GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
82, 23-32 (1991)
The Primary Structure of Glucagon-like Peptide but Not Insulin Has Been Conserved between the American Eel, Anguilla rostrata and the European Eel, Anguilla anguilla J. MICHAEL CONLON,* P.C. ANDREWS,‘F LARSTHIM,S
AND THOMAS W. MOON§
*Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School, Omaha, Nebraska 68178; fDepartment of Biochemistry, Purdue University, West Lafayette, Indiana 47907; $Novo Nordisk Research Institute, Bagsvaerd, Denmark; and $Department of Biology, University of Ottawa, Ottawa, Ontario KlN 6N5, Canada
Accepted May II, 1990 Insulin was isolated from the pancreas of the American eel, Anguilla primary structure was established as A-Chain 5 10 GIVEQCCHKPCSIFDLQNYCN B-Chain 5 IO 15 ASTQHLCGSHLVEALYLVCGSNGFFFNPKD
15
20
rostrata,
and its
20
25
30
Eel insulin contains unusual substitutions at B-21, B-22, and B-26 in the putative receptorbinding region of the molecule compared with other mammalian and fish insulins. The A-chain of insulin from the European eel contains an asparagine rather than a serine residue at position A-12. Similarly, amino acid composition data indicate the B-chain of insulin from the European eel is appreciably different from that from the American eel. The primary structure of glucagon-like peptide (GLP) from the American eel is identical to that from the European eel, Anguilla anguilla. The primary structure of the peptide was established as 5 10 15 20 25 30 HAEGTYTSDVSSYLQDQAAKEFVSWLKTGR Fast-atom bombardment mass spectrometry demonstrated that the COOH-terminal arginyl residue is a-amidated. The strong evolutionary pressure to conserve the structure of GLP provides further support for the assertion that the peptide plays an important regulatory role in teleost fish. 0 1991 Academic Press. Inc.
The physiological role of insulin and glucagon in lower vertebrates is incompletely understood. Numerous studies have described the effects of pancreatic hormones on intermediary metabolism and growth in teleost fish but, until recently, all studies were carried out using either mammalian peptides or impure preparations of fish pancreatic extracts. The isolation in pure form of relatively large amounts of insulin (Plisetskaya er al., 1985), glucagon and gluca-
gon-like peptide (GLP) (Plisetskaya et al., 1986) from the coho salmon, Oncorhyncus kisutch, has enabled biological testing in the species of origin. Salmon insulin was shown to exhibit a more pronounced hypoglycemic effect than bovine insulin when injected into juvenile coho salmon (Plisetskaya et al., 1985). Salmon glucagon was more potent than mammalian glucagon and salmon GLP in stimulating glycogenolysis both in vivo and using liver slices from ju23 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved
24
CONLON
venile salmon (Plisetskaya et al., 1989). In contrast, salmon GLP is a more potent stimulator of gluconeogenesis than salmon glucagon in isolated salmon hepatocyte (Mommsen et al., 1987). Related studies in viva using juvenile salmon have shown that the effects of salmon GLP are markedly seasonal but effects upon glycolytic, lipolytic, and gluconeogenic metabolic pathways were observed (Mommsen and Moon, 1989; Plisetskaya er al., 1989). Human GLP-1 (7-37) is a potent stimulant of insulin release in mammals (Mojsov et al., 1987) but salmon GLP had little or no effect upon plasma insulin levels in salmon. Several studies have investigated the effects of pancreatic hormones on metabolic parameters in the eel (Ince and Thorpe, 1977; Inui and Ishioka, 1983; Foster and Moon, 1989; Foster et al., 1989). Although effects on protein synthesis, glucagon metabolism, and gluconeogenesis were observed, these studies were carried out using insulin and glucagon from heterologous species and so the physiological significance of the data is unclear. A recent study has shown that eel glucagon shows unusual structural features not found in other piscine or mammalian glucagons (Conlon et al., 1988). The aim of the present study was to purify and characterize insulin and GLP from the pancreas of the American eel, Anguilla rostrata. The availability of the eel peptides will enable the establishment of homologous radioimmunoassays and an assessment of their role in metabolic regulation in the eel. MATERIALS
AND METHODS
Approximately 1000 immature (3-t years old, 35-60 cm long) specimens of American eel were collected at the Saunders Hydroelectric Dam, Cornwall, Ontario, on the St. Lawrence River. Examination of the stomach contents indicated that the eels had been feeding prior to capture. Pancreatic tissue (43.8 g) was frozen immediately after dissection and subsequently extracted at 0” with ethanol/O.7 M HCl (3/l ; v/v) (400 ml) using a Waring blender. The homogenate was stirred for 3 hr at 0” and centrifuged (16OOg Extraction
method.
ET
AL.
for 1 hr at 4”). Peptides were isolated from the supernatant using Sep-Pak C-18 cartridges (Waters Associates, Milford, MA) as previously described (Conlon et al., 1988). Extraction of pancreatic tissue (9.6 g) from specimens of adult European eel and isolation of the peptides on Sep-Pak cartridges has been described previously (Cordon et al., 1988). Purification of peptides from A. rostrata. The elBuent from the Sep-Pak cartridges was lyophilized and redissolved in 0.1% v/v trifluoroacetic acid (20 ml). The solution (20% of the total extract) was injected onto a Supelcosil LC-II-DB column (250 X 10 mm; Supelco Inc., Bellefonte, PA) equilibrated with 0.1% trifluoroacetic acid at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 5 min followed by a raise to 49% (v/v) over 75 min using linear gradients. Absorbance was measured at 214 and 280 nm and individual peaks were collected by hand. The peak denoted by the bar (see Fig. 1), which contained insulin and GLP, was rechromatographed on a Vydac 214TP54 C-4 column (250 x 4.6 mm; The Separations Group, Hesperia, CA) equilibrated with acetonitrile/water/ trifluoroacetic acid (2 1.0/78.9/O. 1) at a flow rate of 1.5 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 38.5% (v/v) over 40 min using a linear gradient. Purification of peptides from A. anguilla. The pancreatic extract from the European eel, after partial purification on Sep-Pak cartridges, was chromatographed on the semipreparative Supelcosil LC-18-DB column as previously described (Conlon ef al., 1988). The peak denoted by I (see Fig. 3), which contained insulin, was rechromatographed on a Vydac 218TP54 (C-18) column (250 x 4.6 mm) equilibrated with acetonitrile/water/trifluoracetic acid (21.0/78.9/O. 1) at a flow rate of 1.5 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 35% (v/v) over 40 min using a linear gradient. The peak denoted by II, which contained GLP, was rechromatographed on the Vydac 218TP54 column under the same conditions. Insulins from A. rostrata (approximately 4 nmol) and A. an&la (approximately 10 nmol) were reduced (dithiothreitol) and pyridylethylated (4-vinylpryridine) according to the method of Cordon et al. (1989). The derivatized A-chains and B-chains were separated by reversed-phase HPLC under the conditions shown in Fig. 1. Peptide characterization. Amino acid compositions were determined using approximately 1 nmol peptide by precolumn derivatization with phenylisothiocyanate as previously described (Conlon et al., 1990). The primary structures of the A-chain and B-chain of insulin and GLP from the American eel were determined by automated Edman degradation at Creighton University using an Applied Biosystems model 471A sequenator modified for on-line detection of phenyl-
STRUCTURES
OF
EEL
thiohydantoin (PTH) amino acids under gradient elution conditions. The primary structures of the A-chain and B-chain of insulin and GLP from the European eel were determined at the Novo Nordisk Research Institute using an Applied Biosystems model 470A sequenator. In both cases, the manufacturers standard operating procedures were used and the detection limits for PTH-amino acids was 0.5 pmol. Positive ion fast-atom bombardment mass spectrometry was carried out as described by Andrews and Dixon (1987) using a Kratos MS-50 mass spectrometer. GLP from the European eel (500 pmol) was digested with (a) trypsin and (b) endopeptidase Glu-C from Staphy/ococcus aureus V8 at an enzyme to substrate ratio of 1:20. The digests were applied directly to the probe tip without isolation of peptide fragments. The accuracy of mass determinations was 20.5 Da. Califomium-252 plasma desorption time-of-flight mass spectrometry was carried out on intact GLP from the European eel using a BIO-ION Nordic BIN-2OK instrument as described previously (Andrews et al., 1988). The accuracy of mass determinations was 20.1%. Calibration spectra using human neuropeptide Y and somatostatin-14 were run at the same time.
