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I want to do scheme for assays for N. meningitidis Capsule Polymerases from these papers.

Glycobiology vol. 24 no. 2 pp. 150–158, 2014 doi:10.1093/glycob/cwt102 Advance Access publication on November 20, 2013 Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: A new perspective for vaccine development Timm Fiebig2, Francesco Berti3, Friedrich Freiberger2, Vittoria Pinto3, Heike Claus4, Maria Rosaria Romano3, Daniela Proietti3, Barbara Brogioni3, Katharina Stummeyer2,5, Monika Berger2, Ulrich Vogel4, Paolo Costantino3, and Rita Gerardy-Schahn1,2 Keywords: capsule polymerases / Neisseria meningitidis serogroup X / NMR / recombinant protein production / vaccine development 2 Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany; 3Novartis Vaccines and Diagnostics Research, Via Fiorentina 1, 53100 Siena, Italy; and 4Institute for Hygiene and Microbiology, University of Würzburg, 97080 Würzburg, Germany Introduction Received on October 4, 2013; revised on November 11, 2013; accepted on November 11, 2013 Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is NmX for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of NmX called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the NmX capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with NmX-specific antibodies. Moreover, the chemical identity of in vitro produced NmX polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the NmX CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs. 1 To whom correspondence should be addressed: Tel: +49-511-532-9802; Fax: +49-511-532-8801; e-mail: 5 Present address: GRS - Gesellschaft für Anlagen- und Reaktorsicherheit, Schwertnergasse 1, 50667 Köln, Germany. Bacterial meningitis remains a serious threat to global health, accounting for an estimated annual 170,000 deaths worldwide. Importantly, Neisseria meningitidis (Nm) is the only bacterium capable of generating epidemics of meningitis (http://www., last accessed 26 November 2013). Based on different immunological properties of their capsular polysaccharides (CPSs), 12 serogroups have been identified, of which six (A, B, C, W, X and Y) are responsible for the majority of life-threatening diseases (Harrison et al. 2013). The fact that antibodies against the capsular polysaccharides are capable of killing meningococci in the presence of complement (Gotschlich et al. 1969) led to the development of polysaccharide and polysaccharide-protein conjugate vaccines (Jódar et al. 2002). The CPSs of all pathophysiologically relevant serogroups are negatively charged linear polymers. Although negative charge is introduced by the nonulose sialic acid in NmB, -C, -W and –Y, negative charge in the CPS of NmA and NmX results from phosphodiester bonds linking together N-acetyl-mannosamine 1-phosphate units ([→6)-α-D-ManpNAc-(1 → OPO3→]n) (Liu et al. 1971) and N-acetyl-glucosamine 1-phosphate ([→4)-α-DGlcpNAc-(1 → OPO3→]n), respectively (Bundle et al. 1973; Bundle, Smith, et al. 1974). Globally, NmA has the highest incidence rate and has in the recent past been responsible for large outbreaks in the African meningitis belt (Stephens et al. 2007). For this reason, a monovalent conjugate vaccine, MenAfriVac®, consisting of the NmA capsular polysaccharide coupled to tetanus toxoid, has been specifically designed for Africa (Roberts 2010). NmX has historically been associated with only sporadic or small clusters of disease, but outbreaks of NmX occurred in Niger (Boisier et al. 2007), Uganda, Kenya (Mutonga et al. 2009), Togo and Burkina Faso between 2006 and 2010 (Delrieu et al. 2011; Xie et al. 2013). Probably due to the colonization of niches that emerged by efficient vaccination against other serogroups, NmX, for which no vaccine exists, could gain increasing prevalence in the African countries. With an incidence of at least 120 cases per 100,000 residents in Burkina Faso in 2009 (Delrieu et al. 2011), NmX is subject to close surveillance, giving additional evidence for the need of an effective vaccine. © The Author 2013. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 150 The capsule polymerase of Neisseria meningitidis serogroup X A logic candidate for vaccine development is the NmX capsular polysaccharide (CPSX). If tested in rabbits, the purified CPSX in fact demonstrated immunogenicity (Bundle, Jennings, et al. 1974) and the conjugate of CPSX with CRM197 (CrossReacting Material 197, an enzymatically inactive and nontoxic form of diphtheria toxin) induced bactericidal antibodies in mice (Micoli, Romano, et al. 2013). On the background of these promising results, the goal of the current study was to clone and functionally express the capsule polymerase (CP) of NmX and to use the recombinant enzyme for the in vitro production of CPSX. As the preparation of conjugate vaccines so far depends on CPS isolated from bacterial cultures, production in an enzyme-catalyzed in vitro reaction from simple and highly pure compounds would provide an attractive, safe and economic alternative. The chromosomal locus cps containing the genetic information for capsule biosynthesis and surface transport is substructured into six regions: A–D, D′ and E (Figure 1). Regions B and C are highly conserved and encode the proteins necessary for export and assembly of the polysaccharide on the cell surface. Region A in contrast is serogroup-specific and encodes the glycosyltransferase(s) that catalyze the polymerization of the CPS (Harrison et al. 2013). In NmX, region A comprises three open reading frames called csxA, -B and -C (former xcbA, -B and -C). Based on bioinformatics information and a deletion–insertion mutational approach, csxA is believed to encode the CP CsxA (Tzeng et al. 2003). Here, we describe the molecular cloning and functional expression of csxA. The recombinant CsxA, carrying epitope tags at the N- and/or the C-terminus was soluble and could be purified in good quantity and quality. In the presence of short oligosaccharide primers derived from the CPSX, CsxA produced long polymers with immunological and chemical properties identical to the natural polymer. Results Functional identification and recombinant expression of CsxA To ensure that the protein encoded in csxA is a functional CP, the protein was expressed in Escherichia coli BL21(DE3) as MBP (maltose-binding protein) fusion protein with C-terminal His6-tag under the control of a T7-promoter (MBP-CsxA-His6). After cell lysis, the supernatant was prepared as described (Material and methods) and directly used to test MBP-CsxAHis6 for its capacity to elongate CPSX oligomers of an average degree of polymerization (avDP) of 15. In the radioactive incorporation assay used, supernatants isolated from bacteria expressing MBP-CsxA-His6 efficiently elongated the added primers, whereas supernatants obtained from mock transformed bacteria were negative (Figure 2). Based on this strong evidence that csxA encodes the NmX CP CsxA, we next concentrated on identifying suitable conditions for the recombinant expression of CsxA. As shown in Figure 3A, a spectrum of conditions was tested by varying the added fusion parts, the promoters used to drive protein expression and the host bacterial strains. As anti-CsxA antibodies are not available, all constructs were cloned with C-terminal His6-tags, to facilitate analytical steps and assist purification of recombinant proteins. The N-terminus was either let free, extended with a short StrepII-tag or fused to MBP, a large protein known to support solubility and folding of fusion partners (Sørensen and Mortensen 2005). At the promoter level, we tested tac and T7 and the host bacteria were varied between E. coli B [BL21 and BL21(DE3)] and E. coli K-12 [HMS174 (DE3) and M15( pREP4)] strains. The choice for these strains was based on the observation that E. coli B strains (but not E. coli K-12 strains) contain most of the components of the capsule gene cluster (Andreishcheva and Vann 2006) common to group II CPS expressing bacteria (i.e. N. meningitidis) and products of these genes may alter the expression levels and/or solubility of heterologous expressed CPs. The levels of recombinant protein as well as the distribution between soluble and insoluble bacterial fraction was analyzed by western blotting (Figure 3A). Though soluble protein could be detected under all conditions, expression under the control of the tac promoter significantly increased the amount of recombinant protein. Under these test conditions, highest expression levels were seen in M15( pREP4) cells. The two most promising conditions [CsxA-His6 and MBP-CsxA-His6, both under tac-promoter control in M15( pREP4)] were then upscaled to 500 mL of cultures and recombinant proteins were enriched by immobilized metal ion affinity chromatography (IMAC) via the His6-tag. From 500 mL of bacterial cultures, 2 mg of CsxA-His6 and 7 mg of MBP-CsxA-His6 were isolated. In western blot analyses, the isolated protein fractions displayed, however, N- and C-terminal degradations. A trial undertaken to eliminate truncated proteins by two consecutive affinity chromatography steps (first IMAC, second amylose resin) was not successful, suggesting that MBP-CsxA-His6 (and most probably also CsxA-His6) forms oligomers that include truncated protein forms. To interrogate this possibility, recombinant MBP-CsxA-His6 was loaded onto a preparative size exclusion chromatography (SEC) column. With low salt buffer (25 mM NaCl), MBP-CsxA-His6 (99.2 kDa) appeared in the void volume (9.32 mL, Figure 3B). If run in the presence Fig. 1. Schematic representation of the chromosomal locus (cps) of NmX. Products of genes encoded in region A are involved in the synthesis of the capsule polysaccharide and are serogroup-specific. For further information, see text [figure adapted from Harrison et al. (2013)]. 