Characterization of glycosaminoglycan-binding domains present in insulin-like growth factor-binding protein-3.
Matrix metalloproteinase 3 cleaves insulin-like growth factor-binding protein-3 (IGFBP-3) into six fragments, four of which bind heparin-Sepharose (Fowlkes, J. L., Enghild, J. J., Suzuki, K., and Nagase, H. (1994) J. Biol. Chem. 269, 25742-25746). Sequence analysis of IGFBP-3 heparin-binding fragments shows that all fragments contain at least one of two highly basic, putative heparin-binding consensus sequences present in IGFBP-3. Epitope-specific antibodies generated against synthetic peptides containing these domains recognized IGFBP-3, yet were significantly inhibited from binding in the presence of heparin, demonstrating that these regions of IGFBP-3 contain functional heparin-binding domains. IGFBP-3 peptides containing one of the two heparin-binding consensus sequences bound heparin in a solid phase binding assay in a dose-dependent and saturable manner. However, the IGFBP-3 peptide containing the heparin-binding consensus sequence 149KKGHA153 bound heparin with approximately 4-fold less affinity than the IGFBP-3 peptide containing the longer heparin-binding consensus sequence 219YKKKQCRP226. Examination of several well characterized glycosaminoglycans to inhibit the binding of heparin to both heparin-binding IGFBP-3 peptides revealed that the most potent inhibitors were heparin, heparan sulfate, and dermatan sulfate; chondroitin sulfate A and hyaluronic acid were intermediate in their inhibitory activities; and chondroitin sulfate C caused no inhibition. These studies identify and characterize the glycosaminoglycan-binding domains in IGFBP-3, providing a basis for the better understanding of IGFBP-3-glycosaminoglycan interactions at the cellular and extracellular interface.
Fowlkes
J L
JL
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Serra
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli; D006025:Glycosaminoglycans / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives; D014162:Transfection
D000906:Antibodies; D000939:Epitopes; D006025:Glycosaminoglycans; D018972:Insulin-Like Growth Factor Binding Protein 3; D010446:Peptide Fragments; D011994:Recombinant Proteins; C037405:heparin-sepharose; D012685:Sepharose; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3