RESULTS
Purification of insulin and GLP from A. rostrata. The elution profile on a semipreparative C- 18 reversed-phase HPLC column of the pancreatic extract from the American eel is shown in Fig. 1. The retention time of the peak denoted by the bar was comparable to that of porcine insulin and so this was selected for further purifi-
PANCREATIC
25
HORMONES
I
40
33
20 I‘ c 10
lo
20
30
-I 40
TMEW
2. Reversed-phase HPLC on a Vydac 214TP54 column of a mixture of insulin and GLP from A. rostrata after partial purification under the conditions shown in Fig. 1. The dashed line shows the concentration of acetonitrile in the eluting solvent. FIG.
cation on a Vydac C-4 wide-pore column. Under the conditions of chromatography shown in Fig. 2, the peak was resolved into two major components which were subsequently identified as insulin and GLP. The approximate final yields of the pure peptides (starting from 20% of the original extract) were 18 nmol insulin and 26 nmol GLP. Purification of insulin and GLP from A. anguilla. As shown in Fig. 3, reversedphase HPLC of the pancreatic extract from the European eel, under the same elution conditions used for chromatography of the 2.0
$l
FIG. 1. Reversed-phase HPLC on a Supelcosil LC18DB column of an extract of pancreas from A. rostrata after partial purification on Sep-Pak cartridges. Details of the elution conditions are given in the text. The peak denoted by the bar contained a mixture of insulin and GLP and was purified further. The dashed line shows the concentration of acetonitrile in the eluting solvent.
0
FIG. 3. Reversed-phase HPLC on a Supelcosil LC18-DB column of an extract of pancreas from A. anguilla after partial purification on Sep-Pak cartridges. Full details of the chromatographic procedure are given in Conlon et al. (1988). Peak I contains insulin and peak II contains GLP.
CONLON
26
American eel extract, resulted in partial resolution of insulin (peak I) from GLP (peak II). The peptides were identified by a determination of their amino acid compositions. The peptides in peaks I and II were purified to apparent homogeneity on an analytical Vydac C-18 column (chromatograms not shown). The approximate final yields of pure peptides were 16 nmol insulin and 3 nmol GLP. Structural characterization of eel insulins. The amino acid compositions of Achains and B-chains of insulins from A. rostrata and A. anguilla are shown in Table 1.
The data indicate that the A-chain from the American eel differs from that from the European eel by the single substitution of a Ser residue by an Asx residue. The compositions of the B-chains, however, are appreciably different and indicate at least four amino acid substitutions between the species. It was possible to determine the complete primary structures of the A-chain and B-chain of insulin from A. rostrata by au-
ET AL.
tomated Edman degradation (Table 2). The A-chain comprised 21 amino acid residues and the B-chain 30 amino acid residues. PTH-derivatives were unambiguously identified at each cycle of degradation and the good agreement between the sequence analysis data and the amino acid compositions demonstrated that the full sequences of the peptides had been obtained. The complete primary structure of the Achain of insulin from A. anguilla was obtained by Edman degradation (Table 2). Consistent with the amino acid composition data, the structure of the A-chain from the European eel differed from that from the American eel by the substitution of a serine residue by an asparagine residue at position 12. It was not possible to obtain the amino acid sequence of the pyridylethylated Bchain of insulin from A. anguilla by Edman degradation. On two occasions approximately 4 nmol of peptide was loaded onto the sequencer disc but only very low yields of PTH amino acids were detected. It is not
TABLE 1 AMINO ACID COMPOSITIONSOF THE A-CHAIN AND B-CHAIN OF INSULINS FROM A. rostrata
AND A. anguilla
Amino acid composition (mol of residue/mol) Amino acid Asx Glx Ser GUY His Arg Thr Ala Pro ‘W Val Met Be Leu Phe LYS
Insulin A-chain A. rostrata
Insulin B-Chain A. anguilla
3.0 (3) 2.7 (3)
4.0 (4) 3.3 (3)
1.0 (1) 1.3 (1) 0.8 (1)
1.3 (3) 0.9 (1)
0.9 (I) 0.8 (1) 0.4 (1)
0.7 0.9 0.4
1.4 (2)
1.4 0.7
1.0 (1) 0.8 (1) 1.3 (1)
(1) (1) (1)
(1) (1) 1.0 (1) 1.1 (1)
A. rostrata 3.3 2.1 3.1 3.2 1.9
A. anguilla
(3) (2) (3) (3) (2)
3.0 1.2 2.0 4.0 1.9
1.0 (1)
1.9
2.2 (2)
1.0 1.5
1.1 (1) 0.8 (1) 1.6 (2)
0.9 1.9
3.6 (4) 2.8 (3) 1.3 (1)
3.2 2.8 2.0
Note. Determinations were carried out in duplicate and the values in parentheses represent the number of residues expected from the proposed sequences.
STRUCTURES
AIJTOMATEDEDMAN
DEGRADATION
OF
OFTHE a-CHAIN
EEL
PANCREATIC
TABLE 2 A-CHAIN ANDB-CHAIN OF INSULIN
FROM
27
HORMONES
OF INSULIN
FROM
A.rostrata
Insulin A-chain
Insulin B-chain
A. rostrata Cycle
No.
Amino acid
I
GlY
2 3
Ile Val
4
au
5
Gin PE-Cys PE-Cys His
6 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Note.
