Bhattacharya Rupa, Devi Manju S, Dhople Vishnu M, Jesudasan Rachel A
Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India.
BMC Cell Biol. 2013 Nov 20;14:50. doi: 10.1186/1471-2121-14-50.
Acrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done.
One of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice.
We have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study.
顶体蛋白在受精生理过程中发挥着关键作用。鉴定定位于顶体的蛋白是理解其对受精作用的基础。仍不断有新的顶体蛋白被报道。为了捕获尚未报道的定位于顶体尤其是精子整体的蛋白，对小鼠精子蛋白进行了二维聚丙烯酰胺凝胶电泳（2D-PAGE）和质谱分析。
上述研究中鉴定出的一个蛋白点在NCBI数据库中被报道为来自日本理化学研究所cDNA 1700026L06的假定蛋白，定位于2号染色体。使用针对重组蛋白的兔抗体制备的抗体进行免疫荧光研究表明，该蛋白定位于小鼠顶体和精子尾部。基于该蛋白的定位，将其命名为小鼠顶体和精子尾部蛋白（MAST，[Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]）。该蛋白与大鼠精子细胞特异性蛋白RSB66有96%的同源性。蛋白质印迹法显示MAST在睾丸中特异性表达。使用MAST抗体进行的免疫共沉淀研究鉴定出两种钙结合蛋白，钙树突蛋白和钙网蛋白为MAST的相互作用伴侣。钙树突蛋白和钙网蛋白基因分别定位于小鼠5号和8号染色体。在一只Yq缺失突变小鼠中，该小鼠生育力低下且Y染色体长臂的三分之二缺失，MAST未能定位于顶体。然而，蛋白质印迹分析显示野生型和突变型小鼠睾丸中MAST的表达量相同。Yq缺失小鼠精子中MAST免疫沉淀复合物中存在的顶体钙结合蛋白上调。
我们鉴定出一种小鼠顶体蛋白MAST，其在睾丸中特异性表达。MAST不包含任何已知的蛋白相互作用基序；然而它与定位于顶体的钙结合蛋白形成复合物。Yq缺失小鼠中复合物中鉴定出的所有蛋白的错误表达引发了关于由Y染色体调控的假定途径的假说。然而，从当前研究中尚不清楚Y染色体在该复合物调控中的作用。
