Biochemical Studies of Semenogelins with Emphasis on Zinc-Binding Properties

Abstract: Semenogelins I and II (SgI and SgII) are homologous and rapidly evolving proteins that have a repetitive primary structure and are synthesized mainly in the seminal vesicles. Upon ejaculation, a gelatinous coagulum is formed when the semenogelin-rich secretion from the seminal vesicles is mixed with the prostate secretion, which contains high concentrations of zinc(II) and proteases. These proteases, primarily prostate-specific antigen (PSA), begin to cleave the semenogelins, which results in liquefaction of the gel within 10 to 20 minutes. The research findings reported in this thesis show that human SgI and SgII bind zinc(II) with a dissociation constant of 5 µM and a stoichiometry of at least ten ions per protein molecule. A truncated SgI lacking 60 amino acid residues, encoded by a polymorphic SgI-gene, was confirmed to have zinc(II)-binding capacity similar to that of the wild-type protein. Circular dichroism spectroscopy revealed a heat stable secondary structure, and computer-based prediction of the spectra indicated that SgI (wild-type) contains 5?10% alpha-helices and 10?20% beta-sheets. The tertiary structure of SgI is changed upon binding of zinc(II), a property that may affect the ability of SgI to form a gel. Furthermore, it was discovered that the zinc(II)-binding properties of SgI enable direct measurements of the interaction between SgI and zinc(II) by surface plasmon resonance analysis. Besides playing a structural role, zinc(II) binding to the semenogelins may have a regulatory function. It has been suggested that, by modulating the level of free zinc, the semenogelins control the activity of PSA (which is inhibited by zinc(II)). PSA can also be regulated by protein C inhibitor. Thus, a characterization of the binding between SgI and protein C inhibitor in the presence and absence of zinc(II) was carried out.