LYS
Pro PE-Cys Ser Ile Phe Asp Leu Gln Asn W
PE-Cys Asn
A. anguilla
A. rostrata
Yield (pmol)
Amino acid
Yield (pm4
Amino acid
1273 1124 1093
GlY Ile Val GlU Gln PE-Cys PE-Cys His Lys Pro PE-Cys Asn Be Phe Asp Leu Gin Asn
6800 7100 6400 5100
Ala Ser Thr Gin His Leu PE-Cys QY
697 775 683
715 482 969 617 538 107
616 594 242
505 447 294 493 313 203
4600 4400 3700
1400 5400 3600 2900 3000 3600 3700 1800 3200 2500
2locl
‘Or
2400
PE-Cys Asn
1400 900
Ser
His Leu Val Glu
Ala Leu Vr
Leu Val PE-Cys GUY Ser Asn GUY
Phe Phe Phe Asn Pro Lys Asp PE-Cys refers to the vinylpyridine
ANDTHE
A.anguih
Yield
(pmol) 1577 413 213 977 679 1099 889 856 230 545 734 674 525 687 589 588 578
518 400 367
9s 321 271 298 390 409 252 180
181 110
derivative of cysteine.
clear whether this was the result of sequencer malfunction or a consequence of the fact that the a-amino group of the NH,terminal residue was blocked. An insulin with a derivatized NH,-terminal residue has not been described but such blockages can arise artifactually during the extraction and purification procedure. Structural characterization of eel GLP. The amino acid composition of GLP from the American eel was identical to that of GLP from the European eel (Table 3). The
data indicated a peptide of 30 amino acid residues. Identities of the two peptides were confirmed by automated Edman degradations carried out in two different laboratories (Table 4). The primary structure of GLP from both species was established as a 30 amino acid-residue peptide and no PTH derivative was detected during cycle 31 of the Edman degradation. Agreement between the sequence analysis and the amino acid composition data was good. The primary structure of GLP from the European
28
CONLON TABLE
AMINO ACID COMPOSITIONS PEFTIDE FROM A. rostrum Amino
ET AL.
3
TABLE
OF GLUCAGON-LIKE AND A. anguilla
acid
Asx Glx Ser GlY His Ax Thr Ala ‘W Val Leu Phe LYS
A. rostrata
2.2 (2) 4.1 (4) 4.1 (4) 2.2 (2) 1.1 (I) 1.3 (I) 2.7 (3) 3.1 (3) 1.I (2) 2.0 (2) 2.0 (2) 1.0 (1) 2.1 (2)
OF
acid composition
(mol of residue/mol) Amino
4
AUTOMATED EDMAN DEGRADATION GLUCAGON-LIKE PEPTIDE FROM A. rostrata AND A. anguilla A. rostrata
A. anguilla
2.1 (2) 4.3 (4) 3.9 (4) 2.0 (2) 1.1 (1) 1.0 (1) 2.8 (3) 3.0 (3) 1.8 (2) 1.9 (2) 2.2 (2) 1.0 (1) 2.3 (2)
Note. Figures in parentheses represent the number of residues expected from the proposed sequences.
eel was confirmed by mass spectrometry and it was demonstrated that the COOHterminal arginine residue was a-amidated. When a tryptic digest of eel GLP was subjected to fast-atom bombardment mass spectrometry, prominent molecular ions (MH+) were detected at masses 907.7 and 2170.7 Da. These molecular ions correspond to the predicted monoisotopic masses (M+) of GLP-(21-27)-peptide (907.5) and GLP-(1-20)-peptide (2170). A molecular ion corresponding to the (28-30) fragment was not identified in the spectrum. Following digestion of GLP with endoproteinase Glu-C, MH+ at 1962.8 and 1092.6 were observed. The masses of these ions are in close agreement with the calculated Mf of 1961.9 for GLP-(4-21)-peptide and 1091.6 for the amidated form of GLP(22-30)-peptide. The experimental mass of intact eel GLP, determined by plasma desorption time-of-flight mass spectrometry, was 3377.2 k 3.4 Da compared with a calculated average mass of 3374.6 for the amidated form of the peptide. Thus, agreement between the sequence analysis data and the mass spectrometry data is good.
Amino Cycle
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26 21 28 29 30
acid His Ala Glu GUY Thr ‘Or Thr Ser Asp Val Ser Ser Tyr Leu Gln Asp Gin Ala Ala LYS Glu Phe Val Ser Trp Leu LYS Thr GUY Arg
A. anguilla
Yield (PmoU 731 1447 911 881 266 900
226 150 291 559 104
120 362 330 278 172 262 326 403 262 214 216 223 36 26 126 112 20 86 65
acid
Yield (pmol)
His Ala Glu GUY Thr Tyr Thr Ser Asp Val Ser Ser ‘M Leu Gln Asp Gin Ala Ala LYS Glu Phe Val Ser Trp Leu LYS Thr GUY Ax
249 1168 756 922 382 871 328 331 381 606 250 257 328 365 202 349 218 210 265 194 139 162 135 58 18 211 144 59 62 64
Amino
DISCUSSION
The primary structure of insulin from the American eel is compared with that of insulin from another teleost (tuna) and from a mammal (human) in Table 5. Determination of the 3-dimensional structure of porcine insulin by X-ray crystallography has demonstrated that residues (2-g) and (13-19) in the A-chain and residues (9-19) in the B-chain from stable a-helices. These regions have been quite well conserved in eel insulin. In particular, the three cystine bridges, the glycine residues at B-8 and B-23, and the
STRUCTURES
OF
EEL
PANCREATIC
TABLE A
29
HORMONES
5
COMPARISONOFTHEPRIMARY STRUCTUREOF INSULINS FROMTHEAMERICAN EEL,TUNA,AND
HUMAN
A-Chain Eel Tuna Human
5 10 GIVEQCCHKPCSIFDLQNYCN ----------N------TSI--LYQ-E----
15
20
--------
B-Chain Eel Tuna Human
5 ASTQHLgCGSHLV VAPP-e--p---FVN-m-e-----
10
hydrophobic core residues at A-2, A-16, B12, B-15, and B-24, which are important in determining the conformation of the insulin monomer (Dafgard et al., 1985), have been retained. Comparative studies of the binding of insulins from a variety of species have identified the B-21-B-26 region of insulin as important in interacting with its specific cell-surface receptor. With the exception of the hystricomorph rodents, this region has been well conserved during evolution. Eel insulin, however, contains a serine residue rather than an acidic residue at position B-21, an asparagine for an arginine at position 22 and a phenylalanine for a tryosine at position 26. Amino acid substitutions in this region of insulin in the guinea pig and other hystricomorphs lead to reduced binding affinity and biological potency. Studies are now underway to determine the effects of the purified eel insulin on eel hepatocytes. Amino acid residues involved in the formation of the insulin dimer are B-12, B-16, B-20, B-24, B-26, and B-28 (Dafgard et al., 1985). The substitution of tyrosine by phenylalanine at B-26 may impair the ability of eel insulin to dimerize but the other residues involved have been conserved. Similarly, the histidine residue at B-10, which is important in determining the ability of insulin to aggregate further into zinc-containing hexamers, is present in eel insulin. Hagfish insulin lacks this histidine residue and does
15 EALYLVCGSNGF
20
25 F
30 FNPKD
--D-------DR---y-p --ER---y---A
not form hexamers but several teleost insulins have been shown to form hexamers and zinc is present in high concentrations in teleost Brockmann bodies (CutIield et al., 1986). Residues B-14 (Ala), B-17 (Leu) and B-18 (Val) are also involved in hexamer formation and have been conserved in eel insulin. Despite the fact that the NH,terminal region of the B-chain may be involved in hexamer formation, evolutionary pressure to conserve this region of insulin has been weak and the B-chain of eel insulin contains a novel NH,-terminal sequence. An unexpected result from this study is the demonstration that the primary structure of insulin from the American eel, A. rostrata is different from that of insulin from the European eel, A. anguilla. In order to reproduce, the European eel migrates to a definite zone of the Sargasso Sea that partially overlaps the breeding area of the American eel (Schmidt, 1923). It has been proposed that the two Atlantic eel populations originate from the same gene pool and are not distinct species by merely ecophenotypes (Tucker, 1959). Later work, however, in which the electrophoretic mobilities of a variety of enzymes from specimens of both populations were measured, clearly demonstrated that A. anguilla and A. rostrata are distinct species (Comparini and Rodino, 1980). Although further work is necessary to determine the amino acid
30
CONLON
sequence of the B-chain of insulin from the European eel, the partial characterization presented in this study indicates that the structures of the two insulins are appreciably different and suggests that the divergence into different species in not a recent event. In this context, it is interesting to note that an antibody raised against rat atria1 natiuretic factor detects immunoreactive material in the heart of A. anguilla but not A. juponica (U. O’Sullivan, unpublished data). In contrast to insulin, the primary structure of GLP has been conserved between the American and European eels. GLP is a product of the posttranslational processing of proglucagon and has been isolated and characterized from several species of fish: anglerfish, Lophius americanus (Andrews et al., 1986; Nichols et al., 1988), catfish, Ictalurus punctata (Andrews and Ronner, 1985), coho salmon, Oncorhynchus kisutch (Plisetskaya et al., 1986), daddy sculpin, Cottus scorpius (Conlon et al., 1987), alligator gar, Lepisosteus spatula (Pollock et al., 1988), and Pacific ratfish, Hydrolagus colliei (Conlon et al., 1989). Little is known about structure-activity relationships in the GLP molecule. Consequently, a comparison of the sequences of teleost GLPs permits identification of conserved domains and so provides some insight into which regions of the molecule may be important for receptor binding and biological activity in fish. Table 6 indicates that evolutionary pressure has acted more strongly to conTABLE A COMPARISON
OF THE
AL.