151 T Fiebig et al. Fig. 2. To analyze if CsxA contains an active CP, E. coli BL21(DE3) were transformed with plasmids driving the expression of MBP-CsxA-His6 or MBP alone (mock). Cultures were adjusted to identical OD600, cells were lysed and equal volumes of the soluble fractions tested for activity in a radioactive incorporation assay. of 700 mM NaCl, the peak containing most protein eluted with 11.65 mL, corresponding to 350 kDa and thus to tri- to tetrameric protein assemblies. N-terminal sequences determined for the His6-tagged bands of 56 and 48 kDa (Figure 3B) identified CsxA missing 2 and 45 amino acids at the N terminus, respectively. In summary, the Coomassie stained polyacrylamide gel electrophoresis (PAGE) as well as the western blot analysis of the peak fractions (collected in the presence of 700 mM salt) demonstrated that MBP-CsxA-His6 could be isolated in high quality and quantity. MBP-CsxA-His6 needs divalent cations for activity To evaluate the relevance of divalent cations for the activity of MBP-CsxA-His6, an adaption of the radioactive incorporation assay developed for polysialyltranferases (Weisgerber and Troy 1990) was used. The assay measured the elongation of oligosaccharide primers (avDP15) derived from CPSX with the labeled donor sugar UDP-[14C]-GlcNAc. Since enzymatically produced long chains are immobile in descending paper chromatography, the radioactivity retained at the loading spot could be correlated with enzymatic activity. As shown in Figure 4, the enzyme strictly depends on the presence of magnesium ions. Under in vitro conditions, 10 mM MgCl2 were sufficient to install maximal activity, which did not change in the presence of higher MgCl2 concentrations (20 and 50 mM). A replacement of Mg2+ by other divalent cations such as Mn2+ and Ca2+ was not possible and gave background values similar to those measured in the absence of Mg2+ or the presence of ethylenediaminetetraacetate (EDTA) (Figure 4 and data not shown). Dithiothreitol (DTT) up to a concentration of 2 mM had a slight but reproducibly positive effect on MBP-CsxA-His6 activity. In vitro synthesis of NmX capsule polymer and its characterization by immunological and chemical means Studies carried out to determine the size of CPSX isolated from natural source, identified high molecular weights. Depending 152 on the technique used, values between avDP300 and avDP800 have been reported (Berti et al. 2012; Xie et al. 2012). To test if chains of similar size can be produced in vitro with recombinant MBP-CsxA-His6, the enzyme was incubated with N-acetylglucosamine (UDP-GlcNAc) in the presence of CPSX oligomers (avDP3.2). The reaction was allowed to proceed overnight and products were analyzed by high percentage PAGE (Figure 5A). Long polymers ( partly not entering the gel) were obtained. In dot blot analysis, these products were stained by an antibody specifically directed against CPSX (Figure 5B). Of note, when MBP-CsxA-His6 was incubated with UDPGlcNAc in the absence of avDP3.2, low amounts of long polymers were formed (Figure 5A, control 1). The appearance of these products indicated that CsxA has a self-priming capacity. A similar capacity to start the synthesis of their products de novo has been described for other polymerases that are using nucleotide sugars like the heparosan synthases PmHS1 and PmHS2 from Pasteurella multocida (DeAngelis and White 2002, 2004). All other conditions (Figure 5A and B, controls 2–5) did not reveal product signals neither in high percentage PAGE nor dot blot analysis. The 31P NMR spectra recorded for the synthesized CPSX (Figure 5C, reaction and control 1) were in agreement with the spectrum obtained for the marker (avDP15) and with literature data (Berti et al. 2012; Xie et al. 2012). Besides of uridine monophosphate (UMP), the second product of the transfer reaction, small amounts of GlcNAc-1P were seen, which most likely represent the product of CsxA hydrolysis activity, a feature observed in many transferases that use nucleotide sugars (Soya et al. 2009; Sindhuwinata et al. 2010; Sugiarto et al. 2012). Importantly, 100% of the donor sugar was consumed in the presence of primers (with marginal hydrolysis activity), while only 16% of the added UDP-GlcNAc were used in the de novo reaction. Moreover, in the de novo reaction, only half of the used UDP-GlcNAc was incorporated into the growing chain, whereas the second half was hydrolyzed. These data demonstrate that product formation in the primed elongation is significantly advantaged over the self-priming reaction. To further compare in vitro synthesized CPSX (CPSXiv) to CPSX isolated from natural source (CPSXn), CPSXiv was purified by anion exchange chromatography (Figure 6A). In SEC (Figure 6B), highly similar migration profiles were obtained for both polymers and allowed to calculate the molecular weights to 407 kDa for CPSXn and 286 kDa for CPSXiv. 1 H NMR spectra recorded for the long polymers (Figure 6C) were in full congruence and again in agreement with published data (Berti et al. 2012). All protons characteristic for the CPSX repeating unit ( proton at C1 at 5.5 ppm; other protons in the range 4.2–3.7 ppm) were detected for the CPSXiv. Finally, the identity between CPSXiv and CPSXn was confirmed after total hydrolysis of the polymers. Under the strong acidic conditions used [2 M trifluoroacetic acid (TFA), 100°C for 2.5 h] 4P-GlcN was generated. In 1H NMR (Figure 6D), the anomeric region with α and β protons at C1 (signals at 5.4 and 4.9 ppm, respectively) appeared and were in full congruence with the control sample (synthetic 4P-GlcN) and with published data (Micoli, Adamo, et al. 2013). Together, our data demonstrate that recombinant CsxA can be used to produce CPSX in a bio-identical form in high quality and purity. The capsule polymerase of Neisseria meningitidis serogroup X Fig. 3. (A) Western blot analysis to identify the construct suited for recombinant production of CsxA. All constructs were C-terminally tagged with His6. The Nterminus was either free or extended by StrepII or MBP. Constructs were cloned to enable expression under the control of a T7 or a tac promoter and derivatives of E. coli B [BL21 and BL21(DE3)] and E. coli K-12 [HMS174(DE3) and M15( pREP4)] were used as expression hosts. Protein expression was monitored separately in the soluble (s) and insoluble ( p) fractions prepared from cell lysates. The western blots were stained with antibodies recognizing the epitope sequences as indicated. (B) Preparative SEC was used to (i) further purify MBP-CsxA-His6 and (ii) estimate the oligomerization state of the recombinant protein. In the presence of 700 mM NaCl, the majority of the protein eluted with an apparent molecular mass of 350 kDa, corresponding to a tri- to tetramer. With buffer containing 25 mM NaCl, the protein eluted in the void volume. Peak fractions taken in the presence of 700 mM salt were analyzed in Coomassie stained sodium dodecyl sulfate (SDS)– PAGE and western blot using an anti-MBP antibody (red) and the anti-pentaHis (green). Both analyses demonstrated that MBP-CsxA-His6 could be enriched in high quality. Standards used to calibrate the SEC column were: Thyroglobulin (669 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa). Discussion The increasing prevalence of NmX carriage in sub-Saharan Africa and the need of vaccine-based prevention of disastrous NmX outbreaks is an urgent need in public health (Xie et al. 2013). Crucial to the success of vaccination programs in the sub-Saharan meningitis belt is the affordability of a safe and high-quality immunogenic vaccine. MenAfriVac®, a vaccine specifically designed to combat spread and outbreaks of NmA in Africa has impressively demonstrated the success of this scheme (Frasch et al. 2012). At under 50 cent per dose (Roberts 2010), mass vaccination campaigns in Burkina Faso, Mali and Niger in 2010 (LaForce and Okwo-Bele 2011; Djingarey et al. 2012; Caini et al. 2013) installed herd immunity protecting not only vaccinated but also non-vaccinated individuals and young children (Kristiansen et al. 2013). A similar strategy for the production of an efficient NmX vaccine has just been published by the Novartis Vaccines Institute for Global Health (Micoli, Romano, et al. 2013). Until today, the production of conjugate vaccines against Nm strains depends on the use of CPS isolated from bacterial cell culture. Cultivation of pathogenic Nm strains, harvest of bacteria and purification of the capsular polysaccharide for use in humans are thereby cost intensive steps, which, in addition, require a high-tech environment. A way to circumvent these 153 T Fiebig et al. steps in the vaccine production chain would be the enzyme catalyzed synthesis of CPS starting from low cost and ultra-pure reagents. In this study, we describe the molecular cloning and Fig. 4. Activity of MBP-CsxA-His6 depends on the presence of Mg2+. Activity of the recombinant purified MBP-CsxA-His6 was determined in a radioactive incorporation assay in the absence or the presence of MgCl2 concentrations as indicated. functional expression of the CP CsxA of NmX. The protein could be produced in high quality and in vitro produced long CPSX chains from short oligosaccharide primers (avDP3.2) that in terms of immunological and chemical qualities were indiscernible from the polymer isolated from natural source. Together with the recent demonstrations that isolated CPSX— different to CPSA—is extremely stable in aqueous solution (no degradation after 3 weeks at 45°C; Berti et al. 2012) makes stock production of CPSX from cheap starting materials an attractive perspective in future vaccine developmental programs. In the current study, we focused on the use of a recombinant CsxA N-terminally fused to MBP and C-terminally extended with a His6-tag (MBP-CsxA-His6), since the protein could be expressed in significant yields (14 mg per liter culture) and showed no degradation or loss of activity if stored in aliquots at −80°C for >14 months. Also in the absence of the large N-terminal fusion partner (i.e. construct CsxA-His6), good yields of an active enzyme were obtained and future work may identify conditions, that favor expression levels and stability of this latter protein form. While the current study was under review, a manuscript describing the functional expression of CsxA as an N-terminal His6-tagged protein appeared (Muindi et al. 2013). This first study highlights that the expression of CsxA on the background of E. coli BL21(DE3) was sufficient to obtain a functional Fig. 5. (A) Products synthesized by MBP-CsxA-His6 after priming with avDP3.2 and overnight reaction in the presence of UDP-GlcNAc are displayed on high percentage PAGE stained with a combination of Alcian blue/silver. Based on the used marker avDP15, comprising