© 2000 by The Society for Integrative and Comparative Biology
Molecular Evolution of Insulin in Non-Mammalian Vertebrates1
1 Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School, Omaha, Nebraska 68178-0405
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The traditional view, based primarily on X-ray crystallographic data, is that the amino acid residues at positions B12, B16, B23-B26, A1-A5, A19 and A21 in the insulin molecule comprise the receptor-binding domain. More recently, however, it has been proposed that the conformation adopted by insulin in the crystal structure is an inactive one. The results of alanine-scanning mutagenesis studies suggest that GlyB23, PheB24, IleA2, ValA3, and TyrA19 interact directly with the receptor with LeuB6, GlyB8, LeuB11, GluB13 and PheB25, although not part of the binding epitope, being important in maintaining the receptor-binding conformation. A comparison of the primary structures of insulins from a wide range of non-mammalian vertebrates, from hagfish to birds, provides support for this revised view by demonstrating that strong evolutionary pressure has acted to conserve those amino acids postulated to be important in the biologically active conformation. In addition to the cysteine residues, the amino acids at B6, B8, B11, B23, B24, A2, A3, and A19 are invariant in all species yet studied with only conservative substitutions (Glu
Asp) at B13 and (Phe Tyr) at B25. In contrast, several insulins containing substitutions at positions B16, A5 and A21, sites of importance in maintaining the crystal structure conformation, have been identified. Although the amino acid sequences of insulin are not generally useful as molecular markers for inferring phylogenetic relationships between species, the presence of common structural features in insulins from closely related species may permit a valid inference. For example, the presence of an N-terminal pentapeptide extension to the B-chains of insulins isolated from both holarctic and southern hemisphere lampreys supports the monophyletic status of the Petromyzontiformes. |
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Elucidation of the three-dimensional crystal structure of pig insulin by X-ray analysis (Blundell et al., 1972
) provided the first insight into the conformation of the protein and the nature of the surface in the molecule that interacts with the insulin receptor. The A-chain of insulin comprises two 
-helical segments (A2–A-8 and A13–A19). The B-chain can adopt two distinct conformations. In the T-state, residues B9–B19 form an -helix, residues B20–B23 constitute a ß-turn and residues B24–B30 adopt an extended ß-strand conformation. In the R-state, residues B1–B19 form a contiguous -helical region (Baker et al., 1988). Pig insulin readily associates into stable dimers and, in the presence of Zn2+ ions, forms stable hexamers. The traditional view, based primarily on X-ray crystallographic data, is that the receptor-binding region of insulin is related to the dimer-forming surface and comprises a domain formed from B12, B16, B23–B26, A1–A5, A19 and A21. Residues B20 and B28 are also important in dimer formation and residues B6, B10, B14, B17, B18, A13, and A14 are involved in hexamer formation are (Baker et al., 1988).
Several pieces of evidence have suggested that neither of the conformations of insulin adopted in the crystalline state (T- and R-states) is the receptor binding conformation. The crystal structure of insulin from the Agnathan Myxine glutinosa (Atlantic hagfish) is very similar to that of pig insulin (Cutfield et al., 1979) yet hagfish insulin displays only 5% of the potency of pig insulin in stimulating lipogenesis in isolated rat fat cells (Emdin et al., 1977). In the crystal structure of pig insulin, the C-terminal region of the B-chain is located in close proximity to the N-terminal region of the A-chain. Chemical cross-linking of these domains by formation of a peptide bond between LysB29 and GlyA1 produces a molecule whose crystal structure and self-associative properties are very similar to native insulin but is biologically inactive (Derewenda et al., 1991). This observation implies that the active, receptor-binding conformation of insulin requires a separation of the C-terminus of the B-chain and the N-terminus of the A-chain. An alternative model has been provided by the results of a study by Kristensen et al. (1997) who used alanine scanning mutagenesis to identify specific side chains of insulin which strongly influenced binding to its receptor. Substitution of LeuB6, GlyB8, GlyB23, and PheB24, IleA2, ValA3 and TyrA19 by Ala resulted in >20-fold decrease in binding affinity. In contrast, substitutions at LeuB16, TyrB26, GluA4, GlnA5, and AsnA21, residues formerly considered to part of the binding surface, had relatively minor effects on binding affinity. Substitutions at LeuB11, GluB13 and PheB25 resulted in approximately a 10-fold decrease in binding affinity but, unexpectedly, replacement of GlyB20, GluB21 and ArgB22 by Ala produced analogs with appreciably increased binding affinity (2–4-fold). These data have suggested the alternative model in which the receptor binding domain consists of five residues (IleA2, ValA3, TyrA19, GlyB23, and Phe24) forming a patch on the surface of the molecule (Kristensen et al., 1997). Residues LeuB6, GlyB8, LeuB11, GluB13 and PheB26, although not part of the binding epitope, are presumably of importance in maintaining the overall receptor-binding conformation of insulin.
