Epitope expression of gonococcal lipooligosaccharide (LOS). Importance of the lipoidal moiety for expression of an epitope that exists in the oligosaccharide moiety of LOS

doi:10.1016/0161-5890(88)90116-2

Molecular Immunology

Volume 25, Issue 8, August 1988, Pages 799-809

https://doi.org/10.1016/0161-5890(88)90116-2 Get rights and content

Abstract

Antigenic expression of lipooligosaccharide (LOS) of strain F62 of Neisseria gonorrhoeae, was investigated with mouse monoclonal IgM antibody 3F11. F62 LOS was modified in various ways in order to understand structural requirements for expression of the 3F11-defined epitope. When the LOS was partially deacylated by treating it with 50 mM NaOH at 80°C for 20 min or with anhydrous hydrazine at 80°C for 20 min, the binding of 3F11 to those deacylated LOS samples decreased significantly. Removal of phosphate groups by treatment of the LOS with HF (4 days at 4°C) did not affect the antigenicity at all. Neither did reduction of carboxyl groups in the LOS molecule (by activation of carboxyl groups with a carbodiimide followed by treatment with NaBH₄) alter epitope expression. On oxidation with NaIO₄, the LOS lost its antigenicity completely. The presence of Mg²⁺ did not change the circular dichroism (CD) behavior of F62 LOS. However, the partially deacylated LOS samples showed significantly different CD patterns in the 190–200 nm region compared with F62 LOS, which suggests conformational changes of F62 LOS due to the loss of fatty acids in the lipoidal moiety. Oligosaccharide (OS) and lipoidal components obtained after hydrolysis of F62 LOS with 1% acetic acid, were not recognized by the antibody. The antigenicity of OS was not retained by non-stereospecific acylation of OS with decanoyl chloride. We conclude the following: (1) 3F11-defined epitope exists in the OS moiety of F62 LOS; however, for it to be expressed, the carbohydrate moiety must be in a certain conformation that is defined by an overall structure of the LOS molecule. This structure is significantly influenced by some of the fatty acids in the lipoidal moiety of the LOS molecule; (2) the presence of phosphate or 3-deoxy-manno-2-octulosonic acid (dOclA) is not essential for expression of the 3F11-defined epitope; (3) the presence of divalent cations does not affect epitope expression.

