In a previous study, evidence was provided for the presence of a novel plasma-membrane-associated neutral-pH-optimum alpha-L-fucosidase in rat sperm. In the present study, rat sperm alpha-L-fucosidase was characterized during epididymal maturation. The pH 7 activity optimum of alpha-L-fucosidase and its subunit composition (one or two closely spaced immunoreactive protein bands of about 53+/-2 kDa) did not appear to change during transit through the epididymis. Isoelectric focusing of alpha-L-fucosidase indicated the presence of a major isoform (B) with a pI near 7 in sperm from testis, caput, corpus and the proximal half of the cauda. alpha-L-Fucosidase from sperm from the distal half of the cauda, which contained a significant enrichment of sperm and alpha-L-fucosidase activity, contained isoform B and an additional minor isoform (A) with a pI near 5.2. Isoform B and small amounts of isoform A were present in sperm from the vas deferens. The two fucosidase isoforms present in sperm from the distal cauda were separated by isoelectric focusing and comparatively characterized. They had similar pH-activity curves (with optima near pH 7) and comparable apparent KM values (0.4+/-0.04 mM) for 4-methylumbelliferyl alpha-l-fucopyranoside. Preincubation of the isoforms at different temperatures indicated that isoform A is considerably more thermostable than isoform B. Immunoprecipitation studies using polyclonal antibodies against human liver alpha-L-fucosidase indicated that approx. 90% of the enzymic activity for both isoforms was immunoprecipitable under conditions that immunoprecipitated essentially all the human liver enzyme. Neuraminidase treatment of sperm alpha-L-fucosidase from distal cauda (when compared with the appropriate heat-treated control) led to disappearance of isoform A and a concomitant increase in isoform B. The overall results suggest that isoform A is derived by sialylation of isoform B near the end of epididymal maturation.
1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum alpha-L-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for alpha-L-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the alpha-L-fucosidase activity was associated with the 48,000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of alpha-L-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH-activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm-egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm-egg interactions.
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