Antigenic and immunogenic properties of cyanogen bromide peptides from gonococcal outer membrane protein IB. Evidence for the existence of a surface-exposed conserved epitope.

Abstract

Two distinct species of gonococcal porin proteins exist that differ with regard to surface exposure. Protein IB, expressed by strains of the WII/III serogroup, has both termini buried in the outer membrane, leaving a central region of the molecule exposed at the cell surface. We have attempted to define this region of protein IB in detail by studying the antigenic and immunogenic properites of peptides derived from protein IB. Treatment of gonococcal protein IB (serotope 5) with cyanogen bromide resulted in cleavage of protein IB into three major fragments of Mr of 15,000, 13,000, and 8,000. The location of these peptides in the intact protein was determined by analysis of partial cleavage products. The 8,000 Mr peptide (CB2) was found to be located in the central region of the protein. Chymotrypsin cleavage of protein IB revealed a cleavage site near one of the cyanogen bromide cleavage sites. Trypsin was found to cleave the protein, either in outer membranes complexes (OMC) or in detergent micelles, in the central CB2 fragment. These results suggest that CB2 is a part of the surface-exposed region of protein IB. Immunization of mice with purified protein IB (serotype 5) induced antibodies against all three CB-peptides. Absorption of the sera with homologous OMC resulted in a complete removal of antibodies against CB2, supplying further evidence for its surface-exposed nature. Antibodies against the 13,000 Mr peptide (CB1) could not be absorbed with intact OMC, suggesting that this peptide is buried within the outer membrane. Antisera raised against CB2 of serotype 5 demonstrated a considerable cross-reactivity with heterologous outer membranes. On the contrary, intact OMC induced mainly type-specific antibodies. These data demonstrate the presence of conserved epitopes on the surface-exposed CB2 peptide. These conserved epitopes are generally not very immunogenic when present in intact OMC.