Studies on human pregnancy-induced insulin-like growth factor (IGF)-binding protein-4 proteases in serum: Determination of IGF-II dependency and localization of cleavage site

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4), a consistent inhibitor of IGF action, is subject to proteolytic cleavage by the IGF-II-d ependent IGFBP-4 protease. However, regulation of the IGF-II-dependent IGFB P-4 protease in vivo is not known. As IGFBP proteases are known to be trigg ered during pregnancy, we systematically evaluated the changes in IGFBP-4 p roteolysis by serum collected throughout human pregnancy. Results from in v itro protease assays using recombinant IGFBP-4 revealed that IGFBP-4 proteo lysis determined in both the presence and absence of exogenous IGF-II signi ficantly increased during the first and second trimesters and reached a pla teau by the third trimester. However, in the absence of IGF-II, IGFBP-4 pro teolysis by pregnancy serum was only observed after prolonged incubation. I GF-II dose dependently increased IGFBP-4 proteolysis by pregnancy serum, wi th maximal stimulation observed at a concentration of 0.7 mol/L relative to IGFBP-4. In contrast, IGF-II at an equimolar dose had little effect on pro teolysis of recombinant human IGFBP-3, whereas excess IGF-II reproducibly i nhibited recombinant human IGFBP-3 proteolysis by pregnancy serum. Although IGF-II enhanced IGFBP-4 proteolysis, results from N-terminal sequence and mass spectrometric analyses of IGFBP-4 proteolytic fragments demonstrate th at the cleavage site (Met(135)-Lys(136)) in human IGFBP-4 was not altered b y IGF-II. Deletion of the residues 121-141 containing this cleavage site bl ocked IGFBP-4 proteolysis. These findings demonstrate that the increase in IGFBP-4 proteolysis during pregnancy was accounted for mainly by the IGF-II -dependent IGFBP-4 proteolysis. Because IGFBP-4 is a potent inhibitor of IG F actions, it can be speculated that the pregnancy-induced IGFBP-4 protease s may play an important role in regulating fetal growth.