The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)

• Published in: Biochemical journal 2005-07, Vol.389 (Pt 1), p.63-72
• Main Authors: Saksouk, Nehmé, Pelosi, Ludovic, Colin-Morel, Pierre, Boumedienne, Manel, Abdian, Patricia L, Geremia, Roberto A
• Format: Article
• Language: English
• Subjects: Bacterial Capsules; Bacterial Capsules - biosynthesis; Bacterial Capsules - metabolism; Biochemistry, Molecular Biology; Carbohydrate Sequence; Cell Membrane; Cell Membrane - metabolism; Cloning, Molecular; Escherichia coli; Escherichia coli - cytology; Escherichia coli - genetics; Escherichia coli - metabolism; Galactosyltransferases; Galactosyltransferases - chemistry; Galactosyltransferases - isolation & purification; Galactosyltransferases - metabolism; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glycosylation; Life Sciences; Polysaccharides, Bacterial; Polysaccharides, Bacterial - biosynthesis; Protein Structure, Quaternary; Streptococcus pneumoniae; Streptococcus pneumoniae - classification; Streptococcus pneumoniae - enzymology; Streptococcus pneumoniae - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20050217

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one alpha-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->4)-beta-D-Glcp-(1-->]n). Expression of WciS-His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/DeltagalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [beta-D-GlcAp-(1-->4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by alpha-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS-His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.