Identification of the major TG4 cross-linking sites in the androgen-dependent SVS I exclusively expressed in mouse seminal vesicle.
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.
Tseng
Huan-Chin
HC
Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan.
Lin
Han-Jia
HJ
Tang
Jyh-Bing
JB
Gandhi
P S Sudhakar
PS
Chang
Wei-Chao
WC
Chen
Yee-Hsiung
YH
D000588:Amines / Q000494:pharmacology; D000595:Amino Acid Sequence; D000728:Androgens / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D055162:Biocatalysis / Q000187:drug effects; D001710:Biotin / Q000031:analogs & derivatives / Q000494:pharmacology; D019921:Biotinylation / Q000187:drug effects; D003062:Codon / Q000235:genetics; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012669:Seminal Vesicles / Q000187:drug effects* / Q000201:enzymology*; D011503:Transglutaminases / Q000378:metabolism*
D000588:Amines; D000728:Androgens; D003062:Codon; D003432:Cross-Linking Reagents; D008562:Membrane Glycoproteins; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C521511:SVS-1 protein, mouse; C076022:5-(biotinamido)pentylamine; D001710:Biotin; D012343:RNA, Transfer; C418702:transglutaminase 4; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins