Structural and Immunochemical Characterization of the Lipooligosaccharides Expressed byNeisseria subflava44

Abstract

Neisserial lipooligosaccharides (LOSs) are a family of complex cell surface glycolipids. We used mass spectrometry techniques (electrospray ionization, collision-induced dissociation, and multiple step), combined with fluorophore-assisted carbohydrate electrophoresis monosaccharide composition analysis, to determine the structure of the two low-molecular-mass LOS molecules (LOSI and LOSII) expressed by Neisseria subflava 44. We determined that LOSI contains one glucose on both the ␣ and ␤ chains. LOSII is structurally related to LOSI and differs from it by the addition of a hexose (either glucose or galactose) on the ␣ chain. LOSI and LOSII were able to bind monoclonal antibody (MAb) 25-1-LC1 when analyzed by Western blotting experiments. We used a set of genetically defined Neisseria gonorrhoeae mutants that expressed single defined LOS epitopes and a group of Neisseria meningitidis strains that expresses chemically defined LOS components to determine the structures recognized by MAb 25-1-LC1. We found that extensions onto the ␤-chain glucose of LOSI block the recognition by this MAb, as does further elongation from the LOSII ␣ chain. The LOSI structure was determined to be the minimum structure that is recognized by MAb 25-1-LC1.

Biochemical Analysis of Lpt3, a Protein Responsible forPhosphoethanolamine Addition to Lipooligosaccharide ofPathogenic Neisseria

“…Structure of neisserial LOS. The structure presented in this figure represents the LOS molecules that can be synthesized by the gonococcus that is the epitope for MAb 2-1-L8 (modified with permission from Tong et al [36]). LOS biosynthetic genes are italicized.…”

Analysis of Lipooligosaccharide Biosynthesis in the Neisseriaceae

“…Structural analysis showed that the major LOSs expressed by N. sublflava 44 possess one glucose on the ␤ chain (42). Banerjee et al (3) recently described gene lgtG, whose product adds a glucose onto Hep II.…”

“…We chose to examine the genetic potential of N. subflava 44 to make LOS in greater detail because its LOS was able to bind MAb 1B2 on a colony blot yet failed to express a detectable amount of the lacto-N-neotetraose LOS component when its LOS was analyzed by SDS-PAGE. Furthermore, mass spectrometry combined with fluorophore-assisted carbohydrate electrophoresis monosaccharide composition analysis showed that N. subflava 44 expresses two major LOS components (LOSI and LOSII) (42). LOSI contains one glucose on both the ␣ and ␤ chains.…”

“…␣ chain [16]), MAb 3G9 (marker for a lactosyl group on the ␤ chain [17]), MAb 17-1-L1 (marker for an alternate ␣-chain extension terminating in galactose [16]), MAb 25-1-LC1 (marker for the presence of ␤-chain glucose and ␣-chain lactose or sucrose [42]), and MAb B5 (marker for the presence of phosphoethanolamine on Hep II [36]). Of the nonpathogenic strains examined, N. lactamica 5841 contained LOS components that bound MAbs 1B2 and 2-1-L8 (Table 3).…”

Importance of Antibodies to Lipopolysaccharide in Natural and Vaccine-Induced Serum Bactericidal Activity against Neisseria meningitidis Group B

“…The monoclonal antibody 21-1-LC1 (49) appears to have these characteristics (see Fig. S2 in the supplemental material) in that it was made against an L5 strain expressing mostly the truncated L8-like form of the LPS (L8-5), as judged by silver-stained SDS-PAGE.…”