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 by modulating IGF binding to its receptors. The major IGFBP in serum is IGFBP-3, which transports 70-90% of the circulating IGFs. In target cell systems, it sequesters IGFs and inhibits their hormonal actions, but may potentiate IGF activity or exert IGF-independent effects under specific conditions. IGFBP-3 can be modified by IGFBP-3 proteases, which degrade it into smaller fragments. IGFBP-3 fragments generated by proteolysis have reduced affinity for IGFs, thereby modifying IGF action. To study IGFBP-3 fragments in vivo and in vitro, we constructed six different IGFBP-3 fragments by use of a baculovirus expression system and generated 8 different monoclonal IGFBP-3 antibodies. Based on the known cleavage 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-terminal IGFBP-3(98-264) fragment. By stepwise truncation from the C-terminal end, we created IGFBP-3(98-232), IGFBP-3(98-206), IGFBP-3(98-179), and IGFBP-3(98-159). A strong recognition of the C-terminus and the intermediate 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 and two additional mAbs recognized the N- as well as the C-terminal parts and lacked immunoreactivity for the intermediate part of IGFBP-3. The 15 kDa IGFBP-3 fragment resulting from plasmin digestion was found to 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 IGFBP-3.
Vorwerk
P
P
Dept. Pediatrics, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Oh
Y
Y
Lee
P D
PD
Khare
A
A
Rosenfeld
R G
RG
D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D005822:Genetic Vectors; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D018972:Insulin-Like Growth Factor Binding Protein 3; D010446:Peptide Fragments; D011994:Recombinant Proteins