This review compares the amino acid sequences of insulins from a wide range of non-mammalian vertebrates with a view to correlating the residues in the molecule have been strongly conserved during evolution with those residues that structure-activity studies have shown are important in mediating its biological actions.
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The primary structures of reptilian insulins are known for a crocodilian, the American alligator Alligator mississipiensis (Lance et al., 1984
); the chelonians, the red-eared turtle Psuedemys scripta (Conlon and Hicks, 1990) and the Brazilian slider turtle, Chrysemys dorbigni (Cascone et al., 1991); and for three species of snakes, the rattlesnake Crotalus atrox (Kimmel et al. 1976), the colubrid snake Zoacys dhumndades (Zhang et al., 1981) and the Burmese python Python molurus (Conlon et al., 1997b). These amino acid sequences are compared with the corresponding sequences of avian insulins (the common structures from chicken/turkey/ostrich and from duck/goose [Evans et al., 1988]) and human insulin in Figure 1.
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The data demonstrate that there is no direct correlation between the frequency of amino acid substitutions and the generally accepted time of divergence of the species. Although the fossil record shows that birds and crocodilians developed from a common branch of the reptilian group (Gauthier et al., 1988
), turtle and chicken insulins have the same amino acid sequence whereas alligator insulin differs from chicken insulin by 3 substitutions in the B-chain. The traditional view is that Testudines (tortoises and turtles) are considered to be a monophyletic group that is basal to other groups of living reptiles but recent cladistic analysis has challenged this placement (Rieppel and deBraga, 1996).
The primary structure of insulin has not been particularly well conserved among the reptiles. Python insulin contains 5 amino acid substitutions compared with the more highly derived rattlesnake and colubrid snake. However, the substitution (Phe Tyr) at position B25 and a Glu residue at position A8 are found in all three insulins. Those residues on the surface of human insulin that were shown by alanine-scanning mutagenesis to be of importance in defining the receptor-binding region (B6, B8, B11, B13, B23, B24, A2, A3 and A19) (Kristensen et al., 1997) have been conserved in all amniote insulins except for alligator insulin which contains the conservative substitution (Glu Asp) at B13. Those residues in human insulin shown by X-ray analysis to be involved in dimer formation (B12, B16, B20, B24–B26 and B28) and those residues involved in hexamer formation (B6, B10, B14, B17, B18, A13, and A14) (Baker et al., 1988) are fully conserved in python insulin but insulins from the other snakes contain substitutions at B16 and B18. Consistent with this analysis, the affinity of python insulin for the human insulin receptor is not appreciable different from that of human insulin (Conlon et al., 1997b). Python insulin was slightly less potent (1.5-fold) than human insulin for inhibiting the binding of radiolabeled human insulin to the secreted extracellular domain of the recombinant human insulin receptor (Williams et al., 1995) but slightly more potent (1.8-fold) than human insulin in inhibiting binding to the soluble full-length receptor. Chicken insulin binds to insulin receptors on rat liver membranes with a 2–3-fold increased binding affinity compared with pig insulin (Simon et al., 1977). This increase in affinity has been ascribed to the presence of the HisA8 residue, that is also present in alligator and turtle insulins. This residue may form stabilizing structural motifs in the active conformation of insulin monomer that are of critical importance for receptor recognition (Derewenda et al., 1991). Conversely, the residue may destabilize the inactive conformation that is adopted in the crystal structure of insulin.
