Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, VIC 3800, Australia.
Biol Cell. 2009 Nov 16;102(2):93-106. doi: 10.1042/BC20090099.
CRISP2 (cysteine-rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca2+ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2-binding partner, which we have designated SHTAP (sperm head and tail associated protein).
Using yeast two-hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2-binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (approximately 20-87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the approximately 26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two-hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related-1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri-acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co-localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP-CRISP2 complex appeared to be redistributed within the head.
The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP-CRISP2 complex play a role in the attainment of sperm functional competence.
CRISP2（富含半胱氨酸的分泌蛋白 2）是一种精子顶体和尾部蛋白，具有调节肌醇 1,4,5-三磷酸受体（ryanodine receptor）中钙离子流动的能力。基于这些特性，CRISP2 通过调节顶体反应和精子运动中的离子信号，在受精中具有潜在作用。本研究旨在确定一种新的 CRISP2 结合伴侣 SHTAP（精子头部和尾部相关蛋白）的表达、亚细胞定位及其在精子发生中的作用。
通过对成年睾丸表达文库的酵母双杂交筛选，我们鉴定出 SHTAP 是一种新的小鼠 CRISP2 结合伴侣。对所有 Shtap cDNA 克隆的序列分析表明，小鼠 Shtap 基因嵌入在编码不相关蛋白 NSUN4（NOL1/NOP2/Sun 结构域家族成员 4）的基因中。在公共数据库中注释了 Shtap 基因的五个直系同源物。SHTAP 及其直系同源物与任何已知的蛋白质或功能基序（包括 NSUN4）没有显著的序列相似性。使用 SHTAP 抗血清，在睾丸、精子和各种体组织中检测到多个 SHTAP 同工型（约 20-87 kDa）。有趣的是，只有大约 26 kDa 的 SHTAP 同工型能够与 CRISP2 相互作用。此外，酵母双杂交试验表明，CRISP2 与 SHTAP 的最大结合需要 CAP（CRISP/抗原 5/发病相关蛋白 1）和 CRISP 结构域。SHTAP 蛋白定位于圆形精子细胞的顶体周围区域，以及伸长精子的头部和尾部，在那里它与 CRISP2 共定位。在精子获能期间，SHTAP 和 SHTAP-CRISP2 复合物似乎在头部内重新分布。
本研究首次报道了小鼠 Shtap 基因的鉴定、注释和表达分析。在精子获能过程中观察到的重新分布提出了 SHTAP 和 SHTAP-CRISP2 复合物在获得精子功能能力方面发挥作用的可能性。
