鯉魚硫胱胺酸蛋白酶cathepsin Z在卵子成熟過程中的研究

Abstract

此研究從鯉魚卵巢cDNA基因庫中選殖出一個新型的酵素，此酵素從未在鯉魚身上發表，也缺乏功能性的研究，經過比對，發現它屬於papain family家族的一員，與哺乳類的cathepsin Z相類似。cathepsin Z在分類上屬於cysteine protease。經過比對，此酵素有關於活性調控與形成三級結構的重要區域都與其它cysteine proteases類似。 觀察此酵素在鯉魚卵子最後成熟過程中的變化，發現cathepsin Z的mRNA在鯉魚注射GnRH-analogue 的8-12小時後表現量最多，稍後在排卵時又降到未注射前的水準，推測與卵細胞內卵黃的分解有關，也與先前的研究認定卵細胞內蛋白質分解在成熟過程中8-16小時達到最高峰相吻合。 此外，cathepsin Z存在於成熟大卵的表層顆粒與卵黃細胞質附近。在卵子發生表層反應時，會釋放到圍卵腔中，進而形成受精膜的一部分。最後，實驗表現的重組蛋白具有活性，可以分解gelatin，並且可以切割鯉魚的卵黃前質素(vitellogenin)，證明cathepsin Z具有內切酶的特性。

A cDNA encoding cathepsin Z (CTPZ) was cloned from a carp ovarian cDNA library. It is homologous to mammalian CTPZ. The amino acid residues important for protein folding and enzymatic activity of cysteine proteases are conserved in carp CTPZ. During oocyte maturation, cathepsin Z transcripts increased and reached peak at 8 h post-treatment of GnRH analogue by 9-folds compared to that of untreated oocytes, then declined near to the level of untreated oocytes in ovulated eggs. Cathepsin Z was discharged from cortical granules to perivitelline space after cortical reaction and became new component of fertilization envelope. Inside the oocyte, cathepsin Z was stained in the cytoplasm surrounding the yolk granules. The recombinant cathepsin Z has an enzymatic activity toward vitellogenin, and inhibited by PMSF or E-64, a specific inhibitor of thiol protease. The potential roles of cathepsin Z in carp eggs are discussed.