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Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases.

Freiberger F, Claus H, Günzel A, Oltmann-Norden I, Vionnet J, Mühlenhoff M, Vogel U, Vann WF, Gerardy-Schahn R, Stummeyer K - Mol. Microbiol. (2007)

Bottom Line: The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST).Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1.Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

ABSTRACT
The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in alpha-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. Subsequent structure-function analyses of NmB-polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E-D/E-G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

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PolySia biosynthesis of purified NmB-polyST. A. Purified NmB-polyST was assayed for 30 min in the presence of 0.1 mM pentameric α2,8-linked sialic acid (DP5) and 1 mM CMP-[14C]-Neu5Ac. Subsequently, half of the sample was digested with polySia-specific endoN. Radiolabelled reaction products were separated by acrylamide gel electrophoresis (10%) and detected by autoradiography. The following dyes were used as standards and correspond to polySia chain length given in brackets: trypan blue (DP100), xylene cyanol (DP52), bromophenol blue (DP19), bromcresol purple (DP11). B. Dependence of chain length distribution on acceptor concentration. The assay was performed as described in (A) but DP5 concentrations were varied as indicated. Samples were separated on a 25% polyacrylamide gel. To display single oligomeric-reaction products at the highest acceptor concentration more clearly, less sample volume was applied in lane 4.
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fig05: PolySia biosynthesis of purified NmB-polyST. A. Purified NmB-polyST was assayed for 30 min in the presence of 0.1 mM pentameric α2,8-linked sialic acid (DP5) and 1 mM CMP-[14C]-Neu5Ac. Subsequently, half of the sample was digested with polySia-specific endoN. Radiolabelled reaction products were separated by acrylamide gel electrophoresis (10%) and detected by autoradiography. The following dyes were used as standards and correspond to polySia chain length given in brackets: trypan blue (DP100), xylene cyanol (DP52), bromophenol blue (DP19), bromcresol purple (DP11). B. Dependence of chain length distribution on acceptor concentration. The assay was performed as described in (A) but DP5 concentrations were varied as indicated. Samples were separated on a 25% polyacrylamide gel. To display single oligomeric-reaction products at the highest acceptor concentration more clearly, less sample volume was applied in lane 4.

