Molecular cloning of NILE glycoprotein and evidence for its continued expression in mature rat CNS

Abstract

The NILE glycoprotein is a rat neuronal cell adhesion molecule which has been reported to be very similar in structure, function, and distribution to the mouse L1 glycoprotein. Here we report the complete nucleotide sequence of the NILE message (5,208 nucleotides) and the deduced amino acid sequence of the NILE polypeptide (1,257 amino acids). The predicted NILE protein is 96% identical to L1 at the amino acid level, confirming that the two molecules are homologues. The sequence information shows that NILE is a transmembrane molecule with an extensive ectodomain and a much smaller cytoplasmic domain. The extracellular portion of the molecule contains six immunoglobulin C-2 type domains followed by five fibronectin type III repeats. These two structural motifs are characteristic of several other cell adhesion molecules. The cytoplasmic tails of NILE and L1 are identical to each other and distinct from the cytoplasmic regions of all other cell adhesion molecules except Ng-CAM and neuroglian. Several possible sites for phosphorylation are present in the cytoplasmic tail of NILE. Antisera were produced against two NILE-beta-galactosidase fusion proteins containing distinct segments of the NILE polypeptide: the cytoplasmic domain and the segment containing fibronectin type III repeats. Immunoblots with these antisera and Northern blots with a NILE cDNA probe indicate that NILE continues to be expressed in most areas of the mature rat brain. This contradicts previous immunofluorescence data, which suggested that NILE was substantially down-regulated in maturing nerve fiber tracts. This raises the possibility that NILE could be masked in situ by interactions with other cell surface molecules.

Axon-Regenerating Retinal Ganglion Cells in Adult Rats Synthesize the Cell Adhesion Molecule L1 but Not TAG-1 or SC-1

“…Immunoblots were performed with the antiserum against mouse L1 as described by Vielmetter et al (1991) with detergent-extracted rat brain membrane proteins. The emerging bands at approximately 200, 140, and 80 kDa were typical for L1 in rodents (Rathjen and Schachner, 1984;Prince et al, 1991).…”

“…The following plasmids were used: pBKSII(2) containing a 1.6-kb insert of TAG-1 (kindly provided by A. J. W. Furley et al, 1990); pGEM-3 containing a 750-bp insert of GAP-43 (kindly provided by J. H. P. Skene); pBKSII(1) containing a 3.5-kb insert of SC-1 (kindly provided by T. M. Jessel); and pBKSII M 13 containing a 5.2-kb insert of NILE (kindly provided by W. Stallcup), the rat homolog of L1 (Bock et al, 1985;Prince et al, 1991). From the 58 terminal region of NILE/L1 1.3 kb was subcloned in pBSKII(1) using the unique sites of BamHI/XhoI.…”

Differential Effects of Two Hydrocephalus/MASA Syndrome-related Mutations on the Homophilic Binding and Neuritogenic Activities of the Cell Adhesion Molecule L1

“…Substitutions with another charged or polar residue are likely to be tolerated. Indeed, this residue is less conserved in L1 homologs, and His-210 is replaced by Asn in mouse and rat (2,3) and by Ser in the Drosophila homolog (21). This is also supported by the less deleterious effects of the H210Q mutation.…”

“…L1 is a 200-kDa transmembrane glycoprotein and a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. It contains six Ig-like domains in the amino-terminal region, followed by five fibronectin type III repeats, one transmembrane domain, and a cytoplasmic domain (2,3). L1 can undergo homophilic interactions with L1 (4, 5), as well as heterophilic interactions with other adhesion molecules, such as NCAM 1 (6), TAG-1/axonin-1 (7,8), F3/F11 (9), glia (10), and components of the extracellular matrix (11,12).…”

Molecular Characterization of a Novel Human Hybrid-type Receptor That Binds the α2-Macroglobulin Receptor-associated Protein

“…5), and liver, lung, bone marrow, and adrenals (not shown). DISCUSSION We characterize a new receptor with structural modules described previously in different gene families: the segment containing 10 cysteine residues present in yeast Vps10p (12) and human gp95/sortilin, 2 the YWTD and class A repeats of the LDLR gene family, and the fibronectin type III repeats (23) present in several proteins including certain neural adhesion proteins (24,25). SorLA-1 may therefore be classified as a hybrid receptor and, according to its domain structure, a potential target for multiple ligands.…”