CLONING OF A RAT HOMOLOG OF MOUSE GLYCAM-1 REVEALS CONSERVATION OF STRUCTURAL DOMAINS

Recently we described the isolation of a mouse cDNA clone encoding a m ucin-like endothelial glycoprotein that appears to function as an adhe sive ligand for L selectin. This ligand has been named GlyCAM 1 (Gly-c osylation-dependent Cell Adhesion Molecule 1) because its adhesive int eractions with the L selectin lectin domain require that the GlyCAM 1 polypeptide chain be appropriately modified with carbohydrates. These carbohydrate modifications include the addition of sialic acid as well as sulfate residues to O-linked carbohydrate side chains that are clu stered in two serine/threonine-rich domains of the mucin. An additiona l interesting structure that may have relevance to the association of GlyCAM 1 with the lumenal surface of the endothelium was a potential a mphipathic helix at the C terminus of the glycoprotein. In order to ex amine the importance of the postulated O-linked domains as well as the potential amphipathic helix, we have cloned the rat homologue of GlyC AM 1. The sequence of this clone reveals a serine/threonine-rich prote in that is highly homologous with the mouse GlyCAM 1. As was found for the mouse GlyCAM 1, the rat homologue shows a clustering of these pot ential O-linked carbohydrate acceptors in two domains of the protein. Interestingly, many of the serines and threonines are found to be spac ed identically in the two homologues, consistent with the possibility that both density and position of the O-linked side chains may be impo rtant for appropriate L selectin-mediated adhesion. In support of its postulated functional importance, the C-terminal potential amphipathic helix is conserved in the rat homologue. Finally, immunoprecipitation analysis of [S-35]sulfate-labeled rat lymph nodes with either a mouse L selectin IgG chimera or a peptide antiserum directed against a rela tively conserved portion of mouse GlyCAM 1 demonstrates a approximatel y 45-kDa sulfated ligand in rat lymph nodes that is analogous to that previously described for mouse lymph nodes.