title: Evidence that the interaction between insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-4 is essential for the action of the IGF-II-dependent IGFBP-4 protease.
abstract: A variety of human cell types, including human osteoblasts (hOBs), produce an IGFBP-4 protease, which cleaves IGFBP-4 in the presence of IGF-II. Recently, the pregnancy-associated plasma protein (PAPP)-A has been determined to be the IGF-II-dependent IGFBP-4 protease produced by human fibroblasts. This study sought to define the mechanism by which IGF-II enhances IGFBP-4 proteolysis. Addition of PAPP-A antibody blocked the IGFBP-4 proteolytic activity in hOB conditioned medium (CM), suggesting that PAPP-A is the major IGFBP-4 protease in hOB CM. Pre-incubation of IGFBP-4 with IGF-II, followed by removal of unbound IGF-II, led to IGFBP-4 proteolysis without further requirement of the presence of IGF-II in the reaction. In contrast, prior incubation of the partially purified IGFBP-4 protease from either hOB CM or human pregnancy serum with IGF-II did not lead to IGFBP-4 proteolysis unless IGF-II was re-added to the assays. To further confirm that the interaction between IGF-II and IGFBP-4 is required for IGFBP-4 protease activity, we prepared IGFBP-4 mutants, which contained the intact cleavage site (Met135-Lys136) but lacked the IGF binding activity, by deleting the residues Leu72-His74 in the IGF binding domain or Cys183-Glu237 that contained an IGF binding enhancing motif. The IGFBP-4 protease was unable to cleave these IGFBP-4 mutants, regardless of whether or not IGF-II was present in the assay. Conversely, an IGFBP-4 mutant with His74 replaced by an Ala, which exhibited normal IGF binding activity, was effectively cleaved in the presence of IGF-II. Taken together, these findings provided strong evidence that the interaction between IGF-II and IGFBP-4, rather than the direct interaction between IGF-II and IGFBP-4 protease, is required for optimal IGFBP-4 proteolysis.
authors.affiliation: Musculoskeletal Disease Center, J. L. Pettis Memorial Veterans' Medical Center, Loma Linda, California 92357, USA. Xuezhong.Qin@med.va.gov
authors.forename: X
authors.initials: X
authors.lastname: Qin
authors.forename: D
authors.initials: D
authors.lastname: Byun
authors.forename: K H
authors.initials: KH
authors.lastname: Lau
authors.forename: D J
authors.initials: DJ
authors.lastname: Baylink
authors.forename: S
authors.initials: S
authors.lastname: Mohan
mesh_terms: D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000378:metabolism; D005260:Female; D006801:Humans; D015151:Immunoblotting; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism* / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009154:Mutation; D010006:Osteoblasts / Q000201:enzymology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011247:Pregnancy; D011266:Pregnancy-Associated Plasma Protein-A / Q000276:immunology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins
chemical_list: D000906:Antibodies; D017077:Culture Media, Conditioned; D018974:Insulin-Like Growth Factor Binding Protein 4; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A