. The expression of neurolin correlates with early events of retinal ganglion cell (RGC) differentiation in zebrafish embryos. Neurolin mRNA first appears [28 h postfertilization, (PF)] in nasoventral cells, representing the first RGCs, then in dorsal, central (34 to 40 h PF) and temporal RGCs. After differentiation of RGCs in the central portion of the retina, RGCs exhibiting neurolin mRNA form rings. These rings move toward the retinal periphery and encompass older (central) RGCs. Thereafter, such as at 3.5 days PF, neurolin mRNA expressing RGCs are confined to the annular growth zone at the retinal peripheral margin. Two hours after onset of mRNA expression, RGCs acquire antineurolin immunoreactivity on the surface of their somata and on their axons as they extend to the tectum. The mRNA signal in RGCs decreases significantly within 20 h after its appearance, which correlates with the arrival of axons in the tectum. This is followed by weakening of neurolin immunoreactivity on RGCs and axons. This pattern of RGC differentiation in zebrafish revealed by the expression of neurolin, is unique among vertebrates. The spatiotemporal expression pattern of neurolin suggests a functional significance of this cell adhesion protein in RGC recognition and RGC axon growth (Laessing, 1996).

In contrast to the spinal sensory ganglia that reiterate a basic organizational and functional unit, each cranial ganglion mediates a distinct sensory modality and exhibits a characteristic pattern of peripheral and central neuronal connectivity. Protein molecules responsible for establishment and maintenance of the cranial ganglion-specific networks are not known. Hamster monoclonal antibody 802C11 strongly stains neurons and their processes of the VIIIth cranial ganglion (hearing and equilibrium), but not the Vth cranial (somatosensory) or spinal ganglia in the mouse embryo. The cellular staining pattern of positive neurons suggests that the antigen is associated with the cell membrane, and biochemical analyses of the antigen from adult mouse brain show the antigen to be a glycosylated intrinsic membrane protein of approximately 100 kDa. The antigen was purified, and based on the partial amino acid sequences, its entire cDNA was cloned. The deduced amino acid sequence reveals that the antigen belongs to the immunoglobulin superfamily with a significant homology (73.5% identity) to chicken SC1 protein. Chicken SC1 has been shown to be a cell-cell adhesion proteins in vitro with a proposed role in neurite extension of spinal motor neurons. These results suggest that murine SC1-related protein (MuSC) is involved in the pathfinding and/or fasciculation of specific cranial sensory nerve fibers (Sekine-Aizawa, 1998).

SC1 is a secreted glycoprotein with a high amino acid sequence similarity to SPARC (Secreted Protein, Acidic, Rich in Cysteine). SC1 transcripts are first detected in mouse embryos after day 8.5 post coitus in somites at the medial lip of the dermomyotome. Expression of SC1 transcripts by the progenitor cells continues as they begin involuting under the dermomyotome and during their migration along the lateral wall of the dermomyotome. After myotome migration is completed, SC1 mRNA expression is downregulated in the trunk region. The data indicate that SC1 expression is restricted to the initial stages of epaxial myotome differentiation and migration, undergoing rapid downregulation prior to myotome emigration from the somitic environment (Ringuette, 1998).

It is well established that the notochord influences the development of adjacent neural and mesodermal tissue. Involvement of the notochord in the differentiation of the dorsal pancreas has been demonstrated. However, knowledge of the signals involved in pancreatic development is still incomplete. In order to identify proteins potentially implicated during pancreatic differentiation, monoclonal antibodies against previously established embryonic pancreatic ductal epithelial cell lines (BUD and RED) were raised and characterized. Using the MAb 2117, the cell surface antigen 2117 (Ag 2117) was cloned. The predicted sequence for Ag 2117 is the rat homolog of BEN. Initially reported as a protein expressed on epithelial cells of the chicken bursa of Fabricius, BEN is expressed in a variety of tissues during development and described as a marker for the developing central and peripheral chicken nervous systems. A role has been suggested for BEN in the adhesion of stem cells and progenitor cells to the blood-forming tissue microenvironment. BEN, initially expressed exclusively in the notochord during the early development of rat, is implicated in pancreatic development. Ag 2117 regulates the pancreatic epithelial cell growth through the ras and Jun kinase pathways. In addition, Ag 2117 is able to regulate the expression of the transcription factor PDX1, required for insulin gene expression, in embryonic pancreas organ cultures (Stephan, 1999).

