Purification and Structural Characterization of Insulin from the Lesser Siren, Siren Intermedia (Amphibia: Caudata)

Home , Habrosaurus, Lesser siren, Siren (genus)

General and Comparative Endocrinology 106, 295-300 (1997) Article No. GC976912

Purification and Structural Characterization of Insulin from the Lesser Siren, Siren intermedia (Amphibia: Caudata)

J. Michael Conlon,*-^ Stanley E. Trauth,t and David M. Severl^

*Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School, Omaha, Nebraska 68178; ^Department oj Biological Sciences, Arkansas State University, State University, Arkansas 72467; and ^Department of Biology, Saint Mary's College, Notre Dame, Indiana 46556

Accepted March 4, 1997

Insulin has been isolated from an extract of the pancreas and Kezer, 1993). Sirens are eel-like salamanders with of a salamander, the lesser siren Siren intermedia, and its tiny forelimbs but no hindlimbs or pelvic girdle and primary structure was established as: A-chain, Gly-Ile-Val- are generally found in muddy pools in the southeast• Glu-Gln-Cys-Cys-His-Asn-Thri''-Cys-Ser-Leu-Tyr-Gln-Leu- ern and central United States and Northern Mexico Glu-Asn-Tyr-Cys^°-Asn, and B-chain, Val-Pro-Asn-Lys-Pro- (Martof, 1974; Villela and Brandon, 1992). The Sireni• Leu-Cys-Gly-Ala-Hisio-Leu-Val-Glu-Val-Met-Tyr-Phe-Val- dae are usually assigned to a separate suborder, Siren• Cys-Gly^o-Asp-Arg-Gly-Phe-Phe-Tyr-Pro-Ser-Ser-Thr ^o. oidea, within the order Caudata (Duellman and Trueb, Although those amino acid residues considered to consti• 1986). However, the Sirenidae and Salamandridae tute the receptor-binding region of insulin have been have been placed together in the Salamandroidea retained, siren insulin contains several substitutions (Estes, 1981) and some taxonomists have proposed (Gin Lys at B4, Ser Ala at B9, Ala Val at B14, placing the species in their own order, Trachystoma, Leu -> Met at B15, Leu Phe at B17, Pro — Ser at B28, within the subclass Lissamphibia (Cope, 1989; Zug, and Lys Ser at B29) of amino acid residues that are 1993). Cladistic analysis based upon a combination of conserved in insulins from species of other amphibian molecular and morphological characters places Sireni• orders. The biological activity of siren insulin was not dae as the sister group of all other families of sala• investigated in this study but the substitutions at B28 manders (Larson and Dimirdck, 1993). Although the fossil (involved in dimer formation) and at B14 and B17 record is incomplete, salamanders probably evolved dur• (involved in hexamer formation) may be expected to ing the later Permian and Triassic and the radiation of the influence conformation and therefore biological potency. modem families must have occurred by the late Mesozoic The data are consistent with the view that the Sirenoidea (Naylor, 1980). The extinct Sirenid genus Habrosaurus is represent an early divergence from the ancestral stock of known from the late Cretaceous (Carroll, 1984). salamanders. © 1997 Academic Press Despite the importance of Amphibia in phylogeny and in our understanding of the evolution of physi• The family Sirenidae comprises two genera. Siren ological processes, our knowledge of the structures and Pseudobranchus, each containing two species (Moler and biological activities of amphibian regulatory pep• tides is largely confined to the Anura (frogs and toads)

^ To whom correspondence and reprint requests should be ad• (Andersen et al, 1992). Previous studies have led to the dressed. Fax; (402) 280-2690. isolation and structural characterization of insulin

