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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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Influence of N-terminal fusion tags on NmB-polyST expression and activity. A. Schematic representation of NmB-polyST fusion proteins. NmB-polyST is shown as white box while short epitope tags (His, T7, Strep II) and large fusion partners (MBP, NusA) are given as black and grey boxes respectively. The length of the black ruler represents 200 amino acids. B. Western blot analysis of NmB-polyST fusion proteins. Proteins were expressed in E. coli BL21(DE3) and equal amounts of soluble (s) and insoluble (i) fractions were separated by SDS-PAGE. C-terminally epitope-tagged fusion proteins were detected by Western blot analysis with anti-His-tag antibody. C. Enzymatic activity of NmB-polyST fusion proteins in the soluble fractions. PolyST activity was analysed using the radiochemical activity assay. Reactions were incubated at room temperature and aliquots were assayed for radiolabelled polySia at the indicated time points. Each value represents the average of three independent determinations with the standard deviation indicated. D. Specific activities of NmB-polyST fusion proteins were standardized by NmB-polyST expression levels, which were determined by immunoblotting and infrared fluorescence detection.
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fig01: Influence of N-terminal fusion tags on NmB-polyST expression and activity. A. Schematic representation of NmB-polyST fusion proteins. NmB-polyST is shown as white box while short epitope tags (His, T7, Strep II) and large fusion partners (MBP, NusA) are given as black and grey boxes respectively. The length of the black ruler represents 200 amino acids. B. Western blot analysis of NmB-polyST fusion proteins. Proteins were expressed in E. coli BL21(DE3) and equal amounts of soluble (s) and insoluble (i) fractions were separated by SDS-PAGE. C-terminally epitope-tagged fusion proteins were detected by Western blot analysis with anti-His-tag antibody. C. Enzymatic activity of NmB-polyST fusion proteins in the soluble fractions. PolyST activity was analysed using the radiochemical activity assay. Reactions were incubated at room temperature and aliquots were assayed for radiolabelled polySia at the indicated time points. Each value represents the average of three independent determinations with the standard deviation indicated. D. Specific activities of NmB-polyST fusion proteins were standardized by NmB-polyST expression levels, which were determined by immunoblotting and infrared fluorescence detection.

Mentions: Detailed structure–function analyses of bacterial polysialyltransferases were hitherto prevented by insufficient supply of the enzymes. Expression levels were described to be low and polyST activity was found associated with bacterial membranes (Shen et al., 1999; Steenbergen and Vimr, 2003; Vionnet et al., 2006). As attempts to solubilize polyST were either unsuccessful or accompanied by inactivation of the enzyme, all analyses have been performed with crude membrane fractions as enzyme source. With the aim of obtaining purified enzyme in yields sufficient to perform structure–function studies, we began the current work with a systematic search for conditions that would allow production of recombinant NmB-polyST. To test the influence of N-terminal fusion partners on NmB-polyST expression and activity, constructs were generated either with short N-terminal epitope tags (T7, Strep II) or with additional large fusion parts like NusA and maltose-binding protein (MBP) (Fig. 1A). The constructs were expressed in E. coli BL21 (DE3) and soluble and insoluble fractions of the bacterial lysates were analysed for expression and activity of NmB-polyST. Although soluble protein could be detected for all constructs (Fig. 1B), the addition of large N-terminal fusions considerably increased the amount of active protein in the soluble fractions (Fig. 1C). Compared with polySTs carrying only short N-terminal epitope tags, additional fusion of NusA or MBP increased the soluble activity of NusA– and MBP–polyST two- and threefold, respectively. Also, the specific activity of both fusion proteins was increased twofold compared with enzymes with short tags (Fig. 1D). Subsequent experiments were therefore carried out with either NusA or MBP fusion proteins.


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)

Influence of N-terminal fusion tags on NmB-polyST expression and activity. A. Schematic representation of NmB-polyST fusion proteins. NmB-polyST is shown as white box while short epitope tags (His, T7, Strep II) and large fusion partners (MBP, NusA) are given as black and grey boxes respectively. The length of the black ruler represents 200 amino acids. B. Western blot analysis of NmB-polyST fusion proteins. Proteins were expressed in E. coli BL21(DE3) and equal amounts of soluble (s) and insoluble (i) fractions were separated by SDS-PAGE. C-terminally epitope-tagged fusion proteins were detected by Western blot analysis with anti-His-tag antibody. C. Enzymatic activity of NmB-polyST fusion proteins in the soluble fractions. PolyST activity was analysed using the radiochemical activity assay. Reactions were incubated at room temperature and aliquots were assayed for radiolabelled polySia at the indicated time points. Each value represents the average of three independent determinations with the standard deviation indicated. D. Specific activities of NmB-polyST fusion proteins were standardized by NmB-polyST expression levels, which were determined by immunoblotting and infrared fluorescence detection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169525&req=5

fig01: Influence of N-terminal fusion tags on NmB-polyST expression and activity. A. Schematic representation of NmB-polyST fusion proteins. NmB-polyST is shown as white box while short epitope tags (His, T7, Strep II) and large fusion partners (MBP, NusA) are given as black and grey boxes respectively. The length of the black ruler represents 200 amino acids. B. Western blot analysis of NmB-polyST fusion proteins. Proteins were expressed in E. coli BL21(DE3) and equal amounts of soluble (s) and insoluble (i) fractions were separated by SDS-PAGE. C-terminally epitope-tagged fusion proteins were detected by Western blot analysis with anti-His-tag antibody. C. Enzymatic activity of NmB-polyST fusion proteins in the soluble fractions. PolyST activity was analysed using the radiochemical activity assay. Reactions were incubated at room temperature and aliquots were assayed for radiolabelled polySia at the indicated time points. Each value represents the average of three independent determinations with the standard deviation indicated. D. Specific activities of NmB-polyST fusion proteins were standardized by NmB-polyST expression levels, which were determined by immunoblotting and infrared fluorescence detection.
Mentions: Detailed structure–function analyses of bacterial polysialyltransferases were hitherto prevented by insufficient supply of the enzymes. Expression levels were described to be low and polyST activity was found associated with bacterial membranes (Shen et al., 1999; Steenbergen and Vimr, 2003; Vionnet et al., 2006). As attempts to solubilize polyST were either unsuccessful or accompanied by inactivation of the enzyme, all analyses have been performed with crude membrane fractions as enzyme source. With the aim of obtaining purified enzyme in yields sufficient to perform structure–function studies, we began the current work with a systematic search for conditions that would allow production of recombinant NmB-polyST. To test the influence of N-terminal fusion partners on NmB-polyST expression and activity, constructs were generated either with short N-terminal epitope tags (T7, Strep II) or with additional large fusion parts like NusA and maltose-binding protein (MBP) (Fig. 1A). The constructs were expressed in E. coli BL21 (DE3) and soluble and insoluble fractions of the bacterial lysates were analysed for expression and activity of NmB-polyST. Although soluble protein could be detected for all constructs (Fig. 1B), the addition of large N-terminal fusions considerably increased the amount of active protein in the soluble fractions (Fig. 1C). Compared with polySTs carrying only short N-terminal epitope tags, additional fusion of NusA or MBP increased the soluble activity of NusA– and MBP–polyST two- and threefold, respectively. Also, the specific activity of both fusion proteins was increased twofold compared with enzymes with short tags (Fig. 1D). Subsequent experiments were therefore carried out with either NusA or MBP fusion proteins.

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