Purification and partial characterization of acrosome reaction inhibiting glycoprotein from human seminal plasma.
The human acrosome reaction (AR; sperm exocytosis) is absolutely required for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human seminal plasma was purified by differential centrifugation, carboxymethyl cellulose chromatography, chromatofocusing, and Sephacryl S300 gel filtration. A highly purified protein with a molecular weight of 74,000 was obtained as determined by gel filtration and SDS-PAGE. ARIG eluted in a narrow pH range (6.2-5.4) during chromatofocusing, corresponding to a pl of 5.8 +/- 0.4. It had covalent modifications, including internal disulfide bonds, and both complex N-linked and O-linked oligosaccharide chains. Lectin analysis suggested that sialic acid was absent and that the complex oligosaccharide chains had sequences containing galactose, galactosamine, and/or glucosamine in a beta 1-4 linkage. Mannose residues were also present. When ARIG was added to in vitro-capacitated human spermatozoa 30 min prior to the calcium ionophore A23187, the AR was significantly inhibited (ID50 = 8.5 micrograms/ml). In addition, ARIG reduced sperm exocytosis in response to atrial natriuretic peptide (a guanylate cyclase activator) and to the protein kinase C activators phorbol myristate acetate and dioctanoylglycerol. The ability of ARIG to block the human AR induced by a variety of agonists and the fact that biological activity of the protein was lost after removal of its sugar moieties suggests that it may function as a general inhibitor of sperm exocytosis and that its interaction with spermatozoa may be mediated by carbohydrate-binding proteins on the sperm cell.
Drisdel
R C
RC
Department of Biochemistry, Rush University, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA.
Mack
S R
SR
Anderson
R A
RA
Zaneveld
L J
LJ
D000177:Acrosome / Q000187:drug effects* / Q000502:physiology*; D000581:Amidohydrolases / Q000494:pharmacology; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000032:analysis; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000187:drug effects; D006023:Glycoproteins / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology*; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012661:Semen / Q000737:chemistry*; D013094:Spermatozoa / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002241:Carbohydrates; D004075:Diglycerides; D006023:Glycoproteins; C044295:1,2-dioctanoylglycerol; D000001:Calcimycin; D009320:Atrial Natriuretic Factor; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium