Mountjoy Jeremi R, Xu Wei, McLeod Dave, Hyndman David, Oko Richard
Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L3N6.
Biol Reprod. 2008 Aug;79(2):223-32. doi: 10.1095/biolreprod.107.065060. Epub 2008 Apr 9.
The perinuclear theca (PT) of mammalian sperm is a unique subcellular structure encapsulating the nucleus. Compositionally, the PT is made up of at least six prominent polypeptides (60, 36, 31, 28, 24, and 15 kDa), of which only two have been sequence identified, as well as many less prominent ones. As an ongoing process in unveiling the protein composition of the PT, we have uncovered the sequence identity of the prominent 24-kDa polypeptide (PT24). Initial N-terminal sequence analysis obtained by Edman degradation suggested that PT24 is a RAB2 protein. This was corroborated by mass spectrometric analyses of trypsin-digested fragments of PT24, identifying RAB2A of the RAB2 subfamily as the best sequence match. Quadrapole/time-of-flight analysis identified 72%% sequence coverage between PT24 and bull, human, mouse, or rabbit RAB2A. Since a genome search only identified two RAB2 subfamily members, RAB2A and RAB2B, the 72%% sequence coverage of PT24 provides assurance that this protein is RAB2A and not a new RAB2 subfamily member. Furthermore, commercial RAB2A antibodies, raised against oligopeptide fragments in the unique C-terminal region of RAB2A, specifically labeled PT24 on Western blot analysis of PT extracts. These anti-RAB2A antibodies, along with immune serum that we raised and affinity purified against isolated PT24, demonstrated at both light and electron microscope levels that RAB2 is associated with the periphery of the growing proacrosomic and acrosomic vesicles in the Golgi and cap phases of spermiogenesis and consequently assembled as part of the PT. This pattern of subacrosomal assembly is reminiscent of the pathway used by SubH2Bv (PT15), another prominent and exclusive subacrosomal protein, indicating a common route for subacrosomal-PT assembly. Traditionally somatic RAB2 proteins are involved in vesicular transport between the endoplasmic reticulum and the cis-side of the Golgi apparatus. Our study suggests an unprecedented direction of RAB2A-mediated vesicular transport in spermatids during acrosomal biogenesis, from the trans-side of the Golgi apparatus to the nuclear envelope.
哺乳动物精子的核周鞘（PT）是一种包裹细胞核的独特亚细胞结构。在组成上，核周鞘由至少六种主要多肽（60、36、31、28、24和15 kDa）组成，其中只有两种已被测序鉴定，还有许多不太显著的多肽。作为揭示核周鞘蛋白质组成的一个持续过程，我们已经确定了主要的24 kDa多肽（PT24）的序列同一性。通过埃德曼降解进行的初始N端序列分析表明PT24是一种RAB2蛋白。对PT24胰蛋白酶消化片段的质谱分析证实了这一点，确定RAB2亚家族的RAB2A是最佳序列匹配。四极杆/飞行时间分析确定PT24与牛、人、小鼠或兔RAB2A之间的序列覆盖率为72%。由于基因组搜索仅鉴定出两个RAB2亚家族成员，即RAB2A和RAB2B，PT24的72%序列覆盖率确保了该蛋白是RAB2A而不是一个新的RAB2亚家族成员。此外，针对RAB2A独特C端区域的寡肽片段产生的商业RAB2A抗体，在对PT提取物的蛋白质印迹分析中特异性标记了PT24。这些抗RAB2A抗体，以及我们针对分离的PT24产生并亲和纯化的免疫血清，在光学显微镜和电子显微镜水平上均显示，RAB2与精子发生的高尔基体期和帽期正在生长的前顶体囊泡和顶体囊泡的周边相关联，并因此作为核周鞘的一部分组装起来。这种顶体下组装模式让人联想到另一种主要且独特的顶体下蛋白SubH2Bv（PT15）所采用的途径，表明顶体下 - 核周鞘组装存在一条共同途径。传统上，体细胞RAB2蛋白参与内质网和高尔基体顺面之间的囊泡运输。我们的研究表明，在顶体生物发生过程中，RAB2A介导的囊泡运输在精子细胞中出现了前所未有的方向，即从高尔基体反面到核膜。