CPSX chains that range in size between DP10 and DP60, the synthesized polymers (reaction) reach chain lengths significantly longer. In control 1, MBP-CsxA-His6 was incubated with UDP-GlcNAc in the absence of priming oligomers. A faint band at the start of the gel indicates that MBP-CsxA-His6 is able to synthesize high molecular weight CPSX also in the absence of acceptor molecules. All other controls (controls 2–5), in which reactants were missing as indicated, remained negative. (B) CPSX produced in the reaction as well as in control 1 was in dot blot analysis recognized with an antibody specifically directed against the CPSX. (C) 31P NMR spectra obtained for reaction products and substrates of the enzyme reaction. In the top panel, the marker avDP15 was used to determine the positions of the phosphor-monoester (Pme) on the non-reducing end of the hydrolyzed CPSX, the inorganic phosphates (Pi) and the internal phosphor-diester (Pde). After overnight incubation in the presence of the primer avDP3.2 (bottom), 96% of the added nucleotide donor sugar were inserted into the polymer. A strong signal indicates UMP as the second product of the enzyme reaction and a small peak indicating the generation of GlcNAc-1P as a side product. In the absence of the primer avDP3.2 (middle panel, control 1), a small product peak indicates that MBP-CsxA-His6 is able to start CPSX production de novo. However, with 84% of the nucleotide sugar remaining and a massively increased hydrolysis activity (half of the used nucleotide donor), it can be stated that the de novo reaction is drastically disadvantaged in comparison with the primed chain elongation. 154 The capsule polymerase of Neisseria meningitidis serogroup X Fig. 6. (A) In vitro synthesized capsule polysaccharide (CPSX) was purified by anion exchange chromatography. With the NaCl gradient used (dotted line), the polymer eluted as a single sharp peak well separated from other components of the reaction mixture. (B) The size of polymers synthesized in vitro (CPSXiv) or isolated from natural source (CPSXn) was comparable as determined by SEC. (C) 1H NMR spectra recorded from CPSXiv and CPSXn confirmed the identity of the two samples. The sharp peaks at 3.4 and 2.6 ppm in the CPSXn and CPSXiv spectra, respectively, represent process-related impurities. (D) 1H NMR spectra recorded from CPSXiv and CPSXn after total acidic hydrolysis were identical and in accordance with the control spectrum of chemically synthesized 4P-GlcN (synthetic). protein. In our screen of best expression systems, we identified the E. coli K-12 derivative M15( pREP4) as perfectly suited expression system, which, in contrast to E. coli B strains, does not possess genes that are part of the capsule gene cluster (Blattner et al. 1997; Andreishcheva and Vann 2006). This finding is of significance because the use of E. coli K-12 derivatives excludes the risk of heterologous capsule expression and thus should facilitate the development of economic production conditions. In high percentage PAGE and SEC, we showed that CPSX synthesized by MBP-CsxA-His6 is comparable in size to CPSX harvested from cultivated NmX (Berti et al. 2012; Xie et al. 2012). In addition, we have demonstrated the immunological and chemical identity by dot blot and NMR analyses. Only trace amounts of GlcNAc-1P were identified as side product and represent by all likelihood the result of facilitated UDP-GlcNAc hydrolysis in the presence of CsxA. This type of site activity has been described for many glycosyltransferases (Soya et al. 2009; Sindhuwinata et al. 2010; Sugiarto et al. 2012). Similar to UMP, GlcNAc-1P could be easily cleaned out from the preparation in a single chromatographic step. Using oligosaccharide primers with a fluorescent group added to the reducing end sugar, Muindi et al. (2013) showed that the trisaccharide is the minimal acceptor for recombinant His6-CsxA. Differently, we demonstrate that the recombinant MBP-CsxA-His6 produces CPSX also in the absence of any priming oligosaccharide (Figure 3; control 1). A similar selfpriming activity has been shown for other glycopolymerases (DeAngelis and White 2002, 2004) and is in line with the starting reaction suggested by Tzeng et al. (2003). These authors suggest that CsxA forms a phosphodiester between two UDPGlcNAc residues, whereby the cleavage of the diphosphatebond provides the driving energy. In this case, a UDP would remain at the reducing end sugar. Another possible substrate to start the reaction is GlcNAc-1P. However, as free GlcNAc-1P accumulated in the absence of primers (control 1; Figure 5C), it 155 T Fiebig et al. is likely not an efficient primer. Future analyses must be designed to interrogate the priming reaction in more detail. The next steps planed in our research are to up-scale the production of recombinant CsxA and to use the enzyme for large-scale production of CPSX and conjugation to protein carrier(s) to demonstrate immunogenicity equivalent to the polysaccharide extracted from NmX (Micoli, Romano, et al. 2013). Materials and methods General cloning of csxA Region A of serogroup X strain α388 was amplified with primers BS6 (5′-CCA CCA CCA AAC AAT ACT GCC GGC GGC GCT TCC C-3′, NMB0071: pos. 320-287) and BS7 (5′-GCT AAA CAC GCC CGC ACG CGC CAT TTC TTC CGC C-3′, NMB0064: pos. 357-324) and cloned into pCRscript (Stratagene) to generate plasmid pHC19. With the plasmid pHC19 as a template, csxA was amplified by polymerase chain reaction (PCR) using the primer pair KS423 (5′-GC GGA TCC ATT ATG AGC AAA ATT AGC AAA TTG-3′) and KS424 (5′-CCG CTC GAG TTG TCC ACT AGG CTG TGA TG-3′), containing BamHI and XhoI sites (underlined), respectively. The obtained PCR product 1 was ligated into the expression vector pET22b-Strep (Schwarzer et al. 2009) using the corresponding BamHI/XhoI sites. The resulting plasmid pStrepII-csxA-His6 (T7), allowed the expression of a recombinant protein with StrepII- at the N- and His6-tag at the C-terminus, under the control of the T7 promoter. To express csxA in fusion with MBP, the vector pMBP-csxA-His6 (T7) was constructed by inserting PCR product 1 into the BamHI/XhoI sites of the vector pMBP-Strep (Freiberger et al. 2007). To obtain plasmids in which the expression of csxA was under tac promoter control, a short sequence encoding an S3N10-linker (Kavoosi et al. 2007) at the 3′-end followed by BamHI/AvrII/HindIII sites was introduced into the pMal-c vector (NEB). Therefore, the gene encoding MBP was amplified with the primers TF14 (5′-GCA TCT CAT ATG AAA ACT GAA GAA GGT AAA CTG G-3′) binding the 5′-end of the MBP gene and TF12 (5′-AAG TTC AAG CTT TTA CCT AGG GGA TCC GTT ATT GTT ATT GTT GTT GTT ATT GTT ATT GGA GCT CGA ATT AGT CTG CGC GTC TTT CAG) binding the 3′-end of the MBP coding sequence and encoding the linker region. The resulting PCR product was ligated via HindIII/ BlpI into the corresponding sites of pMal-c resulting in pMal-c-S3N10. The region encoding CsxA-His6 was subsequently subcloned from pMBP-csxA-His6 (T7) via BamHI/AvrII into the corresponding sites of pMal-c-S3N10 giving the construct pMBP-csxA-His6 (tac). pcsxA-His6 (tac) was generated from PCR product 2 that was amplified with TF44 (5′-GCA TCT CAT ATG ATT ATG AGC AAA ATT AGC-3′) and KS424 and was cloned via NdeI/XhoI into a modified version of pMBPcsxA-His6 (tac). In the modified vector, an NdeI site was introduced 5′- of the MBP gene and an already existing NdeI was removed from the non-coding region of the vector backbone via site-directed mutagenesis using the primers TF28 (5′-TCACA CCGCCTATGGTGCACTCTCAG-3′) and TF29 (5′-TGCACC ATAGGCGGTGTGAAATAC-3′). Expression and purification of recombinant CsxA Freshly transformed E. coli expression strains were grown at 37°C in PowerBroth medium (Molecular Dimensions). At an 156 optical density of OD600 = 1.0 protein expression was induced by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside. Simultaneously, the incubation temperature was reduced to 15° C for a period of 20 h. For test expression experiments, cultures were adjusted to an identical OD600 = 3.0 and cells from 0.2 mL of culture volume were pelleted with 16,000 × g for 2 min. Five hundred milliliter of cultures used for protein purification were first pelleted by centrifugation at 6000 × g for 10 min at 4° C. After a washing step with Tris-buffer (50 mM Tris, pH 8.0, 500 mM NaCl), cells were resuspended in lysis buffer [50 mM Tris, pH 8.0, 500 mM NaCl, 2 mM DTT, 0.2 mg/mL DNase (Roche), 0.1 mg/mL RNaseA (Roche), 0.1 mg/mL lysozyme (Serva) and EDTA free protease inhibitor (complete EDTA-free, Roche)] and disrupted by 3–4 cycles of freeze-thawing, whereby the thawing step was facilitated by sonication in a sonication bath (Bandelin Sonorex sonicator). Cell pellets obtained from 0.2 mL of test cultures were lysed with 0.1 mL and pellets from 500 mL culture with 15 mL lysis buffer and aliquoted into 1.5 mL Eppendorf tubes. In test expressions, soluble and insoluble fractions were separated by centrifugation (16,000 × g, 10–20 min, 4°C), the supernatant mixed (1:1) with Laemmli buffer and used for PAGE as described below. If used for protein purification, the soluble fractions were directly loaded onto affinity matrices (HisTrap, GE Healthcare, for IMAC) or pre-swollen amylose resin (New England Biolabs). After loading, columns were washed with binding buffer (50 mM Tris, pH 8.0, 500– 700 mM NaCl, 1 mM DTT) without (in the case of amylose resin) or with 25 mM imidazol (in the case of HisTrap columns). Elution of recombinant proteins was achieved with a single-step maltose gradient (10 mM maltose) from amylose resin and a linear imidazole gradient (50–500 mM imidazole over 20 min) from HisTrap columns. Fractions containing recombinant protein were pooled and for further purification by SEC applied to a Superdex 10/300 GL column (GE Healthcare). Depending on the experimental requests, proteins were eluted with high salt buffer (50 mM Tris, pH 8.0, 500–700 mM NaCl, 1 mM DTT) or low salt buffer (50 mM Tris, pH 8.0, 25 mM NaCl, 1 mM DTT). Isolated proteins were concentrated to 8 mg/mL using Amicon Ultra centrifugal devices (Millipore, 30 or 50 MWCO). After separation into aliquots, samples were snap frozen in liquid nitrogen and stored at −80°C. SDS–PAGE and immunoblotting Sodium dodecyl sulfate (SDS)–PAGE was performed under