title: The Polybasic Region of the Polysialyltransferase ST8Sia-IV Binds Directly to the Neural Cell Adhesion Molecule, NCAM.
abstract: Polysialic acid (polySia) is a unique post-translational modification found on a small set of mammalian glycoproteins. Composed of long chains of α2,8-linked sialic acid, this large, negatively charged polymer attenuates protein and cell adhesion and modulates signaling mediated by its carriers and proteins that interact with these carriers. PolySia is crucial for the proper development of the nervous system and is upregulated during tissue regeneration and in highly invasive cancers. Our laboratory has previously shown that the neural cell adhesion molecule, NCAM, has an acidic surface patch in its first fibronectin type III repeat (FN1) that is critical for the polysialylation of N-glycans on the adjacent immunoglobulin domain (Ig5). We have also identified a polysialyltransferase (polyST) polybasic region (PBR) that may mediate substrate recognition. However, a direct interaction between the NCAM FN1 acidic patch and the polyST PBR has yet to be demonstrated. Here, we have probed this interaction using isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. We observe direct and specific binding between FN1 and the PBR peptide that is dependent upon acidic residues in FN1 and basic residues of the PBR. NMR titration experiments verified the role of the FN1 acidic patch in the recognition of the PBR and suggest a conformational change of the Ig5-FN1 linker region following binding of the PBR to the acidic patch. Finally, mutation of residues identified by NMR titration experiments impacts NCAM polysialylation, supporting their mechanistic role in protein-specific polysialylation.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago , Chicago, Illinois 60607, United States.
authors.forename: Gaurang P
authors.initials: GP
authors.lastname: Bhide
authors.affiliation: Center for Structural Biology, Research Resources Center, University of Illinois at Chicago , Chicago, Illinois 60607, United States.|Department of Microbiology and Immunology, University of Illinois at Chicago , Chicago, Illinois 60612, United States.
authors.forename: Gerd
authors.initials: G
authors.lastname: Prehna
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago , Chicago, Illinois 60607, United States.|Center for Structural Biology, Research Resources Center, University of Illinois at Chicago , Chicago, Illinois 60607, United States.
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Ramirez
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago , Chicago, Illinois 60607, United States.
authors.forename: Karen J
authors.identifier: 0000-0002-3801-071X
authors.initials: KJ
authors.lastname: Colley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D000071238:Fibronectin Type III Domain / Q000235:genetics*; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012794:Sialic Acids / Q000737:chemistry* / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C471213:His-His-His-His-His-His; D019006:Neural Cell Adhesion Molecules; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D012794:Sialic Acids; C021319:polysialic acid; D006639:Histidine; D012799:Sialyltransferases; C515117:ST8SIA4 protein, human