Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii.
The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.
Ching
W M
WM
Infectious Diseases Department, Naval Medical Research Institute, Bethesda, MD 20889-5055.
Carl
M
M
Dasch
G A
GA
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology*; D003488:Cyanogen Bromide; D000939:Epitopes; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D012283:Rickettsia prowazekii / Q000276:immunology*; D012286:Rickettsia typhi / Q000276:immunology*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C055764:S-layer proteins; D003488:Cyanogen Bromide