0016-6480/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 295 296 Conlon, Trauth, and Sever from the toad Xenopus laevis (Anura: Pipidae) (Shulinder et Vydac 214TP54 C-4 reversed-phase column equili• al, 1989), from a salamander, the three-toed amphiuma, brated with acetonitrile/water/trifluoroacetic acid Amphiuma tridactylum (Caudata: Amphiumidae) (Conlon (210/789/1) at a flow rate of 1.5 ml/min. The concen• et al, 1996) and from the caedlian, Typhlonectes nutans tration of acetonitrile in the eluting solvent was raised (Gymnophiona: Typhlonectidae) (Conlon et al, 1995). We to 42% over 40 min using a linear gradient. Siren now extend these studies to describe the purification and insulin was purified to near homogeneity, as assessed characterization of iiisulin from an extract of the pancreas by peak symmetry, by chromatography on a 0.46 X of the lesser siren. Siren intermedia Le Conte. 25-cm Vydac 219TP54 phenyl column under the same conditions used for the C-4 column. Structural characterization. Siren insulin (approxi• MATERIALS AND METHODS mately 2 nmol) was incubated for 6 hr at room tempera- tiire with dithiothieitol (2 mg) in 0.1 M Tris-HCl/6 M guanidine hydrochloride buffer, pH 7.5 (0.4 ml) imder an Tissue extraction. Sirens (18 specimens of both atmosphere of argon. Cysteine residues were derivatized sexes; weight 31-135 g; length 15-30 cm) were col• by addition of 4-vinylpyridine (3 ml) and the pyridylethyl- lected between February and April of 1996 at sites in ated A- and B-chains of insulin were separated on a 25 X Craighead County and Clay County, Arkansas. The 0.46-cm Vydac C-4 column under the conditions used animals had been actively feeding and were in breed• for the purification of intact insulin (Fig. IB). ing condition (large ovarian follicles in females). Pan• Amino acid compositions were determined in dupli• creatic tissue (1.4 g), taken from animals sacrificed by cate by precolumn derivatization with phenylisothio- immersion in tricaine methanesulfonate, was extracted cyanate (Bidlingmeyer et al, 1984) using an Applied with ethanol/0.7 M HCl (3/1 v/v; 900 ml) as previ• Biosystems Model 420A derivatizer followed by re• ously described (Conlon et al, 1996). After centrifuga- versed-phase HPLC with an Applied Biosystems Model tion (1600g for 1 hr at 4°), ethanol was removed from 130A separation system. Hydrolysis (24 hr at 110° in 5.7 the supernatant under reduced pressure. Peptide mate• M HCl) of approximately 1 nmol of sample was carried rial was isolated from the extract using Sep-Pak C-18 out. Cysteine and tryptophan residues were not deter• cartridges (Waters Associates, Milford, MA). Bound mined. The primary structures of the siren peptides were material was eluted with acetonitrile/water/trifluoro- determined by automated Edman degradation in an Ap- acetic acid (70/29/1, v/v/v) and freeze-dried. pUed Biosystems Model 471A sequenator modified for Radioimmunoassay. Insulin-like immunoreactiv- detection of phenylthiohydantoin (PTH) amino add deriva• ity was measured using an antiserum raised against pig tives under gradient elution conditions. Approximately insulin as previously described (Flatt and Bailey, 1981). l-mnol portions of the peptides were used. The detec• Purification of siren insulin. The pancreatic ex• tion limit for the PTH derivatives was 1 pmol. tract, after partial purification on Sep-Pak cartridges, was redissolved in 0.1% (v/v) trifluoroacetic acid/ water (5 ml) and injected onto a 1 X 25-cm Vydac 218TP510 C-18 reversed-phase HPLC column (Separa• RESULTS tions Group, Hesperia, CA) equilibrated with 0.1% (v/v) trifluoroacetic acid/water at a flow rate of 2 Purification of Siren Insulin ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min and The crude extract of the S. intermedia pancreas then raised to 49% (v/v) over 60 min using linear contained only a very low concentration (approxi• gradients. Absorbance was measured at 214 and 280 mately 1 pmol/g tissue weight) of insulin-like immu• nm and individual peaks were collected by hand. noreactivity measured with an antiserum raised against Fractions (2 ml) were collected and assayed for insulin• pig insulin, and the immunoreactivity in serial dilu• like immunoreactivity at a dilution of 1:30. The peak tions of the extract did not diminish in parallel with the designated I (containing insulin-like immunoreactiv• pig insulin standard. The pancreatic extract, after ity) (Fig. 1) was rechromatographed on a 0.46 X 25-cm partial purification on Sep-Pak cartridges, was injected

