Analyses of gonococcal lipopolysaccharide in whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: stable association of lipopolysaccharide with the major outer membrane protein (protein I) of Neisseria gonorrhoeae

Publisher Website
Google Scholar
Abstract
The lipopolysaccharide (LPS) of N. gonorrhoeae whole-cell lysates and proteinase K-digested lysates was examined and compared with purified homologous LPS by a method which preferentially stains LPS in polyacrylamide gels. The Ag-stained profile of gonococcal LPS in the proteinase K-digested lysate was similar to that of homologous purified LPS; the LPS profile in whole-cell lysates was much smaller than that of digested lysates or purified LPS. Conditions of solubilization did not affect these differences. Since it is known that LPS migrates in a unique fashion in 2nd-dimension electrophoresis, the location of LPS in the whole-cell lysates was probed by 2nd-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a variety of stains and radiolabels. Results from these experiments indicated a stable and reproducible association of LPS with proteins ranging between 23,000-36,000 in MW in particular major outer membrane protein I. In addition to staining with the Ag method, which preferentially stains LPS, the putative LPS was resistant to digestion by proteinase K, did not stain with Coomassie brilliant blue, and was not labeled extrinsically with 125I (Iodogen method) or intrinsically with [35S]methionine. Analysis of 2 dimensional gels by immunoblotting with rabbit antisera prepared from protein I bands removed from a polyacrylamide gel revealed the presence of antigens in the same area of the gel (below proteins that were 23,000-36,000 in MW). Antibodies to constituents which migrated below the diagonal were essentially removed by adsorption of antisera with purified LPS, as were antibodie to homologous LPS and LPS in proteinase K-digested whole-cell lysates. Immunoblotting with a monoclonal antibody specific for LPS demonstrated reactivity of the antibody with LPS and with the protein I band. Evidently, protein I and perhaps other proteins in the whole-cell lysate are stably associated with LPS; this complex is resistant to dissociation in sodium dodecyl sulfate at high temperature (.apprx. 100.degree. C) but does, for unknown reasons, dissociate with electrophoresis in the 2nd dimension. The association of LPS with protein antigens in sodium dodecyl sulfate-polyacrylamide gels adds another dimension of complexity to analysis of these antigens by immunoelectroblotting. The tight association of LPS with the major outer membrane protein I may alter the nature of the immune response generated by purified protein I vaccine antigens. The possible role of protein-LPS complexes in the pathogenesis of gonorrhea is discussed.