PURIFICATION AND MICROSEQUENCING OF THE INTRAACROSOMAL PROTEIN-SP-10 - EVIDENCE THAT SP-10 HETEROGENEITY RESULTS FROM ENDOPROTEOLYTIC PROCESSES
PURIFICATION AND MICROSEQUENCING OF THE INTRAACROSOMAL PROTEIN-SP-10 - EVIDENCE THAT SP-10 HETEROGENEITY RESULTS FROM ENDOPROTEOLYTIC PROCESSES
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DOI：
10.1095/biolreprod47.1.11
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发表时间：
1992-07-01
影响因子：
3.6
通讯作者：
FLICKINGER, CJ
中科院分区：
文献类型：
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作者：
HERR, JC;KLOTZ, K;FLICKINGER, CJ
人精子抗原SP-10已被证明是睾丸特异性的顶体内蛋白，其与顶体囊泡的膜和基质相关。用SP-10单克隆抗体进行蛋白质印迹分析的精子提取物显示SP-10肽的异质性范围为17.5-34 kDa。尽管从SP-10 cDNA测序中推导出了265个氨基酸（28.3kDa）的整个SP-10氨基酸序列，但对多个SP-10肽带的性质还不完全了解。在这项研究中，我们开发了一个三步纯化SP-10肽的方法，使用单克隆抗体亲和层析，反相高效液相色谱法，和制备凝胶电泳。通过该方案分离并使用Edman降解测序的8个SP-10肽显示对应于推断的SP-10氨基酸序列上的区域的氨基末端。表观质量逐渐降低的肽进一步向羧基末端排列。基于SP-10序列上的假定切割位点，预测作用于五个不同肽键的内切蛋白酶切割SP-10：这些内切蛋白酶在精氨酸（胰蛋白酶样蛋白酶，可能是顶体蛋白酶）之后水解，并且在丝氨酸、脯氨酸、甘氨酸和谷氨酸（先前未描述的顶体内蛋白酶特异性）之后水解。本研究1）提供了SP-10肽的纯化方法; 2）证实了先前测序的SP-10 cDNA编码真实的SP-10;和3）产生了内切蛋白酶有助于SP-10异质性的间接证据。
The human sperm antigen SP-10 has been shown to be a testis-specific, intra-acrosomal protein that is associated with the membranes and matrix of the acrosomal vesicle. Sperm extracts, analyzed on Western blots with a monoclonal antibody to SP-10, have shown heterogeneity of SP-10 peptides ranging from 17.5-34 kDa. Although the entire SP-10 amino acid sequence of 265 amino acids (28.3 kDa) has been deduced from sequencing SP-10 cDNAs, the nature of multiple SP-10 peptide bands is incompletely understood. In this study we developed a three-step purification method for SP-10 peptides using monoclonal antibody affinity chromatography, reverse-phase HPLC, and preparative gel electrophoresis. Eight SP-10 peptides separated by this protocol and sequenced using Edman degradation showed amino termini that corresponded to regions on the deduced SP-10 amino acid sequence. Peptides with progressively lower apparent mass aligned further toward the carboxy terminus. On the basis of putative cleavage sites on the SP-10 sequence, endoproteases that act at five different peptide bonds are predicted to cleave SP-10: these hydrolyze following arginine (a trypsin-like protease, possibly acrosin), and following serine, proline, glycine, and glutamic acid (previously undescribed intra-acrosomal protease specificities). The present studies 1) provide a purification method for SP-10 peptides; 2) confirm that the SP-10 cDNAs previously sequenced encode authentic SP-10; and 3) yield indirect evidence that endoproteases act to contribute to SP-10 heterogeneity.