Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen

Abstract

In semen, the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 mol (mol of protein)(-1) and with an average dissociation constant of approx. 5 microM per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.

Figure 1. Purified semenogelin proteins (SgI and SgII), and seminal plasma separated by SDS/PAGE (12% gel) and (A) visualized by radio-ligand blotting (⁶⁵ZnCl₂) and (B) staining with Coomassie Blue

M1, low-molecular-mass standard stained with Coomassie Blue; M2, low-molecular-mass standard as a part of the radio-ligand blot; SgI, 5 μg of purifed SgI; SgII, 5 μg of purifed SgII; Sem pl liq., 0.4 μl of liquefied seminal plasma; Sem pl nonliq., 0.04 μl of non-liquefied seminal plasma; SgI+PSA, 5 μg of SgI digested by PSA; SgII+PSA, 5 μg of SgII digested by PSA.

Figure 2. Fluorescence of 15 μM FluoZin-2 titrated with Zn²⁺ in the presence of different amounts of intact semenogelins

(A) Zn²⁺ titrations with the following concentrations of SgI: 0 (▲), 0.075 (△), 0.15 (●), 0.30 (○), 0.60 (■) and 1.2 (□) μM. (B) Zn²⁺ titrations with the following concentrations of SgII: 0 (▲), 0.074 (△), 0.14 (●), 0.28 (○), 0.56 (■) and 1.1 (□), μM. (C) Computer fitting to the first nine steps in the 0.30 μM SgI Zn²⁺-titration. With a K_D of approx. 5 μM, the three fitting curves represent a stoichiometry of 1 (…), 10 (−) and 20 (---). (D) Computer fitting to the first nine steps in the 0.28 μM SgII Zn²⁺-titration. With a K_D of approx. 5 μM, the three fitting lines represent a stoichiometry of 1 (…), 12 (−) and 20 (---).

Figure 3. Fluorescence of 15 μM FluoZin-2 titrated with Zn²⁺ in the presence of (A) PSgI(28–37) and (B) PSgI(421–430)

The peptides were used at concentrations of 0 (▲), 8 (△), 16 (●) and 32 (○) μM.

Figure 4. ¹H NMR spectra of PSgI(28–37) and PSgI(421–430) in the presence and absence of Zn²⁺

Figure 5. Gel filtration of the semenogelin peptides PSgI(28–37) (A) and PSgI(421–430) (B) in the presence (---) and absence (−) of Zn²⁺

Figure 6. Activation of Zn²⁺-inhibited PSA by the addition of increasing amounts of different Zn²⁺ binders

The activity was studied using a synthetic chromogenic substrate for PSA. The following Zn²⁺ binders were used: SgI (▲), SgII (△), EGTA (+), PSgI(99–113) (●), PSgI(421–430) (○), PSgI(190–199) (■) and PSgI(28–37) (□).