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MS of intact LOS of the group C strain, 89I, did find multiple lipid phosphoforms (42, 43), but this might have been an exception. What effects, if any, the different lipids might have on glycose antigen formation (48, 49) were not explored. Also surprising was that the nLc4 terminal Gal appeared to protect L3,7 but not L2,4 strains from bactericidal activity.
Antibodies that initiate complement-mediated killing of Neisseria meningitidis as they enter the bloodstream from the oropharynx protect against disseminated disease. Human IgGs that bind the neisserial L7 lipooligosaccharide (LOS) are bactericidal for L3,7 and L2,4 meningococci in the presence of human complement. These strains share a lacto-N-neotetraose (nLc₄) LOS α chain. We used a set of mutants that have successive saccharide deletions from the nLc₄ α chain to characterize further the binding and bactericidal activity of nLc₄ LOS IgG. We found that the nLc₄ α chain conforms at least four different antigens. We separately purified IgG that required the nLc₄ (non-reducing) terminal galactose (Gal) for binding and IgG that bound the truncated nLc₃ α chain that lacks this Gal residue. IgG that bound the internal nLc₃ α chain killed both L3,7 and L2,4 strains, whereas IgG that required the nLc₄ terminal Gal residue for binding killed L2,4 stains but not L3,7 strains. These results show that the diversity of LOS antibodies in human serum is as much a function of the conformation of multiple antigens by a single glycoform as of the production of multiple glycoforms. Differences in sensitivity to killing by human nLc₄ LOS IgG may account for the fact that fully two-thirds of endemic group B meningococcal disease in infants and children is caused by L3,7 strains, but only 20% is caused by L2,4 stains.
Characterization of a peptide vaccine candidate mimicking an oligosaccharide epitope of Neisseria gonorrhoeae and resultant immune responses and function
2006, Vaccine
The 2C7 epitope is a conserved oligosaccharide structure, a part of lipooligosaccharide (LOS) on Neisseria gonorrhoeae, present in 95% of clinical gonococcal isolates. 2C7 may represent a potential candidate for an anti-gonococcal vaccine. To circumvent the limitations of saccharide immunogens in producing long lived immune responses, we identified a peptide that mimics the 2C7 epitope using a random peptide library, characterizing linear and cyclic forms and formulating a multiple antigenic peptide. The multiple antigenic peptide Octa-MAP1 was used for immunization, and elicited ≥4-fold increase in cross-reactive anti-LOS antibodies in 26 of 30 mice (87%). IgG anti-LOS antibody elicited by Octa-MAP1 immunization possessed dose-responsive direct complement (C)-dependent bactericidal activity against gonococcal strains that expressed the 2C7 epitope. These data indicate that a peptide can mimic an oligosaccharide epitope and may form the basis for the development of a vaccine candidate for human immunization against N. gonorrhoeae.
Selection of peptides that bind to the core oligosaccharide of R-form LPS from a phage-displayed heptapeptide library
2001, FEMS Microbiology Letters
To characterize common sites within the core oligosaccharide of the R-form lipopolysaccharide (LPS), we screened peptides from a phage-displayed heptapeptide library by using the most truncated form of R-LPS, Re-LPS (S. Typhimurium SL1165) as a ligand. After three rounds of biopanning/amplification and subsequent screening by phagemid enzyme-linked immunosorbent assay (ELISA), we selected three distinct clones that bind to the ligand LPS. We characterized the binding sites of the three clones by ELISA and thin-layer chromatography immunostaining and found that the three clones bind the two Re-LPSs (SL1165 and S. Minnesota Re595) and Rb₂-LPS. In addition, one of the clones also bound to S-form LPS (S. Enteritidis). Current data show that those clones bind to common carbohydrate structure(s) expressed in the core oligosaccharides of those LPS samples.
Structural relationships and sialylation among meningococcal L1, L8, and L3,7 lipooligosaccharide serotypes
2000, Journal of Biological Chemistry
Citation Excerpt :
We dephosphorylated LOS and OS by hydrolysis with HF. LOS and OS were reacted with a 48% (aqueous) HF solution (10 μg of LOS per μl) in the dark at 4 °C for 16–20 h (OS and O-deacylated LOS) or 36–40 h (intact LOS) (9). After evaporation of HF, the residues were resuspended in H2O and lyophilized.
Eighteen of 34 endemic meningococcal case strains were of the L8 lipooligosaccharide (LOS) type; four of these were both L3 and L7 (L3,7), and seven were L1. L1 structures arose by alternative terminal Gal substitutions of lactosyl diheptoside L8 structures, as determined by electrospray ionization and other mass spectrometric techniques, and enzymatic and chemical degradations (Structures L1 and L1a).The more abundant molecule, designated L1, had a trihexose globosyl α chain; the less abundant one, designated L1a, had a β-lactosyl α chain and a parallel α-lactosaminyl γ chain. A P ^k globoside (Galα1→4Galβ1→4 Glc-R) monoclonal antibody bound 9/10 L1 strains, but a P₁ globoside (Galα1→4Galβ1→4GlcNAc-R) mAb bound none of them. α-Galactosidase caused loss of both L1 structures and creation of L8 structures; β-galactosidase caused loss of the L8 determinant. The L1/P ^k glycose was partially sialylated. Some LOS also had unsubstituted basal β-GlcNAc additions. These structural relationships explain co-expression of L8, L1, and L3,7 serotypes.
Separation of lipooligosaccharides by linear gradient gel electrophoresis
2000, Analytical Biochemistry
Lipooligosaccharides (LOSs) are one of the major antigenic and immunogenic components on the outer membrane of mucosal Gram-negative bacteria. These glycolipid antigens are in the M_r range of 3–7 kDa, and SDS/PAGE has been used as an analytical tool. Although we are able to separate relatively higher M_r LOS components by mini-PAGE, we encounter difficulties in resolving LOS components below 3.6 kDa present in heterogeneous LOS preparations. In the present study, we selected PID2 LOS consisting of six LOS components of 3.0–5.1 kDa as a model LOS and examined mini-PAGE conditions not only to resolve smaller M_r LOS components but also to retain resolving capability of higher LOS components. We found that mini-PAGE with stepwise and linear gradient gels (glycine–SDS) resolved smaller M_r LOS components. Mini-PAGE with linear gradient gels gave the best resolution, and LOS components of 3.0–5.1 kDa were separated as tight and even bands. Because of the resolution, LOS components were stained chemically and immunochemically much better than those on continuous or stepwise gradient gels. Our study also showed that preformed tricine–SDS (TSDS) minigels such as 16.5 and 10–20% (linear gradient) did not resolve PID2 LOS, which indicated that heterogeneous LOS preparations may not be fully analyzed by using these TSDS minigels. By using glycine–SDS linear gradient mini-PAGE, we should be able not only to screen expression of LOSs but also to characterize smaller M_r LOS components present in heterogeneous LOS preparations whose identities may have been neglected in the past.
Structural and immunochemical characterization of a Neisseria gonorrhoeae epitope defined by a monoclonal antibody 2C7; the antibody recognizes a conserved epitope on specific lipo-oligosaccharides in spite of the presence of human carbohydrate epitopes
1999, Journal of Biological Chemistry
Lipo-oligosaccharides (LOS) produced byNeisseria gonorrhoeae are important antigenic and immunogenic components of the outer membrane complex. Previously, we showed that murine monoclonal antibody (mAb) 2C7 did not cross-react with human glycosphingolipids but identified the LOS epitope that is widely expressed in vivo and in vitro (Gulati, S., McQuillen, D. P., Mandrell, R. E., Jani, D. B., and Rice, P. A. (1996) J. Infect. Dis. 174, 1223–1237). In the present study, we analyzed the structure of gonococcal strain WG LOS containing the 2C7 epitope and investigated the structural requirements for expression of the epitope. We determined that the WG LOS components are Hep[1]-elongated forms of 15253 LOS that have a lactose on both Hep[1] and Hep[2] (Yamasaki, R., Kerwood, D. E., Schneider, H., Quinn, K. P., Griffiss, J. M., and Mandrell, R. E. (1994) J. Biol. Chem. 269, 30345–30351). In addition, we found that expression of the 2C7 epitope within the LOS is blocked when the Hep[2]-lactose is elongated. Based on the structural data of these LOS and the results obtained from immunochemical analyses, we conclude the following: 1) mAb 2C7 requires both the 15253 OS minimum structure and theN-linked fatty acids in the lipoidal moiety for expression of the epitope; 2) mAb 2C7 binds to the LOS that elongates the lactose on Hep[1] of the 15253 OS, but not the one on Hep[2]; and 3) the 2C7 epitope is expressed on gonococcal LOS despite the presence of human carbohydrate epitopes such as a lactosamine or itsN-acetylgalactosaminylated (globo) form. Our study shows that the conserved epitope defined by mAb 2C7 could potentially be used as a safe site for the development of a vaccine candidate.

