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Purification and characterization of rat epididymal-fluid alpha-D-mannosidase: similarities to sperm plasma-membrane alpha-D-mannosidase.

ABSTRACT: We have previously reported the occurrence and partial characterization of a novel alpha-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and Orgebin-Crist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar alpha-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12% recovery to a state approaching homogeneity using: (1) (NH4)2SO4 precipitation; (2) affinity chromatography on immobilized mannan and D-mannosamine; (3) ion-exchange (DE-52) column chromatography; (4) molecular-sieve chromatography. The enzyme was eluted from the final column (Sephacryl S-400) at an apparent molecular mass of 460 kDa. When resolved by SDS/PAGE (under denaturing conditions), the enzyme showed a major protein band (115 kDa) and few very minor bands. The polyclonal antibody raised against the major protein band was found to cross-react with the alpha-D-mannosidase activity present in epididymal fluid (soluble) and detergent-solubilized spermatozoa from the rat and mouse. This result suggested that the soluble and membrane-bound enzyme activities shared a common antigenic site(s). The antibody was used to characterize further the alpha-D-mannosidase activity(ies) present in the rat epididymal fluid and rat sperm plasma membranes. Data from these studies show that the two forms are similar in (a) subunit molecular mass, (b) substrate specificity and (c) inhibitory effect of several sugars. These similarities suggest that the soluble and membrane-bound alpha-D-mannosidase activities are isoforms. Immunoprecipitation studies after solubilization of the testis and epididymal particulate fraction from sexually immature rats show that the testis (but not the epididymis) contains the immunoreactive alpha-D-mannosidase activity. This result and the fact that spermatozoa from the rat rete testis show alpha-D-mannosidase activity indicate that the sperm enzyme is synthesized in the testis during spermatogenesis.

SUBMITTER: Tulsiani DR

PROVIDER: S-EPMC1132291 | biostudies-other | 1993 Mar

REPOSITORIES: biostudies-other

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Project description:Previous studies from this laboratory have identified rat epididymal luminal fluid acid beta-D-galactosidase activity which also optimally hydrolyses a glycoprotein substrate at neutral pH [Skudlarek, Tulsiani and Orgebin-Crist (1992) Biochem. J. 286, 907-914]. We have now separated the luminal fluid beta-D-galactosidase into two molecular forms by ion-exchange chromatography on a column of DE-52. The separated enzyme activities were purified to an apparent homogeneity by molecular-sieve chromatography followed by affinity chromatography on a column of immobilized p-nitrophenyl beta-D-thiogalactopyranoside. The purified forms, when resolved by SDS/PAGE under reducing conditions, showed apparent molecular masses of 84 and 97 kDa. Kinetic studies, including a pH-dependent substrate preference and pH-dependent association/dissociation, disclosed no differences between these two forms. The two forms had identical N-terminal amino acid sequences. However, the 97 kDa form contained much more total carbohydrate and sialic acid than the 84 kDa form. The carbohydrate moieties in the two forms were assessed by comparing their size on SDS/PAGE before and after treatment with endo-enzymes. The removal of N-linked glycans by treatment with N-glycanase or endoglycosidase F generated de-N-glycosylated polypeptides of an apparent molecular mass of 70 kDa, and indicated that the two forms contained varying amounts of asparagine (N)-linked high mannose/hybrid-type and biantennary complex-type oligosaccharides. This result and the fact that the two molecular forms had identical N-terminal amino acid sequences indicated that the two forms probably have identical or very similar polypeptides. The potential role of the enzyme in modification of sperm plasma membrane (PM) glycoproteins was examined by resolving caput sperm PM proteins (before and after treatment in vitro of the membranes with the purified beta-D-galactosidase) on SDS/PAGE, followed by staining with peanut agglutinin (PNA), a lectin which preferentially binds to Gal beta 1,3GalNAc-linkages found in O-linked glycoproteins. The evidence presented in this report has indicated that a PNA-positive glycoprotein of an apparent molecular mass of 135-150 kDa present on the caput (but not cauda) sperm PM is degalactosylated by the digestion in vitro of the membranes with purified luminal fluid beta-D-galactosidase. This result suggests a possible role for the epididymal luminal fluid beta-D-galactosidases.

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Purification and characterization of rat epididymal-fluid alpha-D-mannosidase: similarities to sperm plasma-membrane alpha-D-mannosidase.

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