title: Characterization and acceptor preference of a soluble meningococcal group C polysialyltransferase.
abstract: Vaccines against Neisseria meningitidis group C are based on its α-2,9-linked polysialic acid capsular polysaccharide. This polysialic acid expressed on the surface of N. meningitidis and in the absence of specific antibody serves to evade host defense mechanisms. The polysialyltransferase (PST) that forms the group C polysialic acid (NmC PST) is located in the cytoplasmic membrane. Until recently, detailed characterization of bacterial polysialyltransferases has been hampered by a lack of availability of soluble enzyme preparations. We have constructed chimeras of the group C polysialyltransferase that catalyzes the formation α-2,9-polysialic acid as a soluble enzyme. We used site-directed mutagenesis to determine the region of the enzyme necessary for synthesis of the α-2,9 linkage. A chimera of NmB and NmC PSTs containing only amino acids 1 to 107 of the NmB polysialyltransferase catalyzed the synthesis of α-2,8-polysialic acid. The NmC polysialyltransferase requires an exogenous acceptor for catalytic activity. While it requires a minimum of a disialylated oligosaccharide to catalyze transfer, it can form high-molecular-weight α-2,9-polysialic acid in a nonprocessive fashion when initiated with an α-2,8-polysialic acid acceptor. De novo synthesis in vivo requires an endogenous acceptor. We attempted to reconstitute de novo activity of the soluble group C polysialyltransferase with membrane components. We found that an acapsular mutant with a defect in the polysialyltransferase produces outer membrane vesicles containing an acceptor for the α-2,9-polysialyltransferase. This acceptor is an amphipathic molecule and can be elongated to produce polysialic acid that is reactive with group C-specific antibody.
authors.affiliation: Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Building 29, Room 103, 8800 Rockville Pike, Bethesda, MD 20892, USA.
authors.forename: Dwight C
authors.initials: DC
authors.lastname: Peterson
authors.forename: Gayathri
authors.initials: G
authors.lastname: Arakere
authors.forename: Justine
authors.initials: J
authors.lastname: Vionnet
authors.forename: Pumtiwitt C
authors.initials: PC
authors.lastname: McCarthy
authors.forename: Willie F
authors.initials: WF
authors.lastname: Vann
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology; D016667:Bacterial Capsules / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D038542:Neisseria meningitidis, Serogroup C / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D012794:Sialic Acids; C021319:polysialic acid; D012799:Sialyltransferases