MOLECULAR CHAR OF HOST CELL RECEPTORS FOR TRYPANOSOMA CRUZI

本研究的长期目标是阐明作为克氏锥虫受体的哺乳动物细胞的分子结构。克氏锥虫是恰加斯病的病原体，该病影响数百万人，导致心脏骤停，经常伴有死亡。尽管在这一领域取得了重大进展，但人们对克氏锥虫的宿主细胞受体知之甚少。我们发现从心肌细胞中纯化的表面74 kDa糖蛋白和该分子的抗体抑制克氏锥虫与心肌细胞的结合和内化。该糖蛋白与侵袭性锥马鞭毛体结合，而与非侵袭性锥马鞭毛体不结合，并且仅在锥马鞭毛体可侵袭的细胞上表达。重组形式的克氏T. gp83表面反式唾液酸酶与心肌母细胞结合，Kd为1x10- 4mum，介导锥虫与心肌母细胞的结合，识别74 kDa的糖蛋白。单抗4A4可以消除锥虫与心肌母细胞和其他细胞的结合，单抗4A4可以识别gp83反式唾液酸酶或其重组形式的表位，这是锥虫与宿主细胞结合所必需的。该单克隆抗体还能消除4-gp83与74 kDa糖蛋白的结合或可溶性gp83反式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糖蛋白可能作为克氏锥虫的受体，介导锥虫结合以促进其进入。在这个提议中，我们将测试这个假设，我们将研究这个受体的分子结构。为此，我们提出了以下具体目标：a)从心脏细胞cDNA文库中鉴定表达74 kDa蛋白的克隆，对编码74 kDa蛋白的全长cDNA进行测序并预测其氨基酸序列；b)表达和纯化重组74 kDa糖蛋白，用于与锥虫的配体结合研究，并评估重组分子及其抗体抑制克氏锥虫感染的能力。c)来测试的能力74 kDa糖蛋白作为受体的t . cruzi使转染哺乳动物细胞系不能入侵通过t . cruzi cDNA 74 kDa蛋白质编码,d)来确定该地区在t . cruzi gp83配体结合74 kDa糖蛋白这个提议将产生新的信息和见解的分子结构膜蛋白可能作为受体的细胞入侵的t . cruzi形式。这些研究将有助于建立宿主细胞识别克氏锥虫以促进锥虫进入的分子基础，这对分子干预尚未治愈的疾病可能具有重要意义。

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.