Moncrief J S, Obiso R, Barroso L A, Kling J J, Wright R L, Van Tassell R L, Lyerly D M, Wilkins T D
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0305.
Infect Immun. 1995 Jan;63(1):175-81. doi: 10.1128/iai.63.1.175-181.1995.
During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.
在过去十年中，产生肠毒素的脆弱拟杆菌菌株已被认为与动物和人类的腹泻病有关。细胞外肠毒素已被纯化，并被鉴定为一种单一多肽（相对分子质量约为20,000）。使用单特异性引物PCR克隆了脆弱拟杆菌肠毒素基因的一部分。通过酶联免疫吸附测定和免疫印迹分析，克隆基因片段表达的重组蛋白与针对脆弱拟杆菌肠毒素的单特异性抗体发生反应。推导的氨基酸序列揭示了一种特征性的锌结合共有基序（HEXXHXXGXXH/甲硫氨酸转折），这是金属蛋白酶（称为金属锌蛋白酶）的特征。序列比较显示在锌结合和甲硫氨酸转折区域与基质金属蛋白酶（如人成纤维细胞胶原酶）有密切的同一性。纯化的肠毒素每分子含有1克原子的Zn2+，并能水解明胶、偶氮胶原、肌动蛋白、原肌球蛋白和纤维蛋白原。肠毒素还会进行自身消化。两种自身消化产物的N端氨基酸序列与重组肠毒素推导的氨基酸序列相同，并显示在半胱氨酸-亮氨酸和丝氨酸-亮氨酸肽键处发生切割。通过凝胶分级分离和酶谱分析，明胶酶（IV型胶原酶）活性与毒素迁移一致，表明蛋白酶活性归因于肠毒素而非污染的蛋白酶。最佳蛋白水解活性发生在37℃和pH 6.5。确定了肌动蛋白中的主要蛋白水解切割位点，显示在甘氨酸-甲硫氨酸和苏氨酸-亮氨酸肽键处发生切割。酶活性受到金属螯合剂的抑制，但不受其他类蛋白酶抑制剂的抑制。此外，乙酰氧基甲基酯EDTA抑制了肠毒素对人癌HT-29细胞的细胞毒性活性。肠毒素的金属蛋白酶活性提示了一种可能的肠毒性机制，并且可能在脆弱拟杆菌引起的疾病研究中有其他意义。
