Journal of Andrology
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Published-Ahead-of-Print May 28, 2009, DOI:10.2164/jandrol.108.007146

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Identification of Acrosomal Matrix-Specific Hydrolases Binding Proteins of Bovine Cauda Epididymal Spermatozoa

Subir K Nagdas *, Shatasha L Hamilton , and Samirsubas Raychoudhury

* To whom correspondence should be addressed. E-mail: snagdas{at}uncfsu.edu.

Previously, we described the isolation of a detergent-stable complex from bovine sperm acrosome, termed the outer acrosomal membrane-associated matrix complex (OMC). This stable matrix assembly is associated with the luminal surface of the outer acrosomal membrane and exhibits specific binding activity for acrosin. The present study was undertaken to identify the matrix proteins that specifically interact to acrosomal hydrolases. The OMC fraction exhibited polypeptides of 54, 50, and 45kDa and a major polypeptide family between 38-19kDa by SDS-PAGE. In this study, we purified 45kDa polypeptide, termed OMC45 from the high-pH insoluble fraction of OMC and the polyclonal antibody was raised against 45kDa polypeptide. Anti-OMC45 polyclonal antibody reacts strongly on immunoblots with the OMC45 band. Using immunofluorescence anti-OMC45 localizes specifically to the acrosomal cap. Two-dimensional PAGE and immunoblot analysis of OMC identified a set of approximately 5~6 isoelectric variants of 45 kDa in the pH range of 5.5-7.2. To identify matrix-specific hydrolase-binding proteins, OMC32 (32kDa polypeptide isolated from high pH soluble fraction of OMC) and OMC45 polypeptides were coupled to AminoLink Plus resin separately and incubated with soluble acrosomal hydrolases. Acrosin and N-acetylglucosaminidase bound the OMC32 polypeptide in a concentration-dependent fashion. In contrast, OMC45 polypeptide exhibited stronger affinity to acrosin than N-acetylglucosaminidase. The binding specificity of acrosomal matrix proteins to hydrolases strongly suggests that the matrix polypeptides play an important role in the regulation of hydrolases release during the acrosome reaction and could also function during acrosome assembly to target and/or segregate hydrolases within the acrosome interior.


Key words: Epididymis • Sperm • acrosomal hydrolases • acrosomal matrix • acrosome • protein-protein interaction






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