文昌鱼CT/CGRP家族和内源性大麻素系统的预测与进化分析
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摘要
文昌鱼(amphioxus)是现存与脊椎动物祖先最接近的头索动物，是介于无脊椎动物和脊椎动物之间的过渡类型，一直被认为是研究脊椎动物起源和进化的重要模式动物。文昌鱼基因的序列和表达模式已被广泛用于不同物种之间的比较基因组学研究和发育同源性分析。作为脊椎动物的重要调节系统，降钙素(CT)/降钙素基因相关肽（CGRP）家族和内源性大麻素系统（ECS）具有重要研究价值和广阔应用前景。本文对文昌鱼CT/CGRP家族和ECS的生物大分子进行研究分析，进而探讨并揭示了这两个系统在脊椎动物进化过程中的发展与分化。
CT/CGRP家族是脊椎动物的一大类蛋白家族，包括一类在组成和结构上相近的多肽。最近，发现无脊椎动物玻璃海鞘（Ciona intestinalis）也存在CT/CGRP家族成员Ci-CT，并被认为是脊椎动物CT/CGRP家族的原始类型。但CT/CGRP家族在脊椎动物进化过程中是如何分化成不同的家族成员迄今仍不清楚。本文首先在佛罗里达文昌鱼（Branchiostoma florida）基因组库中BLASTP搜索CT/CGRP家族的蛋白序列，发现了3条类CT/CGRP家族的氨基酸序列，并分别命名为Bf-CTL1、Bf-CTL2和Bf-CTL3，其长度分别为31aa、33aa和32aa。通过序列比对表明：上述3条Bf-CTL的蛋白前体拥有与其他脊索动物CT/CGRP家族成员相同的蛋白修饰位点，具体包括类降钙素结构域两侧典型内切蛋白酶解位点(KR/RR和RXXR)以及C末端Gly信号。通过一致性比较、系统进化树构建、基因结构分析、基因共线性分析以及蛋白三级结构预测等方法都表明Bf-CTL1、Bf-CTL2和Bf-CTL3分别与脊椎动物的AM1/4亚家族、CT/CGRP亚家族和AM2/3有类似的特征。在佛罗里达文昌鱼基因组数据库中，我们还发现了文昌鱼降钙素受体样受体(Bf-CRLR)及其受体活性修饰蛋白(Bf-RAMP)，并推测它们对CT/CGRP家族生物学功能的发挥有必不可少的作用。
通过进一步对文昌鱼CT/CGRP家族成员及其受体预测分析，表明文昌鱼中已经形成CT/CGRP家族系统，这很好地解释了脊椎动物CT/CGRP家族的起源，并表明脊椎动物早期的CT/CGRP家族已在头索动物中发生基因分化。
本文随后对内源性大麻素系统(ECS)进行了系统的分析，作为一个重要的神经调节系统，ECS参与生物体许多生理和病理过程。在脊椎动物ECS中，目前已知存在8种受体(CNR1，CNR2，GPR55，PPARA，PPARG，TRPV1，TRPV4，TRPA)与大麻素相结合起作用，其中CNR1还需要大麻素受体活性互作蛋白CRIP1的活化。内源性大麻素合成路径中有5种合成酶(ABHD4，DAGLA，DAGLB，NAPE-PLD，PTPN22)，并通过5种代谢酶(ASAHL，FAAH1，FAAH2，MGLL，PTGS2)代谢。但在无脊椎动物ECS中，相关受体和代谢合成酶的种类相对较少。
本文从佛罗里达文昌鱼基因组库中搜索到5种类ECS受体（CNR，PPAR，TRPVL1，TRPVL2，TRPA）、3种类ECS合成酶（ABHD4/5，ABHD4/5L，DAGLA）、6种类ECS代谢酶(ASAHL，FAAH1，FAAH2，MGLL，PTGSL1，PTGSL2)和类大麻素受体活性互作蛋白CRIP1。通过与其他脊索动物ECS基因在蛋白结构域、蛋白功能位点图谱、基因结构和系统发生分析等方面进行比较，判定所提取基因都是脊椎动物ECS基因的原始类型。最后对佛罗里达文昌鱼和玻璃海鞘以及脊椎动物（人、鼠、斑马鱼和河豚）之间的ECS基因选择压力(Ka/Ks)进行计算和分析。通过分析，我们发现所有文昌鱼、海鞘和脊椎动物之间的ECS基因选择压力均小于1，这说明ECS基因在物种进化过程中进行了较强的纯化选择作用。通过相对进化速率(Ka+Ks)和选择压力间的相关性检验，再次验证了ECS基因在进化过程中的相对进化速率与选择压力无相关性。通过比较同义替代率(Ks)发现文昌鱼ECS基因Ks值达到饱和状态相对海鞘ECS基因Ks值较少，所以我们推测文昌鱼比海鞘ECS基因进化程度更高。
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 cephalochordates
The 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.
