title: The polysialyltransferase ST8Sia II/STX: posttranslational processing and role of autopolysialylation in the polysialylation of neural cell adhesion molecule.
abstract: The presence of alpha2,8-linked polysialic acid on the neural cell adhesion molecule (NCAM) is known to modulate cell interactions during development and oncogenesis. Two enzymes, the alpha2,8-polysialyltransferases ST8Sia IV()/PST and ST8Sia II()/STX are responsible for the polysialylation of NCAM. We previously reported that both ST8Sia IV/PST and ST8Sia II/STX enzymes are themselves modified by alpha2,8-linked polysialic acid chains, a process called autopolysialylation. In the case of ST8Sia IV/PST, autopolysialylation is not required for enzymatic activity. However, whether the autopolysialylation of ST8Sia II/STX is required for its ability to polysialylate NCAM is unknown. To understand how autopolysialylation impacts ST8Sia II/STX enzymatic activity, we employed a mutagenesis approach. We found that ST8Sia II/STX is modified by six Asn-linked oligosaccharides and that polysialic acid is distributed among the oligosaccharides modifying Asn 89, 219, and 234. Coexpression of a nonautopolysialylated ST8Sia II/STX mutant with NCAM demonstrated that autopolysialylation is not required for ST8Sia II/STX polysialyltransferase activity. In addition, catalytically active, nonautopolysialylated ST8Sia II/STX does not polysialylate any endogenous COS-1 cell proteins, highlighting the protein specificity of polysialylation. Furthermore, immunoblot analysis of NCAM polysialylation by autopolysialylated and nonautopolysialylated ST8Sia II/STX suggests that the NCAM is polysialylated to a higher degree by autopolysialylated ST8Sia II/STX. Therefore, we conclude that autopolysialylation of ST8Sia II/STX, like that of ST8Sia IV/PST, is not required for, but does enhance, NCAM polysialylation.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Illinois College of Medicine, 1819 West Polk Street M/C 536, Chicago, IL 60612, USA.
authors.forename: B E
authors.initials: BE
authors.lastname: Close
authors.forename: J M
authors.initials: JM
authors.lastname: Wilkinson
authors.forename: T J
authors.initials: TJ
authors.lastname: Bohrer
authors.forename: C P
authors.initials: CP
authors.lastname: Goodwin
authors.forename: L J
authors.initials: LJ
authors.lastname: Broom
authors.forename: K J
authors.initials: KJ
authors.lastname: Colley
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D018076:DNA, Complementary / Q000235:genetics; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; C103469:polysialyl neural cell adhesion molecule; D001216:Asparagine; D012799:Sialyltransferases