Semisynthesis and biological activity of porcine [LeuB24]insulin and [LeuB25]insulin.

Abstract

Two analogs of porcine insulin with substitutions of leucine for phenylalanine in the COOH-terminal region of the insulin B chain have been prepared by a combination of solid-phase synthesis and semisynthesis. Solid-phase synthesis of the substituted octapeptides B23-B30 bearing the trifluoroacetyl group on lysine-B29, enzymatic coupling of the octapeptides to bis(tertiaiy-butyloxycarbonyl)desoctapeptide insulin by trypsin, and deprotection of the corresponding adducts in formic acid and piperidine resulted in two insulin derivatives, one with leucine at position B24 and the other with leucine at position B25. These analogs had only about 10% and 1%, re- These results suggest that the antagonistic activity of a human insulin variant having leucine at position B24 or B25 can be assigned to the molecule with the sequence Gly-Leu-Phe-Tyr (residues B23-B26) in its active site.Studies of naturally occurring and chemically modified insulins have shown that the COOH-terminal region of the B chain lies within the site conferring activity to the hormone (1-5). Although residues B23-B26 (Gly-Phe-Phe-Tyr) have remained invariant during animal evolution, we recently identified an abnormal human insulin in which this portion of the molecule had been modified: the variant, isolated in equimolar amounts with normal insulin from the pancreas of a diabetic but hyperinsulinemic patient, appeared to contain a substitution of leucine for phenylalanine at position 24 or 25 of the insulin B chain (6). The mixture of normal and abnormal hormones had lower than expected activity both in competing for the binding of [1251]iodoinsulin to membrane receptors on rat adipocytes and in stimulating 2-deoxyglucose uptake and glucose oxidation by the same cells (6). Importantly, the biological activity of the hormone preparation (15% of normal) was much lower than its apparent binding activity (45% of normal). These results, and others on the biological effectiveness of the insulin mixture present in plasma (7) and of the insulin variant purified by cellular adsorption (8), were consistent with the patient's clinical features (7) and suggested that the abnormal hormone was an active antagonist of insulin action. Further studies on placing the amino acid substitution in sequence and on determining the mechanism by which the variant might interfere with a cellular response to insulin were prevented, however, by the small amount of material available.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3181The pioneering work of Inouye et al. (9), which describes the semisynthesis of insulin derivatives by trypsin-catalyzed peptide bond formation at Arg-B22, now permits the preparation of analogs containing amino acid substitutions in the COOHterminal region of the insulin B chain. In this report we describe the solid-phase synthesis of the [LeuB , trifluoroacetyl (TFA)-LysB29] and [...

Aromatic Anchor at an Invariant Hormone-Receptor Interface

“…3C Table 2). The affinity of the Leu B24 variant was reduced by 2-fold (K d 0.10 Ϯ 0.02 nM) in accordance with past studies (19,51); the affinity of the Ile B24 variant was reduced by 3-fold (K d 0.15 Ϯ 0.02 nM). Such small differences are consistent with natural variation among mammalian insulins (species variants (52)).…”

[LeuB24]insulin and [AlaB24]insulin: altered structures and cellular processing of B24-substituted insulin analogs.

“…[LeuB"]insulin, [LeuB25]_ insulin, and [MaBlB]insulin (all derivatives of the porcine hormone) were prepared essentially as described (8). The final preparations gave the expected amino acid compositions after hydrolysis in 6 M HCl for 20 hr at 110°C and were shown to contain <10% monodesamido and mono-protected forms by polyacrylamide gel electrophoresis at pH 8.7 (21).…”

“…The recent identification of an abnormal insulin variant with leucine replacing phenylalanine at position B24 (5)(6)(7) emphasizes the special importance of the COOHterminal B-chain domain in conferring biological activity on the hormone. In particular, investigations of semisynthetic [LeuB"]insulin as well as of [LeuB"]insulin have shown major alterations in the receptor binding and biological activities of the analogs (8)(9)(10), in the abilities of the analogs to affect the rate of [1mI]iodoinsulin ("mI-insulin) dissociation from cell-surface receptors (11,12), and in the self-association of the B24 derivative (13,14). Structure-function relationships in the cellmediated degradation of insulin [both to low molecular weight products by lysosomal mechanisms (15)(16)(17)(18)(19) and to peptide intermediates (20)], however, have not been studied in detail.…”

Structure of Monomeric Porcine DesB1–B2 Despentapeptide (B26–B30) Insulin at 1.65 Å Resolution

“…In fact, amino-acid substitutions within these regions have been identified in the insulins from © 1997 International Union of Crystallography Printed in Great Britain -all rights reserved three individuals in whom genetic mutations resulted in the secretion of abnormal hormones associated with diabetes (Tager et al, 1979(Tager et al, , 1980Shoelson, Fickova et al, 1983;Haneda, Chan, Kowk, Rubenstein & Steiner, 1983), insulin Chicago (B25-Phe--~B25-Leu), insulin Los Angeles (B24-Phe--->B24-Ser), and insulin Wakayama (A3-Val--->A3-Leu). The structural analysis of a A1-B29 cross-linked insulin, which was completely inactive, indicates that its conformation is nearly same as that of native insulin (Derewenda et al, 1991).…”