Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Abstract

SummaryA two-dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surfaceexposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site-directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T-cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (ٌ) from Semliki Forest virus were inserted into Bgl II restriction sites created by site-directed mutagenesis. The ٌ epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while ٌ epitopes in the N-terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface-exposed loops which can be used to insert foreign epitopes.

Crystal structure of the OpcA integral membrane adhesin from Neisseria meningitidis

“…One end of the barrel consists of much shorter turns and forms the periplasmic end of the molecule, whereas the exterior is made up of five loop regions of varying length that would protrude above the membrane. These assignments are similar to a two-dimensional topology map of OpcA proposed previously, except that the location of the periplasmic turns within the sequence differ by up to four amino acids (26). The loops correspond to regions that are known to be exposed on the cell surface from antibody reactivity against an inserted antigen sequence (26).…”

“…These assignments are similar to a two-dimensional topology map of OpcA proposed previously, except that the location of the periplasmic turns within the sequence differ by up to four amino acids (26). The loops correspond to regions that are known to be exposed on the cell surface from antibody reactivity against an inserted antigen sequence (26). The ␤-strands extend well above the predicted membrane surface, with this regular secondary structure contributing to the formation of the loop regions.…”

Immunization with Recombinant Opc Outer Membrane Protein from Neisseria meningitidis : Influence of Sequence Variation and Levels of Expression on the Bactericidal Immune Response against Meningococci

“…The amino acid sequences of strains MC58 and MC131 were identical to that of strain H44/76. Strain MC122 showed two amino acid changes in Opc compared to MC58, from S to G at position 37 and K to T at position 227, which corresponds to the apex of loop 5 in the predicted model of the Opc protein structure (16). Strain MC119 showed four amino acid changes, occurring within predicted loops 3, 4, and 5, while strain MC139 showed five amino acid changes compared to MC58, four of which occurred within predicted loops 3, 4, and 5.…”

“…Opc protein has been shown to play an important role in meningococcal adhesion and invasion of both epithelial and endothelial cells and perhaps represents a common virulence factor (29,30). Although the protein is not a porin, it is also believed to adopt a ␤-sheet structure in the outer membrane, with six surface-exposed loops (16). In this paper, we report the cloning of the opc gene using the E. coli expression system that has been effective with the class 1 protein, immunization with renatured recombinant protein using adjuvant formulations compatible with human immunization, and the effect of both sequence variation and degree of expression on the potential protective effect.…”

ConformationalEpitopes Recognized by Protective Anti-Neisserial Surface Protein AAntibodies

“…The epitopes recognized by MAbs reactive with Opc, a 10-stranded ␤-barrel (17), and Opa, postulated to have an 8-stranded ␤-barrel topology similar to that of NspA, have been mapped using overlapping synthetic peptides (10,12). With the idea of performing a similar mapping study of anti-NspA MAbs, overlapping synthetic peptides corresponding to the entire mature sequence of NspA from strain 8047 were synthesized on solid supports and tested for binding by using MAbs AL12 and 14C7.…”