Studies on the primary structure and antigenic determinants of pilin isolated from Pseudomonas aeruginosa K

Abstract

The complete amino acid sequence of P. aeruginosa K (PAK) pilin was determined using a combination of automated and manual Edman degradation techniques. Suitable peptides were derived from cyanogen bromide, tryptic, chymotryptic, peptic, thermolytic and citraconylated tryptic cleavages of unmodified or carboxymethylated pilin. The protein, a single polypeptide chain, has N-methylphenylalanine at the NH2-terminus, a total of 144 residues, a MW of 15,013 and an equal number of acid and basic amino acids. The NH2-terminal region (residues 1-43) is very hydrophobic with only 3 charged residues, suggesting a possible role in subunit-subunit interaction. The 2 half-cystines, residues 129 and 142, are linked through a disulfide bridge in the native protein. To delineate the antigenic regions of pilin, the protein was cleaved at Arg-30, Arg-53 and Arg-120 to produce peptide fragments cTI (residues 1-30), cTII (residues 31-53), cTIII (residues 54-120) and cTIV (residues 121-144), cTIII and cTIV were further degraded into several subfragments. The purified peptides were subjected to immunological analysis using direct and competitive enzyme-linked immunosorbent assay procedures. A major antigenic determinant was delineated in a region of the protein encompassing residues 82-101. Three other epitopes were also identified, but reacted with only minor amounts of antibody in the rabbit polyclonal antiserum.