Neurolin is a growth-associated cell surface glycoprotein from goldfish and zebra fish that has been shown to be involved in axonal path-finding in the goldfish retina and suggested to function as a receptor for axon guidance positively proteins. Being a member of the immunoglobulin superfamily of cell adhesion proteins, neurolin consists of five N-terminal extracellular immunoglobulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. Repeated injections of polyclonal Fab fragments against neurolin and of monoclonal antibodies against any of the Ig domains cause path-finding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structure-function determination, recombinant neurolin with the complete extracellular part but lacking the transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neurolin was carried out by Ni-affinity chromatography and subsequent high-performance liquid chromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and 60.9 kDa were revealed, which suggests that approximately 10 kDa are due to glycosylation. The predicted molecular mass is 51.5 kDa, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yields an apparent molecular mass of 72 kDa. Gel shift assays using SDS-PAGE and Western blot analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns were identified using specific lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with specific glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation has revealed heterogeneous glycosylations at all five N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure: they are fucosylated with alpha(2,3)-terminal neuraminic acid. These data serve as a first detailed model to characterize the molecular recognition structures exhibited by the extracellular domains (Denzinger, 1999).

In an effort to identify aberrantly expressed genes in v-rel-induced tumors, monoclonal antibodies were developed that react selectively with avian B-cell tumors. One antibody, HY78, immunoprecipitates a 120-kDa glycoprotein (p120) from cells that express v-rel. N-terminal amino acid sequencing of p120 has identified a 27-amino-acid sequence that is also present in DM-GRASP, an adhesion protein belonging to the immunoglobulin superfamily. Evidence from tissue distribution, immunological cross-reaction, PCR amplification, cDNA cloning, and DNA sequence shows that p120 is indeed DM-GRASP. Northern (RNA) analysis using a probe from the DM-GRASP gene has identified a 5.3-kb transcript in mRNA from bursa, thymus, and brain as well as from v-rel-induced B-cell lymphomas but not from bursal B cells. The induction of this protein by v-rel during the development of bursal B-cell lymphomas appears, therefore, to be ectopic in nature. Overexpression of v-rel or c-rel in chicken embryonic fibroblasts, B-cell lines, and spleen mononuclear cells induces the expression of DM-GRASP. The ratio of DM-GRASP to v-Rel is fivefold higher than that of DM-GRASP/c-Rel in a B-cell line, DT95. Interestingly, the presence of HY78 antibody inhibits the in vitro proliferation of v-rel-transformed cells but not cells that are immortalized by myc. These data suggest that DM-GRASP is one of the genes induced during v-rel-mediated tumor development and that DM-GRASP may be involved in the growth of v-rel tumor cells (Zhang, 1995).

DM-GRASP is an immunoglobulin superfamily cell adhesion protein that is expressed in both the developing nervous and immune systems. Specific populations of neurons that respond to DM-GRASP substrates appear to require homophilic interactions between DM-GRASP proteins. It was of interest to determine whether DM-GRASP interacts heterophilically with other ligands as well. Eleven proteins from embryonic chick brain membranes have been found to consistently bind to and elute from a DM-GRASP-Sepharose affinity column. One of these proteins is DM-GRASP itself, consistent with its known homophilic binding. Another protein, at 130 kD, is immunoreactive with monoclonal antibodies to NgCAM. Other neural cell adhesion proteins were not detected in the eluate. The DM-GRASP-Sepharose eluate also contains a potent neurite stimulating activity, which cannot be accounted for by either DM-GRASP or NgCAM. To investigate the interaction of DM-GRASP and NgCAM, antibodies against DM-GRASP were added to neuronal cultures extending neurites on an NgCAM substrate. The presence of antibodies to DM-GRASP decreases neurite extension on laminin, suggesting that the antibody is not toxic or generally an inhibitor of motility. Two possible models are presented for the DM-GRASP-NgCAM association and a hypothesis is presented for neural cell adhesion function that features the dimerization of cell adhesion proteins (DeBernardo, 1996).

Examination was carried out of the expression and function of a cell membrane protein in the developing chick retinotectal system identified by a monoclonal antibody (mAb 4H5) and the corresponding antiserum. The protein shares a series of properties, including the N-terminal amino acid sequence, with a cell adhesion protein termed DM-GRASP, SC1, BEN, and JC7. It can therefore be considered identical with this molecule and is referred to as SC1/DMGRASP. In early development of the retinotectal system, SC1/DMGRASP is exclusively expressed on growing, far-projecting, tract-forming axons. Expression begins at the onset of retina ganglion cell axogenesis and its maximum expression overlaps with the phase of maximal axon extension. Later in development, SC1/DMGRASP appears on distinct laminae within plexiform layers in spatiotemporal correlation with synaptogenesis. In an in vitro assay system designed to study the elongation of RGC axonal processes on preexisting RGC axons, addition of SC1/DMGRASP antiserum specifically reduces lengths of axonal processes. In contrast, axonal growth on laminin or basal lamina preparations is not SC1/DMGRASP-dependent. Taken together, the data provide evidence for a role of SC1/DMGRASP in axonal elongation of SC1/DMGRASP-positive axons on such axons, thereby possibly contributing to the pathway and target finding mechanisms of far-projecting, tract-forming central nervous system neurons (Pollerberg, 1994).

