Identification of angiotensin I in several vertebrate species: its structural and functional evolution

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Abstract

In order to delineate further the molecular evolution of the renin–angiotensin system in vertebrates, angiotensin I (ANG I) has been isolated after incubation of plasma and kidney extracts of emu (Dromiceus novaehollandiae), axolotl (Ambystoma mexicanum), and sea lamprey (Petromyzon marinus). The identified sequences were [Asp1, Val5, Asn9] ANG I in emu, [Asp1, Val5, His9] ANG I in axolotl, and [Asn1, Val5, Thr9] ANG I in sea lamprey. These results confirmed the previous findings that tetrapods have Asp and fishes including cyclostomes have Asn at the N-terminus, and that the amino acid residue at position 9 of ANG I was highly variable but, those at other positions were well conserved among different species. Since Asp and Asn are convertible during incubation, angiotensinogen sequences were searched in the genome and/or EST database to determine the N-terminal amino acid residue from the gene. The screening detected 12 tetrapod (10 mammalian, one avian, and one amphibian) and seven teleostean angiotensinogen sequences. Among them, all tetrapods have [Asp1] ANG except for Xenopus, and all teleosts have [Asn1] ANG, thereby confirming the above rule. Comparison of the vasopressor activity in the eel revealed that [Asn1] ANG I and II were more potent than [Asp1] peptides, which was opposite to the previous results in mammals and birds, in which [Asp1] ANG I and II were more potent. Collectively, the present results support the general rule that tetrapods have [Asp1] ANG and fishes including cyclostomes have [Asn1] ANG. However, an aquatic anuran (Xenopus) has [Asn1] ANG in its gene despite another aquatic urodele (axolotl) has [Asp1] ANG. From the functional viewpoint, homologous [Asn1] ANG was more potent in fish as is homologous [Asp1] ANG in tetrapods, suggesting that ANG II molecule has undergone co-evolution with its receptor during vertebrate phylogeny.

Introduction

It has become increasingly clear that the renin–angiotensin system is a phylogenically old hormonal system that is present throughout the vertebrate species including elasmobranchs (Takei et al., 1993) and cyclostomes (Rankin et al., 2004). With respect to ANG I molecules, their sequences are generally well conserved with variations only at positions 1, 3, 5, and 9. All tetrapod species from amphibians to mammals have aspartate (Asp) at position 1, while fish ANG has asparagine (Asn) instead of Asp except for the bowfin, from which [Asp1] ANG I was isolated (Takei et al., 1998). A small amount of [Asp1] ANG I was also identified together with [Asn1] ANG I in the eel (Hasegawa et al., 1983); however, this may be due to the conversion of Asn to Asp during the isolation process (Khosla et al., 1985). The amino acid residue at position 3 is Val in all animals thus far examined except for the dogfish, Triakis scyllia, where Val is replaced by Pro (Takei et al., 1993). The fifth amino acid is Ile in all mammalian species except for the cattle, in which Val is present at this position. By contrast, most non-mammalian species have Val at position 5 except for the dogfish and flounder that have Ile as in mammals (Balment et al., 2003). Compared with the minor variation at positions 3 and 5, the ninth amino acid is highly variable and it differs even among species of the same class (Brown and Balment, 1997; Kobayashi and Takei, 1996; Nishimura, 1987).

The activity of plasma renin–angiotensin system is dependent mostly on the formation of the active principle, ANG II (Campbell, 1987). In view of the evolution, the amino acid residue at position 9 of ANG I may affect the performance of converting enzyme, because the enzyme cleaves off ANG II between eighth and ninth position. On the other hand, the activity of produced ANG II appears to be dependent on its N-terminal amino acid residue as [Asp1] ANG II exhibits greater vasopressor effects in mammals and birds (Takei and Hasegawa, 1990; Watanabe et al., 1977). This suggests that the ANG II molecule has evolved in parallel with its receptor during the transition from aquatic to terrestrial life. In order to substantiate the above hypothesis on the molecular and functional evolution of the renin–angiotensin system, more ANG I sequences should be determined in various vertebrate species and the effects of [Asp1] and [Asn1] ANG should be compared in fishes.

In the present study, we isolated and sequenced ANG I from three vertebrate species; emu, axolotl, and sea lamprey. The emu belongs to Rattitae, which includes non-flying avian species that holds a unique evolutionary position. Thus far, avian ANG I has been identified only in the galliformes birds (Nakayama et al., 1973; Takei and Hasegawa, 1990). The axolotl was chosen because this neotenic urodele is suggested to have unique ANG II molecule (Kloas and Hanke, 1993); [Asp1] ANG II did not bind the kidney of this species although ANG II receptors are present in the kidney of all species thus far examined including frogs (Kloas and Hanke, 1992). The sea lamprey was used to compare the ANG I sequence with the recently identified ANG I of another lamprey, Lampetra fluviatilis (Rankin et al., 2004).

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Animals

The emus, Dromiceu novaehollandiae (n=8), were kept under natural conditions in the Fauna Park at Macquarie University, until they were approximately 6 months old and weighing ca. 15 kg. The axolotls, Ambystoma mexicanum, weighing 80–120 g (n=125) were obtained from the animal stock of the Department of Zoology II, University of Karlsruhe. They were maintained in tanks of aerated tap water at 18 °C under a 12L–12D photoperiod. The sea lampreys, Petromyzon marinus, weighing 618–1141 g (n=8) were

Purification of ANG I

An ANG I-like vasopressor material was produced after incubation of emu plasma with the kidney extracts. The material was purified by cation-exchange, gel-permeation, and reverse-phase HPLC in this order (Fig. 1A). The ANG I-like material was eluted near the position of chicken ANG I in all HPCLs. Since the final yield was estimated to be only 480 pmol as estimated by the peak height of chicken ANG I at 220 nm, the material in the peak was subjected to sequence analysis without further

Discussion

In the present study, we have isolated ANG I from the sea lamprey, P. marinus, confirming our previous finding that the renin–angiotensin system is present in the most primitive vertebrate group, cyclostomes (Rankin et al., 2004). Although the sea lamprey belongs to a genus different from the river lamprey, L. fluviatilis, the two species have an identical ANG I sequence. In invertebrates, all components of the renin–angiotensin system seem to be present in the leech, including renin-like

Acknowledgments

The authors thank Drs. K. Nakajima and T.X. Watanabe of Peptide Institute, for peptide synthesis and cooperation. This work was supported in part by Grant-in-Aid for Creative Basic Research (12NP0201) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and for Scientific Research (13304063) from the Japan Society for the Promotion of Science to Y.T.

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