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Epitope expression of gonococcal lipooligosaccharide (LOS). Importance of the lipoidal moiety for expression of an epitope that exists in the oligosaccharide moiety of LOS

Abstract

Carbohydr. polymers

Anal. Biochem.

J. biol. Chem.

J. Immun. Methods

J. biol. Chem.

Anal. Biochem.

Monoclonal antibody analysis of lipopolysaccharide from Neisseria gonorrhoeae and Netsseria meningitidis

Infect. Immun.

Phenotypic variation in epitope expression of the Neisseria gonorrhoeae lipooligosaccharide

Infect. Immun.

Micro determination of phosphorus

Anal. Chem.

Colorimetric method for determination of sugars and related substances

Anal. Chem.

Possible orientation of the fatty acid chains in lipopolysaccharide

Z. Naturforsch. Teil C

Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms

Eur. J. Biochem.

Nature and heterogeneity of the antigens of Neisseria gonorrhoeae involved in the serum bactericidal reaction

Infect. Immun.

Toxic activity of purified lipopolysaccharide of Neisseria gonorrhoeae for human fallo- pian tube mucosa

J. Infect. Dis.

Physical heterogeneity of neisserial lipooligosaccharides reflects oligosaccharides that differ in apparent molecular weight, chemical composition, and antigenic expression

Infect. Immun.

Chemical modification and serological properties of the 3-deoxy-α-d-manno-2-octulosonic acid-containing polysaccharide from Escherichia coli LP 1092

Carbohydr. Res.