Dissecting the substrate activity of tandem repeats QXK(S/T) in the transglutaminase-catalytic site of mouse SVSⅢ
Date Issued
2006
Date
2006
Author(s)
Yen, Hsiang-Yu
DOI
zh-TW
Abstract
Mouse seminal vesicle secretion protein SVSⅢ is a glycoprotein containing 265 amino acid residues, in which residues 108-145 is compound of 5 tandem repeats of an oligopeptide QXK(S/T), where X represents an aliphatic amino acid residue. This region has previously demonstrated as a transglutaminase (TGase)-catalyzed site in the formation of an ε-(γ-glutamyl) lysine bond. This work was conducted to assess the impact of amino acid residue on the NH2 side of Q on the TGase substrate activity of QXK(S/T).
We prepared a recombinant polypeptide of GST fused to either QSQIKSQTQVKS (F1) or AQLKSQPGQLKT (F2), and assayed their TGase substrate activity. We found that both F1 and F2 but not GST alone could be cross-linked by TGase. The enzyme cross-linked rate of F1was much faster than that of F2, stronger manifestation of QSQIKSQTQVKS than AQLKSQPGQLKT as a good TGase substrate. Meanwhile, a truncated protein that contains residues 27-278 in the exotoxin from Pseudomonas aeruginosa in which V48 is mutated to E was fused to R19L, a mutated P12 in which R19 of the parent protein is replaced by L. This recombinant protein was tentatively designated as EX-R19L. Another recombinant protein LEX-R19L, was prepared by ligation of QSQIKSQTQVKS to EX-R19L, and it was cross-linked by TGase. Female mice were immunized with EX-(R19L)2, LEX-R19L or the TGase-crosslinked LEX-R19L. Among the three antigens, the last one generated the antisera which contained the highest titer of antibody against R19L.
Subjects
轉麩胺醯胺酶
交聯作用區
substrate activity
transglutaminase
mouse SVSⅢ
Type
other
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