reducing conditions using 2.5% (v/v) β-mercaptoethanol and 1.5% (w/v) SDS. Proteins were stained using Roti-Blue (Carl Roth GmbH) according to the manufacturer’s guidelines. For western blot analysis, samples and standard proteins were blotted onto polyvinylidene fluoride membranes (Millipore). His-tagged proteins were detected with 1 μg/mL of anti-penta-His antibody (Qiagen) and goat anti-mouse IR680 or goat anti-mouse IR800 antibody (LI-COR) as a second antibody. Second antibodies were used in a 1:20,000 dilution. For the display of MBP-fusion proteins, the anti-MBP (antibodiesonline) was used and followed by a goat anti-rabbit IR680 antibody (LI-COR) or a goat anti-rabbit IR800 antibody (LI-COR). The StrepII-tag was detected with non-modified Strep-Tactin (IBA) conjugated to IRDye 800CW NHS Ester Infrared Dye The capsule polymerase of Neisseria meningitidis serogroup X (LI-COR) in a 1:2000 dilution. Western blot bands were detected with the Odyssey infrared imaging system (LI-COR). Preparation of CPSX oligosaccharides CPSX oligosaccharide samples with an avDP of 3.2 and 15 were generated by acidic hydrolysis of long CPSX. Therefore, solutions containing 2.5 mg CPSX/mL sodium acetate buffer (50 mM sodium acetate, pH 4.8) were incubated at 80°C for 6 h and two pool fractions (avDP 3.2 and 15, respectively) were purified by anionic exchange chromatography (Q-Sepharose column, GE Healthcare) using sodium acetate gradient. The avDP of oligosaccharide samples was determined by 31P NMR analysis (Berti et al. 2012). If used in enzymatic reactions, hydrolyzed CPSX was dephosphorylated using acid phosphatase (Sigma) according to the manufacturer’s guidelines. Activity testing of CsxA by use of a radioactive assay system CsxA activity was analyzed using an adaptation of a radioactive incorporation assay previously described for polysialyltransferases (Weisgerber and Troy 1990; Freiberger et al. 2007). Briefly, assays were carried out with 5 pmol MBP-CsxA-His6 in a total reaction volume of 25 μL of assay buffer (50 mM Tris, pH 8.0, containing 1 mM DTT). Divalent cations were added from stock solutions. To prime the reactions, 4 μL of a CPSX oligosaccharide solution (370 ng/μL, avDP15) was used. Reactions were started by the addition of 2 mM UDPGlcNAc (Calbiochem) containing 0.05 μCi UDP-[14C]-GlcNAc (American Radiolabeled Chemicals). Samples were incubated at 37°C and 5 µL of aliquots spotted onto Whatman 3MM CHR paper after 0, 10, 20 and 30 min. Following descending paper chromatography, the chromatographically immobile 14C-labled CsxA reaction products were quantified by scintillation counting. Purification of CPSXiv CPSXiv was purified using a MonoQ HR 5/5 column (Pharmacia Biotech) at a flow rate of 1 mL/min and a linear sodium chloride gradient. CPSXiv eluted at 600 mM NaCl. The respective fractions were pooled, dialyzed (ZelluTrans, Roth, 1 kDA MWCO) against water and freeze dried for further analysis. Acidic hydrolysis of CPSX For the total hydrolysis of CPSX to 4P-GlcN, 0.5 mg samples of polysaccharide were treated for 2.5 h with 2 M TFA at 100° C (Micoli, Adamo, et al. 2013). Immunological and physicochemical analysis of CsxA reaction products To produce sufficient CsxA reaction product for dot blot, PAGE and NMR analyses, 39 pmol MBP-CsxA-His6 in reaction buffer (50 mM Tris, pH 8.0, 1 mM DTT and 20 mM MgCl2) were incubated with 5 mM UDP-GlcNAc and 0.11 mg of the CPSX oligosaccharide mixture with avDP3.2. The total reaction volume was adjusted to 750 μL. Reactions as well as control samples (containing reactants as indicated in Figure 3A) were incubated over night at 37°C. 10 μL of each sample was then used for separation on high percentage (25%) PAGE and visualized by a combined Alcian blue/silver staining procedure (Min and Cowman 1986). For dot blot analyses, 5 µL of aliquots of the reaction mixtures was spotted onto nitrocellulose (Whatman) and detected with a polyclonal serum (Remel/Oxoid; in 1:20,000 dilution) specifically recognizing the NmX capsule polysaccharide. Blots were developed with a goat anti-rabbit IR800 antibody (LI-COR) in 1:20,000 dilution. The residual sample (735 μL) was freeze-dried, resolved in deuterium oxide (D2O, 99.9% atom D; Aldrich) to give a concentration of 1–10 mg saccharide in 0.75 mL D2O and used for product characterization by NMR. 1 H and 31P NMR experiments were recorded on a Bruker Avance III 400 MHz spectrometer, equipped with a high precision temperature controller, using a 5-mm broadband probe (Bruker). For data acquisition and processing, the TopSpin version 2.6 software (Bruker) was used. 1H NMR spectra were collected at 25 ± 0.1°C with 32k data points over a 10 ppm spectral width. The transmitter was set at the water frequency which was used as the reference signal (4.79 ppm).31P NMR spectra were recorded at 161.9 MHz at 25 ± 0.1°C, with 32k data points over a 20 ppm spectral width. 85% phosphoric acid in deuterium oxide was used as an external standard (0 ppm). All spectra (1H and 31P NMR spectra) were obtained in quantitative manner using a total recycle time to ensure a full recovery of each signal (5 × Longitudinal Relaxation Time T1). In order to assign all 31P NMR peaks, bidimensional 1H-31P Heteronuclear Multiple-Bond Correlation (HMBC) experiments were acquired with a standard pulse program. 4096 and 512 data points were collected in F2 and F1 dimension, respectively. The average molecular weight of polysaccharides was estimated by SEC using an Ultimate 3000 system (Dionex) on PolySep GFC-P 6000 analytical column with GFC-Guard column (Phenomenex) calibrated with a series of defined pullulans standards (Polymer) of average molecular weights ranging from 20,000 to 800,000 Da. The void volume and total volume were determined with dextran and sodium azide, respectively. The polysaccharide samples were analyzed at a concentration of 1 mg/mL, using sodium phosphate buffer (10 mM, pH 7.2) as a mobile phase, at a flow rate of 0.5 mL/min. Funding This work was supported by the Deutsche Forschungsgemeinschaft (DFG) in the framework of DFG Research Unit 548 (Ge801/10-1). Acknowledgements We thank Timothy G. Keys and David Schwarzer for many helpful discussions and Christa Litschko and Andrea Bethe for help with experimental work. Conflict of interest The authors F.B., V.P., M.R.R., D.P., B.B. and P.C. are full-time employees of Novartis Vaccines. Abbreviations avDP, average degree of polymerization; CP, capsule polymerase; CPS, capsular polysaccharide; CPSA, capsular polysaccharide of 157 T Fiebig et al. NmA; CPSX, capsular polysaccharide of NmX; CPSXiv, in vitro synthesized CPSX; CPSXn, CPSX isolated from natural source; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetate; IMAC, immobilized metal ion affinity chromatography; IPTG, isopropyl β-D-1-thiogalactopyranoside; MBP, maltose-binding protein; Nm, Neisseria meningitidis; NmX, Neisseria meningitidis serogroup X; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; SEC, size exclusion chromatography; TFA, trifluoroacetic acid; UDP-GlcNAc, N-acetylglucosamine; UMP, uridine monophosphate. References Andreishcheva E, Vann W. 2006. Escherichia coli BL21(DE3) chromosome contains a group II capsular gene cluster. Gene. 384:113–119. 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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 49, pp. 33945–33957, December 5, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Dissection of Hexosyl- and Sialyltransferase Domains in the Bifunctional Capsule Polymerases from Neisseria meningitidis W and Y Defines a New Sialyltransferase Family* Received for publication, July 24, 2014, and in revised form, October 15, 2014 Published, JBC Papers in Press, October 23, 2014, DOI 10.1074/jbc.M114.597773 Angela Romanow‡, Timothy G. Keys‡, Katharina Stummeyer‡1, Friedrich Freiberger‡2, Bernard Henrissat§¶, and Rita Gerardy-Schahn‡3 From the ‡Institute of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany, §UMR 7257, Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille, France, and the ¶Department of Biological Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia Crucial virulence determinants of disease causing Neisseria meningitidis species are their extracellular polysaccharide capsules. In the serogroups W and Y, these are heteropolymers of the repeating units (36)-!-D-Gal-(134)-!-Neu5Ac-(23)n in NmW and (36)-!-D-Glc-(134)-!-Neu5Ac-(23)n in NmY. The capsule polymerases, SiaDW and SiaDY, which synthesize these highly unusual polymers, are composed of two predicted GT-B fold domains separated by a large stretch of amino acids (aa 399 –762). We recently showed that residues critical to the hexosyl- and sialyltransferase activity are found in the predicted N-terminal (aa 1–398) and C-terminal (aa 763–1037) GT-B fold domains, respectively. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain extends well beyond the predicted C-terminal GT-B domain and defines a new glycosyltransferase family, GT97, in CAZy (Carbohydrate-Active enZYmes Database). Bacterial capsules are a protective layer of extracellular polysaccharides that are firmly attached to the cell surface (1). Capsules both enhance bacterial survival in the environment by providing a highly hydrated physical barrier surrounding the cell (2) and contribute to symbiotic and pathogenic interactions with their host (1, 3). Capsules are divided into four groups based on characteristics of the polysaccharide and export * This work arose as a new research aspect in the frame of Deutsche Forsch- ungsgemeinschaft (DFG) Research Unit 548. This work was supported in part through DFG Grant Ge801/10-1 and in part through impact-derived resources of the Institute for Cellular Chemistry. 1 Present address: Gesellschaft für Anlagen- und Reaktorsicherheit, Schwertnergasse 1, D-50667 Köln, Germany. 2 Present address: Abbott Laboratories GmbH, Justus-von-Liebig Strasse 33, D-31535 Neustadt am Rübenberge, Germany. 