Functional relationships of the sialyltransferases involved in expression of the polysialic acid capsules of Escherichia coli K1 and K92 and Neisseria meningitidis groups B or C.
Polysialic acid (PSA) capsules are cell-associated homopolymers of alpha2,8-, alpha2,9-, or alternating alpha2,8/2,9-linked sialic acid residues that function as essential virulence factors in neuroinvasive diseases caused by certain strains of Escherichia coli and Neisseria meningitidis. PSA chains structurally identical to the bacterial alpha2,8-linked capsular polysaccharides are also synthesized by the mammalian central nervous system, where they regulate neuronal function in association with the neural cell adhesion molecule (NCAM). Despite the structural identity between bacterial and NCAM PSAs, the respective polysialyltransferases (polySTs) responsible for polymerizing sialyl residues from donor CMP-sialic acid are not homologous glycosyltransferases. To better define the mechanism of capsule biosynthesis, we established the functional interchangeability of bacterial polySTs by complementation of a polymerase-deficient E. coli K1 mutant with the polyST genes from groups B or C N. meningitidis and the control E. coli K92 polymerase gene. The biochemical and immunochemical results demonstrated that linkage specificity is dictated solely by the source of the polymerase structural gene. To determine the molecular basis for linkage specificity, we created chimeras of the K1 and K92 polySTs by overlap extension PCR. Exchanging the first 52 N-terminal amino acids of the K1 NeuS with the C terminus of the K92 homologue did not alter specificity of the resulting chimera, whereas exchanging the first 85 or reciprocally exchanging the first 100 residues did. These results demonstrated that linkage specificity is dependent on residues located between positions 53 and 85 from the N terminus. Site-directed mutagenesis of the K92 polyST N terminus indicated that no single residue alteration was sufficient to affect specificity, consistent with the proposed function of this domain in orienting the acceptor. The combined results provide the first evidence for residues critical to acceptor binding and elongation in polysialyltransferase.
Steenbergen
Susan M
SM
Laboratory of Sialobiology, Department of Pathobiology, University of Illinois, Urbana, Illinois 61802, USA. e-vimr@uiuc.edu
Vimr
Eric R
ER
D000595:Amino Acid Sequence; D016667:Bacterial Capsules / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D038541:Neisseria meningitidis, Serogroup B / Q000201:enzymology / Q000378:metabolism; D038542:Neisseria meningitidis, Serogroup C / Q000201:enzymology / Q000378:metabolism; D010957:Plasmids; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D016415:Sequence Alignment; D012794:Sialic Acids / Q000096:biosynthesis*; D012799:Sialyltransferases / Q000737:chemistry* / Q000378:metabolism
D011135:Polysaccharides, Bacterial; D012794:Sialic Acids; C021319:polysialic acid; D012799:Sialyltransferases