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-dependent IGFBP-4 protease. However, regulation of the IGF-II-dependent IGFBP-4 protease in vivo is not known. As IGFBP proteases are known to be triggered during pregnancy, we systematically evaluated the changes in IGFBP-4 proteolysis by serum collected throughout human pregnancy. Results from in vitro protease assays using recombinant IGFBP-4 revealed that IGFBP-4 proteolysis determined in both the presence and absence of exogenous IGF-II significantly increased during the first and second trimesters and reached a plateau by the third trimester. However, in the absence of IGF-II, IGFBP-4 proteolysis by pregnancy serum was only observed after prolonged incubation. IGF-II dose dependently increased IGFBP-4 proteolysis by pregnancy serum, with 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 proteolysis of recombinant human IGFBP-3, whereas excess IGF-II reproducibly inhibited 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 that the cleavage site (Met135-Lys136) in human IGFBP-4 was not altered by IGF-II. Deletion of the residues 121-141 containing this cleavage site blocked 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 IGF actions, it can be speculated that the pregnancy-induced IGFBP-4 proteases may play an important role in regulating fetal growth.
Byun
D
D
J.L. Pettis Veterans Administration Medical Center, Loma Linda, California 92357, USA.
Mohan
S
S
Kim
C
C
Suh
K
K
Yoo
M
M
Lee
H
H
Baylink
D J
DJ
Qin
X
X
D000328:Adult; D005260:Female; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000097:blood; D008970:Molecular Weight; D010006:Osteoblasts / Q000201:enzymology; D011247:Pregnancy / Q000378:metabolism*; D011266:Pregnancy-Associated Plasma Protein-A; D011863:Radioimmunoassay
D007202:Indicators and Reagents; D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4; D007335:Insulin-Like Growth Factor II; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A