3 To whom correspondence should be addressed. Tel.: 49-511-532-9802; Fax: 49-511-532-8801; E-mail: DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 machinery (4, 5). Group II capsules are characterized by polysaccharides with a high charge density and export via an ATPbinding cassette transporter-dependent system (6). Neisseria meningitidis (also referred to as meningococcus), a group of strictly human pathogens, produce group II capsules and are divided into 12 serogroups based on the chemical properties of the capsular polysaccharides (CPSs)4 (7). Six of these serogroups (A, B, C, W, X, and Y) are important pathogens (8) (the serogroup nomenclature is simplified according to Harrison et al. (7)). In serogroups B, C, W, and Y, the negative charge of the CPS results from the building block sialic acid (Sia or Neu5Ac), a nonasugar carrying a carboxylate function in position 1 (9). Whereas CPSs in NmB and NmC are homopolymers with Neu5Ac in !2,8- and !2,9-glycosidic linkage, respectively, the CPSs of NmW and NmY represent heteropolymers of the repeating units (36)-!-D-Gal-(134)-!-Neu5Ac-(23)n in NmW and (36)-!-D-Glc-(134)-!-Neu5Ac-(23)n in NmY (10). These latter structures are highly unusual because sialic acid occurs as an internal sugar. As a rule, sialic acid is a terminal (non-reducing end) sugar and, if internal, is conjugated with other sialic acid residues (11). We recently succeeded with the molecular cloning and functional characterization of the capsule polymerases of NmW (SiaDW) and NmY (SiaDY) and demonstrated bifunctionality for both enzymes (12). The large proteins with molecular masses of 120 kDa are 98% identical at the amino acid level and were predicted to comprise two glycosyltransferase (GT) domains of the GT-B fold (13). Bioinformatics and rational mutagenesis showed that the N-terminal domains encompass hexosyltransferase (HexTF) activity and that the C-terminal domains encode the sialyltransferase (SiaTF) activity (12). 4 The abbreviations used are: CPS, capsular polysaccharide; CAZy, Carbohydrate-Active enZYmes Database; GT, glycosyltransferase; HexTF, hexosyltransferase; 4-MU, 2!-(4-methylumbelliferyl); Sia, sialic acid; SiaTF, sialyltransferase; FD, fluorescence detection; DP, degree of polymerization; aa, amino acids; NulO, nonulosonic acid. JOURNAL OF BIOLOGICAL CHEMISTRY 33945 Downloaded from by guest on February 17, 2017 Background: Capsule polymerases of Neisseria meningitidis serogroups W and Y comprise hexosyl- and sialyltransferase activity. Results: Hexosyltransferase activity is encoded by the predicted N-terminal GT-B fold. Sialyltransferase activity requires 168 additional amino acids upstream of the predicted C-terminal GT-B fold. Conclusion: The sialyltransferase domains of NmW/Y define a new glycosyltransferase (CAZy) family. Significance: The new CAZy family comprises sequences from distantly related species. The Capsule Polymerases of N. meningitidis W and Y EXPERIMENTAL PROCEDURES Generation of Truncation Mutants and Expression of Recombinant Proteins—To separate HexTF and SiaTF domains, truncated variants of SiaDW and SiaDY (Fig. 4) were designed with care not to destroy secondary structure elements as predicted by the Phyre2 server (13). These constructs were generated by PCR using pHC4 (SiaDW) and pHC5 (SiaDY) (15) as templates. Hot Start Phusion-DNA-Polymerase (Thermo Scientific/Fermentas) was used in these experiments, and PCR conditions were as follows: 1 cycle of 98 °C/120 s; 30 cycles of 98 °C/15 s, 65 °C/30 s, 72 °C/30 s; and 1 cycle of 72 °C/300 s. Used primers, containing NdeI or XhoI sites, together with the obtained truncation variants are listed in Table 1. PCR products after digestion with NdeI and XhoI (New England Biolabs, Inc.) were purified and ligated into the respective sites of the expression vector pET22-b (Novagen). After transformation into Escherichia coli XL-1 Blue (Stratagene), transformed colonies were selected on ampicillin, and constructs were controlled by restriction analysis and sequencing. Expressed proteins carried a C-terminal 33946 JOURNAL OF BIOLOGICAL CHEMISTRY His6 epitope. If recombinant proteins were purified, the protocol described by Romanow et al. (12) was used. Synthesis and Purification of Fluorescently Labeled Acceptors to Prime SiaDW and SiaDY Reactions—Because activity testing in the classical radioactive incorporation assay depends on polymer formation (short oligosaccharides are mobile in the paper chromatography step (16)), the reliable testing of monofunctional mutants required a test system in which single sugar transfers could be clearly observed. We exploited the capability of SiaDW and SiaDY to extend sialic acid derivatives carrying the fluorescent label 2!-(4-methylumbelliferyl) (4-MU) at the reducing end. The recombinant C-terminally His6-tagged monofunctional full-length enzymes NmW-(S972A)-His6 and NmY(S972A)-His6 were used as HexTFs, and NmW-(E307A)-His6 and NmY-(E307A)-His6 were used as SiaTFs. 4-MU-Sia-Gal/ Glc were obtained by mixing 4 mM 4-MU-Sia (Sigma-Aldrich or Iris Biotech GmbH) with 4 mM donor sugar UDP-Gal/UDPGlc (Sigma) and 20 "g ml"1 of the respective HexTF (NmW(S972A)-His6 or NmY-(S972A)-His6) in reaction buffer (20 mM Tris/HCl, pH 8.0, 20 mM MgCl2, and 2 mM DTT). After 24 h at 25 °C, enzymes were removed by ultrafiltration (Amicon!Ultra 10 molecular weight cut-off, Millipore), and filtrates containing the reaction products (4-MU-Sia-Gal or 4-MU-Sia-Glc) were lyophilized (Alpha 1-2 LD Plus, Martin Christ). After dissolution in water, samples were desalted on P2-gel filtration columns (Bio-Rad). Reaction products were identified by high performance liquid chromatography using an UFLC-RX system (Shimadzu) coupled to a fluorescence detector (RF-10A XL). Samples were excited at 315 nm and monitored at 375 nm. Although, under the conditions used, this reaction did not give product yields of #90% (see Fig. 2), further product purification was omitted because the acceptor quality increased significantly from 4-MU-Sia to 4-MU-Sia-Hex, making 4-MU-Sia an irrelevant contaminant. The obtained 4-MU-Sia-Gal/Glc were then the starting material for Sia transfer to obtain 4-MU-SiaGal/Glc-Sia. This reaction was carried out with either NmW(E307A)-His6 or NmY-(E307A)-His6 in the presence of 2 mM CMP-Neu5Ac (Nacalai Tesque). Reaction conditions were identical to those described in the first reaction step. However, due to the significantly improved acceptor, the sialylation in this step was complete after 1 h of incubation. After the removal of enzymes and desalting, obtained compounds were used in iterative rounds to synthesize primers of the needed size. Enzyme Testing—Optimal buffer conditions (pH and metal ion concentration) for SiaDW/Y activity were established using a radioactive incorporation assay (12). Identical to other neisserial capsule polymerases, SiaDW/Y activity was optimal at slightly basic pH. Conditions identified with the wild type enzymes were maintained for all assays in this study. For activity testing with fluorescent compounds, reactions were carried out in a total volume of 25 "l. Mixtures contained 50 mM Tris/ HCl, pH 8.0, 20 mM MgCl2, 2 mM DTT, 1 mM acceptor (4-MUSia-Gal/Glc, 4-MU-Sia-Gal/Glc-Sia, or 4-MU-Sia-Gal/GlcSia-Gal/Glc) plus 2 mM of the nucleotide sugar (UDP-Gal/Glc (Sigma) and/or 2 mM CMP-Neu5Ac (Nacalai Tesque)), depending on the tested enzyme. Reactions were started by the addition of 20 "g ml"1 purified enzyme or, if the soluble fraction of bacterial lysates was used as the enzyme source, with VOLUME 289 • NUMBER 49 • DECEMBER 5, 2014 Downloaded from by guest on February 17, 2017 Point mutations introduced to destroy one catalytic function fully inactivated the capsule polymerases SiaDW and SiaDY. However, if the single mutant enzymes were combined in the same reaction tube, 70% of wild-type activity was restored. Beyond the demonstration that the two functional domains are capable of complementing each other in trans, these data indicated that chain elongation needs the successive activity of HexTF and SiaTF domains (12). Additional proof for the independence of the HexTF and SiaTF domains was obtained by saturation transfer difference NMR, showing the simultaneous binding of both sugar-nucleotides (CMP-Neu5Ac and UDPGal) and the priming oligosaccharide acceptor (12). The N-terminal HexTF domains of SiaDW and SiaDY are classified in CAZy family GT4 (14). On the other hand, due to the absence of similarity to known sialyltransferase families and the lack of defined boundaries for the SiaTF domains in SiaDW and SiaDY, these domains could not be assigned to a CAZy family. Here we aimed to determine whether the putative SiaTF domains of SiaDW and SiaDY could be physically separated from the respective HexTF domains and to delineate the boundaries of the SiaTF and HexTF domains. Crucial toward this goal was the availability of a sensitive assay system that would enable the unequivocal detection of single sugar transfers. To this end, we synthesized fluorescently labeled acceptor substrates with which HexTF and SiaTF could be specifically primed. High performance liquid chromatography separation and fluorescence detection (HPLC-FD) of the labeled reaction products provided the single-product resolution necessary to delineate the two glycosyltransferase activities. We show that the N-terminal GT-B folds contain all of the information for HexTF activity, whereas the predicted C-terminal GT-B domains were insufficient to generate active SiaTFs. Using a mutational approach, the SiaTFs were shown to require an additional 168 amino acids immediately upstream of the predicted GT-B domains. Bioinformatics analyses demonstrate that the newly identified SiaTF domains are the first characterized members of a new CAZy family, GT97. The Capsule Polymerases of N. meningitidis W and Y TABLE 1 Bacterial strains, plasmids, and primers used in this study Strains/Plasmids/Primers E. coli strains BL21(DE3) XL1-Blue Description/Sequence Source Novagen Stratagene B; F"ompT hsdSB(rB"mB") gal dcm (DE3) RecA1 ebdA1 gyrA96 thi-1 hsdR17 supE44 Plasmids pET-22b(&) Novagen 5!-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! Primer pairs used for the introduction of mutation in SiaDW KS350/KS351 E307A KS370/KS371 S972A 5!-CTGATCATGACATCAGAAAGTGCGGGATTTCCATATATATTTATG-3! 5!-CATAAATATATATGGAAATCCCGCACTTTCTGATGTCATGATCAG-3! 5!-ATCTCGCGTTGCTGTAGGTGTTTATGCAACTAGCTTATTTG-3! 5!-CAAATAAGCTAGTTGCATAAACACCTACAGCAACGCGAGAT-3! Primer pairs used for the introduction of mutation in SiaDY AR11/AR12 E307A KS370/KS371 S972A 5!-ATACAGATATCCTAATCATGACATCTCAAAGCGCAGGCTTTGGTTATATAT-3! 5!-atatataaccaaagcctgcgctttgagatgtcatgattaggatatctgtat-3! 5!