Mentions: Capsular polysaccharide purified from serogroup B N. meningitidis was shown to exhibit a high degree of polymerization (DP) (Gotschlich et al., 1969; Frosch and Müller, 1993). This implies that NmB-polyST is able to produce large polymers when the enzyme is part of the native capsule biosynthesis complex associated with the inner bacterial membrane. To analyse products synthesized by purified NmB-polyST in more detail, pentameric (DP5) α-2,8-linked oligosialic acid was used as the primer for the recombinant enzyme and the polymerization reaction was started by addition of radiolabelled sugar donor substrate CMP-[14C]-Neu5Ac. Products were separated by high percentage polyacrylamide gel electrophoresis and visualized by autoradiography. Because polySia can be specifically degraded with bacteriophage endosialidases (endoN) (Mühlenhoff et al., 2003; Stummeyer et al., 2005), the nature of the synthesized products was controlled by treating one of two parallel samples with endoN prior to electrophoresis. As shown on the gel in Fig. 5A, which separates polymerization products from DP 10 to > 100, a large fraction of the synthesized polySia was longer than 100 residues per chain. All reaction products were specifically degraded by endoN. This demonstrates that the recombinant MBP–NmB-polyST is able to synthesize long polySia chains starting with short oligomeric acceptors in vitro. However, in addition to the high-molecular-weight polySia, also shorter oligosialic acid reaction products were detectable (Fig. 5A). To validate the presence of short reaction products and analyse the mode of chain elongation in more detail, we studied the influence of acceptor concentrations on chain length distribution. Therefore, DP5 concentrations were altered over three orders of magnitude starting with 1 μM, while the concentration of enzyme and donor substrate CMP-Neu5Ac was kept constant. As shown in Fig. 5B, increasing acceptor concentrations augment the concentration of short- and medium-sized reaction products until – at the point of equimolar concentrations of DP5 and CMP-Neu5Ac (lanes 4 and 5) – virtually all radiolabelled products remain below DP11 (Fig. 5B). An identical correlation of chain length distribution and acceptor concentration was obtained for trimeric (DP3) α-2,8-linked oligo sialic acid (Fig. S1). These data argue against processivity of NmB-PolyST in vitro, as in case of a highly processive mechanism synthesis of few long chains should be favoured over synthesis of many short chains. To evaluate these data in a second assay system, we used our recently developed fluorescence-based polyST assay (Vionnet and Vann, 2007) that utilizes the trisialylganglioside analogue GT3-FCHASE as artificial acceptor substrate. In a first experiment GT3-FCHASE was incubated with purified MBP–NmB-polyST and increasing concentrations of the donor substrate CMP-Neu5Ac (0, 5, 50, 500 μM). After an incubation time of 30 min the synthesized reaction products were analysed by ion exchange HPLC. As shown in Fig. 6, the chain length of synthesized polySia increased with increasing concentrations of donor substrate and resulted in a complete conversion of the acceptor substrate to high-molecular-weight polymer at a CMP-Neu5Ac concentration of 500 μM. This clearly demonstrates synthesis of long polySia chains. To verify the suggested non-processive elongation mode of NmB-polyST (Fig. 5B and Fig. S1), we performed a time-course experiment under reaction conditions that, as shown above, result in synthesis of long polySia chains (Fig. 7). If the reaction was stopped after 2 min predominantly short oligosialic acid-containing products were found (Fig. 7A) that were further elongated as the polymerization progressed (Fig. 7B and C) to finally yield high-molecular-weight polymer (Fig. 7D). This is reflected by a shift of the detected product peaks to longer retention times (Fig. 7). The occurrence of intermediates in the polymerization reaction argues, in agreement with the data obtained from the radioactive assay system, for non-processive chain elongation by MBP–NmB-polyST and indicates that the enzyme dissociates from the product after each addition of sialic acid.


Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases.

Freiberger F, Claus H, Günzel A, Oltmann-Norden I, Vionnet J, Mühlenhoff M, Vogel U, Vann WF, Gerardy-Schahn R, Stummeyer K - Mol. Microbiol. (2007)

PolySia biosynthesis of purified NmB-polyST. A. Purified NmB-polyST was assayed for 30 min in the presence of 0.1 mM pentameric α2,8-linked sialic acid (DP5) and 1 mM CMP-[14C]-Neu5Ac. Subsequently, half of the sample was digested with polySia-specific endoN. Radiolabelled reaction products were separated by acrylamide gel electrophoresis (10%) and detected by autoradiography. The following dyes were used as standards and correspond to polySia chain length given in brackets: trypan blue (DP100), xylene cyanol (DP52), bromophenol blue (DP19), bromcresol purple (DP11). B. Dependence of chain length distribution on acceptor concentration. The assay was performed as described in (A) but DP5 concentrations were varied as indicated. Samples were separated on a 25% polyacrylamide gel. To display single oligomeric-reaction products at the highest acceptor concentration more clearly, less sample volume was applied in lane 4.
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Related In: Results  -  Collection

