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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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In vivo activity of NmB-polyST fused to maltose-binding protein (MBP). A. MBP–NmB-polyST and native NmB-polyST carrying no additional tags were cloned into a neisserial expression vector and transformed into the polyST-deficient Neisseria strain 2517. PolySia capsules of parental strain (mock) and transformants were analysed by whole-cell ELISA using mab 735. Equal loading of the wells with Neisseria was controlled using mab P1.2 for detection of the meningococcal major outer membrane protein PorA. Each value represents the average of three independent determinations with the standard deviation indicated. B. Lysates of the parental strain (mock) and transformants were additionally analysed by SDS-PAGE and Western blotting using mab 735 before and after treatment with polySia-degrading endoN (top). Equal sample loading was confirmed in a parallel Western blot immunostained with anti-PorA antibody P1.2 (bottom).
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fig03: In vivo activity of NmB-polyST fused to maltose-binding protein (MBP). A. MBP–NmB-polyST and native NmB-polyST carrying no additional tags were cloned into a neisserial expression vector and transformed into the polyST-deficient Neisseria strain 2517. PolySia capsules of parental strain (mock) and transformants were analysed by whole-cell ELISA using mab 735. Equal loading of the wells with Neisseria was controlled using mab P1.2 for detection of the meningococcal major outer membrane protein PorA. Each value represents the average of three independent determinations with the standard deviation indicated. B. Lysates of the parental strain (mock) and transformants were additionally analysed by SDS-PAGE and Western blotting using mab 735 before and after treatment with polySia-degrading endoN (top). Equal sample loading was confirmed in a parallel Western blot immunostained with anti-PorA antibody P1.2 (bottom).

Mentions: Our efforts to express recombinant NmB-polyST in the E. coli expression strain BL21 (DE3) clearly revealed beneficial effects of large N-terminal fusion parts on the expression of active, soluble enzyme. However, to analyse if polyST fusion proteins maintain enzymatic activity also in vivo, wild-type and MBP–NmB-polyST were subcloned into a neisserial expression vector and transformed into the polyST-deficient neisserial strain 2517. Parental strain and transformants were analysed for capsular polySia in a quantitative whole-cell ELISA. Equal loading of the microtitre plates with bacteria was confirmed in a parallel ELISA directed against the meningococcal major outer membrane protein PorA. Interestingly, no difference in capsule expression was observed between strains complemented with wild-type or the MBP fusion construct (Fig. 3A). Moreover, Western blot analysis of the neisserial lysates revealed a similar polySia staining of wild-type and fusion protein that was not detectable in samples treated with endoN (Fig. 3B), which is a bacteriophage-derived enzyme that degrades polySia with high substrate specificity (Mühlenhoff et al., 2003; Stummeyer et al., 2005). This clearly demonstrates that MBP–NmB-polyST is enzymatically active in vivo.


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)

In vivo activity of NmB-polyST fused to maltose-binding protein (MBP). A. MBP–NmB-polyST and native NmB-polyST carrying no additional tags were cloned into a neisserial expression vector and transformed into the polyST-deficient Neisseria strain 2517. PolySia capsules of parental strain (mock) and transformants were analysed by whole-cell ELISA using mab 735. Equal loading of the wells with Neisseria was controlled using mab P1.2 for detection of the meningococcal major outer membrane protein PorA. Each value represents the average of three independent determinations with the standard deviation indicated. B. Lysates of the parental strain (mock) and transformants were additionally analysed by SDS-PAGE and Western blotting using mab 735 before and after treatment with polySia-degrading endoN (top). Equal sample loading was confirmed in a parallel Western blot immunostained with anti-PorA antibody P1.2 (bottom).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169525&req=5

fig03: In vivo activity of NmB-polyST fused to maltose-binding protein (MBP). A. MBP–NmB-polyST and native NmB-polyST carrying no additional tags were cloned into a neisserial expression vector and transformed into the polyST-deficient Neisseria strain 2517. PolySia capsules of parental strain (mock) and transformants were analysed by whole-cell ELISA using mab 735. Equal loading of the wells with Neisseria was controlled using mab P1.2 for detection of the meningococcal major outer membrane protein PorA. Each value represents the average of three independent determinations with the standard deviation indicated. B. Lysates of the parental strain (mock) and transformants were additionally analysed by SDS-PAGE and Western blotting using mab 735 before and after treatment with polySia-degrading endoN (top). Equal sample loading was confirmed in a parallel Western blot immunostained with anti-PorA antibody P1.2 (bottom).
Mentions: Our efforts to express recombinant NmB-polyST in the E. coli expression strain BL21 (DE3) clearly revealed beneficial effects of large N-terminal fusion parts on the expression of active, soluble enzyme. However, to analyse if polyST fusion proteins maintain enzymatic activity also in vivo, wild-type and MBP–NmB-polyST were subcloned into a neisserial expression vector and transformed into the polyST-deficient neisserial strain 2517. Parental strain and transformants were analysed for capsular polySia in a quantitative whole-cell ELISA. Equal loading of the microtitre plates with bacteria was confirmed in a parallel ELISA directed against the meningococcal major outer membrane protein PorA. Interestingly, no difference in capsule expression was observed between strains complemented with wild-type or the MBP fusion construct (Fig. 3A). Moreover, Western blot analysis of the neisserial lysates revealed a similar polySia staining of wild-type and fusion protein that was not detectable in samples treated with endoN (Fig. 3B), which is a bacteriophage-derived enzyme that degrades polySia with high substrate specificity (Mühlenhoff et al., 2003; Stummeyer et al., 2005). This clearly demonstrates that MBP–NmB-polyST is enzymatically active in vivo.

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