-ATCTCGCGTTGCTGTAGGTGTTTATGCAACTAGCTTATTTG-3! 5!-CAAATAAGCTAGTTGCATAAACACCTACAGCAACGCGAGAT-3! Primer pairs used for the generation of SiaDW truncations KS422/KS421 C'639 AR2/KS273 N'398 AR1/KS273 N'562 AR9/AR273 N'609 AR10/KS273 N'639 KS433/KS273 N-'676 KS434/KS273 N'729 KS435/KS273 N'777 5!-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3! 5!-CCGCTCGAGGCTGCGCGGAAGAATAGTG-3! 5!-GCATCTCATATGTTTAATAACGTATCATTATCGTC-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGACTGATGATAATTTAATACCTAT-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGAAATATTCTTATAAATATATCTA-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGTCTTGGGAACTTATTCGTGCCTC-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGGGTAAGCGTTCGATGGATG-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGTCACTGAAAAGTAATGTAGTTG-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGAATATCGAAGCATTTCTAAAACC-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! Primer pairs used for the generation of SiaDY truncations KS422/KS421 C'639 AR2/KS273 N'398 AR1/KS273 N'562 KS433/KS273 N'676 KS434/KS273 N'729 5!-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3! 5!-CCGCTCGAGGCTGCGCGGAAGAATAGTG-3! 5!-GCATCTCATATGTTTAATAACGTATCATTATCGTC-3! 5-´CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGACTGATGATAATTTAATACCTAT-3! 5!-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!- GCATCTCATATGGGTAAGCGTTCGATGGATG-3! 5-´CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 5!-GCATCTCATATGTCACTGAAAAGTAATGTAGTTG-3! 5-´CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3! 72–100 "g ml"1 total protein. After appropriate incubation times, reactions were stopped by shock freezing in liquid nitrogen. Synthesized oligo- and polymers were analyzed and quantified via the fluorescent tag using an ultrafast HPLC system (UFLC-RX, Shimadzu) with coupled FD (detector RF-10A XL). The separation of 4-MU-labeled products was achieved with an anion exchange column (CarboPac! PA-100 column, Dionex). Before loading onto the column, samples were diluted 500-fold in water. The buffers A (20 mM NaNO3) and B (1 M NaNO3) were used to establish a curved elution gradient, reaching 21.65% buffer B over 35 min at 0.6 ml min"1 and a column temperature of 50 °C. The curved portion of the gradient is described by the following formula, in which the index "1.425 describes the slope (LS Solution, Shimadzu) (17–19). DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 B% # 21.65 $ $ e "1.425t/ 25 % 1% $e"1.425 % 1% Downloaded from by guest on February 17, 2017 Primer pairs used for cloning KS422/KS273 WT NmW KS422/KS273 WT NmY (Eq. 1) Elution profiles were monitored via fluorescence emission at 375 nm with 315 nm as the excitation wavelength. Under these conditions, the separation of polysaccharides up to a degree of polymerization (DP) of 21 was easily achieved (see “Results”). The HPLC profiles can be quantified to determine reaction progress. Peak areas represent the relative amount of each DP in the product profile and were calculated by integration of HPLC chromatograms with the LC Solution software (Shimadzu). Normalizing peak areas to the total area under the curve, weighting each peak according to the number of transfers required to form this product, and summing over all peaks gives the “normalized and weighted area” according to the formula, JOURNAL OF BIOLOGICAL CHEMISTRY 33947 The Capsule Polymerases of N. meningitidis W and Y ! ( Normalized and weighted area # n#0 " An ! An ( n#0 # $ $n% (Eq. 2) RESULTS Synthesis of Fluorescently Labeled Oligosaccharide Substrates— With the intention to delineate HexTF and SiaTF activities and functionally express the individual enzyme domains, it was first necessary to have defined acceptors to prime each of these activities. As detailed under “Experimental Procedures,” this was achieved using the previously described active site mutants bearing only the HexTF or SiaTF activity (12). The commercially available, but poorly used, fluorescent substrate 4-MUSia was first extended by a single hexose using either NmW(S972A)-His6 or NmY-(S972A)-His6 to give 4-MU-Sia-Gal or 4-MU-Sia-Glc, respectively (henceforth referred to as 4-MUSia-Hex or 4-MU-DP2). The 4-MU-DP2 was then extended by the respective SiaTF (NmW-(E307A)-His6 or NmY-(E307A)His6) to give 4-MU-Sia-Hex-Sia (4-MU-DP3), which proved to be an efficient acceptor for the HexTFs. Structures of the new acceptors (as an example shown for oligosaccharides with Gal as hexose) are given in Fig. 1. Anion exchange separation of 4-MU-Sia and the elongated products 4-MU-DP2 to 4-MU-DP4 was achieved by HPLC with a CarboPac PA-100 column and NaNO3 gradient. Product pro- 33948 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 49 • DECEMBER 5, 2014 Downloaded from by guest on February 17, 2017 where A is the peak area, and n is the number of transfers required to form this product. The normalized and weighted area is directly proportional to the total number of sugar transfers at that reaction time point. SDS-PAGE and Immunoblotting—SDS-PAGE was performed under reducing conditions using 2.5% (v/v) &-mercaptoethanol. Western blot analysis was done on a PVDF membrane (Millipore). For detection of the hexahistidine (His6) tag, penta-His antibody (Qiagen) was used as primary antibody at a concentration of 1 "g ml"1 and subsequently detected with 0.05 "g ml"1 IgG1 anti-mouse IR Dye800 antibody (Odyssey Infrared Imaging!). Protein bands were visualized and quantified with the infrared fluorescence detection system (LI-COR!) according to the manufacturer’s instructions. Bioinformatics—The most similar homologs of the identified SiaTF domain were found by iterative PSI-BLAST searches of the non-redundant protein sequences database using the PSIBLAST server (20). In each round, sequences with identity of #20% and coverage of #70% were included in the following iteration. After four iterations, the collected sequences were filtered for an E-value of better than 1e"50 and for the presence of conserved motifs (see “Results”). These sequences were aligned using the MAFFT server (G-INS-i strategy; BLOSUM30 scoring matrix; unalignlevel: 0.4) (21). Poorly fitting sequences were removed with MaxAlign (22) and by visual inspection of alignments and preliminary trees. The maximum likelihood tree was calculated from 359 ungapped positions using the PhyML software with bootstrapping (23). The tree was visualized with Archaeopterix (24). files for the NmW and NmY substrates show baseline resolution of all compounds (Fig. 1, A and B). Although the elution behavior of each product is dominated by the total charge, charge density also plays a role. This interplay is manifested as large increases in retention time upon the addition of a negatively charged sialic acid and a smaller reduction in retention time upon the addition of a neutral hexose. The subtle effect of different Gal and Glc stereochemistries on the conformation of NmW and NmY oligosaccharides also results in small differences in the observed retention times. Although transfer of the first hexose onto 4-MU-DP1 remained incomplete under the conditions used, subsequent transfer reactions proceeded to completion. Notably, the wild-type enzymes were unable to initiate polymerization on 4-MU-Gal and 4-MU-Glc. The finding suggests that the SiaTF domain requires at least a disaccharide to prime activity or, alternatively, recognition of the acceptor may require the presence of at least one sialic acid residue. The Fluorescent Substrates Enable Sensitive and Quantitative Detection of Capsule Polymerase Activity—To observe the initial steps in chain elongation, the wild-type SiaDW/Y were primed with 1 mM 4-MU-DP3 in the presence of a 4-fold excess of donor sugar (2 mM CMP-Sia and 2 mM UDP-Gal/Glc). Reactions were sampled over 40 min, and products were examined by HPLC-FD (Fig. 2). Shown are representative results obtained with SiaDW. Under these conditions, we observed the successive elongation of 4-MU-DP3 up to a maximum of DP19 (DP16 in the case of SiaDY). The new assay system enabled a precise quantification of SiaDW/Y activity. The normalized and weighted area (see “Experimental Procedures”) is directly proportional to the total number of sugars transferred at a given reaction time point. Progress curves are generated by plotting these values against reaction time (Fig. 2C). The congruence of reaction progress curves obtained in three independent experiments demonstrates the reliability of the assay system. Observation of Single Sugar Transfers Confirms Monofunctionality of Active Site Mutants—We previously generated active site mutants that disabled either the SiaTF or HexTF function of SiaDW (12). However, monofunctionality was tested in a complementation assay using paper chromatography to detect incorporation of radiolabeled sugars into long polysaccharides. In order to unequivocally observe the transfer of single sugar residues, we decided to test the single point mutant enzymes with the new fluorescent acceptor substrates. In separate reactions, NmW-(S972A)-His6 and NmW-(E307A)-His6 were incubated with both donor sugars and primed with either 4-MU-DP2 or 4-MU-DP3 (Fig. 3). As expected, each enzyme transferred a single sugar residue onto the appropriate acceptor, clearly demonstrating the monofunctionality of these enzymes. These monofunctional enzymes served as positive controls in the subsequent experiments to delineate the HexTF and SiaTF domains. Truncation Mutagenesis to Separate HexTF and SiaTF in SiaDW and SiaDY—A schematic representation of the linear sequence of SiaDW/Y (Fig. 4A) shows the previously predicted HexTF and SiaTF domains and a stretch of 365 amino acids linking these domains (12). In bioinformatics analyses, neither sequence homologies nor structural folds could be identified in The Capsule Polymerases of N. meningitidis W and Y Downloaded from by guest on February 17, 2017 FIGURE 1. Fluorescently labeled primers for the separate testing of hexosyl- and sialyltransferase activity and chromatographic behavior in anion exchange chromatography. Starting from 4-MU-Sia, the monofunctional active site mutants of SiaDW/SiaDY were used in iterative steps to generate labeled acceptors of defined length. Reaction samples were analyzed on a CarboPac" PA-100 column, and products eluted with a curved NaNO3 