serve the NH,-terminal and central regions of the molecule than the COOH-terminal region. Preliminary data (B. Gallwitz, W. E. Schmidt, J. M. Conlon, and W. Creutzfeldt, unpublished data) indicate that the COOH-terminal region of human GLP1(7-36)-amide is the domain responsible for binding to its receptor on insulinoma cells whereas the NH,-terminal region may be responsible for activation of adenylate cyclase. The lack of conservation of amino acid sequence in the COOH-terminal region of the fish GLPs again emphasizes the necessity of evaluating biological activity in a homologous species. Fish GLPs do not appear to employ CAMP or cGMP as the intracellular messenger in hepatic tissue (Mommsen et al., 1987) and their mechanism of action is unknown. The COOH-terminal arginine residue of GLP from the European eel was shown to be a-amidated by mass spectrometry. Anglerfish GLP was isolated in three biosynthetically related molecular forms which terminate in (a) -Arg3’-Gly-Arg-Arg-Glu, (b) Arg3’-Gly, and (c) Arg3’-NH, (Andrews et al., 1986). It was concluded that the three forms arose from alternative pathways of posttranslational processing of proglucagon II. Only one molecular form of GLP was isolated in this study but the existence of additional COOH-terminally extended forms is not excluded. In particular, the yield of GLP from the European eel was much less than the yield of GLP from the American eel compared with the corre6
SEQUENCES OF GLUCAGON-LIKE CATFISH, SALMON AND 5
Eel AF-I AF-II Cattish Salmon Sculpin
ET
10
15
HAEGTYTSDVSSYLQDQAAKEFVSWLKTGR.NHz --D--F--------K---I-D--A-QV
--D-----------we-----D---W-e.,Av..mGRRE --D-----------------D-IT---s-QpKpE
------------D--F--------N---I-D--AK--S-KV
T--------D--.----S--A
PEPTIDES SCULPIN
FROM THE
20
EEL,
25
ANGLERFISH,
30
STRUCTURES
OF
EEL
sponding yields of insulin. This difference between the species may reflect a difference in the levels of expression of the proglucagon and proinsulin genes or it may reflect the fact that the amidated form of GLP represents only a minor component in A. anguilla but the major component in A. rostrata. ACKNOWLEDGMENTS
REFERENCES Andrews, P. C., and Dixon, J. E. (1987). Isolation of products and intermediates of pancreatic prosomatostatin processing: Use of fast atom bombardment mass spectrometry as an aid in analysis of prohormone processing. Biochemistry 26, 48534861. Andrews, P. C., and Ronner, P. (1985). Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas. J. Biol. Chem. 260, 39103914. Andrews, P. C., Alai, M., and Cotter, R. J. (1988). The use of plasma desorption time-of-flight mass spectrometry to screen for products of prohormone processing in crude tissue extracts. Ann/. Biochem. 174, 23-31. Andrews, P. C., Hawke, D. H., Lee, T. D., Legesse, K., Noe, B. D., and Shively, J. E. (1986). Isolation and structure of the principal products of preproglucagon processing, including an amidated glucagon-like peptide. J. Biol. Chem. 261, 8128 8133. Comparini, A., and Rodino, E. (1980). Electrophoretic evidence for two species of Anguilla leptocephali in the Sargasso Sea. Nature (London) 287, 435 437. Conlon, J. M., Falkmer, S., and Thim, L. (1987). Primary structures of three fragments of proglucagon from the pancreatic islets of the daddy sculpin scorpius).
Eur.
J. Biochem.
164,
117-122.
Conlon, J. M., Deacon, C. F., Hazon, N., Henderson, I. W., and Thim, L. (1988). Somatostatin-
31
HORMONES
related and glucagon-related peptides with unusual structural features from the European eel (An&la
anguilla).
Gen.
Comp.
Endocrinol.
72,
181-189. Conlon, J. M., Goke, R., and Thim, L. (1989). Multiple molecular forms of insulin and glucagon-like peptide from the Pacific ratfish (Hydrolagus colliei). Gen. Comp. Endocrinol. 73, 136-146. Conlon, J. M., Hicks, J. W., and Smith, D. D. (1990). Isolation and biological activity of a novel kinin ([Thr6]bradykinin) from the turtle, Pseudemys scripta.
This work was supported by the Natural Sciences and Engineering Council of Canada and by the Ontario Ministry of Natural Resources. Dr. P. C. Andrews was supported in part by a research and development award from the American Diabetes Association. The authors wish to thank Mr. Cl. Foster, University of Ottawa, Canada, and Dr. N. Hazon, University of St. Andrews, U.K., for help in tissue collection.
(Cottus
PANCREATIC
Endocrinology
126, 985-991.
Cutfield, J. F., Cutfield, S. M., Carne, A., Emdin. S. 0.. and Falkmer, S. (1986). The isolation, purification and amino-acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin). Eur. J. Biochem. 158, 117-123. Dafgard, E., Bajaj, M., Honegger, A. M., Pitts, J., Wood, S., and Blundell, T. (1985). The conformation of insulin-like growth factors: Relationship with insulins. J. Cell Sci. 3 (SuppI.), 53-64. Foster, G. D., and Moon, T. W. (1989). Insulin and athe regulation of glycogen metabolism and gluconeogenesis in American eel hepatocytes. Gen. Comp. Endocrinol. 73, 37&381. Foster, Cl. D., Storey, K. B., and Moon, T. W. (1989). The regulation of 6-phosphofructo1-kinase by insulin and glucagon in isolated hepatocytes of the American eel. Gen. Comp. Endocrinol.
