Comparative Characterization of the Purified a-L-fucosidases that occur in the Human Sperm Plasma Membrane versus the Seminal Plasma.
1S. Khunsook, 2J.A. Alhadeff, 1B.S. Bean.Departments of 1Biological Sciences and 2Chemistry, Lehigh University, Bethlehem, PA.
This investigation identified the biochemical differences between the purified
a-L-fucosidases of the human sperm membrane and those of the seminal plasma.
Methods were developed for purification of the a-L-fucosidases of the seminal plasma and thea-L-fucosidases of the sperm cell membrane. Semen specimens were obtained from informed volunteers following approved human subjects protocols.Purification to near homogeneity was accomplished for the abundant seminal plasma a-L-fucosidase by centrifugation, dialysis, affinity chromatography on a substrate analog resin, and ultrafiltration.Purification to near homogeneity for the sperm membrane associated a-L-fucosidase was accomplished by selective detergent extraction, dialysis, affinity chromatography, and ultrafiltration.Purified enzyme preparations were analyzed by isoelectric focusing with and without neuraminidase treatment, gel filtration on Sephadex G-200, SDS-PAGE, Western analysis, differential lectin blotting, studies on enzyme kinetics, and pH activity profiles.
Several important differences were found between a-L-fucosidases of the sperm plasma membrane that distinguish it from the notably more abundant seminal fluid enzyme.The extracted a-L-fucosidase of the sperm plasma membrane appears as multimers of a single 51 kDa glycoprotein subunit that does not contain sialic acid, and contains a single membrane-associated hydrophobic region per subunit.The a-L-fucosidase of the seminal plasma appears in several multimeric forms of a 56 kDa glycoprotein subunit that does contain terminal sialic acid residues, and is a soluble enzyme with little or no affinity for lipid materials.Compared with the human liver (lysosomal) enzyme, both seminal a-L-fucosidases demonstrate broad pH optima with notable activity extending through the acid and neutral ranges.Both seminal fluid and sperm a-L-fucosidases have Km values near 0.07 mM with 4-methylumbelliferyl-a-L-fucopyranoside as substrate, suggesting a higher affinity for fucosylated glycoconjugates than seen for the liver enzyme (0.22 mM).
Human semen contains a large amount of a-L-fucosidase activity, the great majority of which is found in the seminal plasma while a small amount is present on the sperm plasma membrane. Comparative characterization of unpurified human seminal fluid and sperm a-L-fucosidases indicates a broad pH optimum between pH 4-7.4 with the maxima at pH 6.2 and 4.6 for seminal fluid fucosidase while sperm fucosidase hasmajor optima at pH 7.3, 6.1, and 4.4. Isoelectric focusing indicates that seminal fluid a-L-fucosidase contains five to seven isoforms with pIs between ~5-7 while sperm fucosidase contains two isoforms with closely-spaced pI values of 6.7 and 6.9. Western blot analysis indicates that the seminal fluid fucosidase contain a major protein band of 56 kDa while sperm fucosidase contains a major protein band of 51 kDa.
The seminal fluid a-L-fucosidase has been purified 18,000-fold to very high purity and specific activity (80,000 nmol/min/mg protein). The purified a-L-fucosidase appears to contain a single subunit of 56-57 kDa (as determined by SDS-PAGE and Western blot analysis).By gel filtration, purified seminal fluid a-L-fucosidase appear to contain three multimeric forms of 110, 236, and 314 kDa, respectively and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl-a-L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0-4.5) with a second neutral optimum (pH 6.4-7.4) with 60-80 % of maximal activity. Apparent KM and Vmax values for the 4MU-Fuc were determined to be 0.06±0.004 mM and 92±7.5 ?mol/min/mg protein, respectively, using Lineweaver-Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment, and lectin blotting studies indicated that the purified seminal plasma a-L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5-7. The acidic isoforms between pI values 5-6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.
The sperm membrane-associated a-L-fucosidase has been purified 8,600-fold to a specific activity of 35,000 nmol/min/mg protein. Isoelectric focusing, neuraminidase treatment, and lectin blotting studies indicated that the purified sperm a-L-fucosidase is not a sialoglycoprotein. The purified sperm a-L-fucosidase appears to consist of a single subunit of 51 kDa, contains two multimeric forms of 230 and 86 kDa and therefore probably exists in tetrameric and dimeric forms. Kinetic analysis with the 4MU-Fuc substrate indicated a broad pH optimum in the neutral range (pH 6.8-7.3) but with retention of 80% of maximal activity at pH 5.5-6 and 77% at pH 4-4.5. Apparent KM and Vmax values for the 4MU-Fuc substrate of the sperm a-L-fucosidase were determined to be 0.08±0.04 mM and 6.8±1.32 ?mol/min/mg protein respectively, using Lineweaver-Burk double reciprocal plots.
N-glycanase treatment studies indicated that both purified human seminal fluid and sperm plasma membrane-associated a-L-fucosidases contain N-linked carbohydrate