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Caudata and Gymnophiona
The primary structure of insulin has been determined for two species of salamander, the three-toed amphiuma Amphiuma tridactylum (Amphiumidae) (Conlon et al., 1996
) and the lesser siren Siren intermedia Le Conte (Sirenidae) (Conlon et al., 1997c), and for the caecilian Typhlonectes natans (Typhlonectidae) (Conlon et al., 1995b). Their amino acid sequences are compared with human insulin and with insulins from the African lungfish Protopterus annectens (Dipnoi) (Conlon et al., 1997a) and Australian lungfish Neoceratodus forsteri (Conlon et al., 1999) in Figure 2. Although sarcopterygian phylogeny, particularly the evolutionary relationships between the coelacanth, lungfishes and tetrapods, continues to be a matter of controversy, paleontological, morphological, and molecular analyses generally favor a close relationship between lungfish and amphibia.
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Despite the fact that the lines of evolution leading to salamanders and mammals diverged least 300 million years ago, the primary structure of amphiuma insulin is remarkably similar to that of human insulin. The amino acid residues considered to comprise the receptor-binding region as well as involved in dimer and hexamer formation have been conserved between the two proteins. Amphiuma insulin, like chicken insulin, contains a HisA8 residue that is presumed to be responsible for its approximately 5-fold higher affinity than pig insulin for binding to the soluble human insulin receptor (Conlon et al., 1996
). An unusual feature of the amphiuma insulin structure is the presence of a dipeptide (Ala-Arg) extension to the N-terminus of the A-chain, which suggests an anomalous pathway of post-translational processing of proinsulin in the region of the C-peptide/A-chain junction, but this extension does not appear to reduce binding affinity.
The biological activity of insulins from the siren and the caecilian have not been investigated. Although those amino acid residues considered to comprise the receptor-binding region of insulin have been conserved in both peptides, siren insulin contains substitutions such as B28 (Pro Ser), involved in dimer formation, and B14 (Leu Val) and B17 (Leu Phe), involved in hexamer formation, that may be expected to influence conformation and bioavailability. Caecilian insulin contain the substitution A5(Gln Lys), a residue that was formerly considered to be a component of the receptor binding region (Baker et al., 1988), but now thought not to influence binding affinity appreciably (Kristensen et al., 1997). The strongly conserved GlyB20 residue has been replaced by Ala in both the caecilian and lungfish insulins. Although this site in the molecule may be involved in dimer formation, the substitution B20 (Gly Ala) in human insulin results in a 2–3-fold increase in binding affinity at the insulin receptor (Kristensen et al., 1997).
Anurans
The primary structure of insulin is known for the cane toad Bufo marinus (Bufonidae) (Conlon et al., 1998a), the South American horned frog Ceratophrys ornata Leptodactylidae) (White et al., 1999), four species of the family Ranidae: the American bullfrog Rana catesbeiana (Conlon et al., 1998c), the European marsh frog Rana ridibunda (Conlon et al., 1998c), the wood frog Rana sylvatica (Conlon et al., 1998c), the African bullfrog Pyxicephalus adspersus (unpublished data, J. M. Conlon), and two molecular forms from the South African clawed toad Xenopus laevis (Pipidae) (Shuldiner et al., 1989). Their amino acid sequences are compared with human insulin in Figure 3.
