6966
Biochemistry 1987, 26, 6966-697 1
Possible Involvement of the A20-A21 Peptide Bond in the Expression of the Biological Activity of Insulin. 1. [ 2 1-Desasparagine,20-cysteinamide-A]insulin and [ 2 1-Desasparagine,20-~ysteineisopropylamide-A]insulint Ying-Chi Chu, Run-Ying Wang, G. Thompson Burke, Jacob D. Chanley, and Panayotis G. Katsoyannis* Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York,New York, New York 10029 Received .April 21, 1987; Revised Manuscript Received June 23, 1987
A chain of insulin has been shown to play a significant role in the expression of the biological activity of the hormone. To further delineate the contribution of this segment, we have synthesized [2 1-desasparagine,20-cysteinamide-A] insulin and [21-desasparagine,2O-~ysteineisopropylamide-A]insulin, in which the C-terminal amino acid residue of the A chain of insulin, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an amide and an isopropylamide, respectively. Both insulin analogues display biological activity, 14-1 5% for the unsubstituted amide analogue and 20-2276 for the isopropylamide analogue, both relative to bovine insulin. In contrast, a [21-desasparagine-A]insulinanalogue has been reported to display less than 4% of the activity of the natural hormone [Carpenter, F. (1966) Am. J. Med. 40, 750-7581, The implications of these findings are discussed, and we conclude that the AZ0-A2’ amide bond plays a significant role in the expression of the biological activity of insulin. ABSTRACT: The C-terminal region of the
M a n y insulin analogues have been prepared by :nodification of the natupal hormone or by chemical synthesis. For these analogues, receptor binding affinities and in vitro biological potencies relative to those of natural bovine insulin were the same. These data were interpreted to indicate that the biological activities of the analogues were wholly a consequence of their binding affinity to the insulin receptor; thus, insulin’s “binding site” and the region associated with the initiation of cellular processes, the “message region”, were indistinguishable (Freychet et al., 1974; Gliemann & Gammeltoft, 1974; Cosmatos et al., 1978). Recent studies, however, from this laboratory, concerning two synthetic insulin analogues as well as the behavior of two naturally occurring insulins, have provided evidence that the region of the insulin molecule responsible for binding to the insulin receptor may be distinguished from that involved in the initiation of the physiological activity of the hormone. Thus, [2 1-asparaginamide-Alinsulin exhibited a biological potency of ca. 13% whereas in receptor binding assays it displayed a potency of ca. 60% relative to insulin (Burlie et al., 1980). A similar discrepancy between biological potency, 4-5%, and receptor binding, ca. 25% has been reported for porcupine (Horuk et al., 1980) and hagfish (Emdin et al., 1980) insulins. In striking contrast, [21-proline-Blinsulin (Schwartz et al., 1983) exhibited an inverse behavior, namely, ca. 33% potency in stimulating lipogenesis while in binding assays it showed ca. 17% potency relative to the natural hormone. In this and the following two papers (Chu et al., 1987a,b), we present evidence that implicates a segment of the insulin molecule possibly involved in the “message region” of this hormone. In this paper we present data indicating that the A20-A2’ amide moiety is a necessary structural element for the expression of biological activity in insulin. We describe the synthesis and biological evaluation of [21 -desasparagine,20-cysteinamide-A]insulin ([desA2’Asn,AZ0-cysteinamide]insulin) and of [21-desasparaThis work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases, U S . Public Health Service (DK-12925).
0006-2960/87/0426-6966!§01.50/0
gine,20-cysteine isopropylamide- A] insulin ([desA2’Asn,A20cysteineisopropylamide] insulin) In both analogues, the C-terminal asparagine resid;\e of the A chain of insulin has been eliminated, and the resulting free carboxyl group of the Azo cysteine residue has been converted to an amide and to a monosubstituted isopropylamide, respectively.
.’
