No abstract

gp20 is a sialoglycoprotein of the human sperm surface with a core peptide homologous to the leukocyte antigen CD52, a GPI-anchored glycosylated protein which is described by the monoclonal antibody CAMPATH-1. Comparative analyses, by means of CAMPATH and anti-gp20, indicated that they describe it in morphologically and functionally different ways, suggesting that the respective epitopes are different but also casting doubt on the immunological identity of the antigen. In the present study, we used immunodepletion to demonstrate that CAMPATH and anti-gp20 interact with the same antigen, but that anti-gp20 has a much higher avidity for the antigen than CAMPATH. Anion exchange fractionation analysis of the antigen revealed three differently charged gp20-CD52 forms, the least charged of which, was largely without a GPI-anchor. All three forms were associated with freshly ejaculated sperm, whereas capacitated sperm only contained the two GPI-anchored, more charged forms, which were also the ones found in the prostasome fraction of seminal plasma and in leukocytes. The two charged, GPI-anchored forms were described as homogeneous by anti-gp20, since they ran as a singlet; the third form ran as a doublet. When tested for insertion into Jurkat T cells, the medium charged form inserted the most readily and the less charged one could not be inserted at all.

Gp20 is a sialylglycoprotein of the human sperm surface related to maturation and capacitation and is homologous to CD52, a glycosyl- phosphatidyl-inositol (GPI)-anchored protein highly expressed in lymphocytes, monocytes, eosinophils, and epididymal cells, described by the monoclonal antibody family CAMPATH. The CAMPATH antigen is characterized by a very short peptide (12 amino acids) and an N-linked oligosaccharide chain bound to the asparagine located in the third position and a GPI anchor bound to the C-terminal serine. The CAMPATH epitope includes three amino acids at the C-terminus and part of the GPI anchor. It has been suggested that anti-gp20 interacts with the same peptide recognized by CAMPATH antibodies but with a different epitope, since it describes the corresponding antigen in a different way. For example, it localizes the corresponding antigen in the equatorial region of the sperm head when sperm are capacitated, whereas CAMPATH antibodies bind all over the sperm surface. Our results indicate that the anti-gp20 epitope does not include the peptide backbone, the GPI anchor, or the N-glycans but consists of O-linked oligosaccharide chains bound to a unique CD52 glycoform present both in sperm and leukocytes. This is suggested by results obtained using many different approaches, such as immunoblot analysis of gp20 after removal of N- and O-glycans and after jacalin (Artocarpus integrifolia agglutinin)-affinity chromatography.

CD52 is a human glycosylphosphatidylinositol (GPI)-anchored antigen exclusively expressed in leukocytes and epididymal cells. It is also present in sperm, being inserted in their plasma membrane as they pass through the epididymis. In a previous paper we identified a new CD52 form without GPI anchor by fast performance liquid chromatography (FPLC) fractionation of semen components. The form has a lower negative charge than the GPI-anchored form and occurs as the only CD52 form in prostasome-free seminal plasma. It was also found associated with the ejaculated sperm, but in contrast to the GPI-anchored one, it is lost during the capacitation process. In this paper we indicate that (1) the GPI-anchored CD52 of the sperm surface serves as receptor for semenogelin I during clot formation, (2) liquefaction involves cleavage of the GPI anchor from certain CD52 molecules, releasing sperm from the clot and the soluble antigen bound to semenogelin fragments into the seminal plasma and (3) the clot is a sponge-like structure housing sperm. Soluble CD52 was immunopurified from the soluble CD52-containing FPLC fraction using CAMPATH-1G and was found to be complexed with a semenogelin-derived peptide of the carboxyl terminal portion of semenogelin I, having the sequence SQTEKLVAGKQI and starting from amino acid 376. Immunoprecipitation and immunoblot analyses using CAMPATH-1G and anti-semenogelin as immunoprecipitating antibodies and anti-gp20 and anti-semenogelin as immunoblot detectors of the corresponding antigens, confirmed that the soluble CD52 formed a complex with semenogelin. The semenogelin-CD52 soluble form was found to be a direct consequence of the liquefaction process since only the GPI-anchored CD52 was recovered in uniquefied semen after recovering sperm and seminal plasma by urea solubilization of the clot.

In a previous article, we suggested that gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk Unio elongatulus has functional carbohydrate epitopes in common with a human zona pellucida glycoprotein, probably ZP3. We demonstrated that: 1) anti-gp273-purified immunoglobulin G (IgG), which recognizes a carbohydrate gp273 epitope including a Lewisa-like structure, interacts with a zona pellucida protein; 2) human sperm specifically bind to gp273; and 3) binding is reversed by anti-gp273 IgG. In the present study, we confirm this suggestion by demonstrating that heat-solubilized zonae pellucidae reverse gp273-human sperm binding, that gp273-binding sites are restricted to the acrosomal region, and that gp273 induces the acrosome reaction in human sperm. We also demonstrated that gp273-binding sites on human sperm function as signaling receptors because exposure of spermatozoa to this glycoprotein results in significant stimulation of protein kinase C (PKC) activity. Because the PKC inhibitor, bisindolylmaleimide I, reverses both PKC activation and the acrosome reaction, this kinase is a key component of the signal transduction pathway activated by gp273 and leading to the exocytotic event.