Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases of Neisseria meningitidis

Authors

  • Heike Claus,

    Corresponding author
    1. Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Straße 2, 97080 Würzburg, Germany.
    Search for more papers by this author
    • These authors contribute equally to this work.

  • Katharina Stummeyer,

    1. Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, 30625 Hannover, Germany.
    Search for more papers by this author
    • These authors contribute equally to this work.

  • Julia Batzilla,

    1. Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Straße 2, 97080 Würzburg, Germany.
    Search for more papers by this author
    • Present address: Max-von-Pettenkofer-Institut, Ludwig-Maximilians-Universität München, Pettenkoferstraße 9a, 80336 München, Germany.

  • Martina Mühlenhoff,

    1. Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, 30625 Hannover, Germany.
    Search for more papers by this author
  • Ulrich Vogel

    1. Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Straße 2, 97080 Würzburg, Germany.
    Search for more papers by this author

*E-mail hclaus@hygiene.uni-wuerzburg.de; Tel. (+49) 931 201 46036; Fax (+49) 931 201 46445.

Summary

The capsular polysaccharides of serogroup W-135 and Y meningococci are sialic acid-containing heteropolymers, with either galactose or glucose as the second sugar residue. As shown previously, sequences of the predicted enzymes that catalyse capsule polymerization, i.e. SiaDW-135 and SiaDY, differ in only a few amino acids. By in vitro assays with purified recombinant proteins, SiaDW-135 and SiaDY were now confirmed to be the capsule polymerases harbouring both hexosyltransferase and sialyltransferase activity. In order to identify amino acids crucial for substrate specificity of the capsule polymerases, polymorphic sites were narrowed down by DNA sequence comparisons and subsequent site-directed mutagenesis. Serogroup-specific amino acids were restricted to the N-terminal part of the proteins. Exclusively amino acid 310, located within the nucleotide recognition domain of the enzymes' predicted hexosyltransferase moiety, accounted for substrate specificity as shown by immunochemistry and in vitro activity assay. Pro-310 determined galactosyltransferase activity that resulted in a serogroup W-135 capsule and Gly-310 determined glucosyltransferase activity that resulted in a serogroup Y capsule. In silico analysis revealed a similar amino acid-based association in other members of the same glycosyltransferase family irrespective of the bacterial species.

Ancillary