An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated amongdifferent strains of Neisseria gonorrhoeae to correlate structural featuresessential for binding to the MAb 2C7. This epitope is widely expressed andconserved in gonococcal isolates, characteristics essential to an effectivecandidate vaccine antigen. Sample lipooligosaccharides (LOS), was preparedby a modification of the hot phenol-water method from which de-O-acetylatedLOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS andES-MSnin a triple quadrupole and an ion trap mass spectrometer,respectively. Previously documented natural heterogeneity was apparent fromboth LOS and OS preparations which was admixed with fragments induced byhydrazine and mild acid treatment. Natural heterogeneity was limited tophosphorylation and antenni extensions to the alpha-chain. Mild acidhydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidiclinkage of lipid A. OS structures were determined by collisional andresonance excitation combined with MS and multistep MSn which providedsequence information from both neutral loss, and nonreducing terminalfragments. A comparison of OS structures, with earlier knowledge of MAbbinding, enzyme treatment, and partial acid hydrolysis indicates a genericoverlapping domain for 2C7 binding. Reoccurring structural features includea Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on thenonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc(gamma-chain), and an alpha-linked lactose (beta-chain) residue. From thecentral heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain),moiety is required although extensions to this residue appear unnecessary.