Schmidt K H, Gerlach D, Wollweber L, Reichardt W, Mann K, Ozegowski J H, Fleischer B
Institute of Experimental Microbiology, Friedrich Schiller University, Jena, Federal Republic of Germany.
Infect Immun. 1995 Dec;63(12):4569-75. doi: 10.1128/iai.63.12.4569-4575.1995.
M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein.
化脓性链球菌的M蛋白是毒力因子，可阻碍吞噬作用，与多种血浆蛋白结合，并诱导交叉反应性自身免疫抗体的形成。最近，有报道称，一些用胃蛋白酶从链球菌中提取的M蛋白（pep M）是超抗原。其中之一，pep M5，得到了详细研究，结果表明它能刺激带有Vβ2、Vβ4和Vβ8的人T细胞。在本研究中，我们通过不同的生化方法从两株A组5型链球菌菌株中提取并纯化了M5蛋白。粗提物通过亲和色谱和离子交换色谱进行分级分离。所有级分都通过免疫印迹法平行检测M蛋白，并检测其T细胞刺激活性。尽管几种M5蛋白的粗制品对Vβ2和Vβ8 T细胞具有促有丝分裂活性，但无论采用何种提取方法，M5蛋白都能纯化到不再具有促有丝分裂活性的程度。促有丝分裂活性在纯化过程中并未被破坏，而是存在于与M蛋白分离的级分中。在这些级分中，通过蛋白质印迹法和酶联免疫吸附测定法可检测到化脓性链球菌热原性外毒素C和促有丝分裂因子MF。此外，抗M蛋白血清不抑制粗提物的促有丝分裂活性，但含有抗化脓性链球菌热原性外毒素C抗体的血清则表现出抑制作用。用大肠杆菌中产生的重组pep M5证实了M5蛋白无法刺激T细胞。我们的数据有力地表明，M蛋白制剂中的促有丝分裂活性是由不同于M蛋白的微量链球菌超抗原引起的。
