Identification of the major TG₄ cross‐linking sites in the androgen‐dependent SVS I exclusively expressed in mouse seminal vesicle

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 (BPNH(2)) or biotin-TVQQEL (A25 peptide) to each of F1-to-F7 by type 4 transglutaminase (TG(4)) from the coagulating gland secretion. F2 was active to a greater extent than the other fragments during the BPNH(2)-glutamine incorporation, and a relatively low extent of A25-lysine cross link was observed with all of the seven fragments. The MS analysis of BPNH(2)-F2 conjugate identified Q(232) and Q(254) as the two major TG(4) cross-linking sites. This was substantiated by the result that much less BPNH(2) 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.

Copulatory plugs inhibit the reproductive success of rival males

“…A male's ejaculate coagulates to form a copulatory plug inside the female reproductive tract in many different taxa, including nematodes (Abele & Gilchrist, ; Barker, ; Palopoli et al ., ), arachnids (Austad, ; Masumoto, ; Uhl et al ., ), insects (Parker, ; Dickinson & Rutowski, ; Orr & Rutowski, ; Rogers et al ., ), reptiles (Devine, ; Herman, ; Friesen et al ., ) and mammals (Devine, ; Martan & Shepherd, ; Voss, ; Williams‐Ashman, ; Dixson & Anderson, ). In rodents, this coagulation occurs when a prostate‐derived transglutaminase, encoded by the gene transglutaminase IV ( Tgm4 ), cross‐links seminal vesicle‐derived secretions upon mixing in the female's reproductive tract (Notides & Williams‐Ashman, ; Williams‐Ashman et al ., ; Fawell & Higgins, ; Esposito et al ., ; Lundwall, ; Lundwall et al ., ; Lin et al ., ; Tseng et al ., , ; Dean, ; Kawano et al ., ).…”

Seminal vesicle secretory protein 7, PATE4, is not required for sperm function but for copulatory plug formation to ensure fecundity†

“…We also found this peptide sequence in SVS2, but the sequence is not conserved in PATE4 (Supplemental Figure S3B). SVS1 also does not contain the sequence “QXK(S/T)”, but Tseng et al showed that two glutamine residues in SVS1 were the major site for TGM4 crosslinking by mass spectrometry (Supplemental Figure S3A) [8]. As PATE4 also has four glutamine and ten lysine residues (Supplemental Figure S3), our results suggest that PATE4 may have an unidentified target sequence for TGM4 or a function to promote plug formation independent from TGM4.…”

Physiological function of seminal vesicle secretions on male fecundity

“…Although SVS1 does not contain the sequence “QXK(S/T),” Tseng et al . showed that two glutamine residues in SVS1 (Q 232 and Q 254 ) were the major site for TGM4 cross‐linking by mass spectrometry . Recently, we reported that prostate and testis expression 4 (PATE4; also known as SVS7) is the essential factor for copulatory plug formation with Pate4 KO mice (Figure and Table ) .…”

“…In copulatory plug formation, it is thought that transglutaminase 4 (TGM4) from prostates and coagulating glands catalyze the formation of ε‐(γ‐glutamyl)lysine cross‐bridges among seminal vesicle secretion 1 (SVS1) to SVS3 (Table ) . In fact, previous papers showed that single KO males of Tgm4 or Svs2 are subfertile due to plug formation defects .…”