PROTEIN DETERMINANTS FOR SPECIFIC POLYSIALYLATION OF THE NEURAL CELL-ADHESION MOLECULE

Expression of polysialic acid (PSA) involves its specific attachment t o the neural cell adhesion molecule (NCAM), Here we identify the amino acid residues within NCAM that are polysialylated and structural doma ins of the NCAM polypeptide that are required for addition of PSA in c ells, Chicken NCAM cDNAs containing amino acid mutations, domain delet ions, and domain substitutions were expressed in the F11 rat/mouse hyb rid cell line, which can produce polysialylated NCAM, Polysialylation of the chicken NCAM was evaluated by immunopurification and electropho resis, Mutation of all three potential N-glycosylation sites within th e fifth immunoglobulin domain (Ig5) abrogated polysialylation, Analysi s of paired mutations revealed that Asn-459 is heavily polysialylated, Asn-430 has a lower level of substitution, and Asn-404 receives littl e or no PSA, Analysis of domain deletions established that the intrace llular domain, Ig domains 1-3, and the COOH terminal fibronectin-type III (FNIII) repeat are not required for polysialylation, but that dele tion of either the adjacent Ig4 or FNIII-type domain prevented additio n of PSA, Accordingly, a minimal polypeptide for polysialylation was f ound to contain Ig domains 4 and 5, the adjacent FNIII repeat, plus a membrane attachment, These results suggest that although all PSA is lo cated within Ig5, regions outside Ig5 also play a role in PSA addition to NCAM, Furthermore, molecular modeling indicates spatial proximity of Asn-430 and Asn-459 and a tight-locking arrangement between Ig4, Ig 5, and FMIII#1 that would be consistent with their formation of a spat ially discrete enzyme recognition site for polysialylation.