Prediction And Evolution Of The CT/CGRP Family And Endocannabinoid System In Amphioxus

Amphioxus or lancelet, a cephalochordate, has long been regarded as the livinginvertebrate most closely related to the proximate invertebrate ancestor of vertebrates.It is a well known as a model organism which has been widely used for interspeciescomparative genome studies and developmental homology analysis. Calcitonin(CT)/calcitonin gene-related peptides (CGRP) family and endocannabinoidsystem(ECS) are very important control systems in vertebrates, their primitive typesare also found in invertebrates. This study was conducted to screen the genes ofCT/CGRP family and ECS in amphioxus, and to probe into the origin and evolutionof the systems in vertebrates.As an important protein family in vertebrates, CT/CGRP family, which includesseveral polypeptides with structural similarities, has been extensively studied.Recently, a new member of the CT/CGRP family named Ci-CT has been identified inthe ascidian (Ciona intestinalis), which was regarded as an primitive type ofCT/CGRP family in vertebrates. However, the differentiation of CT/CGRP familymembers in the evolution of vertebrates remains unclear so far. In this study, we haveobtained three CT/CGRP peptide-like peptides named Bf-CTL1, Bf-CTL2andBf-CTL3from the Florida amphioxus (Branchiostoma floridae) genome database byBLASTP, which were consisted of31aa,33aa and32aa, respectively. Our resultsshowed that each of the three Bf-CTL precursor proteins had the same modifier locuswith other chordates by sequence comparison including a CT-like domain flanked by a Gly C-terminal amidation signal and typical endoproteolytic sites (KR/RR andRXXR) at N-and C-termini. It was also revealed that Bf-CTL1, Bf-CTL2andBf-CTL3genes was respectively similar to AM1/4, CGRP and AM2/3in vertebrates.Additionally, a CT receptor-like receptor (Bf-CRLR) and a receptoractivity-modifying protein (Bf-RAMP) which were both necessary for functioning ofCT/CGRP peptides, were also identified in the genome database of B. floridae.With the further analysis of the predicted CT/CGRP family, we found that theCT/CGRP family had already formed in amphioxus.We also discovered that the geneof primitive CT/CGRP family had differentiated in cephalochordatesThe endocannabinoid system (ECS) is an important neural control system, andtakes part in many physiological activities in animals. In vertebrate, the ECS consistsof8receptors (CNR1, CNR2, GPR55, PPARA, PPARG, TRPV1, TRPV4, TRPA) ofcannabinoid, as well as a cannabinoid receptor-interacting protein (CRIP1). There is5synthetic enzymes (ABHD4, DAGLA, DAGLB, NAPE-PLD, PTPN22) and5catabolic enzymes (ASAHL, FAAH1, FAAH2, MGLL, PTGS2) in endocannabinoidmetabolic synthesis pathway. However, the ECS has less receptors and enzymes ininvertebrates.We have found5receptors (CNR，PPAR，TRPVL1，TRPVL2，TRPA) ofcannabinoid,3synthetic enzymes (ABHD4/5，ABHD4/5L，DAGLA),6catabolicenzymes (ASAHL，FAAH1，FAAH2，MGLL，PTGSL1，PTGSL2) and a CRIP1inB. floridae genome database. All these genes were identified with a set ofbioinformatics analysis， including phylogenetic prediction analysis, gene structureanalysis, protein structural domain analysis and functional assessment score (FAS)mapping, etc. At last, the ECS genes selection pressure (Ka/Ks) of vertebrates relative to amphioxus and ascidian were compared and analyzed. Finally we found that theECS genes Ka/Ks of vertebrates relative to amphioxus and ascidian were all less than1, meaning that genes underwent relatively strong purified selection in evolution. Bycorrelation analysis, we conclude that all of the relative rate of evolution tempo(Ka+Ks) values for individual pairwise calculations didn’t correlate with theircorresponding Ka/Ks values in ECS genes evolution. Moreover, calculation andcomparative analysis concluded that Ks of ECS genes in amphioxus are less thanthose in sea squirts. So it’s clear that ECS genes in amphioxus are more evolutionarythan those of in sea squirts.