Foster J A, Friday B B, Maulit M T, Blobel C, Winfrey V P, Olson G E, Kim K S, Gerton G L
Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6080, USA.
J Biol Chem. 1997 May 9;272(19):12714-22. doi: 10.1074/jbc.272.19.12714.
The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in acrosomal matrix-zona pellucida interactions during and immediately following the acrosome reaction in the mouse.
豚鼠精子顶体基质是顶体的致密核心，可能在顶体生物发生和受精过程中起重要作用。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，分离出的顶体基质由数量有限的主要条带组成，其中有一个分子量为67,000的蛋白质，我们将其命名为AM67。间接免疫荧光显示，AM67定位于附睾尾精子顶体的顶端部分。免疫电子显微镜进一步将AM67的定位细化到顶体内的M1（背侧隆起）结构域。利用基于AM67胰蛋白酶肽序列的聚合酶链反应产物，筛选了一个λgt11豚鼠睾丸cDNA文库，得到两个编码AM67肽的cDNA克隆。Northern分析表明，AM67转录为一种1.9千碱基的睾丸特异性mRNA。完整的AM67序列编码一个由533个氨基酸组成的前原多肽，计算分子量为59,768。在可能的信号序列被切割后，该多肽预计分子量为56,851，并具有7个天冬酰胺连接糖基化的共有位点。AM67推导的氨基酸序列与小鼠精子蛋白sp56以及来自各种哺乳动物物种的补体成分4结合蛋白的α亚基最为相似。尽管据报道小鼠sp56是鼠透明带糖蛋白ZP3的细胞表面受体，但使用抗sp56单克隆抗体7C5进行的标准免疫电子显微镜检测在小鼠精子顶体内检测到了sp56，但在顶体完整的小鼠精子表面未检测到sp56。此外，在用多聚甲醛固定制备用于免疫荧光的小鼠精子中检测到了顶体标记，但在未固定的活精子中未观察到。因此，sp56存在于顶体内这一发现进一步支持了sp56和AM67是直系同源物，并表明sp56可能在小鼠顶体反应期间及之后立即在顶体基质-透明带相互作用中发挥作用。
