Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane
Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane
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新型肌浆网膜整体钙结合蛋白 junctate 的分子克隆、表达、功能表征、染色体定位和基因结构
DOI：
10.1074/jbc.m005473200
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发表时间：
2000-12-15
影响因子：
4.8
通讯作者：
Zorzato, F
中科院分区：
文献类型：
--
作者：
Treves, S;Feriotto, G;Zorzato, F
用来自连接蛋白的cDNA探针筛选人骨骼肌和心肌的cDNA文库，导致分离两组cDNA克隆，第一组显示与狗心脏连接蛋白的推导的氨基酸序列84%相同的推导的氨基酸序列，而第二组具有编码预测质量为33 kDa的多肽的单个开放阅读框，其前78个NH 2-末端残基与连接蛋白相同，而其COOH末端结构域与α-酮戊二酸依赖性双加氧酶家族的一个成员β-羟化酶相同。北方印迹分析表明，junctate在包括心脏、胰腺、脑、肺、肝、肾和骨骼肌在内的多种人体组织中表达。荧光原位杂交分析表明，junctin和junctate的遗传位点位于人类8号染色体上的同一条细胞遗传带，对基因组BAC克隆的内含子/外显子边界的分析表明，连接蛋白、连接物和β-羟化酶是由同一基因的选择性剪接产生的。在生理浓度KCl存在下平衡Ca-45（2+）结合的Scatchard分析表明，连接物结合21.0 mol Ca 2 +/mol蛋白，k（D）为217 +/-20 μ M（n = 5），用绿色荧光蛋白标记重组连接物并在COS-7中表达嵌合多肽转染的细胞表明，连接物位于内质网膜上，它的存在增加了与InsP偶联的表面膜受体激活所释放的峰值幅度和瞬时钙离子（3）我们的研究表明，相同基因的选择性剪接产生以下功能不同的蛋白质：酶（β-羟化酶）、SR的结构蛋白（连接蛋白）和膜结合钙结合蛋白（连接蛋白）。
Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones, The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH2-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta -hydroxylase, a member of the alpha -ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate, Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle, Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band oh human chromosome 8, Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta -hydroxylase result from alternative splicing of the same gene.The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium Ca-45(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca2+/mol protein with a k(D) of 217 +/- 20 muM (n = 5), Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation.Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta -hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).