73, 382-289.
Ince, B. W., and Thorpe, A. (1977). Plasma insulin and glucose responses to glucagon and catecholamines in the European silver eel. (Anguilla anguillu L.) Gen. Comp. Endocrinol. 33, 453459. Invi, Y., and Ishioka, H. (1983) Effects of insulin and glucagon on the incorporation of [‘?Z]-glycine into the protein of the liver and opercular muscle of the eel in vitro Gen. Comp. Endocrinol. 51, 208-212.
Mojsov, S., Weir, G. C., and Habener, J. F. (1987) Insulinotropin: glucagon-like peptide 1 (7-37) coencoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J. Clin. Invest. 79, 616-619. Mommsen, T. P., Andrews, P. C., and Plisetskaya, E. M. (1987). Glucagon-like peptides activate hepatic gluconeogenesis. FEES Lett. 219, 227-232. Mommsen, T. P., and Moon, T. W. (1989). Metabolic actions of glucagon-family hormones in liver. Fish Physiol.
Biochem.
7, 279-288.
Nichols, R., Lee, T. D., and Andrews, P. C. (1988). Pancreatic proglucagon processing: Isolation and structures of glucagon and glucagon-like peptide from gene I. Endocrinology 123, 263!L2645.
32
CONLON
Plisetskaya, E. M., Ottolenghi, C., Sheridan, M. A., Mommsen, T. P., and Gorbman, A. (1989). Metabolic effects of salmon glucagon and glucagonlike peptide in coho and chinook salmon. Gen. Comp.
Endocrinol.
13, 205-216.
Plisetskaya, E., Pollock, H. G., Rouse, J. B., Hamilton, J. W., Kimmel, J. R., and Gorbman, B. (1985). Characterization of coho salmon (Oncorhynchus kisutch) insulin. Regul. Pepr. 11, 10% 116. Plisetskaya, E., Pollock, H. G., Rouse, J. B., Hamilton, J. W., Kimmel, J. R., and Gorbman, A. (1986). Isolation and structures of coho salmon
ET AL. (Oncorhynchus kisutch) glucagon and glucagonlike peptide. Regul. Pept. 14, 51-67. Pollock, H. G., Kimmel, J. R., Ebner, K. E., Hamilton, J. W., Rouse, J. B., Lance, V., and Rawitch, A. B. (1988). Isolation of alligator gar (Lepisosteus spatula) glucagon, oxyntomodulin, and glucagon-like peptide: Amino acid sequences of oxyntomodulin and glucagon-like peptide. Gen. Comp. Endocrinol. 69, 133-140. Schmidt, J. (1923). Breeding places and migrations of the eels. Nature (London) 111, 51-54. Tucker, D. W. (1959). A new solution to the Atlantic eel problem. Nature (London) 183, 495-501.
AND
COMPARATIVE
ENDOCRINOLOGY
82, 23-32 (1991)
The Primary Structure of Glucagon-like Peptide but Not Insulin Has Been Conserved between the American Eel, Anguilla rostrata and the European Eel, Anguilla anguilla J. MICHAEL CONLON,* P.C. ANDREWS,‘F LARSTHIM,S
AND THOMAS W. MOON§
*Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School, Omaha, Nebraska 68178; fDepartment of Biochemistry, Purdue University, West Lafayette, Indiana 47907; $Novo Nordisk Research Institute, Bagsvaerd, Denmark; and $Department of Biology, University of Ottawa, Ottawa, Ontario KlN 6N5, Canada
Accepted May II, 1990 Insulin was isolated from the pancreas of the American eel, Anguilla primary structure was established as A-Chain 5 10 GIVEQCCHKPCSIFDLQNYCN B-Chain 5 IO 15 ASTQHLCGSHLVEALYLVCGSNGFFFNPKD
15
20
rostrata,
and its
20
25
30
Eel insulin contains unusual substitutions at B-21, B-22, and B-26 in the putative receptorbinding region of the molecule compared with other mammalian and fish insulins. The A-chain of insulin from the European eel contains an asparagine rather than a serine residue at position A-12. Similarly, amino acid composition data indicate the B-chain of insulin from the European eel is appreciably different from that from the American eel. The primary structure of glucagon-like peptide (GLP) from the American eel is identical to that from the European eel, Anguilla anguilla. The primary structure of the peptide was established as 5 10 15 20 25 30 HAEGTYTSDVSSYLQDQAAKEFVSWLKTGR Fast-atom bombardment mass spectrometry demonstrated that the COOH-terminal arginyl residue is a-amidated. The strong evolutionary pressure to conserve the structure of GLP provides further support for the assertion that the peptide plays an important regulatory role in teleost fish. 0 1991 Academic Press. Inc.
The physiological role of insulin and glucagon in lower vertebrates is incompletely understood. Numerous studies have described the effects of pancreatic hormones on intermediary metabolism and growth in teleost fish but, until recently, all studies were carried out using either mammalian peptides or impure preparations of fish pancreatic extracts. The isolation in pure form of relatively large amounts of insulin (Plisetskaya er al., 1985), glucagon and gluca-
gon-like peptide (GLP) (Plisetskaya et al., 1986) from the coho salmon, Oncorhyncus kisutch, has enabled biological testing in the species of origin. Salmon insulin was shown to exhibit a more pronounced hypoglycemic effect than bovine insulin when injected into juvenile coho salmon (Plisetskaya et al., 1985). Salmon glucagon was more potent than mammalian glucagon and salmon GLP in stimulating glycogenolysis both in vivo and using liver slices from ju23 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved
24
CONLON
venile salmon (Plisetskaya et al., 1989). In contrast, salmon GLP is a more potent stimulator of gluconeogenesis than salmon glucagon in isolated salmon hepatocyte (Mommsen et al., 1987). Related studies in viva using juvenile salmon have shown that the effects of salmon GLP are markedly seasonal but effects upon glycolytic, lipolytic, and gluconeogenic metabolic pathways were observed (Mommsen and Moon, 1989; Plisetskaya er al., 1989). Human GLP-1 (7-37) is a potent stimulant of insulin release in mammals (Mojsov et al., 1987) but salmon GLP had little or no effect upon plasma insulin levels in salmon. Several studies have investigated the effects of pancreatic hormones on metabolic parameters in the eel (Ince and Thorpe, 1977; Inui and Ishioka, 1983; Foster and Moon, 1989; Foster et al., 1989). Although effects on protein synthesis, glucagon metabolism, and gluconeogenesis were observed, these studies were carried out using insulin and glucagon from heterologous species and so the physiological significance of the data is unclear. A recent study has shown that eel glucagon shows unusual structural features not found in other piscine or mammalian glucagons (Conlon et al., 1988). The aim of the present study was to purify and characterize insulin and GLP from the pancreas of the American eel, Anguilla rostrata. The availability of the eel peptides will enable the establishment of homologous radioimmunoassays and an assessment of their role in metabolic regulation in the eel. MATERIALS
AND METHODS
Approximately 1000 immature (3-t years old, 35-60 cm long) specimens of American eel were collected at the Saunders Hydroelectric Dam, Cornwall, Ontario, on the St. Lawrence River. Examination of the stomach contents indicated that the eels had been feeding prior to capture. Pancreatic tissue (43.8 g) was frozen immediately after dissection and subsequently extracted at 0” with ethanol/O.7 M HCl (3/l ; v/v) (400 ml) using a Waring blender. The homogenate was stirred for 3 hr at 0” and centrifuged (16OOg Extraction
method.
