Functional Activity of Human ZP3 Primary Sperm Receptor Resides Toward Its C-Terminus
Functional Activity of Human ZP3 Primary Sperm Receptor Resides Toward Its C-Terminus
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DOI：
10.1095/biolreprod.108.074716
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发表时间：
2009-07-01
影响因子：
3.6
通讯作者：
Gupta, Satish K.
中科院分区：
文献类型：
--
作者：
Bansal, Pankaj;Chakrabarti, Kausiki;Gupta, Satish K.
透明带糖蛋白3（ZP 3）被认为是人类受精过程中的初级精子受体。在此，已经尝试描绘人ZP 3的功能结构域。ZP 3已被克隆并在杆状病毒表达系统中表达为N-末端片段（氨基酸[aa]残基1-175 [pAc-ZP 3（（1-175）（aa））]和23-175 [pBg-ZP 3（（23-175）（aa））]）和C-末端片段（aa残基214-305 [pBg-ZP 3（（214-305 aa））]和214-348 [pBg-ZP 3（（214-348 aa））]）。还表达了包含对应于氨基酸残基1-370的N-和C-末端片段的ZP 3（pAc-ZP 3（[1-370 aa]））。这些重组蛋白的凝集素结合分析显示存在N-和O-连接的糖基化。当获能精子与pBg-ZP 3（（214-348 aa））、pBg-ZP 3（（214-305 aa））和pAc-ZP 3（（1-370 aa））孵育时观察到显著的顶体胞吐诱导（P < 0.05），而与pAc-ZP 3（（1-175 aa））和pBg-ZP 3（23-175 aa））孵育在类似的实验条件下不能这样做。然而，N-和C-末端片段标记的异硫氰酸荧光素显示结合获能的人精子的前头部。大肠杆菌表达的ZP 3 C-末端片段和化学去糖基化的pBg-ZP 3（（214-348 aa））未能诱导顶体胞吐的显著增加（P < 0.05），表明糖基化在赋予ZP 3 C-末端片段功能活性中的相关性。pBg-ZP 3（（214-348 aa））介导的顶体胞吐诱导受G（i）蛋白、细胞外钙、GABA（A）[γ-氨基丁酸（A）]受体介导的Cl-通道和T型电压操纵的钙通道调节。总之，这些研究的结果表明，人ZP 3的功能活性存在于其C-末端结构域。
Zona pellucida glycoprotein 3 (ZP3) has been ascribed as a putative primary sperm receptor during fertilization in humans. Herein, attempts have been made to delineate the functional domain of human ZP3. ZP3 has been cloned and expressed in a baculovirus expression system as N-terminal fragments (amino acid [aa] residues 1-175 [pAc-ZP3((1-175) (aa))]and 23-175 [pBg-ZP3((23-175) (aa))]) and as C-terminal fragments (aa residues 214-305 [pBg-ZP3((214-305 aa))] and 214-348 [pBg-ZP3((214-348 aa))]). ZP3 encompassing both N- and C-terminal fragments corresponding to aa residues 1-370 (pAc-ZP3([1-370 aa])) has also been expressed. Lectin-binding analysis with these recombinant proteins revealed the presence of N- and O-linked glycosylation. Significant induction of acrosomal exocytosis was observed when capacitated sperm were incubated with pBg-ZP3((214-348 aa)), pBg-ZP3((214-305 aa)), and pAc-ZP3((1-370 aa)) (P < 0.05), whereas incubation with pAc-ZP3((1-175 aa)]) and pBg- ZP3((23-175 aa)) failed to do so under similar experimental conditions. However, N- and C-terminal fragments labeled with fluorescein isothiocyanate revealed binding to the anterior head of capacitated human spermatozoa. Escherichia coli-expressed ZP3 C-terminal fragments and chemically deglycosylated pBg- ZP3((214-348 aa)) failed to induce a significant (P < 0.05) increase in acrosomal exocytosis, suggesting the relevance of glycosylation in imparting functional activity to ZP3 C-terminal fragments. pBg-ZP3((214-348 aa))-mediated induction of acrosomal exocytosis is regulated by G(i) protein, extracellular calcium, GABA(A) [gamma aminobutyric acid (A)] receptor-mediated Cl- channel, and T-type voltage-operated calcium channels. Taken together, the results of these studies suggest that the functional activity of human ZP3 resides in its C-terminal domain.