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

Abstract

The complete amino acid sequence of Pseudomonas 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 molecular weight 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 three charged residues, suggesting a possible role in subunit–subunit interaction. The two half-cystines, residues 129 and 142, are shown to be 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.