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In all the anuran insulins, those amino acid residues identified by alanine-scanning mutagenesis as contributing to the receptor binding domain have been conserved. In
Bufo insulin, and in the identical insulin from Ceratophrys, those residues in human insulin that are involved in dimer and hexamer formation have also been conserved with the exception of the substitution B25(Phe Tyr). All the anuran insulins yet characterized contain an HisA8 residue and, as previously discussed, the presence of this residue probably accounts for the fact that Bufo insulin was 4-fold more potent than human insulin in inhibiting the binding of [125I-Tyr-A14] insulin to the soluble full-length recombinant human insulin receptor (Conlon et al., 1998a). All three insulins from the genus Rana contain a dipeptide (Lys-Pro) extension to the N-terminus of the A-chain that is analagous to the dipeptide extension (Ala-Arg) in the amphiuma A-chain (Conlon et al., 1996) and probably arises from a related anomalous pathway of post-translational processing of proinsulin in the region of the C-peptide/A-chain junction. This extension is not present in the A-chain of Pyxicephalus insulin but Pyxicephalus insulin shares structural features in common with the other frogs of the Ranidae family such as MetB17 and SerB30. The amino acid sequences of insulins from R. catesbeiana and R. ridibunda differ by only one residue (Asp for Glu at B21) but R. sylvatica insulin differs from R. catesbeiana insulin at B5 (Tyr His), B13 (Glu Asp), A12 (Thr Met), and A-23 (Asn Ser). The substitution at residue A-23 (corresponding to A-21 in human insulin) is unexpected as this site has been otherwise fully conserved during evolution. Formerly, the environment of the asparagine group at position A-21 was considered to be particularly important in determining the conformation and hence the biological activity of insulin. In the crystal structure, the carboxylate group of A-21 forms an ionic bond with B-22 arginine and a hydrogen bond bridges the -nitrogen of A-21 asparagine with the backbone carbonyl group of B-23 glycine. These features are important for the formation and maintenance of the conformation (Baker et al., 1988). However, more recent work (Kristensen et al., 1997) has suggested that residue A21 may not be of a component of the receptor-binding epitope in the biologically active conformation. It has been shown that freeze-tolerance in the wood frog is associated with anomalous regulation of glycogen metabolism, particularly the inability of insulin convert phosphorylase a to the inactive form in the liver (Storey, 1990). As the primary structure of R. sylvatica glucagon is similar to that of other amphibian and mammalian glucagons (Conlon et al., 1998c), it is tempting to speculate that changes in the amino acid sequence of its insulin mediate, at least in part, the atypical metabolic regulation in this species.
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The amino acid sequence of insulin is known for numerous species of teleosts, particular those fish in which purification of the peptide is facilitated by its synthesis in Brockmann bodies (reviewed in [Ferraz de Lima et al., 1999
]) but insulins from the phylogenetically more ancient antinopterygians have been less well studied. In the terminology of Gardiner (1993), the Actinopterygii may be divided into the "lower actinopterygians" which comprise only two extant lineages, the Polypteriformes (bichirs and redfish) and the Acipenseriformes (sturgeons and paddlefish), and the "higher actinopterygians" which comprise the Ginglymodi (gars), Halecomorphi (bowfin) and Teleostei (teleosts) and are collectively referred to as the Neopterygii. The primary structure of insulin is known for the bowfin Amia calva (Amiiformes) (Conlon et al., 1991), the alligator gar Lepisosteus spatula (Lepisosteiformes) (Pollock et al., 1987); the N. American paddlefish Polyodon spathula (Acipenseriformes) (Nguyen et al., 1994) and for the bichir Polypterus senegalis (Polypteriformes) (Conlon et al., 1998b). These sequences are compared with insulin from the coho salmon Oncorhynchus kisutch (Teleostei) (Plisetskaya et al., 1985) and with human insulin in Figure 4.
