Sher A, Hieny S, Joiner K
J Immunol. 1986 Nov 1;137(9):2961-7.
Epimastigotes (EPI) of Trypanosoma cruzi are highly sensitive to lysis in fresh normal human serum by the alternative complement pathway (ACP). In contrast, metacyclic trypomastigotes (CMT) derived from EPI in stationary culture fail to activate the ACP and are thus resistant to serum-mediated lysis. To investigate the nature of the parasitic surface molecules which enable infective metacyclic trypomastigotes to evade the ACP, CMT were treated with a variety of different proteolytic and glycosidic enzymes, and their sensitivity to ACP-dependent lysis was tested. Pretreatment with pronase was found to cause a near complete reversal in the resistance of CMT to serum lysis, whereas trypsin or chymotrypsin induced smaller increases in complement sensitivity. Similarly, pretreatment with N-glycanase or neuraminidase also partially abrogated the resistance of CMT to ACP-dependent lysis. The effect of these enzymes on susceptibility to complement-mediated lysis was paralleled in increased C3 and C9 deposition on the organism. In addition, electrophoretic analysis of parasite-bound C3 indicated that the hemolytically inactive fragment, iC3b, was the major form of the molecule on CMT, while the hemolytically active fragment, C3b, predominated on pronase-treated CMT. Furthermore, when C3 was deposited on the parasite surface by means of purified ACP components, 80% of C3b on pronase-pretreated CMT but only 14% of the C3b on CMT bound the amplification protein factor B with high affinity, a prerequisite for efficient ACP activation. When cultured at 37 degrees C after pronase treatment, CMT gradually regained their resistance to ACP-mediated lysis. This process was blocked if puromycin, cycloheximide, or tunicamycin were included in the culture medium. The above findings suggest that evasion of the ACP by CMT is dependent on the developmentally regulated synthesis of protein as well as N-linked carbohydrate chains. A stage-specific 90,000 to 115,000 m.w. glycoprotein doublet present on the surface of CMT was shown to be uniquely sensitive to pronase digestion. Thus, this complex, which is also recognized by a CMT-specific monoclonal antibody, may be the glycoprotein component responsible for control of ACP activation
克氏锥虫的前鞭毛体（EPI）对新鲜正常人血清中通过替代补体途径（ACP）介导的溶解高度敏感。相比之下，在静止培养中由EPI衍生而来的循环后期锥鞭毛体（CMT）不能激活ACP，因此对血清介导的溶解具有抗性。为了研究使感染性循环后期锥鞭毛体逃避ACP的寄生虫表面分子的性质，用多种不同的蛋白水解酶和糖苷酶处理CMT，并测试它们对ACP依赖性溶解的敏感性。发现用链霉蛋白酶预处理可使CMT对血清溶解的抗性几乎完全逆转，而胰蛋白酶或胰凝乳蛋白酶诱导的补体敏感性增加较小。同样，用N-糖苷酶或神经氨酸酶预处理也部分消除了CMT对ACP依赖性溶解的抗性。这些酶对补体介导的溶解敏感性的影响与生物体上C3和C9沉积的增加平行。此外，对寄生虫结合的C3进行电泳分析表明，溶血无活性片段iC3b是CMT上该分子的主要形式，而溶血活性片段C3b在经链霉蛋白酶处理的CMT上占主导地位。此外，当通过纯化的ACP成分将C3沉积在寄生虫表面时，经链霉蛋白酶预处理的CMT上80%的C3b，但CMT上只有14%的C3b与扩增蛋白因子B高亲和力结合，这是有效激活ACP的先决条件。经链霉蛋白酶处理后在37℃培养时，CMT逐渐恢复其对ACP介导的溶解的抗性。如果在培养基中加入嘌呤霉素、放线菌酮或衣霉素，这个过程就会被阻断。上述发现表明，CMT对ACP的逃避依赖于蛋白质以及N-连接碳水化合物链的发育调控合成。CMT表面存在的一种阶段特异性的分子量为90,