gradient as shown. A, structures of the labeled SiaDW glycans (top) and their running behavior on anion exchange chromatography (bottom) are shown. Note that the retention time increases with the addition of a Sia residue and decreases with the addition of Hex, leading to the elution order DP2, DP1, DP4, and then DP3. B, the elution behavior of labeled SiaDY glycans is similar to that of SiaDW. Of note, the chromatograms show that the conversion of 4-MU-DP1 to 4-MU-DP2 was incomplete under the conditions used, whereas all other reactions proceeded to completion. the central 365 amino acids. Therefore, we initially hypothesized that this sequence is not part of the catalytic domains but is necessary for tertiary organization of the two GT-B domains. To test this hypothesis, constructs harboring the predicted catalytic domains C'639 and N'777 (see Fig. 4A) were expressed and tested with the new fluorescent acceptors. DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 The predicted HexTF domain displayed similar activity to the respective active site mutants (Fig. 5A; see Fig. 3D for comparison), confirming that the predicted N-terminal GT-B fold (NmW-C'639-His6) comprises the active domain. In contrast, no activity could be detected with the predicted SiaTF domain (N'777; Fig. 5E), suggesting that the linker JOURNAL OF BIOLOGICAL CHEMISTRY 33949 The Capsule Polymerases of N. meningitidis W and Y region, or part thereof, might be necessary to form the active SiaTF domain. Thus, the N'398 truncation, comprising the SiaTF domain and the entire linker region (see Fig. 4A) was generated and tested. Indeed, this construct showed SiaTF activity comparable with the positive control (compare Figs. 5B and 3A), confirming that the SiaTF domain extends beyond the predicted GT-B fold. Further truncation constructs were tested to more precisely define the boundary of the SiaTF domain. We found that N'562 and N'609 (Fig. 5, C and D) retained SiaTF activity, but further truncation completely inactivated the enzyme. The results described for the HexTF and SiaTF domains of SiaDW were similarly obtained for the two domains in SiaDY. The expression of SiaDY truncation mutants is displayed in Fig. 4B. Activity data are not shown. Taken together, the truncation studies defined the boundaries of the SiaTF domains of these capsule polymerases and demonstrated that the domains can be separated and expressed as active transferases. As originally predicted, the N-terminal HexTF domain comprises a classic GT-B fold belonging to CAZy family GT4 (25). In contrast, the C-terminal SiaTF 33950 JOURNAL OF BIOLOGICAL CHEMISTRY domain extends beyond the predicted GT-B fold and includes )170 additional amino acids of the linker region. The sequence stretch connecting the two GT-B folds is thus not a linking element but part of the functional SiaTF domains in SiaDW/Y. The Sialyltransferase Domains in SiaDW/Y Define a New Glycosyltransferase Family—BLAST searches carried out with the newly identified SiaTF domain (SiaDW aa 563–1037) failed to reveal similarity to any of the known SiaTF families classified in the CAZy database (14). Further, searching the Pfam database (26) revealed no matches to HMM profiles (27, 28) constructed with the known SiaTF families. However, iterative PSI-BLAST searches of the non-redundant protein sequence database (29) revealed similarity to a number of uncharacterized protein sequences. The identified sequences were filtered for e-value (better than 1e"50) and for the presence of conserved motifs that have previously been identified in SiaDW (12). The presented data support the inclusion of these proteins as a new family of glycosyltransferases, GT97, in the CAZy database. Members of the new family are only found in a handful of taxonomically scattered species of bacteria and archaea. No VOLUME 289 • NUMBER 49 • DECEMBER 5, 2014 Downloaded from by guest on February 17, 2017 FIGURE 2. Time lapse recording of the SiaDW reaction. A, SiaDW was primed with 1 mM 4-MU-Sia-Gal-Sia (4-MU-DP3) in the presence of 2 mM UDP-Gal and 2 mM CMP-Sia. At the indicated time points, product profiles were recorded by HPLC-FD. Gray peaks, 4-MU and 4-MU-DP1, which were left in the priming 4-MU-DP3 pool. Due to the steep increase in acceptor quality from DP2 to DP3, both peaks remained unchanged over the reaction course. B, enlarged display of the HPLC-FD profile obtained at 40 min and labeling of individual DPs. C, reaction progress curves were generated by quantification of each chromatogram and plotting these values against the reaction time point. Three independent experiments (R1–R3) document the high reproducibility of the reaction. The Capsule Polymerases of N. meningitidis W and Y homologues were found in eukaryotes. Notably, the identified sequences are primarily found in two types of organisms with very distinct habitats: (i) commensal bacteria inhabiting humans and animals, including a number of opportunistic pathogens, as well as (ii) extremely halophilic bacteria and archaea isolated from saturated brines, salt lakes, and ponds (Fig. 6). Because archaea are not known to incorporate sialic acid into their glycans but have been shown to use homologous biosynthetic pathways for the incorporation of the related nonulosonic acids (NulOs), legionaminic acid and pseudaminic acid (30, 31), it is possible that GT97 glycosyltransferases exhibit specificity for these CMP-activated sugars. Supporting the putative function of GT97 family members as NulO transferases, the genome sequences of all of the identified organisms were found to harbor homologs of other nonulosonic acid biosynthesis genes, specifically NulO synthases (32, 30) and CMPNulO synthetase (33, 30). Although the role of sialylated structures in commensal organisms and opportunistic pathogens is generally understood to be the avoidance of the immune system by molecular mimicry of host structures (34), the functional significance of GT97 family proteins in extreme halophiles is less clear. The phylogenetic tree for the new family revealed clustering that is largely at odds with the true phylogenetic relationships among these organisms (Fig. 6), suggesting that convergent evolution or horizontal gene transfer may have played a role in evolution of the GT97 family. An alignment of the new family (Fig. 7) reveals slight differences from the known bacterial sialyl motifs (indicated in the alignment in red letters). The (D/E)(D/E)G motif (35), which is part of the catalytic center in bacterial sialyltransferases of CAZy families GT38, GT52, and GT80, was found to be replaced by QYA or QHG in the new family. Two other bacteDECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 rial sialyl motifs, the HP motif (35) and the S(S/T) motif (36), which are involved in binding the nucleotide sugar (35), are also highly conserved in GT97. However, the S(S/T) motif is strictly ST in the GT97 family. The functional importance of the HP and the ST motifs has previously been demonstrated for SiaDW/Y (12). To interrogate the importance of the QYA motif, the point mutant SiaDW-Y836F was prepared and completely abolished activity (data not shown). Taken together, the work presented in the current study (i) defines the minimum boundaries of the SiaTF domains of SiaDW/Y, (ii) shows that these domains are functional in isolation, and (iii) allows us to define a novel glycosyltransferase family, GT97, which is present in commensal bacteria of humans and animals and in extremely halophilic archaea and bacteria. DISCUSSION With the recent demonstration that the capsule polymerases of the N. meningitidis serogroups W and Y (SiaDW and SiaDY) are chimeric proteins comprising family GT4 HexTFs at their N terminus and a SiaTF at their C terminus (12), the question arose of whether the two enzymatic functions depend on their common presence in one polypeptide chain or if the functional subunits can be expressed separately. Here we demonstrate that the latter is the case, and we report a truncation study that delineates the boundary of the functionally active C-terminal SiaTF domain. This functional domain is )170 residues longer than the previously predicted GT-B fold domain (12). However, from our results, it is not clear whether the additional region (amino acids 610 –777) serves a chaperone function ensuring correct folding of the SiaTF domain or if it directly participates in catalysis. JOURNAL OF BIOLOGICAL CHEMISTRY 33951 Downloaded from by guest on February 17, 2017 FIGURE 3. Use of fluorescent primers to monitor the reaction products of single point mutant enzymes. Reaction profiles of the monofunctional mutants NmW-(E307A)-His6 and NmW-(S972A)-His6 were recorded in the presence of both donor sugars using the fluorescent primers as indicated. Reaction samples were taken after 15 s and 30 min. Peaks marked in gray represent 4-MU-DP1 and are unmodified in all reactions. A, NmW-(E307A)-His6 with 4-MU-DP2. The only reaction product formed was 4-MU-DP3. B, NmW-(E307A)-His6 primed with 4-MU-DP3. No product was formed. C, NmW-(S972A)-His6 primed with 4-MU-DP2. No product was formed. D, NmW-(S972A)-His6 primed with 4-MU-DP3. The only product formed was 4-MU-DP4. The Capsule Polymerases of N. meningitidis W and Y Downloaded from by guest on February 17, 2017 FIGURE 4. The capsule polymerases from NmW and NmY and deletion mutants made thereof. A, schematic illustration of the full-length capsule polymerases NmW/NmY. The GT-B folds predicted to comprise HexTF and SiaTF are highlighted in green and purple, respectively (13). The linker region connecting the GT-B folded domains is shown in black. Mutants made in the course of this project were named according to the introduced deletion. All proteins were expressed with an N-terminal His6 tag. B, Western blot to monitor protein expression. Constructs as indicated were expressed in BL21(DE3), transformed cells were lysed, and lysates were separated into soluble and insoluble fractions. After electrophoretic separation on 10% SDS-PAGE and Western blotting, proteins were displayed with an anti-penta-His antibody. Protein variants that were inactive in the in vitro test system are marked in red. This study sees SiaDW/Y classified into two CAZy families, GT4 based on the HexTF domain and GT97 based on the newly delineated SiaTF