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Quantitative analysis of insulin-like growth factor-modulated proteolysis of insulin-like growth factor binding protein-4 and -5 by pregnancy-associated plasma protein-A. 2007

Claus Gyrup, and Claus Oxvig
Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.

The metzincin metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) specifically cleaves insulin-like growth factor binding protein (IGFBP)-4 and -5. Regulation of insulin-like growth factor (IGF) bioavailability through cleavage of these inhibitory binding proteins is an important mechanism for the control of growth and development of vertebrate cells. Although proteolysis of IGFBP-4 and -5 by PAPP-A has been extensively studied in many systems, quantitative analyses have been lacking. We have characterized the cleavage of its natural substrates, IGFBP-4 and -5, in the absence and presence of IGF-I or -II and determined the kinetic parameters (Km and kcat) for the different combinations of IGFBP and IGF. The rate of IGFBP-4 proteolysis is dramatically increased upon addition of IGF-I or -II. Kinetic analysis revealed that IGF-II was a more potent activator of IGFBP-4 proteolysis than IGF-I. Proteolysis of IGFBP-5 is slightly inhibited by IGF, and we find that IGF-I and -II display a similar degree of inhibition of IGFBP-5 cleavage. We show that the mechanism of IGF-modulated proteolysis of IGFBP-4 and -5 involves changes in both the recognition of substrate (Km) and the turnover rate (kcat). In addition, we have devised a novel method of revealing potential consequences of substrate modification for kinetic analysis, and we have used this method to establish that there is no apparent difference in the behavior of radiolabeled IGFBP-4 and -5 compared to the behavior of the unmodified protein substrates. We also propose experimental conditions for the proper analysis of IGFBP proteolysis, and we demonstrate their usefulness by quantitatively evaluating the effect of inhibitory compounds on the rate of proteolysis. Finally, we have compared PAPP-A to other proteinases thought to have IGFBP-4 or -5 as a substrate. This emphasizes the potential of PAPP-A to specifically and efficiently function as a regulator in the IGF system.

UI MeSH Term Description Entries
D007334 Insulin-Like Growth Factor I A well-characterized basic peptide believed to be secreted by the liver and to circulate in the blood. It has growth-regulating, insulin-like, and mitogenic activities. This growth factor has a major, but not absolute, dependence on GROWTH HORMONE. It is believed to be mainly active in adults in contrast to INSULIN-LIKE GROWTH FACTOR II, which is a major fetal growth factor. IGF-I,Somatomedin C,IGF-1,IGF-I-SmC,Insulin Like Growth Factor I,Insulin-Like Somatomedin Peptide I,Insulin Like Somatomedin Peptide I
D007335 Insulin-Like Growth Factor II A well-characterized neutral peptide believed to be secreted by the LIVER and to circulate in the BLOOD. It has growth-regulating, insulin-like and mitogenic activities. The growth factor has a major, but not absolute, dependence on SOMATOTROPIN. It is believed to be a major fetal growth factor in contrast to INSULIN-LIKE GROWTH FACTOR I, which is a major growth factor in adults. IGF-II,Multiplication-Stimulating Activity,Somatomedin MSA,IGF-2,Insulin Like Growth Factor II,Insulin-Like Somatomedin Peptide II,Multiplication-Stimulating Factor,Somatomedin A,Factor, Multiplication-Stimulating,Insulin Like Somatomedin Peptide II,Multiplication Stimulating Activity,Multiplication Stimulating Factor
D007700 Kinetics The rate dynamics in chemical or physical systems.
D011266 Pregnancy-Associated Plasma Protein-A A product of the PLACENTA, and DECIDUA, secreted into the maternal circulation during PREGNANCY. It has been identified as an IGF binding protein (IGFBP)-4 protease that proteolyzes IGFBP-4 and thus increases IGF bioavailability. It is found also in human FIBROBLASTS, ovarian FOLLICULAR FLUID, and GRANULOSA CELLS. The enzyme is a heterotetramer of about 500-kDa. PAPP-A,IGFBP-4 Metalloproteinase,IGFBP-4 Protease,IGFBP-4-Specific Proteinase,Insulin-Like Growth Factor-Dependent IGF Binding Protein-4 Protease,Insulin-Like-Growth Factor Binding Protein-4 Protease,PAPP-alpha,Pregnancy Associated alpha Plasma Protein,Pregnancy-Associated alpha-Plasma Protein,IGFBP 4 Metalloproteinase,IGFBP 4 Protease,Insulin Like Growth Factor Binding Protein 4 Protease,Insulin Like Growth Factor Dependent IGF Binding Protein 4 Protease,Metalloproteinase, IGFBP-4,PAPP alpha,Pregnancy Associated Plasma Protein A,Protease, IGFBP-4
D018974 Insulin-Like Growth Factor Binding Protein 4 One of the six homologous soluble proteins that bind insulin-like growth factors (SOMATOMEDINS) and modulate their mitogenic and metabolic actions at the cellular level. IGF-Binding Protein 4,IGFBP-4,IGF Binding Protein 4,Insulin Like Growth Factor Binding Protein 4
D018975 Insulin-Like Growth Factor Binding Protein 5 One of the six homologous soluble proteins that bind insulin-like growth factors (SOMATOMEDINS) and modulate their mitogenic and metabolic actions at the cellular level. IGF-Binding Protein 5,IGFBP-5,IGF Binding Protein 5,Insulin Like Growth Factor Binding Protein 5

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