MOLECULAR CHAR OF HOST CELL RECEPTORS FOR TRYPANOSOMA CRUZI

这项计划的长期目标是阐明哺乳动物细胞上作为克氏锥虫受体的分子结构。T. cruzi是南美锥虫病的病原体，该疾病影响数百万人，导致心脏骤停，经常伴随死亡。尽管在这一领域取得了重大进展，但对T细胞的宿主细胞受体知之甚少。克鲁兹我们已经发现，从心肌成肌细胞中纯化的表面74 kDa糖蛋白和针对该分子的抗体抑制T。cruzi锥鞭毛体结合和内化到心脏成肌细胞中。这种糖蛋白与侵袭性锥鞭毛体结合，但不与非侵袭性外鞭毛体形式的T。cruzi，并且仅在可被锥鞭毛体侵入的细胞上表达。T. cruzigp 83表面转唾液酸酶识别这种74 kDa的糖蛋白，该酶以1 × 10 -4 μ M的Kd与心脏成肌细胞结合以介导锥虫与成肌细胞的结合。锥虫鞭毛体与心脏成肌细胞和其他细胞的结合被mAb 4A 4消除，mAb 4A 4识别gp 83转唾液酸酶或其重组形式上的表位，并且是锥虫体与宿主细胞结合所需的。该单克隆抗体还消除了4-gp 83与74 kDa糖蛋白的结合或可溶性gp 83转唾液酸酶与成肌细胞的结合。鉴于这些发现，我们假设宿主74 kDa糖蛋白可能作为T. cruzi介导锥虫结合以促进进入。在本提案中，我们将测试这一假设，我们将研究这种受体的分子结构。为此，我们提出了以下具体目标：a）从心脏细胞的cDNA文库中鉴定表达74 kDa蛋白质的克隆，对编码74 kDa蛋白质的全长cDNA进行测序并预测其氨基酸序列，B）表达并纯化重组74 kDa糖蛋白用于对锥虫的配体结合研究，并评估重组分子及其抗体抑制T. c）测试74 kDa糖蛋白作为T. cruzi的方法，通过分离不能被T. cruzi与编码74 kDa蛋白质的cDNA进行比较，d）确定T. cruzi gp 83配体结合74 kDa糖蛋白该提议将产生关于细胞膜蛋白分子结构的新信息和见解，该膜蛋白可能作为侵袭性形式T细胞的受体发挥作用。克鲁兹这些研究将有助于建立T. Cruzi识别宿主细胞，以促进锥虫进入，这可能是重要的分子干预，但无法治愈的疾病。

The long term goal of this proposal is to elucidate the molecular structures on mammalian cells that function as receptors for Trypanosoma cruzi. T. cruzi is the causative agent of Chagas' disease, which affects millions of people causing cardiac arrest, frequently accompanied by death. Despite significant advances in this area very little is known, if any, about host cell receptors for T. cruzi. We have found that the purified surface 74 kDa glycoprotein from heart myoblasts and antibodies to this molecule inhibit T. cruzi trypomastigote binding and internalization into heart myoblasts. This glycoprotein binds to invasive trypomastigote but not to non-invasive epimastigote forms of T. cruzi and is only expressed on cells that can be invaded by trypomastigotes. The recombinant forms of T. cruzi gp83 surface trans-sialidase, which binds to heart myoblasts with a Kd of 1x10-4 muM to mediate trypanosome binding to myoblasts, recognizes this 74 kDa glycoprotein. Binding of trypomastigotes to heart myoblasts and other cells is abolished by mAb 4A4 which recognizes an epitope on the gp83 trans-sialidase or its recombinant form and is required for trypanosome binding to host cells. This monoclonal antibody also abolishes the binding of the 4-gp83 to the 74 kDa glycoprotein or the binding of soluble gp83 trans-sialidase to myoblasts. In view of these findings, we have hypothesized that the host 74 kDa glycoprotein may function as a receptor for T. cruzi to mediate trypanosome binding to facilitate entry. In this proposal we will test this hypothesis and we will investigate the molecular structure of this receptor. To this end, we proposed the following specific aims: a) to identify clones expressing the 74 kDa protein from a cDNA library of heart cells, sequence the full length cDNA encoding the 74 kDa protein and predict its amino acid sequence, b) to express and purify the recombinant 74 kDa glycoprotein for ligand binding studies to trypanosomes, and to assess the ability of the recombinant molecule and its antibodies to inhibit T. cruzi infection, c) to test the ability of the 74 kDa glycoprotein to function as a receptor for T. cruzi by transfecting mammalian cell lines which cannot be invaded by T. cruzi with cDNA coding for the 74 kDa protein, d) to determine the region on the T. cruzi gp83 ligand that binds to the 74 kDa glycoprotein This proposal will generate new information and insights about the molecular structure of a membrane protein of cells that may function as a receptor for invasive forms of T. cruzi. These studies will contributes to establish the molecular basis on T. cruzi recognition by host cells to promote trypanosome entry, which may be important for molecular intervention in yet incurable disease.