domain. Several other bifunctional GTs also belong to different CAZy families, including the Pasteurella multocida heparosan synthases (GT2 and GT45) (37) and the bifunctional human glycosyltransferase LARGE (GT8 and GT49) (38). Successful delineation of the two catalytic units required a novel assay system for the detection of single sugar transfers. To this end, we exploited two monofunctional active site mutants, 33952 JOURNAL OF BIOLOGICAL CHEMISTRY capable of transferring either a single hexose or sialic acid residue, for the controlled synthesis of fluorescently labeled acceptor substrates. The new substrates (4-MU-Sia-Hex for SiaTFs and 4-MU-Sia-Hex-Sia for HexTFs), combined with HPLC-FD analysis of reaction products, provided a highly sensitive and robust assay of these enzymatic activities. The stability and specificity of the new substrates for the target activity enabled activity measurements directly from bacterial lysates, which accelerates mutant testing enormously. In addition, we also demonstrate that the new assay system can be used to quantiVOLUME 289 • NUMBER 49 • DECEMBER 5, 2014 The Capsule Polymerases of N. meningitidis W and Y FIGURE 6. Phylogenetic tree of family GT97. Phylogenetic analysis was carried out using the PhyML software with bootstrapping (23) using the alignment shown in Fig. 7 as input. Protein accession numbers and species names are indicated, and bootstrapping values are given at the nodes. GT97 family members were only found in the bacterial and archaeal domains of life, and the majority of these species are either human or animal commensals (including several opportunistic pathogens) or extreme halophiles, as indicated. tatively monitor the product distribution and time course of the polymerization reaction. The results of the current study reveal some details about the acceptor specificity of the HexTF and SiaTF domains of DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 SiaDW/Y. For the HexTF domain, 4-MU-Sia proved to be a very poor acceptor compared with 4-MU-Sia-Hex-Sia; however, under the reaction conditions used, up to 90% of 4-MU-Sia could be converted to 4-MU-Sia-Hex. In contrast, neither the JOURNAL OF BIOLOGICAL CHEMISTRY 33953 Downloaded from by guest on February 17, 2017 FIGURE 5. Activity testing of truncation mutants. Truncation mutant comprising the HexTF (A) and the SiaTF with variant N-terminal extensions (B–E) were assayed in the presence of both donor sugars and a suited fluorescently labeled acceptor as indicated. Reaction samples were taken after 15 s and 30 min. Peaks marked in gray represent 4-MU-DP1. These peaks are unchanged over the reaction course. A, NmW-(C'639)-His6 with 4-MU-DP3 catalyzes the transfer of UDP-Gal by creating 4-MU-DP4. B, NmW-(N'398)-His6 primed with 4-MU-DP2 catalyzes the transfer of CMP-Sia by producing 4-MU-DP3. C, NmW-(N'562)-His6 primed with 4-MU-DP2 catalyzes the transfer of CMP-Sia by creating 4-MU-DP3. D, NmW-(N'609)-His6 primed with 4-MU-DP2 catalyzes the transfer of CMP-Sia by producing 4-MU-DP3. E, NmW-(N'777)-His6 primed with 4-MU-DP2 does not catalyze the transfer of CMP-Sia. The Capsule Polymerases of N. meningitidis W and Y Downloaded from by guest on February 17, 2017 33954 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 49 • DECEMBER 5, 2014 The Capsule Polymerases of N. meningitidis W and Y wild-type nor monofunctional SiaTFs used 4-MU-Gal/Glc as an acceptor, but these enzymes were highly efficient with 4-MU-Sia-Hex. These results indicate that a minimum of one Sia residue is necessary for recognition by the acceptor binding sites of both the HexTF and SiaTF domain. Further, the addition of the hydrophobic group MU improved acceptor quality because free Sia was not a substrate for the enzymes (12). These findings may reflect the nature of the in vivo priming acceptor. Willis and Whitfield (2) recently demonstrated for a number of Gram-negative bacteria, including N. meningitidis serogroup B, that the capsular polysaccharide is anchored via the reducing end to a unique glycolipid consisting of a lysophosphatidylglycerol moiety &-linked to oligo-3-deoxy-D-manno-oct-2-ulosonic acid. The observed acceptor specificities of SiaDW/Y agree well with the proposition that this universal anchor structure also primes in vivo capsule synthesis in NmW and NmY. This would explain both the requirement for a Sia residue, because this is a structural analog of 3-deoxy-D-manno-oct-2-ulosonic acid, and the improvement of acceptor quality by the addition of a hydrophobic group that may occupy the lipid binding site. Using the newly defined SiaTF domain to search protein databases revealed a new family of glycosyltransferases that is classified as GT97 in the CAZy database. The phylogenetic tree of GT97 sequences showed anomalous clustering, with high similarity occurring between sequences from distantly related species. This may suggest that horizontal gene transfer or convergent evolution has played a role in the history of the GT97 family. A case in point is the grouping of the Bacteroidetes Salinibacter ruber protein (WP_013061143) with those from haloarchaea Natronorubrum sulfidifaciens, Halorhabdus tiamatea, and Halorubrum kocurii. Indeed, the genome sequence of the extremely halophilic bacterium S. ruber revealed that considerable amounts of genetic material have been shared with its haloarchaea coinhabitants in concentrated sea water brines (39). These findings are in agreement with the well studied phylogeny of NulO biosynthetic pathways, which include FIGURE 7. Multiple sequence alignment of family GT97. Protein sequences were aligned with the MAFFT server (G-INS-I strategy; BLOSUM20 scoring matrix; unalignlevel: 0.4) (21) and visualized using BioEdit (42). High conservation is indicated by white letters on a black background, less conserved regions have black letters on a gray background, and the sialyl-motifs are highlighted in red. Accession numbers correspond to SiaDW and uncharacterized proteins from the following organisms: WP_002260055.1 (aa 563–1037/N. meningitidis serogroup W), WP_017271149.1 (aa 545–999/Sinorhizobium meliloti), WP_022901562.1 (aa 653–1117/Humibacter albus), KDS93228.1 (aa 678 –1144/Dermabacter hominis 1368), WP_010549827.1 (aa 651–1116/Brachybacterium paraconglomeratum), WP_017823772.1 (aa 664 –1129/Brachybacterium muris), WP_018949732.1 (aa 18 – 458/Thioalkalivibrio sp. ALMg11), WP_011570252.1 (aa 101–539/ Roseobacter denitrificans), WP_022244478.1 (aa 8 – 439/Roseburia sp. CAG:45), WP_005904190.1 (aa 21– 464/Fusobacterium nucleatum), WP_010200258.1 (aa 9 – 467/Bacillus sp. m3-13), WP_026894340.1 (aa 8 – 467/Clostridiisalibacter paucivorans), WP_013061143.1 (aa 8 – 446/S. ruber), WP_014051977.1 (aa 44 – 483/ halophilic archaeon DL31), WP_008847961.1 (aa 33– 474/H. kocurii), WP_008524267.1 (aa 8 – 446/H. tiamatea), WP_008161724.1 (aa 9 – 445/N. sulfidifaciens), WP_003463620.1 (aa 15– 456/Gracilibacillus halophilus), WP_014552521.1 (aa 15– 457/Halanaerobium praevalens), WP_012956677.1 (aa 15– 497/Methanobrevibacter ruminantium), WP_016357908.1 (aa 13– 470/Methanobrevibacter sp. AbM4). DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33955 Downloaded from by guest on February 17, 2017 FIGURE 7—continued The Capsule Polymerases of N. meningitidis W and Y REFERENCES 1. Willis, L. M., and Whitfield, C. (2013) Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporterdependent pathways. 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E., Knirel, Y. A., Gordon, J. I., Gagneux, P., Nizet, V., and Varki, A. (2009) Innovations in host and microbial sialic acid biosynthesis revealed by phylogenomic prediction of nonulosonic acid structure. Proc. Natl. Acad. Sci. U.S.A. 106, 13552–13557 31. Kandiba, L., and Eichler, J. (2013) Analysis of putative nonulosonic acid biosynthesis pathways in Archaea reveals a complex evolutionary history. FEMS Microbiol. Lett. 345, 110 –120 32. Hao, J., Balagurumoorthy, P., Sarilla, S., and Sundaramoorthy, M. (2005) Cloning, expression, and characterization of sialic acid synthases. Biochem. Biophys. Res. Commun. 338, 1507–1514 33. Gilbert, M., Watson, D. C., and Wakarchuck, W. W. (1997) Purification and characterization of the recombinant CMP-sialic acid synthetase from Neisseria meningitidis. Biotechnol. Lett. 19, 417– 420 34. Severi, E., Hood, D. W., and Thomas, G. H. (2007) Sialic acid utilization by bacterial pathogens. Microbiology 153, 2817–2822 VOLUME 289 • NUMBER 49 • DECEMBER 5, 2014 Downloaded from by guest on February 17, 2017 pathways for sialic acid, pseudaminic acid, and legionaminic acid (30, 31). These pathways are also taxonomically scattered among bacteria and archaea and show complex phylogenetic relationships suggestive of horizontal gene transfer and convergent evolution (30, 31, 40). The presence of GT97 sequences in archaea raises the question of whether this family exhibits specificity for different CMP-activated donor sugars in the different domains of life. Archaea are not known to incorporate sialic acid into their glycans but rather pseudaminic acid and legionaminic acid, which share related biosynthetic pathways (30). Thus, it is possible, if not likely, that some of the identified GT97 proteins are actually pseudaminic acid or legionaminic acid transferases. A notable finding is the presence of GT97 proteins in several extreme halophiles. 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Ser. 41, 95–98 Downloaded from by guest on February 17, 2017 DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33957 Dissection of Hexosyl- and Sialyltransferase Domains in the Bifunctional Capsule Polymerases from Neisseria meningitidis W and Y Defines a New Sialyltransferase Family Angela Romanow, Timothy G. Keys, Katharina Stummeyer, Friedrich Freiberger, Bernard Henrissat and Rita Gerardy-Schahn J. Biol. Chem. 2014, 289:33945-33957. doi: 10.1074/jbc.M114.597773 originally published online October 23, 2014 Access the most updated version of this article at doi: 10.1074/jbc.M114.597773 Click here to choose from all of JBC's e-mail alerts This article cites 42 references, 19 of which can be accessed free at Downloaded from by guest on February 17, 2017 Alerts: • When this article is cited • When a correction for this article is posted
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