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Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads is comprised of 54, 50, 45, and 38–19kDa polypeptides. The objective of this study is to identify and to characterize the 45kDa (OMC45) polypeptide and to define its role in binding acrosomal hydrolases and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI–TOF–TOF yielded 8 peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X–100–permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti–OMC45 and anti–TEKT3. The OMC45 polypeptide was solubilized by RIPA (radio-immunoprecipitation assay) buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti–OMC45 immunoprecipitation pellet. An identical blot stained with anti–TEKT3 exhibited the presence of TEKT3 polypeptide in the anti–OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide; that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N–linked and O–linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N–acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid membrane complex.

Rabbit polyclonal antibodies were raised against beta-glucuronidase purified from mouse liver. This antiserum immunoprecipitated the beta-glucuronidase secreted by mouse fibroblasts but did not cross-react with the same enzyme isolated from human tissue. The beta-glucuronidase present in mouse 3T3 fibroblasts and mouse peritoneal macrophages was clearly identified by indirect immunofluorescence, using the antiserum and an FITC-conjugated second antibody, while human fibroblasts with normal levels of beta-glucuronidase activity did not fluoresce when tested with the same reagents. A range of human fibroblasts, human neuroblastoma and rat glioma cells did not fluoresce when incubated with the antibody but did fluoresce after they had been co-cultured for 24 h with mouse macrophages, showing that mouse beta-glucuronidase had been transferred from adherent macrophages into adjacent recipient cells. Transfer took place even when receptor-mediated endocytosis was blocked with a suitable competitive ligand, the transferred enzyme being visible mainly as a bright punctate fluorescence with a lysosome-like distribution. Macrophages thus have the potential to act as donors of lysosomal enzymes to a wide range of recipient cells and to transfer enzymes to them during direct cell-to-cell contact.

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