Superscript1/SuperscriptH Superscript13/Su

Authors: Veronica Esposito Valeria Musi Daniele Veggi Annalisa Pastore Mariagrazia Pizza

Publish Date: 2010/03/20

Volume: 4, Issue: 1, Pages: 107-109

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Abstract

GNA2132 Genomederived Neisseria Antigen 2132 is a surfaceexposed lipoprotein discovered by reverse vaccinology and expressed by genetically diverse Neisseria meningitidis strains Pizza et al 2000 The protein induces bactericidal antibodies against most strains of Meningococccus and has been included in a multivalent recombinant vaccine against N meningitidis serogroup B Structure determination of GNA2132 is important for understanding the antigenic properties of the protein in view of increased efficiency vaccine development We report practically complete 1H 13C and 15N assignment of the detectable spectrum of a highly conserved Cterminal region of GNA2132 residues 245–427 in micellar solution a medium used to improve the spectral quality The first 32 residues of our construct up to residue 277 were not visible in the spectrum presumably because of line broadening due to solvent and/or conformational exchange Secondary structure predictions based on chemical shift information indicate the presence of an all βprotein with eight β strandsNeisseria meningitis is a human pathogen which induces meningitis and sepsis in children and young adults The bacterium is surrounded by a capsular polysaccharide which defines thirteen different serogroups five of which A B C Y and W135 are the major responsible for disease in humans Harrison 2006 Conjugate vaccines against serogroups A C Y and W135 based on polysaccharides are available or in late phase of development The same strategy cannot however be used for Meningococcus B since its capsular polysaccharide has a structural homology with a polysialic acid α2–8Nacetylneuraminic acid which is present in human cells Stein et al 2005 The only vaccines proven to be effective against serogroup B are based on outer membrane vescicles OMV purified from the bacterium Bjune et al 1991 The main limitation of such vaccines also called “tailor made” is their strain specificity due to the high variability of PorA the main antigenic component of the vesicles Stephens 2007We have used a genomebased approach named “reverse vaccinology” to overcome these limitations and develop a vaccine potentially able to cover the Meningococcus B strain diversity Pizza et al 2000 This strategy has allowed the identification of novel surfaceexposed antigens able to induce bactericidal antibodies Giuliani et al 2006 a property known to correlate with protection in humans Among this is GNA2132 a surface exposed lipoprotein of 48 kDa able to induce bactericidal antibodies and play a protective role in animal modelsBecause of its potential importance in vaccinology GNA2132 is one of the components of a Meningococcus B vaccine which has entered phase III clinical trials Large effort is being dedicated to the definition of the structural and functional properties of this antigen to better characterize its immune response and potential role in pathogenesis We have recently shown that GNA2132 is able to bind heparin through an argininerich region and is cleaved by two different proteases human lactoferrin and meningococcal NalP Serruto et al 2010 Sequence analysis on a panel of strains representative of the meningococcal diversity has allowed the definition of two regions an Nterminal domain with several hypervariable regions and a Cterminal domain highly conserved in sequence Pizza et al 2000The region 245427 of the GNA2132 sequence cloned in a pET21b + vector Novagen was expressed in Escherichia coli BL21DE3pLysS The sequence was preceded by an MA sequence added for cloning reasons and followed by a histidine tag residues LEHHHHHH for purification purposes Uniformly15N13Clabelled samples were produced by growing cells at 37°C in M9 minimal medium enriched with 1 g/L 15Nlabelled ammonium sulfate and 4 g/L U2H13Cglucose Cambridge Isotope Laboratories The protein was induced with 1 mM isopropyl βDthiogalactopyranoside and expressed for 4 h The cell pellet was suspended in 50 mM sodium phosphate buffer at pH 8 and lysed by lysozyme The GNA2132 domain was purified in two steps by affinity chromatography using Ni–NTA and cation exchange columns equilibrated in 50 mM sodium acetate at pH 55 Sample purity and identity were checked by SDS–PAGE and electrospray mass spectrometry respectivelyThe NMR samples used for assignment typically contained 05 mM protein concentrations 95 H2O 5 D2O in 20 mM Tris–HCl at pH 7 50 mM NaCl and in the presence of 30 mM dodecylphosphocholate DPC All spectra were recorded at 40°C on a Bruker Avance 700 MHz spectrometer equipped with a cryoprobe Backbone 1HN 15N 13Cα 1Hα 13C′ and side chain 1H and 13C resonances were assigned using HSQC HNCO HNCACO HNCA HNCOCA CBCANH CBCACONH and HNHA experiments HCCHTOCSY HCCCONH CCCONH 1H15N and 1H13CHSQCNOESY spectra were also performed for sidechain assignments Water suppression was achieved by WATERGATE Data were processed with NMRPipe/NMRDraw Delaglio et al 1995 and analyzed with Sparky Goddard Kneller San Francisco CA Torsion angles φ ψ and experimentally based secondary structure predictions were carried out for the HN Hα N Cα Cβ and C′ chemical shifts using TALOS+ Shen et al 2009 and CSI Wishart and Sykes 1994 software Sequence based secondary structure predictions were obtained using the JPRED website http//wwwcompbiodundeeacuk/wwwjpred/Quality of the NMR spectrum and secondary structure of the Cterminal domain of GNA2132 a 1H15N HSQC spectrum in 20 mM Tris–HCl at pH 7 50 mM NaCl and DPC recorded at 40°C and 700 MHz proton frequency A close up shows the assignment of the most crowded region b Secondary structure predictions for the sequence of the GNA2132 domain as obtained from TALOS+ black arrows and from the JPRED webserver grey arrowsFor the time being we used DPC for assignment The construct used spans residues 244427 of GNA2132 The backbone resonances were fully assigned with the exception of the first 32 residues of M363 K367 F368 and of the region from Y407 to E413 which could not be identified in the spectrum These residues are missing because of line broadening likely due to solvent exchange and/or conformational exchange Of the remaining 142 residues more than 94 of the backbone resonances and more than 87 of the side chains resonances were unambiguously identified including the assignment of 90 of the 1H side chain resonances Chemical shifts were deposited in the BioMagResBank under the access number BMRB 16679Evaluation of the obtained Hα C′ Cα and Cβ chemical shifts by TALOS+ Shen et al 2009 and CSI Wishart and Sykes1994 indicates that GNA2132 contains eight βstrands residues 276279 314324 338346 350355 369376 388394 399402 413418 in reasonable agreement with sequence structure predictions Fig 1bThis article is published under an open access license Please check the Copyright Information section for details of this license and what reuse is permitted If your intended use exceeds what is permitted by the license or if you are unable to locate the licence and reuse information please 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