鸡钙离子结合分子伴侣Calreticulin的结构与功能预测及组织表达特性

doi:10.3864/j.issn.0578-1752.2014.19.016

摘要/Abstract

摘要： 【目的】揭示鸡组织细胞内质网(endoplasmic reticulum, ER)中关键Ca²⁺结合分子伴侣钙网蛋白(calreticulin, CRT)的结构与功能及组织表达特性。【方法】应用Laser Gene软件，通过比对Gene bank已登录的12种脊椎动物CRT核苷酸及氨基酸序列以分析其进化关系，利用生物信息学方法预测鸡CRT蛋白的结构与功能，并采用荧光定量RT-PCR方法检测CRT在鸡30个不同组织中的表达特性。【结果】同源性分析结果显示：鸡与其他11个物种CRT基因核苷酸序列中，鸡与兔核苷酸序列同源性最高，为78.7%，与虹鳟的同源性最低，为70.5%；氨基酸序列中，鸡与蛇的亲缘关系最近，为85.0%，与虹鳟的亲缘关系最远，为69.0%，与鼠、猕猴、人、兔、猪、牛和非洲爪蟾蜍的亲缘关系也相对较近，并均在80.1%及以上。蛋白结构与功能预测结果为：鸡CRT由404个氨基酸组成，相对分子质量46.8802 kD，理论等电点为4.41，负电荷氨基酸残基数为102，正电荷氨基酸残基数为53，分子式为C₂₀₇₄H₃₁₀₇N₅₄₃O₆₈₄S₉，鸡CRT具有22个疏水区域，其C端与N端具有很高的疏水性，而C端的亲水性要强于N端，且形成α_1(7-17)-α_2(22-25)-β₁₍₂₆₎-β_2(38-41)-β_3(50-54)-β_4(69-70)-β_5(75-82)-β_6(92-99)-β_{7 (110-114)}-β_8(129-133)-β_9(144-151)-β_10(171-178)-β_11(183-187)- β_12(314-322)-β_13(326-332)-α_3(337-348)-α_4(350-375)-α_5(377-379)-α_6(395-401)的二级结构。CRT蛋白属于跨膜蛋白，存在信号肽，且为分泌蛋白，酶分类属于EC 3.2.1.55或EC 3.4.24.68。组织表达检测结果表明：CRT基因在鸡各组织中广泛表达，其中在回肠、腺胃和十二指肠等组织中表达量较高，且高出肾脏（对照）20倍以上。【结论】鸡CRT核苷酸及氨基酸序列在12种脊椎动物物种中具有相对保守性，鸡CRT为跨膜分泌蛋白，为酸性蛋白，属于α-N-阿拉伯糖苷酶或Tentoxilysin，催化α-L-阿拉伯糖苷内的终端非还原性α-L-阿拉伯呋喃糖苷残基的水解，作用于α-L-阿拉伯呋喃糖苷、含（1,3）和/或（1,5）糖苷键的阿拉伯聚糖、阿拉伯木聚糖和阿拉伯半乳聚糖，能与糖类分子及Ca²⁺特异性结合，可监控糖蛋白组装折叠及Ca²⁺调控，且在消化系统中发挥重要作用。

关键词: 鸡, Calreticulin, 结构与功能, 组织表达特性, 进化关系

Abstract: 【Objective】 The aim of the current study is to reveal the evolutionary relationships, and investigate the protein structure and functions and the expression profiles of calreticulin (CRT) as a key Ca²⁺ binding molecular chaperone within the endoplasmic reticulum (ER) of chicken.【Method】The nucleotides and amino acids of CRT in 12 species of vertebrates recorded in Gene bank were analyzed for evolutionary relationships by Laser Gene, and the structures and functions of CRT protein in chicken were predicted by bioinformatics, and the expression profiles of CRT in 30 organizations of chicken was analyzed by real-time PCR.【Result】Results of homology analysis showed that compared with the other 11 species of nucleotide sequences of CRT gene in chicken, gallus gallus and oryctolagus cuniculus had the highest nucleotide sequence homology, which was 78.7%, in addition, gallus gallus and oncorhynchus mykiss had the lowest homology, which was 70.5%. In the homology of amino acid sequences, the relationship between gallus gallus and crotalus adamanteus cadam is the closest by 85.0%, and the furthest relationships with gallus gallus is oncorhynchus mykiss which was 69.0% in amino acid sequence, besides, the homology of gallus gallus with cricetulus griseus, macaca mulatta, homo sapiens, oryctolagus cuniculus, sus scrofa, bos taurus, and xenopus (silurana) tropicalisis relatively close to almost above 80.1%. The protein structure and function prediction revealed that the CRT of chicken was constitute with 404 amino acids, and had a relative molecular mass of 46.8802 kD and a theoretical isoelectric point of 4.41, moreover, the negative charge is 102 amino acid residues and the positive charge amino acid residues is 53. The molecular formula of the CRT was C₂₀₇₄H₃₁₀₇N₅₄₃O₆₈₄S₉. The CRT of chicken had 22 hydrophobic regions, which of the C end and the N end had a high hydrophobic, while the hydrophilic of C end is stronger than that of N, and the protein formed a secondary structure as α_1(7-17)-α_2(22-25)-β₁₍₂₆₎- β₂_(38-41)-β_3(50-54)-β₄₍_69-70)-β₅₍_75-82)-β_6(92-₉₉₎-β_7(110-114)-β_8(129-133)-β_9(144-151)-β_10(171-178)-β_11(183-187)-β_12(314-322)-β_13(326-332)-α_3(337-348)-α₄₍₃₅_0-375)- α_5(377-379)-α_{6(395- 401)}. CRT belongs to the transmembrane proteins and secreted protein, has signal peptides. The enzyme classification of CRT was EC 3.2.1.55 or EC 3.4.24.68. Tissue expression assay indicated that the gene of CRT expressed widely in chicken tissues, in which the lleum, glandular stomach and duodenum were highly more than the kidney (control) by 20 times. 【Conclusion】The sequence of nucleotide and amino acid in chicken CRT is relatively conserved in 12 vertebrate species. CRT is a transmembrane secretion protein and acidic protein. It belongs to the alpha-N-Arabia-glucosidase or Tentoxilysin, which catalyzed the hydrolysis of the end non-reducing α-L-arabinofuranosidase residues in the terminal of α-L-arabinoside, which effected the α-L- arabinofuranosidase, Arabic glycans containing (1,3) and/or (1,5) glucosidic bond, Arab xylan and arabinogalactan. CRT combined specifically with the carbohydrate molecules and Ca²⁺, which could monitor glycoprotein assembly and folding and Ca²⁺ regulation, and plays an important role in the digestive system.

Key words: chicken, Calreticulin, structure and functions, expression profiles, evolutionary relationship

王丽丽，李楠，曹嫦妤，龚都强，于东，王伟，李金龙. 鸡钙离子结合分子伴侣Calreticulin的结构与功能预测及组织表达特性[J]. 中国农业科学, 2014, 47(19): 3874-3882.

WANG Li-li, LI Nan, CAO Chang-yu, GONG Du-qiang, YU Dong, WANG Wei, LI Jin-long. Structures and Functions Prediction and Expression Profiles of Calreticulin as Calcium Binding Chaperones in Chicken[J]. Scientia Agricultura Sinica, 2014, 47(19): 3874-3882.

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