小鼠SVSⅢ蛋白的轉麩胺醯胺酶交聯作用區中五個QXK(S/T)重複序列的基質活性

Abstract

轉麩胺醯胺酶(Transglutaminase, TGase)在鈣離子存在的條件下，可以使蛋白質中的離胺酸(Lys, K)和麩胺酸(Gln, Q)進行交聯，形成Lys和Gln之間的isopeptide bond。小鼠儲精囊內的SVSⅢ蛋白質，由265個胺基酸所組成，其中第108-145胺基酸具有5個QXKS/T的重複片段 (X代表脂肪類胺基酸Val、Leu和Ile)，為轉麩胺醯胺酶交聯作用區。已知Lys緊鄰的氮端的胺基酸為脂肪性，而碳端為親水性，符合文獻對TGase基質Lys的歸納。因此這五個QXKS/T序列與TGase的反應能力就決定於另一個基質Gln。因為每個序列的Gln碳端緊鄰的胺基酸都是脂肪性，緊鄰Gln之氮端胺基酸將為影響其作為基質能力的關鍵。 爲了要研究Gln之氮端胺基酸為親水性或疏水性，對於TGase反應能力的影響，依QXKS/T氮端為親水性(Ser和Thr)或疏水性(Ala和Gly)胺基酸，以基因重組技術將SVSⅢ中之QSQIKSQTQVKS和AQLKSQPGQLKT融合於GST，分別命名為F1和F2。比較F1和F2與TGase反應的能力而知，F1較易被TGase催化交聯。利用基因重組的技術，將QSQIKSQTQVKS聯結於表現載體，再利用建構的載體表現exotoxin第27-278 的胺基酸序列和R19L(P12的突變蛋白，將P12蛋白的第19個胺基酸Arg用Leu取代)，命名爲LEX-R19L。將LEX-(R19L)經TGase交聯後，可以加強R19L的免疫抗原性。這項研究證實了利用酵素法來製備多倍體交聯蛋白質是可行的。

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.