CT/CGRP家族是脊椎动物的一大类蛋白家族，包括一类在组成和结构上相近的多肽。最近，发现无脊椎动物玻璃海鞘（Ciona intestinalis）也存在CT/CGRP家族成员Ci-CT，并被认为是脊椎动物CT/CGRP家族的原始类型。但CT/CGRP家族在脊椎动物进化过程中是如何分化成不同的家族成员迄今仍不清楚。本文首先在佛罗里达文昌鱼（Branchiostoma florida）基因组库中BLASTP搜索CT/CGRP家族的蛋白序列，发现了3条类CT/CGRP家族的氨基酸序列，并分别命名为Bf-CTL1、Bf-CTL2和Bf-CTL3，其长度分别为31aa、33aa和32aa。通过序列比对表明：上述3条Bf-CTL的蛋白前体拥有与其他脊索动物CT/CGRP家族成员相同的蛋白修饰位点，具体包括类降钙素结构域两侧典型内切蛋白酶解位点(KR/RR和RXXR)以及C末端Gly信号。通过一致性比较、系统进化树构建、基因结构分析、基因共线性分析以及蛋白三级结构预测等方法都表明Bf-CTL1、Bf-CTL2和Bf-CTL3分别与脊椎动物的AM1/4亚家族、CT/CGRP亚家族和AM2/3有类似的特征。在佛罗里达文昌鱼基因组数据库中，我们还发现了文昌鱼降钙素受体样受体(Bf-CRLR)及其受体活性修饰蛋白(Bf-RAMP)，并推测它们对CT/CGRP家族生物学功能的发挥有必不可少的作用。
通过进一步对文昌鱼CT/CGRP家族成员及其受体预测分析，表明文昌鱼中已经形成CT/CGRP家族系统，这很好地解释了脊椎动物CT/CGRP家族的起源，并表明脊椎动物早期的CT/CGRP家族已在头索动物中发生基因分化。
本文随后对内源性大麻素系统(ECS)进行了系统的分析，作为一个重要的神经调节系统，ECS参与生物体许多生理和病理过程。在脊椎动物ECS中，目前已知存在8种受体(CNR1，CNR2，GPR55，PPARA，PPARG，TRPV1，TRPV4，TRPA)与大麻素相结合起作用，其中CNR1还需要大麻素受体活性互作蛋白CRIP1的活化。内源性大麻素合成路径中有5种合成酶(ABHD4，DAGLA，DAGLB，NAPE-PLD，PTPN22)，并通过5种代谢酶(ASAHL，FAAH1，FAAH2，MGLL，PTGS2)代谢。但在无脊椎动物ECS中，相关受体和代谢合成酶的种类相对较少。
本文从佛罗里达文昌鱼基因组库中搜索到5种类ECS受体（CNR，PPAR，TRPVL1，TRPVL2，TRPA）、3种类ECS合成酶（ABHD4/5，ABHD4/5L，DAGLA）、6种类ECS代谢酶(ASAHL，FAAH1，FAAH2，MGLL，PTGSL1，PTGSL2)和类大麻素受体活性互作蛋白CRIP1。通过与其他脊索动物ECS基因在蛋白结构域、蛋白功能位点图谱、基因结构和系统发生分析等方面进行比较，判定所提取基因都是脊椎动物ECS基因的原始类型。最后对佛罗里达文昌鱼和玻璃海鞘以及脊椎动物（人、鼠、斑马鱼和河豚）之间的ECS基因选择压力(Ka/Ks)进行计算和分析。通过分析，我们发现所有文昌鱼、海鞘和脊椎动物之间的ECS基因选择压力均小于1，这说明ECS基因在物种进化过程中进行了较强的纯化选择作用。通过相对进化速率(Ka+Ks)和选择压力间的相关性检验，再次验证了ECS基因在进化过程中的相对进化速率与选择压力无相关性。通过比较同义替代率(Ks)发现文昌鱼ECS基因Ks值达到饱和状态相对海鞘ECS基因Ks值较少，所以我们推测文昌鱼比海鞘ECS基因进化程度更高。
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 cephalochordates
The 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.
引文
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