title: Sequence Requirements for Neuropilin-2 Recognition by ST8SiaIV and Polysialylation of Its O-Glycans.
abstract: Polysialic acid is an oncofetal glycopolymer, added to the glycans of a small group of substrates, that controls cell adhesion and signaling. One of these substrates, neuropilin-2, is a VEGF and semaphorin co-receptor that is polysialylated on its O-glycans in mature dendritic cells and macrophages by the polysialyltransferase ST8SiaIV. To understand the biochemical basis of neuropilin-2 polysialylation, we created a series of domain swap chimeras with sequences from neuropilin-1, a protein for which polysialylation had not been previously reported. To our surprise, we found that membrane-associated neuropilin-1 is polysialylated at ∼50% of the level of neuropilin-2 but not polysialylated when it lacks its cytoplasmic tail and transmembrane region and is secreted from the cell. This was not the case for neuropilin-2, which is polysialylated when either membrane-associated or soluble. Evaluation of the soluble chimeric proteins demonstrated that the meprin A5 antigen-μ tyrosine phosphatase (MAM) domain and the O-glycan-containing linker region of neuropilin-2 are necessary and sufficient for its polysialylation and serve as better recognition and acceptor sites in the polysialylation process than those regions of neuropilin-1. In addition, specific acidic residues on the surface of the MAM domain are critical for neuropilin-2 polysialylation. Based on these data and pull-down experiments, we propose a model where ST8SiaIV recognizes and docks on an acidic surface of the neuropilin-2 MAM domain to polysialylate O-glycans on the adjacent linker region. These results together with those related to neural cell adhesion molecule polysialylation establish a paradigm for the process of protein-specific polysialylation.
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607.
authors.forename: Gaurang P
authors.identifier: http://orcid.org/0000-0002-6120-3747
authors.initials: GP
authors.lastname: Bhide
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607.
authors.forename: Ninoshka R J
authors.identifier: http://orcid.org/0000-0003-1386-0635
authors.initials: NR
authors.lastname: Fernandes
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607 karenc@uic.edu.
authors.forename: Karen J
authors.identifier: http://orcid.org/0000-0002-3801-071X
authors.initials: KJ
authors.lastname: Colley
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006031:Glycosylation; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000235:genetics / Q000378:metabolism*; D039943:Neuropilin-2 / Q000235:genetics / Q000378:metabolism*; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D039943:Neuropilin-2; C581308:neuropilin-2, human; D012799:Sialyltransferases; D008666:Metalloendopeptidases; C053833:meprin A; C515117:ST8SIA4 protein, human; D019158:N-Acetylneuraminic Acid
keywords: glycosylation; neuropilin; polysialic acid; polysialylation; polysialyltransferase; protein trafficking (Golgi); protein-protein interaction; sialic acid; sialyltransferase