The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II.
A limited number of mammalian proteins are modified by polysialic acid, with the neural cell adhesion molecule (NCAM) being the most abundant of these. We hypothesize that polysialylation is a protein-specific glycosylation event and that an initial protein-protein interaction between polysialyltransferases and glycoprotein substrates mediates this specificity. To evaluate the regions of NCAM required for recognition and polysialylation by PST/ST8Sia IV and STX/ST8Sia II, a series of domain deletion proteins were generated, co-expressed with each enzyme, and their polysialylation analyzed. A protein consisting of the fifth immunoglobulin-like domain (Ig5), which contains the reported sites of polysialylation, and the first fibronectin type III repeat (FN1) was polysialylated by both enzymes, whereas a protein consisting of Ig5 alone was not polysialylated by either enzyme. This demonstrates that the Ig5 domain of NCAM and FN1 are sufficient for polysialylation, and suggests that the FN1 may constitute an enzyme recognition and docking site. Two other NCAM mutants, NCAM-6 (Ig1-5) and NCAM-7 (FN1-FN2), were weakly polysialylated by PST/ST8Sia IV, suggesting that a weaker enzyme recognition site may exist within the Ig domains, and that glycans in the FN region are polysialylated. Further analysis indicated that O-linked oligosaccharides in NCAM-7, and O-linked and N-linked glycans in full-length NCAM, are polysialylated when these proteins are co-expressed with the polysialyltransferases in COS-1 cells. Our data support a model in which the polysialyltransferases bind to the FN1 of NCAM to polymerize polysialic acid chains on appropriately presented glycans in adjacent regions.
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Brett E
BE
Department of Biochemistry and Molecular Genetics, University of Illinois, College of Medicine, Chicago, Illinois 60612, USA.
Mendiratta
Shalu Shiv
SS
Geiger
Kristin M
KM
Broom
Lucy J
LJ
Ho
Li-Lun
LL
Colley
Karen J
KJ
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D039842:Neural Cell Adhesion Molecule L1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012794:Sialic Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism; D012995:Solubility
D039842:Neural Cell Adhesion Molecule L1; D012794:Sialic Acids; C021319:polysialic acid; C103469:polysialyl neural cell adhesion molecule; C046778:CMP-N-acetylneuraminate-poly-alpha-2,8-sialosyl sialyltransferase; D012799:Sialyltransferases