ET
AL.
for 1 hr at 4”). Peptides were isolated from the supernatant using Sep-Pak C-18 cartridges (Waters Associates, Milford, MA) as previously described (Conlon et al., 1988). Extraction of pancreatic tissue (9.6 g) from specimens of adult European eel and isolation of the peptides on Sep-Pak cartridges has been described previously (Cordon et al., 1988). Purification of peptides from A. rostrata. The elBuent from the Sep-Pak cartridges was lyophilized and redissolved in 0.1% v/v trifluoroacetic acid (20 ml). The solution (20% of the total extract) was injected onto a Supelcosil LC-II-DB column (250 X 10 mm; Supelco Inc., Bellefonte, PA) equilibrated with 0.1% trifluoroacetic acid at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 5 min followed by a raise to 49% (v/v) over 75 min using linear gradients. Absorbance was measured at 214 and 280 nm and individual peaks were collected by hand. The peak denoted by the bar (see Fig. 1), which contained insulin and GLP, was rechromatographed on a Vydac 214TP54 C-4 column (250 x 4.6 mm; The Separations Group, Hesperia, CA) equilibrated with acetonitrile/water/ trifluoroacetic acid (2 1.0/78.9/O. 1) at a flow rate of 1.5 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 38.5% (v/v) over 40 min using a linear gradient. Purification of peptides from A. anguilla. The pancreatic extract from the European eel, after partial purification on Sep-Pak cartridges, was chromatographed on the semipreparative Supelcosil LC-18-DB column as previously described (Conlon ef al., 1988). The peak denoted by I (see Fig. 3), which contained insulin, was rechromatographed on a Vydac 218TP54 (C-18) column (250 x 4.6 mm) equilibrated with acetonitrile/water/trifluoracetic acid (21.0/78.9/O. 1) at a flow rate of 1.5 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 35% (v/v) over 40 min using a linear gradient. The peak denoted by II, which contained GLP, was rechromatographed on the Vydac 218TP54 column under the same conditions. Insulins from A. rostrata (approximately 4 nmol) and A. an&la (approximately 10 nmol) were reduced (dithiothreitol) and pyridylethylated (4-vinylpryridine) according to the method of Cordon et al. (1989). The derivatized A-chains and B-chains were separated by reversed-phase HPLC under the conditions shown in Fig. 1. Peptide characterization. Amino acid compositions were determined using approximately 1 nmol peptide by precolumn derivatization with phenylisothiocyanate as previously described (Conlon et al., 1990). The primary structures of the A-chain and B-chain of insulin and GLP from the American eel were determined by automated Edman degradation at Creighton University using an Applied Biosystems model 471A sequenator modified for on-line detection of phenyl-
STRUCTURES
OF
EEL
thiohydantoin (PTH) amino acids under gradient elution conditions. The primary structures of the A-chain and B-chain of insulin and GLP from the European eel were determined at the Novo Nordisk Research Institute using an Applied Biosystems model 470A sequenator. In both cases, the manufacturers standard operating procedures were used and the detection limits for PTH-amino acids was 0.5 pmol. Positive ion fast-atom bombardment mass spectrometry was carried out as described by Andrews and Dixon (1987) using a Kratos MS-50 mass spectrometer. GLP from the European eel (500 pmol) was digested with (a) trypsin and (b) endopeptidase Glu-C from Staphy/ococcus aureus V8 at an enzyme to substrate ratio of 1:20. The digests were applied directly to the probe tip without isolation of peptide fragments. The accuracy of mass determinations was 20.5 Da. Califomium-252 plasma desorption time-of-flight mass spectrometry was carried out on intact GLP from the European eel using a BIO-ION Nordic BIN-2OK instrument as described previously (Andrews et al., 1988). The accuracy of mass determinations was 20.1%. Calibration spectra using human neuropeptide Y and somatostatin-14 were run at the same time.
RESULTS
Purification of insulin and GLP from A. rostrata. The elution profile on a semipreparative C- 18 reversed-phase HPLC column of the pancreatic extract from the American eel is shown in Fig. 1. The retention time of the peak denoted by the bar was comparable to that of porcine insulin and so this was selected for further purifi-
PANCREATIC
25
HORMONES
I
40
33
20 I‘ c 10
lo
20
30
-I 40
TMEW
2. Reversed-phase HPLC on a Vydac 214TP54 column of a mixture of insulin and GLP from A. rostrata after partial purification under the conditions shown in Fig. 1. The dashed line shows the concentration of acetonitrile in the eluting solvent. FIG.
cation on a Vydac C-4 wide-pore column. Under the conditions of chromatography shown in Fig. 2, the peak was resolved into two major components which were subsequently identified as insulin and GLP. The approximate final yields of the pure peptides (starting from 20% of the original extract) were 18 nmol insulin and 26 nmol GLP. Purification of insulin and GLP from A. anguilla. As shown in Fig. 3, reversedphase HPLC of the pancreatic extract from the European eel, under the same elution conditions used for chromatography of the 2.0
$l
FIG. 1. Reversed-phase HPLC on a Supelcosil LC18DB column of an extract of pancreas from A. rostrata after partial purification on Sep-Pak cartridges. Details of the elution conditions are given in the text. The peak denoted by the bar contained a mixture of insulin and GLP and was purified further. The dashed line shows the concentration of acetonitrile in the eluting solvent.
0
FIG. 3. Reversed-phase HPLC on a Supelcosil LC18-DB column of an extract of pancreas from A. anguilla after partial purification on Sep-Pak cartridges. Full details of the chromatographic procedure are given in Conlon et al. (1988). Peak I contains insulin and peak II contains GLP.
CONLON
26
American eel extract, resulted in partial resolution of insulin (peak I) from GLP (peak II). The peptides were identified by a determination of their amino acid compositions. The peptides in peaks I and II were purified to apparent homogeneity on an analytical Vydac C-18 column (chromatograms not shown). The approximate final yields of pure peptides were 16 nmol insulin and 3 nmol GLP. Structural characterization of eel insulins. The amino acid compositions of Achains and B-chains of insulins from A. rostrata and A. anguilla are shown in Table 1.
