Protein determinants for specific polysialylation of the neural cell adhesion molecule.
Expression of polysialic acid (PSA) involves its specific attachment to the neural cell adhesion molecule (NCAM). Here we identify the amino acid residues within NCAM that are polysialylated and structural domains of the NCAM polypeptide that are required for addition of PSA in cells. Chicken NCAM cDNAs containing amino acid mutations, domain deletions, and domain substitutions were expressed in the F11 rat/mouse hybrid cell line, which can produce polysialylated NCAM. Polysialylation of the chicken NCAM was evaluated by immunopurification and electrophoresis. Mutation of all three potential N-glycosylation sites within the fifth immunoglobulin domain (Ig5) abrogated polysialylation. Analysis of paired mutations revealed that Asn-459 is heavily polysialylated, Asn-430 has a lower level of substitution, and Asn-404 receives little or no PSA. Analysis of domain deletions established that the intracellular domain, Ig domains 1-3, and the COOH-terminal fibronectin-type III (FNIII) repeat are not required for polysialylation, but that deletion of either the adjacent Ig4 or FNIII-type domain prevented addition of PSA. Accordingly, a minimal polypeptide for polysialylation was found to contain Ig domains 4 and 5, the adjacent FNIII repeat, plus a membrane attachment. These results suggest that although all PSA is located 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, Ig5, and FNIII#1 that would be consistent with their formation of a spatially discrete enzyme recognition site for polysialylation.
Nelson
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RW
Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Bates
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PA
Rutishauser
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D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001483:Base Sequence; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry* / Q000378:metabolism; D002645:Chickens; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D012794:Sialic Acids / Q000378:metabolism*
D015816:Cell Adhesion Molecules, Neuronal; D012794:Sialic Acids; C021319:polysialic acid; D001216:Asparagine