Opossum insulin, glucagon and pancreatic polypeptide: Amino acid sequences

Peptides, Vol. 10, pp. 1195-1197.PergamonPress plc, 1989. Printedin the U.S.A.

0196-9781/89$3.00 + .00

Opossum Insulin, Glucagon and Pancreatic Polypeptide: Amino Acid Sequences J.-H. YU,* J. E N G , * t S. RATTAN:~ AND R. S. Y A L O W * t 1

*Solomon A. Berson Research Laboratory, Veterans Administration Medical Center, Bronx, NY 10468 "pThe Mount Sinai School of Medicine, CUNY, New York, NY 10029 ~:Beth Israel Hospital, Boston, MA 02215 Received 23 June 1989

YU, J.-H., J. ENG, S. RATTAN AND R. S. YALOW. Opossum insulin, glucagon and pancreatic polypeptide: Amino acid sequences. PEPTIDES10(6) 1195-1197, 1989.--Pancreatichormoneshave been purifiedfrom the opossum, a New Worldmarsupial. Opossum insulincontainsa Leu substitutionat the N-terminusof the B-chainin place of the Phe that is generallypresentin mammalian insulins.In addition,there are two other aminoacid substitutionsin the opossum insulinA-chain(positions8 and 18) comparedto pig insulin. Opossum glucagonis identicalto chicken glucagonwith both differingfrom the usual mammalianglucagonby Ser in place of Ash at its penultimateC-terminalposition. Opossum PP differs from the porcine peptide in only 3 sites (position3, 19 and 30). Amino acid sequence

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purification were monitored using radioimmunoassays (RIA's) in general use in our laboratory (5, 8, 16). Initially beef insulin, pork glucagon and beef PP were used as standards. When the peptides were purified, standard curves were prepared with the authentic peptides and the concentrations redetermined. C 18 Sep-Pak cartridge (Waters Associates, Milford, MA) was prepared by washing with 10 ml ethanol and 10 ml distilled water. The redissolved precipitate was then pumped through at 1 ml/min. The cartridge was washed with 6 ml 0.1% trifluoroacetic acid (TFA) and eluted with 2 ml 0.1% TFA/60% acetonitrile (ACN). The eluate was further purified by three steps of HPLC. The three steps consisted of the following columns and elution conditions: 1. MB C18 radial-pak column (Waters Associates) eluted with a linear gradient from 20-60% ACN in 0.13% heptafluorobutyric acid (HFBA). The peak fractions of immunoreactive insulin and the peak fractions containing both glucagon and PP were separately purified in the subsequent two steps. 2. Mono S HR5/5 strong cation exchange column (Pharmacia, Piscataway, NJ) eluted with a linear gradient from 0.1-0.5 M NaC1 in 0.1 TFA/20% ACN. This step also did not separate glucagon and PP. 3. Nova C18 radial-pak cartridge (Waters Associates) eluted with a linear gradient from 20-40% ACN in 0.1% TFA. This step permitted resolution of glucagon and PP. Portions of the purified peptides were subjected to the following procedures: Insulin was reduced with 2-mercaptoethanol and alkylated with 4-vinyl pyridine (1). Glucagon and PP were separately cleaved with Endoproteinase Arg-C and Endoproteinase Lys-C (Boehringer-Mannheim Biochemicals, Indianapolis, IN),

EVOLUTIONARY history suggests that marsupials entered South America from North America about 75 million years ago (2). Following the extinction of all North American marsupials 15 million years ago, the Virginia opossum (Didelphis virginiana) is thought to have remigrated into nontropical North America when land bridges reconnected the two continents about five to two million years ago (11). Several peptide hormones of the hystricognath rodents, the guinea pig and chinchilla, which are New World mammals, differ uniquely from those of Old World mammals, (3-7, 14, 15, 17). In addition, the insulins of the two New World monkeys differ sufficiently so that they cross-react poorly in the usual assay systems for mammalian insulins (10). Thus it was of interest to determine whether the corresponding peptides of the opossum, a New World marsupial, are more closely related to those of New World or to those of Old World mammals. In this report we describe the purification and sequences of insulin, glucagon and pancreatic polypeptide (PP) from the opossum pancreas and compare their sequences to the corresponding hormones of New and Old World eutherian mammals. METHOD The pancreas from an opossum freshly sacrificed at the end of an unrelated experiment was removed and stored frozen until extraction. The pancreas weighed 13.5 g and was extracted in 5 volumes of acid-alcohol (0.2 N HC1 in 75% ethanol) with a Teflon tissue grinder. The acid-alcohol extract was precipitated with 8 volumes of acetone at -30°(2 overnight. The precipitate was collected by centrifugation and the supernatant discarded. The precipitate was dissolved in 21 ml of 1 M acetic acid. The insulin, glucagon and PP in the original extract and subsequent steps in

tRequests for reprints shouldbe addressed to Dr. RosalynS. Yalow, SolomonA. BersonResearch Laboratory, VeteransAdministrationMedicalCenter, 130 W. KingsbridgeRoad, Bronx, NY 10468.