The data indicate that the A-chain from the American eel differs from that from the European eel by the single substitution of a Ser residue by an Asx residue. The compositions of the B-chains, however, are appreciably different and indicate at least four amino acid substitutions between the species. It was possible to determine the complete primary structures of the A-chain and B-chain of insulin from A. rostrata by au-
ET AL.
tomated Edman degradation (Table 2). The A-chain comprised 21 amino acid residues and the B-chain 30 amino acid residues. PTH-derivatives were unambiguously identified at each cycle of degradation and the good agreement between the sequence analysis data and the amino acid compositions demonstrated that the full sequences of the peptides had been obtained. The complete primary structure of the Achain of insulin from A. anguilla was obtained by Edman degradation (Table 2). Consistent with the amino acid composition data, the structure of the A-chain from the European eel differed from that from the American eel by the substitution of a serine residue by an asparagine residue at position 12. It was not possible to obtain the amino acid sequence of the pyridylethylated Bchain of insulin from A. anguilla by Edman degradation. On two occasions approximately 4 nmol of peptide was loaded onto the sequencer disc but only very low yields of PTH amino acids were detected. It is not
TABLE 1 AMINO ACID COMPOSITIONSOF THE A-CHAIN AND B-CHAIN OF INSULINS FROM A. rostrata
AND A. anguilla
Amino acid composition (mol of residue/mol) Amino acid Asx Glx Ser GUY His Arg Thr Ala Pro ‘W Val Met Be Leu Phe LYS
Insulin A-chain A. rostrata
Insulin B-Chain A. anguilla
3.0 (3) 2.7 (3)
4.0 (4) 3.3 (3)
1.0 (1) 1.3 (1) 0.8 (1)
1.3 (3) 0.9 (1)
0.9 (I) 0.8 (1) 0.4 (1)
0.7 0.9 0.4
1.4 (2)
1.4 0.7
1.0 (1) 0.8 (1) 1.3 (1)
(1) (1) (1)
(1) (1) 1.0 (1) 1.1 (1)
A. rostrata 3.3 2.1 3.1 3.2 1.9
A. anguilla
(3) (2) (3) (3) (2)
3.0 1.2 2.0 4.0 1.9
1.0 (1)
1.9
2.2 (2)
1.0 1.5
1.1 (1) 0.8 (1) 1.6 (2)
0.9 1.9
3.6 (4) 2.8 (3) 1.3 (1)
3.2 2.8 2.0
Note. Determinations were carried out in duplicate and the values in parentheses represent the number of residues expected from the proposed sequences.
STRUCTURES
AIJTOMATEDEDMAN
DEGRADATION
OF
OFTHE a-CHAIN
EEL
PANCREATIC
TABLE 2 A-CHAIN ANDB-CHAIN OF INSULIN
FROM
27
HORMONES
OF INSULIN
FROM
A.rostrata
Insulin A-chain
Insulin B-chain
A. rostrata Cycle
No.
Amino acid
I
GlY
2 3
Ile Val
4
au
5
Gin PE-Cys PE-Cys His
6 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Note.
LYS
Pro PE-Cys Ser Ile Phe Asp Leu Gln Asn W
PE-Cys Asn
A. anguilla
A. rostrata
Yield (pmol)
Amino acid
Yield (pm4
Amino acid
1273 1124 1093
GlY Ile Val GlU Gln PE-Cys PE-Cys His Lys Pro PE-Cys Asn Be Phe Asp Leu Gin Asn
6800 7100 6400 5100
Ala Ser Thr Gin His Leu PE-Cys QY
697 775 683
715 482 969 617 538 107
616 594 242
505 447 294 493 313 203
4600 4400 3700
1400 5400 3600 2900 3000 3600 3700 1800 3200 2500
2locl
‘Or
2400
PE-Cys Asn
1400 900
Ser
His Leu Val Glu
Ala Leu Vr
Leu Val PE-Cys GUY Ser Asn GUY
Phe Phe Phe Asn Pro Lys Asp PE-Cys refers to the vinylpyridine
ANDTHE
A.anguih
Yield
(pmol) 1577 413 213 977 679 1099 889 856 230 545 734 674 525 687 589 588 578
518 400 367
9s 321 271 298 390 409 252 180
181 110
derivative of cysteine.
clear whether this was the result of sequencer malfunction or a consequence of the fact that the a-amino group of the NH,terminal residue was blocked. An insulin with a derivatized NH,-terminal residue has not been described but such blockages can arise artifactually during the extraction and purification procedure. Structural characterization of eel GLP. The amino acid composition of GLP from the American eel was identical to that of GLP from the European eel (Table 3). The
data indicated a peptide of 30 amino acid residues. Identities of the two peptides were confirmed by automated Edman degradations carried out in two different laboratories (Table 4). The primary structure of GLP from both species was established as a 30 amino acid-residue peptide and no PTH derivative was detected during cycle 31 of the Edman degradation. Agreement between the sequence analysis and the amino acid composition data was good. The primary structure of GLP from the European
28
CONLON TABLE
AMINO ACID COMPOSITIONS PEFTIDE FROM A. rostrum Amino
ET AL.
3
TABLE
OF GLUCAGON-LIKE AND A. anguilla
acid
Asx Glx Ser GlY His Ax Thr Ala ‘W Val Leu Phe LYS
A. rostrata
2.2 (2) 4.1 (4) 4.1 (4) 2.2 (2) 1.1 (I) 1.3 (I) 2.7 (3) 3.1 (3) 1.I (2) 2.0 (2) 2.0 (2) 1.0 (1) 2.1 (2)
OF
acid composition
(mol of residue/mol) Amino
4
AUTOMATED EDMAN DEGRADATION GLUCAGON-LIKE PEPTIDE FROM A. rostrata AND A. anguilla A. rostrata
A. anguilla
2.1 (2) 4.3 (4) 3.9 (4) 2.0 (2) 1.1 (1) 1.0 (1) 2.8 (3) 3.0 (3) 1.8 (2) 1.9 (2) 2.2 (2) 1.0 (1) 2.3 (2)
Note. Figures in parentheses represent the number of residues expected from the proposed sequences.
eel was confirmed by mass spectrometry and it was demonstrated that the COOHterminal arginine residue was a-amidated. When a tryptic digest of eel GLP was subjected to fast-atom bombardment mass spectrometry, prominent molecular ions (MH+) were detected at masses 907.7 and 2170.7 Da. These molecular ions correspond to the predicted monoisotopic masses (M+) of GLP-(21-27)-peptide (907.5) and GLP-(1-20)-peptide (2170). A molecular ion corresponding to the (28-30) fragment was not identified in the spectrum. Following digestion of GLP with endoproteinase Glu-C, MH+ at 1962.8 and 1092.6 were observed. The masses of these ions are in close agreement with the calculated Mf of 1961.9 for GLP-(4-21)-peptide and 1091.6 for the amidated form of GLP(22-30)-peptide. The experimental mass of intact eel GLP, determined by plasma desorption time-of-flight mass spectrometry, was 3377.2 k 3.4 Da compared with a calculated average mass of 3374.6 for the amidated form of the peptide. Thus, agreement between the sequence analysis data and the mass spectrometry data is good.
