Characterization and functional analysis of phospholipase C-zeta, a candidate of mammalian egg-activating sperm factor

哺乳动物卵子激活精子因子候选磷脂酶 C-zeta 的表征和功能分析

• 批准号: 16390055
• 负责人: MIYAZAKI Shunichi
• 金额: $ 9.41万
• 依托单位: Tokyo Women's Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390055/
• 关键词: Fertilization; Intracellular Calcium Ion; Mammalian Eggs; Calcium Oscillation; Cytosolic Sperm Factor; Egg-activating Protein; Phospholipase C-zeta; Nuclear transpotation abity; 精子因子; ホスフォリパーゼCゼータ; 核移行

Repetitive transient increase in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) called Ca^<2+> oscillations occur at fertilization of mammalian eggs, and trigger egg activation. Each [Ca^<2+>]_i rise is due to Ca^<2+> release from the endoplasmic reticulum mainly through inositol 1,4,5-trisphosphate (IP_3) receptors. Evidence indicates that Ca^<2+> oscillations are caused by cytosolic sperm factor driven into the ooplasm upon sperm-egg fusion. The Ca^<2+> oscillation-inducing factor is the egg-activating protein (EAP). A current strong candidate of EAP is a novel isozyme of IP_3-producing enzyme, phospholipase C-zeta (PLCζ). This study aims to characterize PLCζ and confirm whether PLCζ operates as EAP at fertilization. The following results were obtained. 1) Injection of recombinant PLCζ that was synthesized by baculovirus/Sf9 cell expression system induced Ca^<2+> oscillations in mouse eggs at low concentrations. 2) PLCζ has such a high Ca^<2+>-sensitivity of PLC activity that the … More enzyme can be active in resting cells at 〜100 nM [Ca^<2+>]_i, suitable for a putative EAP to be introduced into the egg at fertilization. 3) The N-terminal EF-hand domain 1 (EF1) and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca^<2+> sensitivity. 4) C2 domain has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P. 5) Ca^<2+> oscillations were induced by injection of RNA encoding PLCζ fused with a fluorescent protein "Venus". The expressed PLCζ corresponded to the estimated amount contained in a single spermatozoon. 6) Expressed PLCζtranslocated into the formed pronucleus. 7) The nuclear localization signal was located at the X-Y linker region of PLCζ molecule. A highly coordinated three-dimensional structure involving a folding in the middle is thought to be required for not only Ca^<2+> oscillation-inducing activity but also nuclear translocation ability. These results support PLCζ as a strong candidate of EAP. To confirm whether PLCζ operates as EAP at fertilization, PLCζ-knockout mice were produced, and PLCζ-transgenic mice were prepared for reserve PLCζ in the knockout mice sperm. Less

哺乳动物卵子受精时细胞内Ca^2+浓度（[Ca^2+] i）的反复瞬时增加称为Ca^2+振荡，并触发卵子激活。[Ca^<2+>]_i的每一次升高都是由于Ca^<2+>主要通过三磷酸肌醇（IP_3）受体从内质网释放。有证据表明，Ca^<2+>振荡是由精卵融合时胞质精子因子进入卵质引起的。Ca^2+振荡诱导因子是卵子激活蛋白（EAP）。一种新的产生IP_3的同工酶--磷脂酶C-zeta（PLC β）是目前EAP的有力候选者。本研究的目的是描述PLC的特点，并确认PLC是否作为EAP在受精。获得了以下结果。1)用杆状病毒/Sf 9细胞表达系统合成的重组PLC基因，在低浓度下注射小鼠卵细胞可引起Ca^2+振荡。2)PLC的Ca^<2+>敏感性很高， ...更多信息 酶在静息细胞中的[Ca^2+] i为100 nM时具有活性，适合于在受精时将推定的EAP引入卵中。3)N端EF手结构域1（EF 1）和EF 2对PLC的Ca 2+活性具有重要作用，EF 3是其高Ca^2+敏感性的原因。4)C2结构域对PI（3）P有很强的亲和力，对PI（5）P也有较弱的亲和力。5）通过注射编码与荧光蛋白“Venus”融合的PLC β的RNA诱导Ca^<2+>振荡。所表达的PLC酶量对应于单个精子中所含的估计量。6)表达的PLC可转位到形成的原核中。7)细胞核定位信号位于PLC γ分子的X-Y连接区。一个高度协调的三维结构，包括一个折叠在中间被认为是所需要的不仅钙振荡诱导活性，而且核转位能力。这些结果支持PLC作为EAP的强有力的候选者。为了确认PLC β在受精时是否作为EAP起作用，产生PLC β敲除小鼠，并制备PLC β转基因小鼠以在敲除小鼠精子中保留PLC β。少

项目成果

Recombinant phospholipase C-zeta has high Ca^<2+> sensitivity and induces Ca^<2+> oscillations in mouse eggs

重组磷脂酶C-zeta具有高Ca^<2>敏感性并诱导小鼠卵子中Ca^<2>振荡

• 发表时间: 2004
• 期刊: Journal of Biological Chemistry 279
• 作者: Kouchi Z;Fukami K;Shikano T;Oda S;Nakamura Y;Takenawa T;Miyazaki S
• 通讯作者: Miyazaki S

Measuremenr of intracellular IP_3 during Ca^<2+> oscillations in mouse Eggs with GTP-based FRET probe

使用基于 GTP 的 FRET 探针测量小鼠卵子 Ca^<2> 振荡过程中的细胞内 IP_3

• 发表时间: 2006
• 期刊: Biochemical and Biophysical Research Communications 345
• 作者: Shirakawa;H.;Ito;M.;Sato;M.;Umezawa;Y.;Miyazaki;S
• 通讯作者: S

The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase Cζ, a Mammalian Egg-activating Factor*

• DOI: 10.1074/jbc.m603473200
• 发表时间: 2006-09
• 期刊: Journal of Biological Chemistry
• 影响因子: 4.8
• 作者: K. Kuroda;Masahiko Ito;T. Shikano;T. Awaji;A. Yoda;H. Takeuchi;K. Kinoshita;S. Miyazaki

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

EF-hand 结构域和 C2 结构域在磷脂酶 Cz 酶活性调节中的作用

• 发表时间: 2005
• 期刊: J.Biol.Chem. 280
• 作者: Kouchi;Z.;et al.
• 通讯作者: et al.

The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca^<2+> oscillation-inducing activities of phospholipase C a mammalian egg-activating factor

X/Y 连接区和 N 端 EF-hand 结构域在核转位和哺乳动物卵激活因子磷脂酶 C 的 Ca^2 > 振荡诱导活性中的作用

• 发表时间: 2006
• 期刊: Journal of Biological Chemistry 281
• 作者: Kuroda;K.;Ito;M.;Shikano;T.;Awaji;T.;Yoda;A.;Takeuchi;H.;Kinoshita;K.;Miyazaki;S.
• 通讯作者: S.