0.6 50

- 40

lU 30 S

< ui 20 y

- 10

10 20 30 40 50 60 70 TIME (min)

0.6 n B -50 -50

-40 - ' '-40

CM m m < < -20 -20 SS

-10 -10

10 20 30 10 20 30 TIME (min) TIME (min)

FIG. 1. Purification by reversed-phase HPLC of siren insulin on (A) semipreparative Vydac C-18, (B) analytical Vydac C-4, and (C) analytical Vydac phenyl columns. 1 denotes the peaks containing insulin-like immunoreactivity. The arrowfs show where peak collection began and ended and (—) shows the concentration of acetonitrile in the eluting solvent.

directly onto a semipreparative Vydac C-18 column, ambiguity phenylthiohydantoin-coupled amino acids and the elution profile is shown in Fig. lA. Insulin-like for 21 cycles of operation of the sequenator during immunoreactivity was associated with the single promi• sequence analysis of the A-chain and for 30 cycles nent peak designated I. Siren insulin was purified to during analysis of the B-chain. The results of amino near homogeneity (as assessed by symmetrical peak acid analysis demonstrated that the A-chain of siren shape) by successive chromatographies on a Vydac C-4 insulin had the composition Asx 2.9 (3), Glx 4.0 (4), Ser column (Fig. IB) and a Vydac phenyl column (Fig. IC). 1.0 (1), Gly 1.0 (1), His 0.9 (1), Thr 0.8 (1), Tyr 1.7 (2), Val The final yield of the pure peptide was 3 nmol. 0.6 (1), lie 0.6 (1), and Leu 2.0 (2) (mol residue/mol peptide). The composition of the B-chain was deter• mined as Asx 2.3 (2), Glx 1.3 (1), Ser 2.0 (2), Gly 3.0 (3), Structural Characterization of Siren Insulin His 1.0 (1), Arg 1.3 (1), Thr 1.0 (1), Ala 1.1 (1), Pro 2.9 (3), The primary structures of the pyridylethylated A- Tyr 1.7 (2), Val 3.8 (4), Met 0.4 (1), Leu 1.9 (2), Phe 2.8 chains and B-chains of siren insulin were determined (3), and Lys 1.0 (1) (mol residue/mol peptide). The by automated Edman degradation and the results are values in parentheses show the number of residues shown in Table 1. It was possible to identify without predicted from the proposed structures. Agreement