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Despite the considerable evolutionary distance between the Acipenseriformes and mammals, the primary structures of the two molecular forms of paddlefish insulins are remarkably similar to that of human insulin. The amino acid residues comprising the putative receptor-binding region together with residues involved in dimer and hexamer formation been conserved between the two insulins. Paddlefish insulin resembles most closely the peptide from the alligator gar (Pollock et. al, 1988) which is consistent with the classical phylogenetic view, derived from morphological analysis and the fossil record, that the Acipenseriformes are more closely related to the Lepisosteiformes than to the Amiiformes (Gardiner, 1993
). The isolation of two forms of paddlefish insulin, in approximately equimolar amounts, that differ by a single amino acid substitution (Asp His at A15) is suggestive of a recent gene duplication event. Polypterus insulin contains several unusual structural features that are not found in insulins from other jawed fish (ArgB4, AsnB21, IleB27, MetB31, AspA8, and ThrA9) but all those residue in human insulin that are involved in receptor binding, and in dimer and hexamer formation have been conserved.
In general, the biological potencies of insulins from teleost fish are between 30 and 50% of the corresponding values for mammalian insulins when measured in mammalian test systems. For example, insulin from the daddy sculpin Cottus scorpius showed 40% of the potency of pig insulin in stimulating lipogenesis in isolated rat fat cells (Cutfield et al., 1986). However, in a comparison of the abilities of partially purified insulins from a wide range of vertebrate species to increase glucose utilization by the mouse hemidiaphragm, bowfin insulin displayed a remarkably low biological potency suggesting that the protein contain unusual structural features (Falkmer and Wilson, 1967). More recent work (Conlon et al., 1991) has shown that bowfin insulin was approximately 14-fold less potent than porcine insulin in inhibiting the binding of [125I-Tyr-A14] human insulin to the recombinant human insulin receptor. The amino acid sequence of bowfin insulin contains several substitutions (Tyr Phe B16,Arg Ser at B22, and Leu Met at A16) at sites that have been relatively well conserved in other vertebrate species. In addition, the presence of LeuA8 invites the speculation that, in contrast to HisA8, this residue destabilizes the receptor-binding conformation of insulin. These unusual amino acid substitutions are not found in the insulin from the other extant "holostean" fish, the gar (Pollock et al., 1987) whose insulin possesses structural features that are typical of a teleost insulin (Plisetskaya et al., 1985; Ferraz de Lima et al., 1999. This observation supports the hypothesis that the bowfin and the gars are probably not descended from a common Mesozoic ancestor but represent the results of parallel evolutionary development (Gardiner, 1993).
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Insulins from elasmobranch fish have not been studied in the detail and the complete primary structure of an elasmobranch insulin is known only for the ray Torpedo marmorata (Conlon and Thim, 1986
) together with a partial structure from the spiny dogfish Squalus acanthias (Bajaj et al., 1983). In contrast, insulin has been purified and characterized from species of each of three extant families of holocephalan fish, the ratfish Hydrolagus colliei (Conlon et al., 1989), the rabbit fish Chimaera monstrosa (Conlon et al., 1988), and the elephant fish Callorhynchus milii (Berks et al., 1989). The primary structures of these insulins are compared with human insulin in Figure 5.
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The amino acid sequence of Torpedo insulin contains 16 amino acid substitutions compared with human insulin but those amino acid residues considered to be important in receptor binding and self-association have been conserved with the exception of the substitutions (Tyr
Phe) at A14 and (Phe Tyr) at B25. Despite the fact that the lines of evolution leading to the Torpedo (Batoidea) and the spiny dogfish (Squalomorpha) diverged at least 200 million years ago, the structure of Torpedo and dogfish insulin are very similar. Torpedo insulin, however, does not contain the unusual Gln extension to the C-terminus of the A-chain and the (uncharacterized) extension to the B-chain found in dogfish insulin. It has been shown that dogfish insulin gives the same maximum response but is approximately one-third as potent as bovine insulin in stimulating lipogenesis in rat fat cells (Bajaj et al., 1983).
