Epitope analysis of the rat dipeptidyl peptidase IV monoclonal antibody 6A3 that blocks pericellular fibronectin-mediated cancer cell adhesion.
We previously showed that the rat dipeptidyl peptidase IV (rDPP IV) monoclonal antibody (mAb) 6A3 greatly inhibits the pericellular polymeric fibronectin-mediated metastatic cancer cell adhesion to rDPP IV. L(311)QWLRRI in rDPP IV has been proposed as the putative fibronectin-binding site. However, the inhibitory mechanism of 6A3 has been elusive. Epitope mapping of 6A3 may help to understand the interaction between fibronectin and rDPP IV. In the present study, we showed that 6A3 species-specifically recognized rDPP IV but inhibited fibronectin/rDPP IV-mediated cell adhesions of various cancer types and species, which was independent of rDPP IV enzymatic activity. The 6A3 epitope was stably exposed in both native and denatured rDPP IV. On the basis of the resolved structures and the species variations in DPP IV sequences, we finely mapped the 6A3 epitope to a surface-exposed Thr331-dependent motif D(329)KTTLVWN, only 11 amino acids away from L(311)QWLRRI on the same plane as the fifth beta-propeller blade. The functionality of 6A3 epitope in rDPP IV was ultimately demonstrated by the ability of 6A3-recognizable fragments to interfere with the inhibitory effect of 6A3 on full-length rDPP IV binding to pericellular polymeric fibronectin. On the basis of structural analysis, and the fact that the preformed fibronectin fragment/rDPP IV complex was co-immunoprecipitated by 6A3 and fixing the rDPP IV structure with paraformaldehyde did not avert the inhibitory effect, the mechanism of 6A3 inhibition may not be the result of complete competition or conformational change.
Hung
Ting-Ting
TT
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Wu
Jun-Yi
JY
Liu
Ju-Fang
JF
Cheng
Hung-Chi
HC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects*; D045744:Cell Line, Tumor; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D011485:Protein Binding / Q000187:drug effects; D051381:Rats
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005353:Fibronectins; D018819:Dipeptidyl Peptidase 4