cDNA cloning and molecular characterization of a glycoprotein with Ca^<2+> -binding EF-hand motifs.

具有Ca 2+ 结合EF-手基序的糖蛋白的cDNA克隆和分子表征。

• 批准号: 03680152
• 负责人: OZAWA Masayuki
• 金额: $ 1.22万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03680152/
• 关键词: Calcium-binding protein; EF-hand; CDNA cloning; ER-resident protein; Glycoprotein; cDNAクロ-ニング

A novel Ca^<2+>-binding protein, designated reticulocalbin, has been identified and characterized. Reticulocalbin is a luminal protein of the endoplasmic reticulum with an Mr of 44,000 as revealed by biochemical analysis such as cell fractionation and immunoflurescence staining.The cDNA of reticulocalbin encodes a protein of 325 amino acids with an amino-terminal signal sequence of 20 amino acids. The protein has six repeats of a domain containing the high affinity Ca^<2+> -binding motif, the EF-hand. Although oxygencontaining amino acids important for the positioning of Ca^<2+> are conserved in all six domains, the conserved glycine reidues in the central portion of the EF-hand motif are absent in three (in sites II, III, and VI) of them. Calcium blots showed that recombinant reticulocalbin expressed in bacterial cells and purified from them binds Ca^<2+>.The protein has the sequence His-Asp-Glu-Leu (HDEL) at its carboxyl terminus. This sequence is similar to the Lys-Asp-Glu-Leu(KDEL) sequence, which serves as a signal to retain the resident proteins in the endoplasmic reticulum of animal cells. A mutant protein lacking the HDEL sequence produced by in vitro mutagenesis has been shown to be secreted into the medium in transient expression assays.

一种新的钙结合蛋白，命名为网状钙结合蛋白，已被鉴定和鉴定。细胞分级和免疫荧光染色等生化分析表明，网状钙蛋白是内质网的一种腔蛋白，其MR为44,000，编码325个氨基酸的蛋白质，其氨基端信号序列为20个氨基酸。该蛋白含有一个含有高亲和力钙结合基序的结构域的六个重复序列，即EF-Hand。虽然含氧氨基酸对Ca^&lt；2+&gt；的所有六个结构域都是保守的，但其中三个结构域(在II、III和VI位点)缺乏EF-Hand基序中央部分保守的甘氨酸残基。钙印迹显示，重组网状钙结合蛋白在细菌细胞中表达并纯化，在其羧基末端含有His-Asp-Glu-Leu(HDEL)序列。该序列类似于Lys-Asp-Glu-Leu(KDEL)序列，KDEL序列是保留动物细胞内质网中驻留蛋白的信号。体外诱变产生的缺乏HDEL序列的突变蛋白在瞬时表达分析中被分泌到培养基中。

项目成果

Masayuki Ozawa: "Reticulocalbin,a novel endoplasmic reticulum resident Ca^<2+>-binding protein with multiple EF-hand motifs and a carboxyl-terminal HDEL sequence." J.Biol.Chem.268. 699-705 (1993)

Masayuki Ozawa：“Reticulocalbin，一种新型内质网驻留 Ca^2 结合蛋白，具有多个 EF-hand 基序和羧基末端 HDEL 序列。”

Ozawa, M.: "Reticulocalbin, a novel endoplasmic reticulum resident Ca^<2+> -binding protein with multiple EF-hand motifs and a carboxyl-terminal HDEL sequence." J. Biol. Chem. 268. 699-705 (1993)

Ozawa, M.：“Reticulocalbin，一种新型内质网驻留 Ca^2 结合蛋白，具有多个 EF-hand 基序和羧基末端 HDEL 序列。”