SYNTHESIS OF IGFBP-3 FRAGMENTS IN A BACULOVIRUS SYSTEM AND CHARACTERIZATION OF MONOCLONAL ANTI-IGFBP-3 ANTIBODIES

IGFBPs play an important role in IGF biological actions bq modulating IGF binding to its receptors. The major IGFBP in serum is IGFBP-3, whi ch transports 70-90% of the circulating IGFs. In target cell systems, it sequesters IGFs and inhibits their hormonal actions, but may potent iate IGF activity or exert IGF-independent effects under specific cond itions. IGFBP-3 can be modified bu IGFBP-3 proteases, which degrade it into smaller fragments. IGFBP-3 fragments generated by proteolysis ha ve reduced affinity for IGFs, thereby modifying IGF action. To study I GFBP-3 fragments in vivo and in vitro, we constructed six different IG FBP-3 fragments by use of a baculovirus expression system and generate d 8 different monoclonal IGFBP-3 antibodies. Based on the known cleava ge sites of IGFBP-3 for PSA, MMPs, and the predicted plasmin cleavage sites, we expressed a N-terminal IGFBP-3(1-97) fragment and a C-termin al IGFBP-3(98-264) fragment. By stepwise truncation from the C-termina l end, we created IGFBP-3(98-232), IGFBP-3(98-206), IGFBP-3(98-179), a nd IGFBP-3(98-159). A strong recognition of the C-terminus and the int ermediate parts of IGFBP-3 by six antibodies was found. Four of these mAbs were able to recognize the intermediate fragment alone. Two mAbs were found to immunoreact only with the N-terminal IGFBP-3 fragment an d two additional mAbs recognized the N- as well as the C-terminal part s and lacked immunoreactivity for the intermediate part of IGFBP-3. Th e 15 kDa IGFBP-3 fragment resulting from plasmin digestion was found t o only react with N-terminal antibodies, while the 29 kDa fragment in pregnancy serum reacted with both N- and C-terminal antibodies. Thus, these mAbs will be useful tools to determine whether IGFBP-3 fragments found in vivo derive from either the N- or C-terminal domains of IGFB P-3.