PURIFICATION AND PARTIAL CHARACTERIZATION OF ACROSOME REACTION INHIBITING GLYCOPROTEIN FROM HUMAN SEMINAL PLASMA

The human acrosome reaction (AR; sperm exocytosis) is absolutely requi red for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human semin al plasma was purified by differential centrifugation, carboxymethyl c ellulose chromatography, chromatofocusing ocusing, and Sephacryl 8300 gel filtration. A highly purified led protein with a molecular weight of 74 000 was obtained as determined by gel filtration and SDS-PAGE. A RIG eluted in a narrow pH range (6.2-5.4) during chromatofocusing, cor responding to a pi of 5.8 +/- 0.4. It had covalent modifications, incl uding internal disulfide bonds, and both complex N-linked and O-linked oligosaccharide chains. Lectin analysis suggested that sialic acid wa s absent and that the complex oligosaccharide chains had sequences con taining galactose, galactosamine, and/or glucosamine in a beta 1-4 lin kage. Mannose residues were also present. When ARIG was added to in vi tro-capacitated human spermatozoa 30 min prior to the calcium ionophor e A23187, the AR was significantly inhibited (ID50 = 8.5 mu g/ml). In addition, ARIG reduced sperm exocytosis in response to atrial natriure tic peptide (a guanylate cyclase activator) and to the protein kinase C activators phorbol myristate acetate and dioctanoylglycerol. The abi lity of ARIG to block the human AR induced by a variety of agonists an d the fact that biological activity of the protein was lost after remo val of its sugar moieties suggests that it may function as a general i nhibitor of sperm exocytosis and that its interaction with spermatozoa may be mediated by carbohydrate-binding proteins on the sperm cell.