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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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Purification of NmB-polyST. A. The MBP fusion protein of NmB-polyST was expressed in E. coli BL21 (DE3) and purified in a sequence of MBP affinity and size exclusion chromatography. Protein containing fractions were analysed by Coomassie-stained SDS-PAGE (left) and Western blot directed against the N-terminal StrepII-tag (middle) and the C-terminal His-tag (right). Protein bands corresponding to MBP–NmB-polyST are indicated by an arrow. Enzyme activity was monitored by the radiochemical polyST assay and specific activities were calculated as indicated. B. Size exclusion chromatography of MBP–NmB-polyST. Elution positions of standard proteins [Thyroglobulin (669 kDa), β-Amylase (200 kDa)] are indicated. Enzymatic activity of the collected fractions (secondary y-axis, grey circles) was determined using the enzyme-linked polyST assay. C. High-speed centrifugation of purified NmB-polyST. Samples were centrifuged as indicated and equal amounts of supernatant (sn) and pellet (p) fraction, which was re-suspended in the same volume as the supernatant, were analysed by Coomassie-stained SDS-PAGE.
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fig04: Purification of NmB-polyST. A. The MBP fusion protein of NmB-polyST was expressed in E. coli BL21 (DE3) and purified in a sequence of MBP affinity and size exclusion chromatography. Protein containing fractions were analysed by Coomassie-stained SDS-PAGE (left) and Western blot directed against the N-terminal StrepII-tag (middle) and the C-terminal His-tag (right). Protein bands corresponding to MBP–NmB-polyST are indicated by an arrow. Enzyme activity was monitored by the radiochemical polyST assay and specific activities were calculated as indicated. B. Size exclusion chromatography of MBP–NmB-polyST. Elution positions of standard proteins [Thyroglobulin (669 kDa), β-Amylase (200 kDa)] are indicated. Enzymatic activity of the collected fractions (secondary y-axis, grey circles) was determined using the enzyme-linked polyST assay. C. High-speed centrifugation of purified NmB-polyST. Samples were centrifuged as indicated and equal amounts of supernatant (sn) and pellet (p) fraction, which was re-suspended in the same volume as the supernatant, were analysed by Coomassie-stained SDS-PAGE.

Mentions: As MBP–NmB-polyST was shown to be active in vitro and in vivo and because MBP could be directly utilized for affinity chromatography, purification was optimized for the MBP–NmB-polyST construct schematically depicted in Fig. 1A. The fusion construct additionally carries two short epitope tags (N-terminal Strep II-, C-terminal His-tag), which were used for detection of the protein throughout the purification. After overexpression in E. coli BL21(DE3) at 15°C, the recombinant MBP–NmB-polyST was purified in two consecutive steps by MBP affinity and size exclusion chromatography. Trials to also utilize the C-terminal His-tag for purification failed, indicating that the epitope is not accessible in the native enzyme. As shown in Fig. 4A, the applied purification procedure yielded a highly enriched MBP–NmB-polyST (protein of 100 kDa). Major bands of smaller molecular weight still visible in the Coomassie-stained SDS-PAGE of the purified pool were also detected in a Western blot directed against the StrepII-tag, but not in a blot stained with an anti-His-tag antibody. This indicates that these bands represent breakdown products of MBP–NmB-polyST caused by C-terminal degradation.


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)

Purification of NmB-polyST. A. The MBP fusion protein of NmB-polyST was expressed in E. coli BL21 (DE3) and purified in a sequence of MBP affinity and size exclusion chromatography. Protein containing fractions were analysed by Coomassie-stained SDS-PAGE (left) and Western blot directed against the N-terminal StrepII-tag (middle) and the C-terminal His-tag (right). Protein bands corresponding to MBP–NmB-polyST are indicated by an arrow. Enzyme activity was monitored by the radiochemical polyST assay and specific activities were calculated as indicated. B. Size exclusion chromatography of MBP–NmB-polyST. Elution positions of standard proteins [Thyroglobulin (669 kDa), β-Amylase (200 kDa)] are indicated. Enzymatic activity of the collected fractions (secondary y-axis, grey circles) was determined using the enzyme-linked polyST assay. C. High-speed centrifugation of purified NmB-polyST. Samples were centrifuged as indicated and equal amounts of supernatant (sn) and pellet (p) fraction, which was re-suspended in the same volume as the supernatant, were analysed by Coomassie-stained SDS-PAGE.
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Related In: Results  -  Collection

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fig04: Purification of NmB-polyST. A. The MBP fusion protein of NmB-polyST was expressed in E. coli BL21 (DE3) and purified in a sequence of MBP affinity and size exclusion chromatography. Protein containing fractions were analysed by Coomassie-stained SDS-PAGE (left) and Western blot directed against the N-terminal StrepII-tag (middle) and the C-terminal His-tag (right). Protein bands corresponding to MBP–NmB-polyST are indicated by an arrow. Enzyme activity was monitored by the radiochemical polyST assay and specific activities were calculated as indicated. B. Size exclusion chromatography of MBP–NmB-polyST. Elution positions of standard proteins [Thyroglobulin (669 kDa), β-Amylase (200 kDa)] are indicated. Enzymatic activity of the collected fractions (secondary y-axis, grey circles) was determined using the enzyme-linked polyST assay. C. High-speed centrifugation of purified NmB-polyST. Samples were centrifuged as indicated and equal amounts of supernatant (sn) and pellet (p) fraction, which was re-suspended in the same volume as the supernatant, were analysed by Coomassie-stained SDS-PAGE.
Mentions: As MBP–NmB-polyST was shown to be active in vitro and in vivo and because MBP could be directly utilized for affinity chromatography, purification was optimized for the MBP–NmB-polyST construct schematically depicted in Fig. 1A. The fusion construct additionally carries two short epitope tags (N-terminal Strep II-, C-terminal His-tag), which were used for detection of the protein throughout the purification. After overexpression in E. coli BL21(DE3) at 15°C, the recombinant MBP–NmB-polyST was purified in two consecutive steps by MBP affinity and size exclusion chromatography. Trials to also utilize the C-terminal His-tag for purification failed, indicating that the epitope is not accessible in the native enzyme. As shown in Fig. 4A, the applied purification procedure yielded a highly enriched MBP–NmB-polyST (protein of 100 kDa). Major bands of smaller molecular weight still visible in the Coomassie-stained SDS-PAGE of the purified pool were also detected in a Western blot directed against the StrepII-tag, but not in a blot stained with an anti-His-tag antibody. This indicates that these bands represent breakdown products of MBP–NmB-polyST caused by C-terminal degradation.

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