A bactericidal monoclonal antibody elicits a change in its antigen, OspB of Borrelia burgdorferi, that can be detected by limited proteolysis.
mAb CB2, directed against outer surface protein B (OspB), causes bacteriolysis of Borrelia burgdorferi in the absence of complement. How this happens is unknown. We examined the effect of mAb binding on OspB tertiary structure by using limited proteolysis to probe changes in protein conformation. Truncated OspB (tOspB) that lacked N-terminal lipid was cleaved by four enzymes: trypsin, endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C. CB2 affected the cleavage by trypsin and Arg-C, but not by AspN or Glu-C. None of the enzymes cleaved CB2 under these conditions. Both trypsin and Arg-C cleaved tOspB near the N-terminus; CB2 slowed the rate of cleavage, but did not affect the identity of the sites cleaved. Irrelevant mAb had no effect, indicating that the effect was specific. CB2 was active against tOspB of strain B31, but not against tOspB of strain BEP4, to which it does not bind, suggesting that binding was required to elicit the effect on cleavage. With trypsin, CB2 showed a maximal effect at 8 mol of tOspB to 1 mol of mAb. At this ratio, not enough CB2 was present to bind all the tOspB; therefore, either CB2 shows turnover or CB2 acts by binding tOspB and effecting a change in this tOspB such that it, in turn, propagates the effect in other molecules of tOspB. Regardless of the mechanism, these data show that CB2 elicits a change in tOspB that can be measured by its reduced susceptibility to protease cleavage.
Katona
L I
LI
Department of Molecular Genetics, State University of New York, Stony Brook, NY 11794, USA. lkatona@path.som.sunysb.edu
Ayalew
S
S
Coleman
J L
JL
Benach
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000494:pharmacology; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001433:Bacteriolysis / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000235:genetics; D015748:Borrelia burgdorferi Group / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C077030:OspB protein, Borrelia burgdorferi; D010450:Endopeptidases; D012697:Serine Endopeptidases; C060325:arginine endopeptidase; D014357:Trypsin