Diversity of human insulin-like growth factor (IGF) binding protein-2 fragments in plasma: primary structure, IGF-binding properties, and disulfide bonding pattern.
The insulin-like growth factor binding proteins (IGFBPs) play a major role in the regulation of the effects and the bioavailability of the insulin-like growth factors (IGFs). IGFs are released from IGFBP-IGF complexes by proteolysis of IGFBPs generating fragments with reduced ligand-binding properties. To identify naturally occurring fragments of IGFBP-2, a peptide library generated from human hemofiltrate was immunologically screened. Purification of immunoreactive IGFBP-2 fragments was performed by consecutive chromatographic steps. A total of 18 different IGFBP-2 fragments was isolated and characterized. The peptides exhibited different N-terminal amino acid residues that were located in the variable midregion of IGFBP-2. Four major cleavage sites were determined to be between Tyr103 and Gly104, Leu152 and Ala153, Arg156 and Glu157, and Gln165 and Met166. The resulting fragments were further processed by amino and/or carboxy peptidases and comprised 37-185 amino acid residues. Ligand blotting, solution binding assays, and BIAcore analyses revealed that all tested fragments retained low IGF-binding capacity. The most abundant fragment IGFBP-2 (167-279) showed 10% of IGF-II binding compared to recombinant human (rh)IGFBP-2. Furthermore, the disulfide bonding pattern of the C-terminal domain of rhIGFBP-2 was defined, indicating linkages between cysteine residues 191-225, 236-247, and 249-270. This study provides the most comprehensive molecular characterization of human IGFBP-2 fragments formed in vivo, exhibiting both residual IGF-binding capacities and the integrin-binding sequence.
Mark
Silke
S
IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse 31, D-30625 Hannover, Germany.
Kübler
Bernd
B
Höning
Stefan
S
Oesterreicher
Sandra
S
John
Harald
H
Braulke
Thomas
T
Forssmann
Wolf-Georg
WG
Ständker
Ludger
L
D000328:Adult; D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006440:Hemofiltration; D006801:Humans; D006868:Hydrolysis; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D004220:Disulfides; D018971:Insulin-Like Growth Factor Binding Protein 2; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D002918:Chymotrypsin