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(Streptococcus pyogenes)所產生的一種細胞外毒素，根據催化區分析將它歸屬於半光胺酸蛋白酶的一種。SPE B會經過自動催化(autocatalysis)或蛋白酶分解(proteolysis)作用，將40 kDa的酶原(zymogen form SPE B簡稱zSPE B或ProSPE B)去掉118個胺基酸的propeptide變成28 kDa活化態的蛋白酶(簡稱mSPE B)。SPE B在In vitro下可以分解細胞間質的主成份fibronectin及vitronectin、作用於interleukin-1β (IL-1β) precursor並產生活化型的IL-1β而加重發炎反應、誘導吞噬細胞進行apoptosis及降低其吞噬能力等，故SPE B被認為是化膿性鏈球菌的重要毒性因子而成為極具吸引力的治療標的。
為瞭解SPE B結構與功能間的相互關係，我們將SPE B和其突變蛋白分別送入大腸桿菌系統誘發蛋白表現，經過Ni+2-chelate affinity 管柱純化，再利用質譜儀來確認該蛋白的分子量，結果顯示實驗值完全符合計算值的分子量。SPE B在純化過程中會自動轉換成28 kDa的活化態形式，為了瞭解由酶原轉變成活化態的機制，我們成功地用1mM汞離子當抑制劑純化出酶原形式的ProSPE B。接著進行其酵素活性分析，結果發現：SPE B的活化機制會藉由分子間與分子內的自動催化作用而達成。
另一方面，為了知道SPE B對不同受質的專一性，有必要針對酶原與活化態SPE B之間的結構差異做比較。由於ProSPE B的突變蛋白(C192S)已有X-ray的三維結構，於是我們將焦點放在活化態SPE B和其抑制劑(E-64)的複合體溶液結構。目前利用同位素標定蛋白(2H,13C,15N triple- and 13C,15N double-labeled samples)完成數個多維異核核磁共振光譜實驗如：HNCA, HNCOCA, HNCACB, CBCACONH, 15N-edited NOESY- and TOCSY-HSQC等，然後再進行圖譜解析與氨基酸的循序判定(sequential assignment)。此外，為了提供更多資訊做為判定時的參考，我們也使用專一標定的胺基酸來純化SPE B，以得知特定胺基酸在HSQC圖譜上的位置（例如已完成15N-labelled Gly, Ala, Val, Leu, Met, Phe, Tyr, Lys, Gln, Asp, Ile等的HSQC圖譜），而目前我利用這些圖譜已完成大約95 %活化態熱原性鏈球菌外毒素B的氨基酸循序判定。近來由於抗生素的濫用，導致許多致病細菌產生抗藥性，本研究的結果將可做為設計屬於SPE B專一性藥物 (rational drug design)的基礎。

Streptococcal pyrogenic exotoxin B (SPE B) is an extracellular cysteine protease produced by the pathogenic bacterium streptococcus pyogenes. SPE B is initially expressed as a 40 kDa zymogen (ProSPE B), and subsequently converted to 28 kDa active protease (mSPE B) by autocatalysis or proteolysis. SPE B was shown in vitro to cleave human fibronectin and to degrade vitronectin. It also participates in the dissemination, colonization, and invasion of bacteria and the inhibition of wound healing. This suggests that SPE B serves as an important virulence factor in streptococcal infections, making it an attractive therapeutic target.
In order to investigate the structure and function relationships of SPE B, we expressed SPE B and its mutants in E. coli and purified them to homogeneity. The molecular weights of these proteins were confirmed by mass spectrometry. Their observed values are in excellent agreement with the calculated values. Recombinant ProSPE B was expressed as a 42 kDa zymogen and converted to a 28 kDa SPE B during the course of purification. In this study we obtained the ProSPE B by adding 1mM HgCl2 as inhibitor in solution to inhibit the autoprocessing of ProSPE B. We also observed that the maturation of ProSPE B involves seven intermediates with a combination of intra- and inter-molecular processings. To elucidate the diverse substrate specificities of SPE B, it is essential to compare the structural differences between ProSPE B and mature SPE B. Since the 3D structure of the C192S mutant of ProSPE B has been determined by X-ray diffraction, we used NMR spectroscopy to study the solution structure of the complex of mature SPEB and cysteine protease inhibitor E-64. The 1H, 13C, and 15N resonances of SPE B were assigned by 3D heteronuclear HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, and 15N-edited NOESY- and TOCSY-HSQC experiments with 2H/13C/15N- and 13C/15N-labeled samples. In addition, we obtained the 1H-15N HSQC spectra of selective 15N-labeled amino acids (Gly, Ala, Val, Leu, Met, Phe, Tyr, Lys, Gln, Asp, and Ile) to facilitate the sequential assignment. Up to now, these experiments provided about 95 % backbone assignment of 28 kDa C192S SPE B.

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