TABLE 1 and with insulin from a mammal (the human) in Fig. 2. Automated Edman Degradation of the A-Chain and B-Chain of The data show that siren insulin contains several Insulin from a Siren substitutions of amino acid residues that are otherwise Cycle A-chain B-chain well conserved in insulins from these and other verte• brate taxa. Although those residues on the surface of 1 Gly (574) Val (1037) 2 He (614) Pro (1076) the molecule that are considered to constitute the 3 Val (687) Asn (833) receptor-binding region (A1-A3, A5, A19, A21, and 4 Glu (379) Lys (996) B22-B26) (Baker et al, 1988) have been retained, siren 5 Gin (335) Pro (829) 6 Cys (433) Leu (644) insulin contains several substitutions (Gin Lys at B4, 7 Cys (442) Cys (471) Ser ^ Ala at B9, Ala ^ Val at B14, Leu ^ Met at B15, 8 His (235) Gly (592) Leu Phe at B17, Pro — Ser at B28, and Lys Ser at 9 Asn (468) Ala(604) 10 Thr (91) His (216) B29) of amino acids that are identical in insulins from 11 Cys (264) Leu (506) other amphibians and the human. There was not 12 Ser(26) Val (480) sufficient pure material to investigate the biological 13 Leu (145) Glu (249) activity of siren insulin but certain of these structural 14 Tyr(153) Val (464) 15 Gin (125) Met (382) features may be expected to influence conformation 16 Leu (129) Tyr (327) and biological potency relative to human insulin. In 17 Glu (79) Phe (302) particular, among those residues in human insulin 18 Asn (73) Val (395) 19 Tyr(68) Cys (217) involved in dimer formation (B12, B16, B20, B24, B26, 20 Cys (34) Gly (331) and B28), siren insulin contains the substitution Pro 21 Asn (21) Asp (353) Ser at B28. Similarly, among those residues in human 22 Arg (162) 23 Gly (316) insulin involved in hexamer formation (B6, BIO, B14, 24 Phe (226) B17, B18, A13, and A14), siren insulin contains the 25 Phe (239) substitutions Ala Val at B14 and Leu Phe at B17. 26 Tyr (189) On the other hand, siren insulin shares with insulins 27 Pro (146) 28 Ser(19) from the chicken (Simon et al, 1977), Xenopus (Shu- 29 Ser(25) linder et al, 1989), and the amphiuma (Conlon et al, 30 Thr (21) 1996) the presence of a histidine residue at position A8. Note. Cysteine was determined as its pyridylethylated derivative. Values in parentheses are the yields of amino acid phenylthiohydan- toins in picomoles. A-Chain

Siren GIVEQ CCHNT CSLYQ LENYC N between the sequence analysis and the amino add compo• Amphiuma AR N- sition data was good, demonstrating that the fuU sequence Caecilian K —LS- E —S— - of the peptides had been obtained. The sKghtly low values Xenopus I S- F- —S— - for the amoimts of valine and isoleudne in the A-chain are Xenopus II S- F- consistent with the presence of an lie-Val bond in the Human —TSI E molecule that is resistant to hydrolysis and the low value for the methionine composition of the B-chain is indicative B-Chain of oxidation to the sulfoxide derivative. The data Siren VPNKP LCGAH LVEVM YFVCG DRGFF YPSST demonstrated that the siren insulin was >98% pure. Amphiuma IT-QY —S AL -L SPK

Caecilian lA-QH —S AL -L—A TPKS

Xenopus I LV-QH —S AL -L YPKV

DISCUSSION Xenopus II LA-QH —S AL -L YPKI

Human FV-QH S- AL -L E -TPK-

The primary structure of siren insulin is compared FIG. 2. A comparison of the primary structures of amphibian with the structures of the known amphibian insulins insulins and human insulin. (-) denotes residue identity.

