Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase

Abstract

Vaccines against Neisseria meningitidis group C are based on its ␣-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 ␣-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 ␣-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 ␣-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 ␣-2,9-polysialic acid in a nonprocessive fashion when initiated with an ␣-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 ␣-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.

Biochemical Characterization of a Polysialyltransferase from Mannheimia haemolytica A2 and Comparison to Other Bacterial Polysialyltransferases

“…The truncation of amino acids at the N-terminus to remove a putative trans-membrane domain as well as the addition of MalE has previously been shown to improve the solubility and specific activity of PST [22], [23], [30].…”

“…None of the PSTs demonstrated activity on the monosialyl acceptor molecule GM3-FCHASE (data not shown), which is consistent with published evidence that at least two Neu5Ac residues on the acceptor molecule are required [22], [26]. We next wanted to determine the optimal reaction pH for PST Mh in the range between pH 7.0–pH 8.0, since these pH values would likely be most suitable for downstream applications as they are near physiological pH and also since previous studies have shown that PSTs from E. coli and N. meningitidis have a broad range of pH tolerance with an optimum at pH 8.0 [22], [23]. We also analyzed reactions at outlying pH values, including pH 5.5, which was expected to be unsuitable for large-scale reactions because of its acidity, as well as a very basic pH at 9.5.…”

“…In bacteria, genes encoding PSTs in E. coli K1 and K92 as well as from N. meningitidis group B and C are located in the capsule biosynthesis locus of the respective organisms [17], [18]. These enzymes have been characterized in recombinant form [19], [20], [21], [22], [23] and grouped into CAZy family GT-38, which consists exclusively of bacterial PSTs. Bacterial PSTs function at the cytoplasmic side of the inner membrane next to the capsule export machinery, which has posed a challenge for expression and analysis of recombinant forms of these peripheral membrane enzymes.…”

Regulation of capsule inNeisseria meningitidis

“…Csc and Csb share 75% sequence homology and 64% identity and synthesize sialic acid homopolymers with different linkages. Through chimeric fusion between Csb and Csc, the 107-aa N-terminal sequence was found to be sufficient to determine the α2→8 polymer linkage (Peterson et al, 2011). A 95-aa C-terminal sequence extension of Csb (compared to the E. coli K1 homologue that catalyzes an identical α2→8 linked polysialic acid capsule) was critical for Csb’s activity because deletion of this region completely abolished capsule polymerase enzymatic activity (Freiberger et al, 2007).…”

Sialic acid metabolism and sialyltransferases: natural functions and applications

“…The highly homologous capsular polysaccharide (CPS) polymerases (SiaDs) of N. meningitidis serogroups W135 and Y which have both hexosyltransferase (α1–4-galactosyltransferase activity for SiaD W135 and α1–4-glucosyltransferase activity for SiaD Y ) and sialyltransferase activities responsible for the synthesis of sialic acid-containing heteropolymeric CPSs [−6Gal/Glcα1–4Neu5Acα2-] n belong to GT4 along with other glycosyltransferases. In comparison, E. coli K1 α2–8-polysialyltransferase (NeuS), E. coli K92 alternating α2–8/9-polysialyltransferase (Vimr et al 1992; Steenbergen et al 1992; Shen et al 1999), N. meningitidis serogroup B α2–8-polysialyltransferase (SiaD) encoded by siaD / synD , and N. meningitidis serogroup C α2–9-polysialyltransferase encoded by synE are grouped into GT38 (Peterson et al 2011). GT42 includes α2–3-sialyltransferases (Cst-I and Cst-III) and a multifunctional α2–3/8-sialyltransferase (Cst-II) which also has α2–8-sialidase and α2–8- trans -sialidase activities (Cheng et al 2008) from C. jejuni (Gilbert et al 2002; Gilbert et al 2000), a P. multocida α2–3-sialyltransferase (PmST3) encoded by Pm1174 gene (Thon et al 2012), as well as an α2–3-sialyltransferase encoded by lic3A gene (Harrison et al 2005) and a multifunctional α2–3/8-sialyltransferase (Lic3B) from H. influenzae (Fox et al 2006).…”