鯉魚Granzyme B之純化及特性

Abstract

Granzyme B是serine proteinase的一員，主要存在於cytotoxic淋巴細胞的分泌顆粒內。此分泌顆粒中含有穿孔素(perforin）、granzymes和其他蛋白分解?。當cytotoxic淋巴細胞和受病原感染的細胞接觸時，分泌顆粒便會釋放內涵物，granzymes和perforin就會進入目標細胞內，引發細胞自殺機制使細胞死亡。在本篇報告中，我們以肝制凝素（Heparin)親和力管柱從鯉魚鰓組織純化到一個還原後分子量約26kDa的蛋白?，經N端15個胺基酸序列比對後，和mature human Granzyme B的N端15個胺基酸序列有七成的相似度。此酵素活性在pH7.5-8.5間最大，由受質專一性判斷為Asp-ase。因此，確認為鯉魚Granzyme B；它是granzymes在硬骨魚類被發現的首例。實驗結果顯示，鯉魚Granzyme B的活性會被高濃度鋅離子（1 mM, 0.5 mM）抑制住。鎘、鎳、錳、鈷離子在1 mM濃度下也具有抑制效果，但作用較鋅離子弱。此外，也發現肝制凝素和Dermatan sulfate(DS）會增強鯉魚Granzyme B的活性，但是無法恢復受到鋅離子抑制的鯉魚Granzyme B活性。組織分佈調查的結果顯示，鯉魚Granzyme B主要出現在頭腎、腎臟和鰓，表示鯉魚Granzyme B可能參與硬骨魚的免疫反應。由上述結果，我們可以推論，Granzyme B的活性會受到高濃度鋅離子及proteoglycans的調控。

Mammalian granzyme B is a neutral serine proteinase found, together with other granzymes and perform, in the exocytotic granules of the cytotoxic lymphocytes. The cytotoxic proteins are released to initiate typical programmed cell death in the target cells when encountered by the cytotoxic lymphocytes. We reported in this thesis the purification and characterization of granzyme B from carp gill. The protein is around 26 kDa as determined by SDS gel electrophoresis and binds strongly in the heparin affinity column chromatography. N-terminal amino acid sequence was determined from the purified protein for up to 15 residues and was identical to human granzyme B at 11 corresponding positions. In addition, the carp proteinase exhibits optimal enzyme activity at pH 7.5 to 8.5 and prefers cutting scissile bonds right after Asp residues. Therefore, we believe that the purified protein is a fish granzyme B and this is the first report on the purification of a teleost granzyme B. In this thesis, we also examined the effects of metal ions and glycosaminoglycans (GAGs) on the activity of gill granzyme B. Zinc ion at 0.5 mM and higher concentrations greatly reduces the enzyme activity of carp granzyme B. Other divalent metal ions such as Cd2+, Ni2+, Mn2+ and Co2+ may also inhibit granzyme B, but to lesser extents. On the contrary, heparin (0.1-100 μg/ml) and dermatan sulfate (DS, 1-1,000 μg/ml) can enhance the enzyme activity of carp granzyme B. However, heparin and DS cannot reactivate the activity of carp granzyme B suppressed by 1 mM Zn2+. The presence of carp granzyme B in different tissues was examined by immunoblotting with a polyclonal antiserum against the purified protein. The carp granzyme B is present mostly in the head kidney, kidney and gill, all of which are immune organs. Therefore, the data suggest that carp granzyme B may be involved in the function of fish immune system. Furthermore, regulation of carp granzyme B involves both proteoglycans and zinc ion.