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|Title:||Localization of an Insulin-like Growth Factor (IGF) Binding Site of Bovine IGF Binding Protein-2 Using Disulfide Mapping and Deletion Mutation Analysis of the C-terminal Domain|
|Citation:||Journal of Biological Chemistry, 1998; 273(8):4647-4652|
|Publisher:||AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC|
|Abstract:||We have investigated which region(s) of bovine insulin-like growth factor binding protein-2 (bIGFBP-2) interact with insulin-like growth factors (IGFs) using C-terminally truncated forms of bIGFBP-2. Initially to aid in mutant design, we defined the disulfide bonding pattern of bIGFBP-2 C-terminal region using enzymatic digestion. The pattern is Cys186-Cys220, Cys231-Cys242, and Cys244-Cys265. In addition, cyanogen bromide cleavage of bIGFBP-2 revealed that the N- and C-terminal cysteine-rich domains were not linked by disulfide bonds. Taking the disulfide bonding pattern into consideration, C-terminal truncation mutants were designed and expressed in COS-1 mammalian cells. Following IGF binding assays, a region between residues 222 and 236 was identified as important in IGF binding. Specifically, mutants truncated by 14, 36, and 48 residues from the C terminus bound IGFs to the same extent as wild type (WT) bIGFBP-2. Removal of 63 residues resulted in a greatly reduced (up to 80-fold) ability to bind IGF compared with WT bIGFBP-2. Interestingly this mutant lacked the IGF-II binding preference of WT bIGFBP-2. Residues 236-270 also appeared to play a role in determining IGF binding specificity as their removal resulted in mutants with higher IGF-II binding affinity.|
|Keywords:||Animals; Cattle; Disulfides; Trypsin; Somatomedins; Insulin-Like Growth Factor Binding Protein 2; Chromatography, High Pressure Liquid; Peptide Mapping; Mutagenesis; Sequence Deletion; Binding Sites; Amino Acid Sequence; Molecular Sequence Data|
|Appears in Collections:||Biochemistry publications|
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