Acosta-Serrano A, Cole R N, Englund P T
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
J Mol Biol. 2000 Dec 8;304(4):633-44. doi: 10.1006/jmbi.2000.4246.
Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered oligosaccharides compared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.
伴刀豆球蛋白A（Con A）通过与EP-前环素上的N-聚糖结合，杀死布氏锥虫的前循环（昆虫）形式，EP-前环素是一种主要的表面糖基磷脂酰肌醇（GPI）锚定蛋白，富含Glu-Pro重复序列。我们之前分离并研究了两个前循环突变体（ConA 1-1和ConA 4-1），它们比野生型（WT）对Con A杀伤更具抗性。尽管与WT细胞相比，这两个突变体都表达相同的改变后的寡糖，但ConA 4-1对凝集素杀伤的抗性比ConA 1-1强得多。因此，我们寻找其他改变来解释敏感性的差异。通过质谱分析，结合化学和酶处理，我们发现这两个突变体都表达了WT细胞中表达不佳或根本不存在的EP-前环素类型。ConA 1-1主要表达EP1-3，这是一种新型前环素，含有18个EP重复序列，部分进行了N-糖基化，并带有杂合型聚糖。另一方面，ConA 4-1细胞几乎只表达EP2-3，这是一种新型非糖基化前环素异构体，有23个EP重复序列且没有糖基化位点。ConA 4-1细胞中EP2-3的优势解释了它们对ConA杀伤的高抗性。因此，前环素种类的转换，这一可能与寄生虫在昆虫媒介中的发育相关的过程，调节了锥虫对细胞毒性凝集素的敏感性。