DM-GRASP (GRASP) is an integral membrane glycoprotein expressed on a restricted set of axons in the developing chick nervous system. Purified GRASP supports neurite extension from the subpopulation of neurons that express GRASP. Sensory, sympathetic, and ciliary neurons express GRASP and extend neurites on a GRASP substrate. In contrast, tectal, diencephalic, and retinal cells express GRASP at a very low level, if at all, and do not extend neurites on a GRASP substrate. Moreover, during the developmental period in which GRASP is downregulated on sensory neurons, the neurons lose the capacity to extend neurites on a GRASP substrate. Recombinant GRASP produced in a baculovirus expression system is biochemically and functionally identical to GRASP purified from embryonic chick brain. The finding that GRASP selectively supports neurite extension supports the hypothesis that it mediates selective fasciculation via a homophilic binding mechanism (DeBernardo, 1995).

Young axons of new retinal ganglion cells (RGCs) in the continuously growing goldfish retina fasciculate with one another and their immediate forerunners on their path toward the optic disk and along the optic nerve. They express the immunoglobulin superfamily cell adhesion proteins (CAMs) neurolin (DM-GRASP) and the L1-like E587 antigen. Repeated injections of Fab fragments from polyclonal antisera against neurolin (neurolin Fabs), approximately three to four cm in length, into the eyes of rapidly growing goldfish causes highly aberrant pathways for young RGC axon subfascicles in the dorsal retina. Many axons grow in circles and fail to reach the optic disk. In contrast, E587 Fabs, used in parallel experiments, disrupt the fascicles but do not interfere with the disk-directed growth. Neurolin Fabs also disturb axonal fasciculation in vivo as well as in vitro but less severely than E587 Fabs. Coinjections of both Fabs increases defasciculation of the dorsal axons in both aberrant and disk-directed routes. They also disrupt the order of young RGC axons in the optic nerve more severely than E587 Fabs alone. This demonstrates that the development of tight and orderly fascicles in the dorsal retina and in the optic nerve requires both E587 antigen and neurolin. More importantly, these results suggest an involvement of neurolin in RGC axonal guidance from the retinal periphery to the optic disk. Because disrupted fascicles and errant axon routes are found only in the dorsal retinal half, a cooperation with so-called positional markers is possible (Ott, 1998).

BEN/SC1/DM-GRASP is a cell adhesion protein belonging to the Ig superfamily that is transiently expressed during avian embryogenesis in a variety of cell types, including the motoneurons of the spinal cord. The pattern of BEN expression during neuromuscular development of the chick has been characterized. Both motoneurons and their target myoblasts express BEN during early embryonic development, and the protein becomes restricted at neuromuscular contacts as soon as postsynaptic acetylcholine receptor clusters are observed in muscle fibers. Muscle cells grown in vitro express and maintain BEN expression even when they fuse and give rise to mature myotubes. When embryos are deprived of innervation by neural tube ablation, BEN expression is observed in muscle fibers, whereas, in control, the protein is already restricted at neuromuscular synaptic sites. These results demonstrate that all myogenic cells intrinsically express BEN and maintain the protein in the absence of innervation. Conversely, when neurons are added to myogenic cultures, BEN is rapidly downregulated in muscle cells, demonstrating that innervation controls the restricted pattern of BEN expression seen in innervated muscles. After nerve section in postnatal muscles, BEN protein becomes again widely spread over muscle fibers. When denervated muscles are allowed to be reinnervated, the protein is reexpressed in regenerating motor axons, and reinnervation of synaptic sites leads to the concentration of BEN at neuromuscular junctions. These results suggest that the BEN cell adhesion protein acts both in the formation of neuromuscular contacts during development and in the events leading to muscle reinnervation (Fournier-Thibault, 1999).

The optic disk-directed growth of retinal ganglion cell axons is markedly disturbed in the presence of polyclonal antineurolin antibodies, which mildly affect fasciculation. New monoclonal antibodies (mAbs) against goldfish neurolin, an immunoglobulin (Ig) superfamily cell adhesion/recognition protein with five Ig domains, were generated to assign function (guidance versus fasciculation) to specific Ig domains. By their ability or failure to recognize Chinese hamster ovary cells expressing recombinant neurolin with deletions of defined Ig domains, different mAbs were identified as being directed against the different Ig domains 1, 2, or 3. Repeated intraocular injections of a mAb against Ig domain 2 disturb the disk-directed growth: axons grow in aberrant routes and fail to reach the optic disk, but remain fasciculated. mAbs against Ig domains 1 and 3 disturb the formation of tight fascicles. mAb against Ig domain 2 significantly increases the incidence of growth cone departure from the disk-oriented fascicle track, while mAbs against Ig domains 1 and 3 do not. Thus, Ig domain 2 of neurolin is apparently essential for growth cone guidance towards the disk, presumably by being part of a receptor (or complex) for an axon guidance component (Leppert, 1999).