Insulin was isolated from an extract of the pancreas of the Pacific ratfish in multiple, biosynthetically-related forms that contained 38-, 37-, 36- and 31-amino acid residues in the B-chain, indicative of an anomalous pathway of post-translational processing of proinsulin. It was proposed that a mutation in the site linking the B-chain and C-peptide regions (Arg-Arg Ile-Arg) in ratfish results in the generation of multiple forms of insulin each arising from cleavages at different sites within the C-peptide region by a putative enzyme with chymotrypsin-like specificity (Conlon et al., 1989). Rabbit fish (Conlon et al., 1988) and elephant fish (Berks et al., 1989) proinsulins also contain the same putative Arg Ile substitution but their insulins were isolated as single components with 38 and 31 residues in the B-chain, respectively. The amino acid residues important in constituting the receptor-binding domain (Kristensen et al., 1997) have been conserved in the three holocephalan insulins, with the exception of the conservative substitution (Glu Asp) at B13. The presence of the C-terminal extension to the B-chain does not appear to influence the biological properties of ratfish insulin appreciably. Ratfish insulin with the 38 residue B-chain was equipotent with human insulin in inhibiting the binding of human insulin to rat fat cells and in stimulating transport of 3-O-methylglucose into the cells (Conlon et al., 1989).
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The lampreys (Petromyzontiformes) and the hagfishes (Myxiniformes) are the only surviving groups of the agnathan (jawless) phase of early vertebrate evolution (Hardisty, 1982
). Although these two groups were once classified collectively as the Cyclostomata, they are no longer considered to form a monophyletic group. Indeed, recent cladistic analysis has suggested that the lampreys are more closely related to gnathostomes (jawed vertebrates) than either are to the hagfishes (Forey and Janvier, 1993). Current views on Agnathan phylogeny favor the hypothesis that the genera of holarctic lampreys belong to a single family (Petromyzontidae) and form an interrrelated progression in which Petromyzon is near to Ichthomyzon at the base of the phylogenetic tree and Lampetra is the most derived. A stock similar to that of contemporary Ichthomyzon is considered to have given rise to the southern hemisphere lampreys, Geotriidae and Mordaciidae, as a result of divergences at different times in the pre-Tertiary period (Potter and Hilliard, 1987). The primary structure of insulin has been determined for the Atlantic hagfish Myxine glutinosa (Peterson et al., 1975 and revised by Chan et al., 1981), for the sea lamprey Petromyzon marinus (Plisetskaya et al., 1988), the river lamprey Lampetra fluviatilis (Conlon et al., 1995a) and the southern-hemisphere lamprey Geotria australis (Conlon et al., 1995c). These amino acid sequences are compared with human insulin in Figure 6.
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Despite the fact that Lampetra and Petromyzon are not considered to be closely related phylogenetically, their insulins are identical. However, the amino acid sequence of Geotria insulin differs from this common sequence by 17 amino acid residues, which is consistent with the proposed ancient divergence of the holarctic and Southern hemisphere lampreys. Similarly, in agreement with the view that the divergence of lampreys and hagfishes is ancient, Lampetra/Petromyzon insulin contains 17 amino acid substitutions compared with the corresponding region of hagfish insulin but only 14 substitutions compared with human insulin. The amino acids in human insulin molecule that constitute the putative receptor binding domain are fully conserved in lamprey insulin together with those residues (B6, B8, B11, B13, and B26) shown to be important in maintaining the receptor-binding conformation (Kristensen et al., 1997
). Consistent with this analysis, Lampetra/Petromyzon insulin was equipotent with porcine insulin in inhibiting the binding of [3-[125I] iodotyrosine-A14] human insulin to the recombinant human insulin receptor (Conlon et al., 1995a) and to insulin receptors on rat hepatocytes (Leibush et al., 1997). Lamprey insulin was, however, significantly more potent than porcine insulin in inhibiting binding of label to high affinity insulin receptors on Lampetra hepatocytes (Leibush et al., 1997).
