Characterization of PH-30, a protein with a role in sperm-egg fusion

Membrane fusion, both intracellular and extracellular, is vital to all cells. Our working hypothesis is that cellular membrane fusion reactions, like the viral counterparts, are protein mediated. Although the process of viral membrane fusion is mediated by specific well characterized proteins, to date no known bona fide cellular membrane fusion protein has been identified. This dissertation focuses on the guinea pig sperm protein PH-30, which is a good candidate to be a cellular membrane fusion protein mainly because one monoclonal antibody that binds PH-30 inhibits sperm-egg fusion, whereas a second does not.; Biochemical characterization of PH-30 has revealed that PH-30 shares features in common with viral membrane fusion proteins. I showed that PH-30 is an integral membrane glycoprotein composed of two tightly associated and immunologically distinct subunits that are both made as larger precursors. The final processing step of PH-30, which exposes an epitope recognized by the fusion inhibitory monoclonal antibody, coincides with the acquisition of fertilization competence. These results are discussed in terms of the possible role of PH-30 in mediating sperm-egg fusion.; The cloning and sequencing of both mature subunits of PH-30 has lent further support to the proposal that PH-30 is involved in membrane fusion. The N-terminal domain of PH-30 {dollar}beta{dollar} was found to be highly homologous to a family of RGD containing snake venom peptides that competitively antagonize platelet aggregation by binding to the platelet integrin IIb/IIIa. We therefore propose that PH-30 {dollar}beta{dollar} binds to an integrin on the egg plasma membrane. The {dollar}alpha{dollar} subunit of PH-30 contains a hydrophobic domain with features similar to viral fusion peptides. Both subunits are class I integral membrane glycoproteins. Thus, like many viral fusion proteins, PH-30 is predicted to be a complex of a protein possessing binding activity {dollar}(beta){dollar} and a protein possessing fusion activity {dollar}(alpha){dollar}.; Finally I present evidence for a sperm protease with a possible role in sperm maturation, and for at least two accessory proteins of yet unknown function that copurify with immunoaffinity purified PH-30.