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Opossum glucagon and PP were equally cross-reactive in their respective RIA's with pork glucagon and beef PP standards. Opossum insulin is much less cross-reactive than is beef insulin in the RIA employed (Fig. 1). Therefore, an extract of opossum pancreas was used as an arbitrary standard throughout the insulin purification. Following purification the concentrations of insulin in the initial extract and the successive steps of purification were determined. The insulin content of the pancreas was 57.2 nmol. The glucagon and PP contents were 7.4 nmol and 7.6 nmol, respectively. The successive steps in the purification of the peptides are shown in Fig. 2. The amino acid sequences of the three peptides are compared with the corresponding peptides of the pig in Fig. 3. Opossum insulin differs from pig insulin in only 3 amino acids, 2 in the A-chain and 1 in the B-chain. This is to be contrasted with GP insulin which differs from the pig peptide in 20 of the 51 amino acids. Opossum pancreatic glucagon is identical in structure to that of chicken glucagon (8) with both differing from the usual mammalian glucagon by Ser substitution in place of Asn at the penultimate C-terminal position. This sequence is in agreement with the sequence reported for the midportion of opossum glicentin (N- and C-terminally extended proglucagon 68) (13). Opossum PP resembles the mammalian PP's, differing from the pig peptide in only three sites (positions 3, 19, 30). Thus it has a conserved C-terminal hexapeptide sequence which is thought to be important for bioactivity since that fragment is capable of reproducing the effects of intact PP in man and dog (9).

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ACKNOWLEDGEMENTS This work was supported in part by the Medical Research Program of the Veterans Administration. J.-H. Yu is a Fellow of the Solomon A. Berson Fund for Medical Research, Inc. We want to acknowledge with thanks Dr. Ronald Chance of Lilly Research Laboratory (Indianapolis, IN) who provided us with the rabbit antibeef PP antiserum (615-R110-146-10) and beef PP. The opossum pancreas was made available through NIH grant, DK-35385, to Dr. Rattan. REFERENCES 1. Applied Biosystems, User Bulletin No. 1, August 20; 1987. Cysteine/ cystine modification for amino acid analysis. 2. Austad, S. N. The adaptable opossum. Sci. Am. 258:98-104; 1988. 3. Bonato, C.; Eng, J.; Pan, Y.-C. E.; Chang, M.; Hulmes, J. D.; Yalow, R. S. Guinea pig "little" gastrin is a hexadecapeptide. Life Sci. 37:2563-2568; 1985. 4. Du, B.-H.; Eng, J.; Hulmes, J. D.; Chang, M.; Pan, Y.-C. E.; Yalow, R. S. Guinea pig has a unique mammalian VIP. Biochem. Biophys. Res. Commun. 128:1093-1098; 1985. 5. Eng, J.; Huang, C.-G.; Pan, Y.-C. E.; Hulmes, J. D.; Yalow, R. S. Guinea pig pancreatic polypeptide: Structure, pancreatic content and distribution. Peptides 8:165-168; 1987. 6. Fan, Z.-W.; Eng, J.; Miedel, M.; Hulmes, J. D.; Pan, Y.-C, E.; Yalow, R. S. Cholecystokinin octapeptides purified from chinchilla and chicken brains. Brain Res. Bull. 18:757-760; 1987. 7. Huang, C.-G.; Eng, J.; Pan, Y.-C. E.; Hulmes, J. D.; Yalow, R. S. Guinea pig glucagon differs from other mammalian glucagons. Diabetes 35:508-512; 1986. 8. Huang, J.; Eng, J.; Yalow, R. S. Chicken glucagon: Sequence and potency in receptor assay. Horm. Metab. Res. 19:542-544; 1987. 9. Konturek, S. J.; Meyers, C. A.; Kwiecien, N.; Obtulowicz, W.; Tasler, J.; Oleksy, J.; Kopp; B.; Coy, D. H.; Schally, A. V. Effect of human pancreatic polypeptide and its C-terminal hexapeptide on pancreatic secretion in man and in the dog. Scand. J. Gastroenterol. 17:359-399; 1982.

10. Mann, G. V.; Crofford, O. B. Insulin levels in primates by immunoassay. Science 169:1312-1313; 1970. 11. Sarich, V. M.; Cronin, J. E. South American mammal molecular systematics, evolutionary clocks, and continental drift. In: Ciochon, R. L.; Chiarelli, A. B., eds. Evolutionary biology of the New World monkeys and continental drift. New York: Plenum Press; 1980: 399-421. 12. Seino, S.; Steiner, D. F.; Bell, G. I. Sequence of a New World primate insulin having low biological potency and immunoreactivity. Proc. Nail. Acad. Sci. USA 84:7423-7427; 1987. 13. Shinomura, Y.; Eng, J.; Rattan, S. C.; Yalow, R. S. Opossum intestine contains a glucagon-68. Biomed. Res. 9(Suppl. 3):33-38; 1988. 14. Shinomura, Y.; Eng, J.; Yalow, R. S. Chinchilla "big" and "little" gastrins. Biochem. Biophys. Res. Commun. 143:7-14; 1987. 15. Smith, L. F. Species variation in the amino acid sequence of insulin. Am. J. Med. 40:662-666; 1966. 16. Yalow, R. S.; Berson, S. A. Radioimmunoassay of insulin. In: Berson, S. A.; Yalow, R. S., eds. Methods in investigative and diagnostic endocrinology, part III, non-pituitary hormones, chapter 3, section 3.2. Amsterdam: North-Holland Publishing Co.; 1973:864870, 17. Zhou, Z.-Z.; Eng, J.; Pan, Y.-C. E.; Chang, M.; Hulmes, J. D.; Raufman, J.-P.; Yalow, R. S. Unique cholecystokinin peptides isolated from guinea pig intestine. Peptides 6:337--441; 1985.