|Abstract: ||Abstract:SVS I was exclusively expressed in seminal vesicle in which the protein was immunolocalized primarily to the luminal epithelium of mucosal folds. The developmental profile of its mRNA expression was shown to be androgen-dependent, manifesting a positive correlation with the animal's maturation. There are 43 glutamine and 43 lysine residues in one molecule of SVS I, which is one of the seven major monomer proteins tentatively assigned on reducing SDS–PAGE during the resolution of mouse seminal vesicle secretion. Based on the fact that SVS I-deduced protein sequence consists of 796 amino acid residues, we produced 7 recombinant polypeptide fragments including residues 1–78/F1, residues 79–259/F2, residues 260–405/F3, residues 406–500/F4, residues 501–650/F5, residues 651–715/F6, and residues 716–796/F7, and measured the covalent incorporation of 5-(biotinamido)pentylamine (BPNH2) or biotin-TVQQEL (A25 peptide) to each of F1-to-F7 by type 4 transglutaminase (TG4) from the coagulating gland secretion. F2 was active to a greater extent than the other fragments during the BPNH2-glutamine incorporation, and a relatively low extent of A25-lysine cross link was observed with all of the seven fragments. The MS analysis of BPNH2-F2 conjugate identified Q232 and Q254 as the two major TG4 cross-linking sites. This was substantiated by the result that much less BPNH2 was cross-linked to any one of the three F2 mutants, including Q232G and Q254G obtained from single-site mutation, and Q232G/Q254G from double-site mutation.
A25 peptide, Biotin-TVQQEL; BPNH2, 5-(biotinamido)pentylamine; CGS, coagulating gland secretion; DTT, dithiothreitol; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; GST, glutathione S-transferase; HMWC, high molecular weight complex, PBST, phosphate buffer saline containing 0.1% Tween 20; PMSF, phenylmethylsulphonyl fluoride; SVS, seminal vesicle secretion; TG4, type IV transglutaminase.
Seminal vesicle and coagulating gland (anterior prostate) are major male accessory apparatus in many mammalian species. Upon ejaculation, seminal vesicle secretion (SVS) and coagulating gland secretion (CGS) are accumulated in the lumen of the respective reproductive glands. SVS makes up the major portion of plasma in the semen which is coagulated in a substantial number of mammalian species, including many myomorphic rodents, some moles, hedgehogs, marsupials, rabbits, stallions, boars, and several primates [Williams-Ashman, 1984]. The deposition of semen coagulum in animals such as rodents into the vagina at coitus results in the formation of a copulatory plug that occludes the vaginal barrel close to the uterine cervix. Despite that the physiological utilization of semen coagulum in mammalian reproduction is not yet well defined, its importance should not be overlooked, taking into consideration that extirpation of seminal vesicle and/or the coagulating gland from mice and rats prevents the copulatory plug formation resulting in greatly reduced fertility [Pang et al., 1979; Peitz and Olds-Clarke, 1986]. It is generally believed that the cross links among the SVS proteins by transglutaminase (TG; EC184.108.40.206), a Ca2+-dependent enzyme that catalyzes the formation of an isopeptide bond between its protein substrate through ε-(γ-glutamyl) lysine cross-bridges [Folk, 1980], is crucial for the formation of a semisolid gelatinous mass in human semen [Aumuller and Riva, 1992; Peter et al., 1998] or the seminal clotting in rodent semen [Harris et al., 1990; Lundwall et al., 1997]. The key step to initiate the TG-catalyzed protein cross link is thought to be the transformation of a glutamine residue on one protein molecule by deamination to an activated acyl group which facilitates the nucleophilic attack by a ε-amino group of a lysine residue on the other protein molecule [Lorand and Graham, 2003].
Because rodents have proven to be good experimental animals for the molecular study of mammalian reproduction, attempts have been made with some progress on the systematic analysis of the mouse SVS proteins that had been shown to consist of several minor proteins such as SVA [Huang et al., 1999, 2000, 2005], P12 [Chen et al., 1998; Luo et al., 2004; Lin et al., 2006b], Ceacam 10 [Li et al., 2005], and seven well-resolved monomer proteins designated SVS I-VII in the decreasing order of Mr values (95,000–8,000) according to their mobility on reduced SDS–PAGE [Chen et al., 1987; Luo et al., 2001]. Although TG of guinea pig liver (TG2) had been shown to cross-link mouse SVS I-III [Lundwall et al., 1997; Lin et al., 2002], in reality this is not the enzyme involved in seminal coagulation during natural coitus. Rather, the activity of TG4, which has been recently purified from mouse CGS [Tseng et al., 2008], is responsible for this reproductive event. Here, we present data to support that: (i) the androgen-dependent SVS I is exclusively expressed in the luminal epithelium of seminal vesicle; (ii) Gln232 and Gln254 are the major TG4-reactive residues on SVS I and (iii) TG4 is much stronger than TG2 to cross-link the glutamine residues in SVS I.