Cell surface adhesion proteins are thought to play a necessary role in axon guidance and fasciculation in the developing nervous system. A potential adhesion protein has been studied using the zn-5 monoclonal antibody, which recognizes the surfaces of zebrafish spinal motoneurons. Zn-5 recognizes zebrafish DM-GRASP. DM-GRASP is a cell adhesion protein of the immunoglobulin superfamily that mediates homophilic adhesion and neurite outgrowth in vitro. In zebrafish, primary motoneurons pioneer the peripheral motor nerve pathways, and the axons of secondary motoneurons follow the routes established by the primary motoneuron axons. Of the two classes of zebrafish spinal motoneurons, only the later growing secondary motoneurons express DM-GRASP. The secondary motoneurons restrict DM-GRASP protein to their cell bodies and fasciculated segments of their axons. Expression of DM-GRASP is transient: the protein is present during the period of axonal growth and disappears after axons have reached their muscle targets. Thus, homophilic adhesion mediated by DM-GRASP may play a role in fasciculation of secondary motoneuron axons but not in pathfinding by the pioneer axons of the primary motoneurons or in guidance of secondary motoneuron axons to their targets (Fashena, 1999).

During nervous system development, neurons form reproducible synapses onto specific targets. The development of stereotyped synapses of the C. elegans HSNL neuron has been examined in vivo. Postsynaptic neurons and muscles are not required for accurate synaptic vesicle clustering in HSNL. Instead, vulval epithelial cells that contact HSNL act as synaptic guidepost cells that direct HSNL presynaptic vesicles to adjacent regions. The mutant syg-1(ky652) has defects in synapse formation that resemble those in animals that lack vulval epithelial cells: HSNL synaptic vesicles fail to accumulate at normal synaptic locations and form ectopic anterior clusters. syg-1 encodes an immunoglobulin superfamily protein that acts in the presynaptic HSNL axon. SYG-1 protein is localized to the site of future synapses, where it initiates synapse formation and localizes synaptic connections in response to the epithelial signal. SYG-1 is related to Drosophila IrreC and vertebrate NEPH1 proteins, which mediate cell-cell recognition in diverse developmental contexts (Shen, 2003).

SYG-1 is closely related to two Drosophila proteins, Rst/IrreC and Kirre/Duf. Although they have not been characterized in synapse formation, these proteins act in a variety of developmental events that involve specific cell recognition. Rst/IrreC mutants have defects in cell adhesion in the retina (the roughest phenotype), and in axon pathfinding at higher steps of visual processing (the irregular chiasm phenotype). During development, founder cells for each muscle attract and fuse with fusion-competent cells. Recognition between the muscle founder and fusion-competent cells is mediated by Rst/IrreC and Kirre on the founder cells, and Sticks and Stones (Sns) and Hibris (Hib) on fusion-competent cells. Muscle fusion occurs through a characteristic prefusion complex, a tight adhesion between cells associated with alignment of paired vesicles at the cell junctions. Several structural features of the prefusion complex are similar to features of synapses, including tight local asymmetric adhesions between cells and the recruitment of vesicles. Sns and Hib are transmembrane proteins with eight immunoglobulin domains that have heterophilic interactions with the Kirre/Duf protein. A predicted ORF in the C. elegans genome, C26G2.1, shows strong homology to SNS and Hibris, suggesting that similar heterophilic recognition mechanisms could also exist in C. elegans (Shen, 2003 and references therein).

SYG-1 bears similarity to many vertebrate immunoglobulin superfamily members, and strongest similarity to three proteins that are only partly characterized, NEPH1, KIAA1867, and hCP41052. One of these genes, NEPH1, is regionally expressed in the mammalian brain, but its function there is unknown. NEPH1 is required for kidney function and the development of the podocyte slit membrane in mice. A human ortholog of Sns/Hib, Nephrin, is also required for kidney function and development of the podocyte slit membrane, a tight epithelial cell adhesion complex that plays a role in glomerular filtration. The adhesion complexes of the mammalian podocyte slit membrane, a future C. elegans synapse, and a Drosophila muscle prefusion complex appear to be initiated by similar molecular interactions that are interpreted in cell-type specific contexts (Shen, 2003 and references therein).