Characterization of insulin-like growth factor-binding proteins in human serum from patients with chronic renal failure.
Western ligand blotting of human serum reveals proteins of 41.5, 38, 33, 28, and 23 kD which will specifically bind [125I]insulin-like growth factor-I [( 125I]IGF-I). The relationship of these binding proteins (BPs) to the two serum BP-IGF complexes of 150 and 40 kD and their relationship to the two purified and well characterized IGF-BPs, BP-25 and BP-53, are not clear. The studies reported here used gel filtration, Western ligand blotting, deglycosylation, affinity cross-linking, and immunoprecipitation with antisera against BP-25 and BP-53 to explore the relationship among these IGF-BP forms. Serum from children with chronic renal failure was used for this study, since, by Western ligand blot, it was rich in all five IGF-BPs. The 28- and 33-kD BPs, which were hard to detect in normal serum, were elevated in chronic renal failure serum. Using this serum in each experiment, it was found that the large (150-kD) BP-IGF complex contained only the 41.5- and 38-kD BPs. Both of these BPs were precipitated by anti-BP-53 antiserum, but not by anti-BP-25 antisera, and both BPs were deglycosylated by endoglycosidase-F to a single 31-kD protein which retained the ability to bind IGF-I. This molecular mass of 31 kD is similar to the 28.7-kD mass for nonglycosylated BP-53 predicted by cDNA sequence analysis. In contrast, the 33-, 28-, and 23-kD BPs were only detected in the small (40-kD) BP-IGF complex. None of the three BPs was precipitated by anti-BP-53 antiserum, and none was digested by endoglycosidase-F. The 28-kD BP comigrated with pure BP-25 under all conditions and was precipitated by two independent anti-BP-25 antisera. The 33-kD BP was also precipitated by both anti-BP-25 antisera, while the 23-kD BP was not precipitated by any of the antisera tested. Thus, BP-53 consists of a core protein of 28.7 kD which is differentially glycosylated in serum to form two BPs of 41.5 and 38 kD; these BPs circulate exclusively as part of the large BP-IGF complex. BP-25 circulates in the small BP-IGF complex as a nonglycosylated protein with an apparent mol wt of 28 kD.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Pediatrics, Stanford University Medical Center, California 94305.
F
F
Liu
D R
DR
Powell
R L
RL
Hintz
D000328:Adult; D015153:Blotting, Western; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification*; D002478:Cells, Cultured; D002648:Child; D002850:Chromatography, Gel; D004586:Electrophoresis / Q000379:methods; D005260:Female; D006031:Glycosylation; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D018969:Insulin-Like Growth Factor Binding Proteins; D007676:Kidney Failure, Chronic / Q000097:blood* / Q000628:therapy; D008297:Male; D011233:Precipitin Tests; D011863:Radioimmunoassay; D006435:Renal Dialysis
D002352:Carrier Proteins; D007106:Immune Sera; D018969:Insulin-Like Growth Factor Binding Proteins