Studies on the primary structure and antigenic determinants of pilin isolated from Pseudomonas aeruginosa K.
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 15013, 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.
P A
PA
Sastry
J R
JR
Pearlstone
L B
LB
Smillie
W
W
Paranchych
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000032:analysis*; D036781:Fimbriae Proteins; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D011550:Pseudomonas aeruginosa / Q000032:analysis*; D014357:Trypsin
D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D036781:Fimbriae Proteins; D014357:Trypsin