Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis

Trypanosoma cruzi is a protozoan parasite transmitted by a triatomine insect, and causing human Chagas disease in South America. This parasite undergoes a complex life cycle alternating between non-proliferative and dividing forms. Owing to their high energy requirement, replicative epimastigotes of the insect midgut display high endocytic activity. This activity is mainly restricted to the cytostome, by which the cargo is taken up and sorted through the endosomal vesicular network to be delivered to reservosomes, the final lysosomal-like compartments. In African trypanosomes tomato lectin (TL) and ricin, respectively specific to poly-N-acetyllactosamine (poly-LacNAc) and β-D-galactose, allowed the identification of giant chains of poly-LacNAc in N-glycoproteins of the endocytic pathway. We show that in T. cruzi epimastigote forms also, glycoproteins of the endocytic pathway are characterized by the presence of N-linked glycans binding to both ricin and TL. Affinity chromatography using both TL and Griffonia simplicifolia lectin II (GSLII), specific to non-reducing terminal residue of N-acetylglucosamine (GlcNAc), led to an enrichment of glycoproteins of the trypanosomal endocytic pathway. Incubation of live parasites with TL, which selectively bound to the cytostome/cytopharynx, specifically inhibited endocytosis of transferrin (Tf) but not dextran, a marker of fluid endocytosis. Taken together, our data suggest that N-glycan modification of endocytic components plays a crucial role in receptor-mediated endocytosis of T. cruzi.克氏锥虫（Trypanosoma cruzi）是一种由锥蝽（triatomine insect）传播的原生动物寄生虫，可在南美引发人类恰加斯病（Chagas disease）。该寄生虫拥有复杂的生命周期，交替处于非增殖型与增殖型两种形态。由于能量需求较高，昆虫中肠内的增殖型上鞭毛体（epimastigotes）展现出极高的胞吞活性。该活性主要局限于胞口（cytostome）：寄生虫通过胞口摄取胞吞底物，经内体囊泡网络（endosomal vesicular network）完成分选后，输送至储备体（reservosomes）——一类终末溶酶体样区室。在非洲锥虫（African trypanosomes）中，分别靶向聚-N-乙酰乳糖胺（poly-N-acetyllactosamine, poly-LacNAc）与β-D-半乳糖（β-D-galactose）的番茄凝集素（tomato lectin, TL）与蓖麻毒素（ricin），被用于鉴定胞吞通路（endocytic pathway）N-糖蛋白（N-glycoproteins）中的巨型聚-LacNAc链。本研究表明，在克氏锥虫上鞭毛体中，胞吞通路的糖蛋白同样具备可同时结合蓖麻毒素与TL的N-连接聚糖（N-linked glycans）。采用TL与葛藤凝集素II（Griffonia simplicifolia lectin II, GSLII，特异性识别N-乙酰葡糖胺（N-acetylglucosamine, GlcNAc）非还原末端残基）的亲和层析策略，可有效富集锥虫胞吞通路的糖蛋白。将活寄生虫与TL共孵育，TL可选择性结合胞口/胞咽（cytostome/cytopharynx），并特异性抑制转铁蛋白（transferrin, Tf）的胞吞过程，但不影响作为液相胞吞（fluid endocytosis）标志物的葡聚糖（dextran）的摄取。综上，本研究数据表明，胞吞组分的N-聚糖修饰在克氏锥虫的受体介导胞吞（receptor-mediated endocytosis）过程中发挥关键作用。