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fig05: PolySia biosynthesis of purified NmB-polyST. A. Purified NmB-polyST was assayed for 30 min in the presence of 0.1 mM pentameric α2,8-linked sialic acid (DP5) and 1 mM CMP-[14C]-Neu5Ac. Subsequently, half of the sample was digested with polySia-specific endoN. Radiolabelled reaction products were separated by acrylamide gel electrophoresis (10%) and detected by autoradiography. The following dyes were used as standards and correspond to polySia chain length given in brackets: trypan blue (DP100), xylene cyanol (DP52), bromophenol blue (DP19), bromcresol purple (DP11). B. Dependence of chain length distribution on acceptor concentration. The assay was performed as described in (A) but DP5 concentrations were varied as indicated. Samples were separated on a 25% polyacrylamide gel. To display single oligomeric-reaction products at the highest acceptor concentration more clearly, less sample volume was applied in lane 4.
Mentions: Capsular polysaccharide purified from serogroup B N. meningitidis was shown to exhibit a high degree of polymerization (DP) (Gotschlich et al., 1969; Frosch and Müller, 1993). This implies that NmB-polyST is able to produce large polymers when the enzyme is part of the native capsule biosynthesis complex associated with the inner bacterial membrane. To analyse products synthesized by purified NmB-polyST in more detail, pentameric (DP5) α-2,8-linked oligosialic acid was used as the primer for the recombinant enzyme and the polymerization reaction was started by addition of radiolabelled sugar donor substrate CMP-[14C]-Neu5Ac. Products were separated by high percentage polyacrylamide gel electrophoresis and visualized by autoradiography. Because polySia can be specifically degraded with bacteriophage endosialidases (endoN) (Mühlenhoff et al., 2003; Stummeyer et al., 2005), the nature of the synthesized products was controlled by treating one of two parallel samples with endoN prior to electrophoresis. As shown on the gel in Fig. 5A, which separates polymerization products from DP 10 to > 100, a large fraction of the synthesized polySia was longer than 100 residues per chain. All reaction products were specifically degraded by endoN. This demonstrates that the recombinant MBP–NmB-polyST is able to synthesize long polySia chains starting with short oligomeric acceptors in vitro. However, in addition to the high-molecular-weight polySia, also shorter oligosialic acid reaction products were detectable (Fig. 5A). To validate the presence of short reaction products and analyse the mode of chain elongation in more detail, we studied the influence of acceptor concentrations on chain length distribution. Therefore, DP5 concentrations were altered over three orders of magnitude starting with 1 μM, while the concentration of enzyme and donor substrate CMP-Neu5Ac was kept constant. As shown in Fig. 5B, increasing acceptor concentrations augment the concentration of short- and medium-sized reaction products until – at the point of equimolar concentrations of DP5 and CMP-Neu5Ac (lanes 4 and 5) – virtually all radiolabelled products remain below DP11 (Fig. 5B). An identical correlation of chain length distribution and acceptor concentration was obtained for trimeric (DP3) α-2,8-linked oligo sialic acid (Fig. S1). These data argue against processivity of NmB-PolyST in vitro, as in case of a highly processive mechanism synthesis of few long chains should be favoured over synthesis of many short chains. To evaluate these data in a second assay system, we used our recently developed fluorescence-based polyST assay (Vionnet and Vann, 2007) that utilizes the trisialylganglioside analogue GT3-FCHASE as artificial acceptor substrate. In a first experiment GT3-FCHASE was incubated with purified MBP–NmB-polyST and increasing concentrations of the donor substrate CMP-Neu5Ac (0, 5, 50, 500 μM). After an incubation time of 30 min the synthesized reaction products were analysed by ion exchange HPLC. As shown in Fig. 6, the chain length of synthesized polySia increased with increasing concentrations of donor substrate and resulted in a complete conversion of the acceptor substrate to high-molecular-weight polymer at a CMP-Neu5Ac concentration of 500 μM. This clearly demonstrates synthesis of long polySia chains. To verify the suggested non-processive elongation mode of NmB-polyST (Fig. 5B and Fig. S1), we performed a time-course experiment under reaction conditions that, as shown above, result in synthesis of long polySia chains (Fig. 7). If the reaction was stopped after 2 min predominantly short oligosialic acid-containing products were found (Fig. 7A) that were further elongated as the polymerization progressed (Fig. 7B and C) to finally yield high-molecular-weight polymer (Fig. 7D). This is reflected by a shift of the detected product peaks to longer retention times (Fig. 7). The occurrence of intermediates in the polymerization reaction argues, in agreement with the data obtained from the radioactive assay system, for non-processive chain elongation by MBP–NmB-polyST and indicates that the enzyme dissociates from the product after each addition of sialic acid.

Bottom Line: The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST).Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1.Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

ABSTRACT
The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in alpha-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. Subsequent structure-function analyses of NmB-polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E-D/E-G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

Show MeSH
Related in: MedlinePlus