Molecular evolution of kiss2 genes and peptides in vertebrates.

The kiss1 peptide (kisspeptin), a product of the kiss1 gene, is one of the key neuropeptides regulating vertebrate reproduction. In 2009, we identified a paralogous gene of kiss1 in the brain of amphibians and named it kiss2. Currently, the presence of the kiss2 gene and the kiss2 peptide is still obscure in amniotes compared with that in other vertebrates. Therefore, we performed genome database analyses in primates and reptiles to investigate the molecular evolution of the kiss2 gene in vertebrates. Because the mature kiss2 peptide has been identified only in amphibians, we further performed immunoaffinity purification and mass spectrometry to identify the mature endogenous kiss2 peptide in the brains of salmon and turtle that possessed the kiss2 gene. Here we provide the first evidence for the presence of a kiss2-like gene in the genome database of primates including humans. Synthetic amidated human KISS2 peptide activated human GPR54 expressed in COS7 cells, but nonamidated KISS2 peptide was inactive. The endogenous amidated kiss2 peptide may not be produced in primates because of the lack of an amidation signal in the precursor polypeptide. The kiss2-like gene may be nonfunctional in crocodilians because of premature stop codons. We identified the mature amidated kiss2 peptide in turtles and fish and analyzed the localization of kiss2 peptide mRNA expression in fish. The present study suggests that the kiss2 gene may have mutated in primates and crocodilians and been lost in birds during the course of evolution. In contrast, the kiss2 gene and mature kiss2 peptide are present in turtles and fish.

Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates.

Kisspeptin and its receptor GPR54 play important roles in mammalian reproduction and cancer metastasis. Because the KiSS and GPR54 genes have been identified in a limited number of vertebrate species, mainly in mammals, the evolutionary history of these genes is poorly understood. In the present study, we have cloned multiple forms of kisspeptin and GPR54 cDNAs from a variety of vertebrate species. We found that fish have two forms of kisspeptin genes, KiSS-1 and KiSS-2, whereas Xenopus possesses three forms of kisspeptin genes, KiSS-1a, KiSS-1b, and KiSS-2. The nonmammalian KiSS-1 gene was found to be the ortholog of the mammalian KiSS-1 gene, whereas the KiSS-2 gene is a novel form, encoding a C-terminally amidated dodecapeptide in the Xenopus brain. This study is the first to identify a mature form of KiSS-2 product in the brain of any vertebrate. Likewise, fish possess two receptors, GPR54-1 and GPR54-2, whereas Xenopus carry three receptors, GPR54-1a, GPR54-1b, and GPR54-2. Sequence identity and genome synteny analyses indicate that Xenopus GPR54-1a is a human GPR54 ortholog, whereas Xenopus GPR54-1b is a fish GPR54-1 ortholog. Both kisspeptins and GPR54s were abundantly expressed in the Xenopus brain, notably in the hypothalamus, suggesting that these ligand-receptor pairs have neuroendocrine and neuromodulatory roles. Synthetic KiSS-1 and KiSS-2 peptides activated GPR54s expressed in CV-1 cells with different potencies, indicating differential ligand selectivity. These data shed new light on the molecular evolution of the kisspeptin-GPR54 system in vertebrates.