EXPERIMENTAL PROCEDURES AND RESULTS Details of materials and analytical procedures used in this investigation are given in a previous publication (Kitagawa et al., 1984). The homogeneity of all the intermediate peptide derivatives was ascertained by thin-layer chromatography on 6060 silica gel (Eastman chromagram sheet) in two solvent systems: chloroform-methanol-water (89:lO:l and 45: 10:1). For the enzymatic digestion with leucine aminopeptidase, the method of Hill and Smith (1957) was employed with a chromatographically purified enzyme (Worthington Biochemical Corp.). ‘251-InsulinBinding Liver Plasma Membranes. A fraction enriched in plasma membranes was prepared from the livers of fasted rats essentially as previously described (Horvat et al., 1975). Triplicate incubations, 0.2 mL, contained 40-80 pg of membrane protein, ‘251-insulin(Du Pont NEN Products, ca. 100 pCi/pg), varying concentrations of unlabeled bovine insulin or analogue, and sodium phosphate buffer, 0.1 M, pH 7.4, containing 6 mg/mL fraction V bovine serum albumin. After incubation at 24 OC for 45 min, the mixtures were diluted with 2 mL of sodium phosphate buffer, 0.1 M, pH 7.4, I Abbreviations: AcOH, acetic acid: Boc, tert-butoxycarbonyl; Bzl, benzyl; CM, carboxymethyl; DMF, dimethylformamide: DMSO, dimethyl sulfoxide; HMPA, hexamethylphosphoramide; NP, p-nitrophenyl; PMB, p-methoxybenzyl: TFA, trifluoroacetic acid; TFMSA, trifluoromethanesulfonic acid: TEA, triethylamine; Tris-HCI, tris(hydroxymethy1)aninomethane hydrochloride. Compounds designated by Roman numerals are described fully in the supplementary material (see paragraph at end of paper regarding supplementary material). The for IUPAC-IUB name of [21-desasparagine,20-cysteinamide-A]insulin, example, is des-A2l-asparagine-[A2O-cysteinamide]insulin (desAsnA21-[A20-cysteinamide]insulin).
0 1987 American Chemical Society
[21-DESASPARAGINE-A] I N S U L I N
VOL.
ANALOGUES
26, N O . 22,
1987
6967
Chart I OB21
Val
G
cys 1
i
t
I
I
I !
OBzl
PMB
PMB PMB
Ser I
G
,
BOC Boc Boc
E-
1
1
Tyr
Gln I u
OBzl PMB I
H'R
.
.
I 3 I X : I a 3 IXa:
( I or I a )
'R ( I 1 o r I I a )
H'R
NH'R
(I11 or I I I a )
( I V or I V a )
'R ( V o r V a )
'R ( V I o r V I a )
'R ( V I 1 o r V I I a )
I H-Gly-Ile-Val-Glu-G1n-Cys-Cys-Ala-Gly-Val-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-As~-Tyr-Cys-NH
1
Glu Asn T y r Cys
H'R
.R
( IX
( V I 1 1 or V I I I a )
or I X a )
R = H
R = -CH(CH3)2
containing 1 mg/mL fraction V bovine serum albumin, ice cold, and immediately filtered on cellulose-acetate filters (Sartorius 11107). The filters were washed twice with ice-cold buffer, dried, and dissolved in Filtron-X (National Diagnostics, Somerville, NJ) for counting. Nonspecific binding, defined as radioactivity remaining on the filter when incubations M unlabeled insulin, amounted to about contained 1 X 10% of the total '2SI-insulinbound in the absence of competitor and was substracted from all values. Relative potency was obtained as the concentration ratio of bovine insulin to analogue required to displace 50% of the specifically bound '251-insulin. Lipogenesis. Rat adipocytes were obtained by collagenase (1 .O mg/mL, Worthington type 11) digestion of epididymal and perirenal fat pads in Krebs-Ringer bicarbonate buffer containing half the recommended calcium concentration, 0.5 mM D-glucose, and 30 mg/mL fatty acid free bovine serum albumin (Boehringer-Mannheim). The gas phase was 95% 02-5% C02. Isolated cells (1 .O mL, 20-40 mg dry weight) were incubated in triplicate in plastic scintillation vials with varying concentrations of bovine insulin or insulin analogue for 45 min at 37 OC before the addition of the label. [33H]Glucose (0.5 pCi, Du Pont NEN Products) was added, and the incubation was continued for 1 h. The reaction was stopped by the addition of 5 N H2S04(0.2 mL), and corn oil (0.2 mL) was added to aid in the extraction of lipids. Soluscint-0 scintillation fluid (National Diagnostics, Somerville, NJ) was added to the vials, which were shaken at room temperature for 30 min before counting. Under these conditions