Amino Cycle
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26 21 28 29 30
acid His Ala Glu GUY Thr ‘Or Thr Ser Asp Val Ser Ser Tyr Leu Gln Asp Gin Ala Ala LYS Glu Phe Val Ser Trp Leu LYS Thr GUY Arg
A. anguilla
Yield (PmoU 731 1447 911 881 266 900
226 150 291 559 104
120 362 330 278 172 262 326 403 262 214 216 223 36 26 126 112 20 86 65
acid
Yield (pmol)
His Ala Glu GUY Thr Tyr Thr Ser Asp Val Ser Ser ‘M Leu Gln Asp Gin Ala Ala LYS Glu Phe Val Ser Trp Leu LYS Thr GUY Ax
249 1168 756 922 382 871 328 331 381 606 250 257 328 365 202 349 218 210 265 194 139 162 135 58 18 211 144 59 62 64
Amino
DISCUSSION
The primary structure of insulin from the American eel is compared with that of insulin from another teleost (tuna) and from a mammal (human) in Table 5. Determination of the 3-dimensional structure of porcine insulin by X-ray crystallography has demonstrated that residues (2-g) and (13-19) in the A-chain and residues (9-19) in the B-chain from stable a-helices. These regions have been quite well conserved in eel insulin. In particular, the three cystine bridges, the glycine residues at B-8 and B-23, and the
STRUCTURES
OF
EEL
PANCREATIC
TABLE A
29
HORMONES
5
COMPARISONOFTHEPRIMARY STRUCTUREOF INSULINS FROMTHEAMERICAN EEL,TUNA,AND
HUMAN
A-Chain Eel Tuna Human
5 10 GIVEQCCHKPCSIFDLQNYCN ----------N------TSI--LYQ-E----
15
20
--------
B-Chain Eel Tuna Human
5 ASTQHLgCGSHLV VAPP-e--p---FVN-m-e-----
10
hydrophobic core residues at A-2, A-16, B12, B-15, and B-24, which are important in determining the conformation of the insulin monomer (Dafgard et al., 1985), have been retained. Comparative studies of the binding of insulins from a variety of species have identified the B-21-B-26 region of insulin as important in interacting with its specific cell-surface receptor. With the exception of the hystricomorph rodents, this region has been well conserved during evolution. Eel insulin, however, contains a serine residue rather than an acidic residue at position B-21, an asparagine for an arginine at position 22 and a phenylalanine for a tryosine at position 26. Amino acid substitutions in this region of insulin in the guinea pig and other hystricomorphs lead to reduced binding affinity and biological potency. Studies are now underway to determine the effects of the purified eel insulin on eel hepatocytes. Amino acid residues involved in the formation of the insulin dimer are B-12, B-16, B-20, B-24, B-26, and B-28 (Dafgard et al., 1985). The substitution of tyrosine by phenylalanine at B-26 may impair the ability of eel insulin to dimerize but the other residues involved have been conserved. Similarly, the histidine residue at B-10, which is important in determining the ability of insulin to aggregate further into zinc-containing hexamers, is present in eel insulin. Hagfish insulin lacks this histidine residue and does
15 EALYLVCGSNGF
20
25 F
30 FNPKD
--D-------DR---y-p --ER---y---A
not form hexamers but several teleost insulins have been shown to form hexamers and zinc is present in high concentrations in teleost Brockmann bodies (CutIield et al., 1986). Residues B-14 (Ala), B-17 (Leu) and B-18 (Val) are also involved in hexamer formation and have been conserved in eel insulin. Despite the fact that the NH,terminal region of the B-chain may be involved in hexamer formation, evolutionary pressure to conserve this region of insulin has been weak and the B-chain of eel insulin contains a novel NH,-terminal sequence. An unexpected result from this study is the demonstration that the primary structure of insulin from the American eel, A. rostrata is different from that of insulin from the European eel, A. anguilla. In order to reproduce, the European eel migrates to a definite zone of the Sargasso Sea that partially overlaps the breeding area of the American eel (Schmidt, 1923). It has been proposed that the two Atlantic eel populations originate from the same gene pool and are not distinct species by merely ecophenotypes (Tucker, 1959). Later work, however, in which the electrophoretic mobilities of a variety of enzymes from specimens of both populations were measured, clearly demonstrated that A. anguilla and A. rostrata are distinct species (Comparini and Rodino, 1980). Although further work is necessary to determine the amino acid
30
CONLON
sequence of the B-chain of insulin from the European eel, the partial characterization presented in this study indicates that the structures of the two insulins are appreciably different and suggests that the divergence into different species in not a recent event. In this context, it is interesting to note that an antibody raised against rat atria1 natiuretic factor detects immunoreactive material in the heart of A. anguilla but not A. juponica (U. O’Sullivan, unpublished data). In contrast to insulin, the primary structure of GLP has been conserved between the American and European eels. GLP is a product of the posttranslational processing of proglucagon and has been isolated and characterized from several species of fish: anglerfish, Lophius americanus (Andrews et al., 1986; Nichols et al., 1988), catfish, Ictalurus punctata (Andrews and Ronner, 1985), coho salmon, Oncorhynchus kisutch (Plisetskaya et al., 1986), daddy sculpin, Cottus scorpius (Conlon et al., 1987), alligator gar, Lepisosteus spatula (Pollock et al., 1988), and Pacific ratfish, Hydrolagus colliei (Conlon et al., 1989). Little is known about structure-activity relationships in the GLP molecule. Consequently, a comparison of the sequences of teleost GLPs permits identification of conserved domains and so provides some insight into which regions of the molecule may be important for receptor binding and biological activity in fish. Table 6 indicates that evolutionary pressure has acted more strongly to conTABLE A COMPARISON
OF THE
AL.
serve the NH,-terminal and central regions of the molecule than the COOH-terminal region. Preliminary data (B. Gallwitz, W. E. Schmidt, J. M. Conlon, and W. Creutzfeldt, unpublished data) indicate that the COOH-terminal region of human GLP1(7-36)-amide is the domain responsible for binding to its receptor on insulinoma cells whereas the NH,-terminal region may be responsible for activation of adenylate cyclase. The lack of conservation of amino acid sequence in the COOH-terminal region of the fish GLPs again emphasizes the necessity of evaluating biological activity in a homologous species. Fish GLPs do not appear to employ CAMP or cGMP as the intracellular messenger in hepatic tissue (Mommsen et al., 1987) and their mechanism of action is unknown. The COOH-terminal arginine residue of GLP from the European eel was shown to be a-amidated by mass spectrometry. Anglerfish GLP was isolated in three biosynthetically related molecular forms which terminate in (a) -Arg3’-Gly-Arg-Arg-Glu, (b) Arg3’-Gly, and (c) Arg3’-NH, (Andrews et al., 1986). It was concluded that the three forms arose from alternative pathways of posttranslational processing of proglucagon II. Only one molecular form of GLP was isolated in this study but the existence of additional COOH-terminally extended forms is not excluded. In particular, the yield of GLP from the European eel was much less than the yield of GLP from the American eel compared with the corre6
SEQUENCES OF GLUCAGON-LIKE CATFISH, SALMON AND 5
Eel AF-I AF-II Cattish Salmon Sculpin
ET
10
15
HAEGTYTSDVSSYLQDQAAKEFVSWLKTGR.NHz --D--F--------K---I-D--A-QV
--D-----------we-----D---W-e.,Av..mGRRE --D-----------------D-IT---s-QpKpE
------------D--F--------N---I-D--AK--S-KV
T--------D--.----S--A
PEPTIDES SCULPIN
FROM THE
20
EEL,
25
ANGLERFISH,
30
STRUCTURES
OF
EEL
sponding yields of insulin. This difference between the species may reflect a difference in the levels of expression of the proglucagon and proinsulin genes or it may reflect the fact that the amidated form of GLP represents only a minor component in A. anguilla but the major component in A. rostrata. ACKNOWLEDGMENTS
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