Study of Protein Cross-Links in Copulatory Plughe Major TG4 Cross-linking Sites of SVS I in mouse Seminal Vesicle
Date Issued
2009
Date
2009
Author(s)
Tseng, Huan-Chin
Abstract
Resolution of mouse seminal vesicle secretion (SVS) by reducing SDS-PAGE can clearly identified seven major monomer proteins tentatively designated as SVS I-VII according to the decreasing order of their molecular masses. However, no monomer forms of SVS I-III are present in SVS. Instead, they appear in various high molecular weight complexes (HMWCs) formed by inter-polypeptide disulfide bridges among SVS I, SVS II and SVS III. The HMWCs become the predominant protein in SVS. We were able to immunodetect SVS I-III in cross sections of mouse copulatory plug. Heating the clotted lump in SDS-PAGE sample buffer in the presence of DTT released none of SVS I or SVS II but only a trace of SVS III into the solution, indicative of covalent cross-links in addition to disulfide bonds among the SVS I-III proteins in the plug. Further, we purified type 4 transglutaminase (TG4) from the mouse coagulating gland to homogeneity, and compared it to type 2 transglutaminase (TG2) from guinea pig liver in terms of enzymatic ability to cross-link proteins in the SVS. We found that TG4 showed much greater activity than TG2 in catalyzing the cross-links between HMWC proteins. On the contrary, it was less active than TG2 in cross-linking any of the free SVS I-III proteins released from the reduced HMWC. The reproductive significance of more efficient TG4 catalysis is discussed.ased on the SVSI-deduced protein sequence, SVS I contains 820 amino acid residues in which there are 43 glutamine and 43 lysine 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 TG4. F2 was much more active than the other fragments during the BPNH2-glutamine incorporation, Relatively, a low extent of A25 cross-linking to any one of the seven polypeptide fragments was observed. 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 three F2 mutants, including Q232G and Q254G obtained from single-site mutation, and Q232G/Q254G from double-site mutation.
Subjects
seminal vesicle
coagulating gland
transglutaminase
protein cross link
seminal coagulation
copulatory plug
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