PURIFICATION AND CHARACTERIZATION OF 2 FORMS OF BETA-D-GALACTOSIDASE FROM RAT EPIDIDYMAL LUMINAL FLUID - EVIDENCE FOR THEIR ROLE IN THE MODIFICATION OF SPERM PLASMA-MEMBRANE GLYCOPROTEIN(S)

Previous studies from this laboratory have identified rat epididymal l uminal fluid acid beta-D-galactosidase activity which also optimally h ydrolyses a glycoprotein substrate at neutral pH [Skudlarek, Tulsiani and Orgebin-Crist (1992) Biochem. J. 286, 907-914]. We have now separa ted the luminal fluid P-D-galactosidase into two molecular forms by io n-exchange chromatography on a column of DE-52. The separated enzyme a ctivities were purified to an apparent homogeneity by molecular-sieve chromatography followed by affinity chromatography on a column of immo bilized 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-de pendent 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 co ntained much more total carbohydrate and sialic acid than the 84 kDa f orm. The carbohydrate moieties in the two forms were assessed by compa ring their size on SDS/PAGE before and after treatment with endo-enzym es, The removal of N-linked glycans by treatment with N-glycanase or e ndoglycosidase F generated de-N-glycosylated polypeptides of an appare nt molecular mass of 70 kDa, and indicated that the two forms containe d varying amounts of asparagine (N)-linked high mannose/hybrid-type an d biantennary complex-type oligosaccharides. This result and the fact that the two molecular forms had identical N-terminal amino acid seque nces indicated that the two forms probably have identical or very simi lar polypeptides, The potential role of the enzyme in modification of sperm plasma membrane (PM) glycoproteins was examined by resolving cap ut sperm PM proteins (before and after treatment in vitro of the membr anes with the purified beta-D-galactosidase) on SDS/PAGE, followed by staining with peanut agglutinin (PNA), a lectin which preferentially b inds to Gal beta 1,3GalNAc-linkages found in O-linked glycoproteins. T he 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 digestio n in vitro of the membranes with purified luminal fluid beta-D-galacto sidase. This result suggests a possible role for the epididymal lumina l fluid beta-D-galactosidases.