Identification of residues in the insulin molecule important for binding to insulin-degrading enzyme.
Insulin-degrading enzyme (IDE) hydrolyzes insulin at a limited number of sites. Although the positions of these cleavages are known, the residues of insulin important in its binding to IDE have not been defined. To this end, we have studied the binding of a variety of insulin analogues to the protease in a solid-phase binding assay using immunoimmobilized IDE. Since IDE binds insulin with 600-fold greater affinity than it does insulin-like growth factor I (25 nM and approximately 16,000 nM, respectively), the first set of analogues studied were hybrid molecules of insulin and IGF I. IGF I mutants [insB1-17,17-70]IGF I, [Tyr55,Gln56]IGF I, and [Phe23,Phe24,Tyr25]IGF I have been synthesized and share the property of having insulin-like amino acids at positions corresponding to primary sites of cleavage of insulin by IDE. Whereas the first two exhibit affinities for IDE similar to that of wild type IGF I, the [Phe23,Phe24,Tyr25]IGF I analogue has a 32-fold greater affinity for the immobilized enzyme. Replacement of Phe-23 by Ser eliminates this increase. Removal of the eight amino acid D-chain region of IGF I (which has been predicted to interfere with binding to the 23-25 region) results in a 25-fold increase in affinity for IDE, confirming the importance of residues 23-25 in the high-affinity recognition of IDE. A similar role for the corresponding (B24-26) residues of insulin is supported by the use of site-directed mutant and semisynthetic insulin analogues. Insulin mutants [B25-Asp]insulin and [B25-His]insulin display 16- and 20-fold decreases in IDE affinity versus wild-type insulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Affholter
J A
JA
Department of Pharmacology, Stanford University School of Medicine, California 94305.
Cascieri
M A
MA
Bayne
M L
ML
Brange
J
J
Casaretto
M
M
Roth
R A
RA
D000818:Animals; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D006859:Hydrogen; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives / Q000378:metabolism*; D007339:Insulysin / Q000378:metabolism*; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000378:metabolism; D013552:Swine
D007328:Insulin; D007334:Insulin-Like Growth Factor I; D006859:Hydrogen; D011972:Receptor, Insulin; D010447:Peptide Hydrolases; D007339:Insulysin