In contrast, the receptor binding affinity of hagfish insulin on isolated rat fat cells was 23% of that of pig insulin and hagfish insulin has only 5% of the potency of pig insulin in stimulating lipogenesis in isolated rat fat cells (Emdin et al., 1977). The amino acids that are postulated to constitute the receptor-binding epitope have been conserved in hagfish insulin but a substitution such as Glu Asn at B13 would be expected to result in reduced binding affinity at a mammalian insulin receptor. The earlier hypothesis that the reduced biological activity was a consequence of changes in the B17-B21 region of the molecule (Cutfield et al., 1979) may not be correct in the light of more recent structure-activity studies (Kristensen et al., 1997). Hagfish insulin, unlike Lampetra/Petromyzon insulin, lacks a histidine residue at position B10 and so forms only dimers not zinc-containing hexamers in the crystal structure (Cutfield et al., 1979).
An extract of the islet organ of Geotria contained only a very low concentration of mature insulin but rather a mixture of intact proinsulin and a partially processed form of proinsulin with an intact A-chain/C-peptide junction (Conlon et al., 1995c). Sequence analysis data have demonstrated that Arg-Arg site linking the C-terminus of the B-chain to the C-peptide region is present in Geotria proinsulin but it is not known whether the usual Lys-Arg processing site at C-peptide/A-chain junction is absent in Geotria proinsulin or whether the Geotria islet organ is unable to synthesize the appropriate prohormone convertase.
Comparing the insulin region of Geotria proinsulin with human insulin (Fig. 6), the Geotria protein contains several amino acid substitutions that would be expected to affect its self-associative properties. Among the residues in human insulin involved in dimer formation, the Pro residue at B28 (corresponding to B33 in Geotria insulin) is replaced by Ser. Geotria insulin also contains three substitutions (His Tyr at B6, His Tyr at B10 and Leu Ile at A13) among the residues in human insulin that are involved in hexamer formation. Nevertheless, those amino acid residues shown by alanine-scanning mutagenesis to be important in maintaining high affinity binding of human insulin to its receptor (Kristensen et al., 1997) have been conserved in Geotria insulin.
In general, the amino acid sequences of insulin are not useful as molecular markers for inferring phylogenetic relationships between species as the resulting cladograms based upon their characters contain features that are clearly at variance with the current consensus based upon analysis of morphological characters and the fossil record (Dores et al., 1996). In other words, molecular clocks based upon insulin sequences frequently "tell the wrong time" (Bajaj et al., 1984). However, among closely related species, the presence of unusual structural features in the insulin molecule may permit a valid inference. Although the complete primary structure of insulin from a species from the family Mordaciidae has not yet been determined, a partial amino acid sequence derived from a small amount of material isolated from an extract of the upper gastrointestinal tract of Mordacia mordax ammocetes revealed that the N-terminus of the B-chain of insulin was extended by the pentapeptide sequence Ser-Ala-Leu-Met-Gly (unpublished data, J. M. Conlon and I. C. Potter). Such an extension has never been found in insulins from other classes of vertebrates and so this observation provides strong support for the hypothesis that all extant lampreys arose from a common ancestor (Potter and Hilliard, 1987).
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ACKNOWLEDGMENTS |
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The author thanks Dr Erika Plisetskaya for stimulating his interest in non-mammalian insulins over the course of many years.
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FOOTNOTES |
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1 From the symposium A Tribute to Erika M. Plisetskaya: New Insights on the Function and Evolution of Gastroenteropancreatic Hormones presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 6–10 January 1999, at Denver, Colorado.
2 E-mail: jmconlon{at}creighton.edu
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References |
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SYNOPSISSTRUCTURE-ACTIVITY RELATIONSHIPSINSULINS FROM NON-MAMMALIAN...INSULINS FROM AMPHIBIAINSULINS FROM ACTINOPTERYGIIINSULINS FROM ELASMOBRANCHII AND...INSULINS FROM AGNATHAReferences |
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Bajaj, M., T.L Blundell, J.E. Pitts, S.P. Wood, M.A. Tatnell, S. Falkmer, S.O. Emdin, L.K. Gowan, H. Crow, C. Schwabe, A. Wollmer, and W. Strassburger. 1983. Dogfish insulin. Primary structure, conformation and biological properties of an elasmobranchial insulin.Eur. J. Biochem., 135:535-542.[Medline]
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