Isolation, primary structure characterization and identification of the glycosylation pattern of recombinant goldfish neurolin, a neuronal cell adhesion protein.

Neurolin is a growth-associated cell surface glycoprotein from goldfish and zebra fish which has been shown to be involved in axonal path-finding in the goldfish retina and suggested to function as a receptor for axon guidance molecules. 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 either 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 revealed 60.9 kDa, which suggested 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) yielded 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 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.

Neurolin Ig domain 2 participates in retinal axon guidance and Ig domains 1 and 3 in fasciculation.

The optic disk-directed growth of retinal ganglion cell axons is markedly disturbed in the presence of polyclonal antineurolin antibodies, which mildly affect fasciculation (Ott, H., M. Bastmeyer, and C.A.O. Stuermer, 1998. J. Neurosci. 18:3363-3372). New monoclonal antibodies (mAbs) against goldfish neurolin, an immunoglobulin (Ig) superfamily cell adhesion/recognition molecule 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, mAbs were identified as being directed against Ig domains 1, 2, or 3, respectively. 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. This was demonstrated by time-lapse videorecording of labeled growth cones. 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.

Identification of reggie-1 and reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons.

Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl-phosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogold-labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPI-linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains).

Molecular characterization of E587 antigen: an axonal recognition molecule expressed in the goldfish central nervous system.

The E587 antigen (Ag) is a 200-Kd membrane glycoprotein originally identified by a monoclonal antibody on new and regenerating retinal ganglion cell axons in the adult goldfish. We report the isolation of cDNAs encoding the E587 Ag and identify it as a member of the L1 family of cell adhesion molecules (CAMs). The predicted amino acid sequence of E587 Ag shows an approximately equal identity (40%) to mouse L1, chick neuron-glia CAM, and chick neuron-glia-related CAM. Although the overall similarity is low, there is a high conservation of structural domains and specific sequence motifs. Wholemount in situ hybridizations were performed on goldfish between 34 hours and 3 days postfertilization (pf). A dramatic increase in E587 Ag mRNA was observed between 34 and 48 hours pf. The expression of E587 Ag mRNA in neurons shortly precedes axonogenesis. A marked decrease in expression occurs by 3 days pf, when the axonal scaffold has already been established. Wholemount immunohistochemistry on embryos demonstrates expression of E587 Ag on all major tracts. E587 Ag is absent from mature retinal ganglion cell axons, but its expression is induced by optic nerve transection. A corresponding induction of E587 Ag mRNA in retinal ganglion cells is shown by in situ hybridization. Furthermore, E587 Ag mRNA was detected in the optic nerve, which suggests that nonneuronal cells also express this molecule. E587 Ag was previously shown to promote retinal axon fasciculation and outgrowth in young fish and to mediate axon-glial interactions in vitro. The expression pattern and developmental regulation of E587 Ag in the central nervous system, its reexpression in retinal ganglion cells following optic nerve transection, and its relation to the L1 family indicate that E587 Ag functions as a cell recognition molecule important during axonal growth and regeneration.

Reggie-1 and reggie-2, two cell surface proteins expressed by retinal ganglion cells during axon regeneration.

Fish--in contrast to mammals--regenerate retinal ganglion cell axons when the optic nerve is severed. Optic nerve injury leads to reexpression of proteins, which typically are first expressed in newly differentiated retinal ganglion cells and axons. Here we identified two new proteins of fish retinal ganglion cells, reggie-1 and reggie-2, with monoclonal antibody M802 and molecular cloning techniques. In normal fish, M802 stained the few retinal axons derived from newborn ganglion cells which in fish are added lifelong to the retinal margin. After optic nerve injury, however, M802 labeled all retinal ganglion cells and retinal axons throughout their path into tectum. Consistent with M802 staining, reggie-1 and reggie-2 mRNAs were present in lesioned retinal ganglion cells, as demonstrated by in situ hybridization, but were not detectable in their normal mature counterparts. In western blots with membrane proteins of the adult goldfish brain, M802 recognizes a 48x10(3) Mr protein band. At the amino acid level, 48x10(3) Mr reggie-1 and reggie-2 are 44% identical, lack transmembrane and membrane anchor domains, but appear membrane associated by ionic interactions. Reggie-1 and reggie-2 are homologous to 35x10(3) Mr ESA (human epidermal surface antigen) but are here identified as neuronal surface proteins, present on newly differentiated ganglion cells at the retinal margin and which are reexpressed in mature ganglion cells upon injury and during axonal regeneration.

Spatiotemporal pattern of retinal ganglion cell differentiation revealed by the expression of neurolin in embryonic zebrafish.

The expression of neurolin, the fish homologue of the cell adhesion molecule DM-GRASP/BEN/SC-1, is dynamically regulated. Here we demonstrate that 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 molecule in RGC recognition and RGC axon growth.

Molecular characterization of fish neurolin: a growth-associated cell surface protein and member of the immunoglobulin superfamily in the fish retinotectal system with similarities to chick protein DM-GRASP/SC-1/BEN.

We have used the polymerase chain reaction to isolate cDNAs coding for goldfish and zebrafish neurolin, a previously identified 86 kDa cell surface glycoprotein in the goldfish visual system. Sequence analysis demonstrates that neurolin belongs to the immunoglobulin superfamily and is 51% similar to the chick cell adhesion molecule 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 are 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 molecule, 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.