This residue, by forming stabilizing interactions with ascertain whether meaningful phylogenetic inferences residues in the receptor, is believed to be responsible may be drawn based upon the structures of this for the observed increase in binding affinity (between hormone. Similarly, for those species from which two- and fivefold) of these insulins for mammalian appreciable amounts of pancreatic tissue may be ob• insulin receptors. tained, a study of the biological properties of purified The phylogenetic relationship between the sirenids amphibian insulins will be of value in providing and other salamanders is unclear as sirenids possess a insight into structure-activity relationships. peculiar combination of primitive and derived morpho• logical characters, some of which are associated with paedomorphosis (Duellman and Trueb, 1986). Recent ACKNOWLEDGMENTS analyses focusing on the oviducal and cloacal anatomy of S. intermedia have concluded that sirenids have retained external fertilization, the ancestral state for The authors thank Ben Ball, Arkansas State University, for help in the Lissamphibia consistent with the hypothesis that collecting specimens. This work was supported by a grant from the National Science Foundation (IBN-9418819). they are derived from a basal stock of salamanders (Sever, 1991; Sever et al., 1996). Siren insulin resembles most closely that of the amphiuma, consistent with the placement of these species within the order Caudata, REFERENCES but the presence of 12 amino acid substitutions is indicative of a relatively ancient divergence of the Andersen, A. C, Tonon, M. C, Pelletier, G., Conlon, J. M., Fasolo, A., species. The A-chain of amphiuma insulin contains 23 and Vaudry, H. (1992). Neuropeptides in amphibian brain. Int. amino acid residues, leading to the suggestion that Rev. Cytol. 138,89-210. multiple amino acid substitutions in the region of the Bajaj, M., Blundell, T., and Wood, S. (1984). Evolution of the insulin Lys-Arg cleavage site linking the C-peptide region to family: Molecular clocks that tell the wrong time. In "Molecular the A-chain results in an anomalous pathway of Variants of Proteins—Biosynthesis and Clinical Relevance" (P. N. Campbell and C. Phelps, Eds.), pp. 45-54. Biochem. Soc, London. proinsulin processing (Conlon et al, 1996). The A-chain Baker, E. N., Blundell, T. L., Cutfield, J. E, Cutfield, S. M., Dodson, of siren insulin does not contain this unusual N- E. J., Dodson, G. G., Hodgkin, D. M. C, Hubbard, R. E., Isacs, N. terminal dipeptide extension found in the amphiuma W., Reynolds, C. D., Sakabe, K., Sakabe, N., and Vijayan, N. M. A-chain, indicating that siren proinsulin is processed (1988). The structure 2 Zn pig insulin crystal at 1.5 A resolution. Philos. Trans. R. Soc. London Ser. B 319,369-456. to insulin in the conventional manner by the action of a Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984). Rapid SPC-2 type convertase (Steiner et al, 1992). Phyloge• analysis of amino adds using pre-column derivatization. /. Chro- netic studies comparing DNA sequences of mitochon• matogr. 336, 93-104. drial ribosomal genes (Hedges et al, 1993) have sup• Carroll, R. L. (1984). Modern amphibians. In "Vertebrate Paleontol• ported the conclusion of earlier morphological ogy and Evolution," pp. 180-191. Freeman, New York. investigations (Van der Horst et al, 1991) that the Conlon, J. M., Cavanaugh, E. S., Mynarcik, D. C, and Whittaker, J. (1996). Characterization of an insulin from the amphiuma (Am• Gymnophonia and the Caudata are sister taxa. Siren phibia: Urodela) with an N-terminally extended A-chain and high insulin, however, contains 18 amino acid substitutions receptor-binding affinity Biochem ]. 313,283-287. compared with the corresponding region of a caecilian Conlon, J. M., and Hicks, J. W. (1990). Isolation and primary insulin (Conlon et al, 1995) but only 14 substitutions structures of insulin, glucagon and somatostatin from the turtle, Psuedyms scripta. Peptides 11,461^66. compared with Xenopus insulin II (Shulinder et al, Conlon, J. M., Hilscher-Conklin, C, and Boyd, S. K. (1995). Purifica• 1989) (Fig. 2). This observation illustrates the pitfalls tion and structural characterization of insulin from a caecilian, associated with inferring phylogenetic relationships Typhlonectes nutans (Amphibia: Gymnophiona). Peptides 16, 1385- between species based upon the amino acid sequences 1388. of their insulins (Bajaj et al, 1984; Conlon et al, 1990; Cope, E. D. (1981). The Batrachia of North America. U.S. Natl. Mus. Bull. 34,1-525. Hedges et al, 1990; Dores et al, 1996). Nevertheless, we Dores, R. M., Rubin, D. A., and Quinn, T. W. (1996). Is it possible to feel there is a need to characterize insulins from a construct phylogenetic trees using polypeptide hormone se• much wider range of amphibian species in order to quences? Gen. Comp. Endocrinol. 103,1-12.

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