熱原性鏈球菌外毒素B (Streptococcal pyrogenic exotoxin B, 簡稱SPE B)，它是由化膿性鏈球菌 (Streptococcal pyogenes) 所產生的一種細胞外毒素，根據其催化區的分類，SPE B是屬於一種半光胺酸的蛋白酶 (cysteine protease) 。SPE B是以40kDa的酶原 (Zymogen, ProSPE B) 形式被鏈球菌分泌至細胞外，之後會經由自動催化 (autocatalysis) 或由其他蛋白酶活化形成28kDa具有活性的蛋白酶。過去對於SPE B的研究中指出SPE B對於化膿性鏈球菌在感染宿主時的佔了很重要的角色，它不僅可以分解細胞間質的主成分fibrinectin及vitronectin且作用於interleukin-1β (IL-1β) precuror使其產生活化型的IL-1β而加重發炎反應，並誘導吞噬細胞進行apoptosis及造成其吞噬能力的下降，故SPE B被認為化膿性鏈球菌的重要毒性因子。過去有研究發現在SPE B的活化中心具有兩種胺基酸分別是半光氨酸(Cysteine)和精氨酸 (Histidine) 且其受質具有特異性，為了更深入了解SPE B酵素催化的反應機制，我們利用定點突變的方法構築兩種突變株，Q162N (推測和自動催化機制有關) 和W359A (推測為受質結合殘基)，另外我們在比對兩百多種的SPE B胺基酸序列時發現SPE B在化膿性鏈球菌中具有多型性，而為了解這些多型性的SPE B在化膿性鏈球菌感染宿主過程中所扮演的角色，我們同樣利用定點突變方法構築T137I、D154N、G308S、G308S/A317S、G384D五種突變株，在純化的過程中發現其會自動催化成活化態也就是28kDa的形式，因此我們在純化的過程中加入 2mM氯化汞 (HgCl2)以維持其酶原的形式和抑制其活性。Q162N和W359A皆具有很弱的酵素活性顯示這些胺基酸對於催化的活性是非常重要的。在利用SPE B切割其突變株蛋白C192S時會有八種中間物質，而在Q162N和W359A這兩種突變蛋白其切割C192S只有產生三種中間物，顯示他們也可能參與受質的結合。而在多型性突變株方面，利用特異性受質C192S切割受質的速率如下: pro-SPE B > T137I > G308S > G308S/A317S > D154N > G384D ，他們在切割C192S時的中間物質和SPE B是相似的，表示大部分多型性的SPE B是影響酵素活性而不是改變其受質特異性。只除了G384D，在G384D切割C192S的結果，我們可以看到其似乎沒有太多的中間物，至於是什麼樣的原因造成它的受質特異性改變，目前還不清楚。另外在非特異性的受質偶氮酪蛋白(azocasein)中，實驗結果也顯示多型性SPE B突變株蛋白酵素活性皆比pro-SPE B差。以上結果顯示Q162和W359在催化活性和受質結合扮演重要的角色並可知道由M1型化膿性鏈球菌所分泌的SPE B具有最高的酵素活性。由以上研究讓我們了解到SPE B的分子作用機制和其多型性對其生物功能上影響。

Streptococcal pyrogenic exotoxin B (SPE B) is an extracellular cysteine protease which is secreted by streptococcus pyogenes. SPE B is initially expressed as a 40 kDa zymogen (ProSPE B), and subsequently converted to a 28 kDa active protease (SPE B) by autocatalysis or proteolysis. SPE B also participates in the dissemination, colonization, and invasion of bacteria and the inhibition of wound healing. Since SPE B participates in host-pathogen interactions and is an important virulence factor, it makes it as an attractive therapeutic target. The last decade has witnessed a remarkable change in the epidemiology of group A streptococcal infections. However, there was little basis for understanding the genetic variation in population structure occurring in connection with temporal changes in the frequency or character of streptococcal diseases, such as invasive infections or rheumatic fever outbreaks. In order to investigate the allelic polymorphism and enzyme mechanism of SPE B, seven SPE B mutants have been expressed in E. coli and purified to homogeneity. During the processing of purification, we used 2mM HgCl2 as an inhibitor to obtain the 42kDa zymogen form. Mutants Q162N and W359A nearly lost their protease activity, implicating that these residues are essential for the catalytic activity. It has been shown that digestion of the pro-SPE B C192S mutant by SPE B produced eight intermediates. In contrast, the pro-SPE B C192S mutant cleaved by mutants W359A and Q162N only produced three intermediates. These results suggest that the residues Q162 and W359 maybe involved in the substrate binding. The allelic mutants T137I, D154N, G308S, G308S/A317S and G384D had minor or significant effects on their protease activity. The relative protease activity of these mutants exhibit in the following order: pro-SPE B > T173I > G308S > G308S/A317S > D154N > G384D >> W359A and Q162N. The digestion of the pro-SPE B C192S mutant by these allelic mutants produced eight intermediates that are similar to wild-type SPE B. These suggested that most allelic forms of SPE B don’t change the substrate specificity but affect their protease activity. Taken together, we provide the evidence that the exotoxin SPE B encoded by streptococcus pyogenes M1 strain is the most active protease. The Q162 and W359 may important roles in both catalytic activity and substrate binding. This study will extend our understanding of the molecular mechanism of SPE B and its variants.

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陳秋月 Expression and Characterization of Streptococcal Pyrogenic Exotoxin B 國立成功大學生物化學研究所碩士論文. 1999.

羅世奇 Preparation and Structure Determination of Active Streptococcal Pyrogenic Exotoxin B 國立成功大學生物化學研究所碩士論文.2001.