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Epitope analysis of the rat dipeptidyl peptidase IV monoclonal antibody 6A3 that blocks pericellular fibronectin‐mediated cancer cell adhesion
Author(s) -
Hung TingTing,
Wu JunYi,
Liu JuFang,
Cheng HungChi
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07352.x
Subject(s) - epitope , fibronectin , monoclonal antibody , chemistry , microbiology and biotechnology , dipeptidyl peptidase , epitope mapping , cell adhesion , biochemistry , antibody , rgd motif , cell , biology , enzyme , immunology
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 β‐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. Structured digital abstract• MINT‐7261577 : DppIV (uniprotkb: P14740 ) binds ( MI:0407 ) to FNIII14 (uniprotkb: P04937 ) by anti bait coimmunoprecipitation ( MI:0006 )