BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ENZYMES SECRETED BY LEISHMANIA

该项目正在进行的研究涉及利什曼原虫的细胞生物学、生物化学和分子生物学，利什曼原虫是一组人类的原生动物病原体。所有利什曼原虫都经历一个二态的生命周期：1)在哺乳动物（人类）中，它们在巨噬细胞溶酶体系统中以细胞内形式（无尾虫）繁殖，最终破坏这些细胞；2)在它们的昆虫载体（吸血沙虫）中，它们在消化道内分化和繁殖为细胞外形式（原生虫），并最终迁移到口部传播。据世界卫生组织估计，利什曼原虫每年在全世界造成远远超过1200万例人类疾病（利什曼病）。在受感染的人体内，这些寄生虫破坏皮肤或内脏器官（即脾脏、肝脏和骨髓）内的巨噬细胞，造成大面积毁容的恶性皮肤溃疡（例如由墨西哥乳杆菌引起）或退行性且最常见的致命内脏疾病（例如由多诺瓦氏乳杆菌引起）。我们实验室以前的研究已经确定利什曼原虫组成性地分泌超过40种不同的可溶性蛋白、糖蛋白和碳水化合物成分。由于这些分泌产物可以很容易地渗透并可能改变利什曼原虫居住的宿主微环境，因此了解这些寄生虫产物的性质似乎至关重要。为此，人们正在研究几种寄生虫分泌酶，以确定它们在这些生物体的生存、维持、生长和传播中的功能作用。此外，编码这些蛋白的基因首次被鉴定和表征，从而确定了它们在寄生虫生长、发育和分化过程中的表达和调控。在过去的一年里，我们的研究已经阐明了利什曼原虫多诺瓦尼和墨西哥利什曼原虫几丁质酶的酶活性、基因结构和染色体位点。此外，我们发现所有致病性利什曼原虫都具有高度保守的该基因位点，并且所有这些生物体都表达与结构相关的分泌几丁质酶活性。同样，从多诺瓦尼L. donovani中鉴定出了几个独特的利什曼分泌酸性磷酸酶（SAcP）家族新成员的基因。我们的生化和分子研究表明，该酶家族在所有致病性利什曼物种中都具有功能保守性，包括L. major以及其他与人类有较远亲缘关系的锥虫寄生虫。此外，我们证明了在人类皮肤和内脏疾病的过程中，这些保守的SAcPs是由所有致病性利什曼原虫的无梭菌形式合成的。此外，我们将这些基因与绿色荧光蛋白（-GFP）串联嵌合构建，以确定将这些酶靶向进入寄生虫分泌途径的独特分子信号。涉及基因缺失、突变和过度表达的实验正在进行中，以证明这些分泌酶实际上对这些人类病原体的生存至关重要。此外，还与Olafson博士的实验室合作进行了L. donovani SAcP的物理/结构分析。这些质谱研究的结果表明，SAcP的c端丝氨酸残基被由高负电荷重复单位（n=32）的磷酸双糖组成的o链碳水化合物侧链大量修饰。这些[- po4 - gal - man -]重复单位与构成寄生虫主要的表面膜糖脂，脂磷聚糖的单位相同。这是在分泌糖蛋白和糖脂中存在这种不寻常的磷酸二糖运动的第一个物理证据。此外，我们发现这些[- po4 - gal - man -]重复单元至少在一定程度上解释了每摩尔蛋白质中SAcP中超过700摩尔的己糖，以及该酶对宿主蛋白水解降解的极端抗性。此外，利用GFP构建了几种不同的截断表面膜蛋白，我们在利什曼原虫中定义了一条独特的内体运输途径，该途径与分泌途径相结合，并调节这些生物体的蛋白质过表达。

Ongoing studies in this project concern the cell biology, biochemistry and molecular biology of Leishmania, a group of protozoan pathogens of humans. All Leishmania parasites undergo a dimorphic life cycle: 1) in mammals (humans), they multiply as obligate intracellular forms [amastigotes] within the lysosomal system of macrophages, eventually destroying these cells and 2) within their insect vectors (blood-sucking sandfies), they differentiate and multiply as, extracellular forms [promastigotes] within the alimentary tract and eventually migrate to the mouth parts for transmission. By World Health Organization estimates, Leishmania parasites annually cause well-over 12 million cases of human disease (leishmaniasis) worldwide. In infected humans, these parasites destroy macrophages within the skin or internal organs (i.e. spleen, liver and bone marrow) causing either large and disfiguring, malignant skin ulcers (e.g. caused by L. mexicana) or degenerative and most often fatal visceral disease (e.g. caused by L. donovani).Previous studies from our laboratory have established that Leishmania parasites constitutively secrete over 40 different soluble protein, glycoprotein and carbohydrate constituents. Since such secretory products can readily permeate and presumably alter the host micro-environments in which Leishmania reside, an understanding of the nature of these parasite products seems essential. To that end, several parasite secretory enzymes are being investigated toward defining their functional roles in the survival, maintenance, growth and transmission of these organisms. Further, genes encoding these proteins have been identified and characterized for the first time toward defining their expression and regulation during parasite growth, development and differentiation. During the past year, our studies have elucidated the enzymatic activity, gene structure and chromosomal locus of the leishmanial chitinase in both L. donovani and L. mexicana. Further, we showed that all pathogenic Leishmania possess a highly conserved locus for this gene and all of these organisms express structurally related secretory chitinase activities. Similarly, the genes for several new members of the unique leishmanial secretory acid phosphatase (SAcP) family were identified and characterized from L. donovani. Our biochemical and molecular studies showed that this family of enzymes was functionally conserved among all pathogenic leishmanial species including L. major as well as, other more distantly related trypanosomatid parasites of humans. Further, we demonstrated that these conserved SAcPs are synthesized by amastigote forms of all pathogenic Leishmania species during the course of both human cutaneous and visceral disease. Moreover, we made chimeric constructs of these genes in tandem with the green fluorescent protein (-GFP) to determine the unique molecular signals responsible for targeting these enzymes into the parasite secretory pathway. Experiments involving gene-deletion, -mutation, and -over expression are in progress toward demonstrating that these secretory enzymes are, in fact, essential to the survival of these human pathogens. Further, physical/structural analyses of the L. donovani SAcP, were also carried out in collaboration with Dr. Olafson's lab. Results of these mass spectrometry studies showed that the C-terminal serine residues of SAcP were heavily decorated with O-linked carbohydrate side chains composed of highly negatively charged repeating units (n=32) of phospho-disaccharides. These [-PO4-Gal-Man-] repeat units are the same as those which constitute the parasite's major surface membrane glycolipid, the lipophosphoglcan. This is the first physical proof for the existence of this unusual phospho-disaccharide moeity in both a secretory glycoprotein and a glycolipid. Moreover, we found that these [-PO4-Gal-Man-] repeat units accounted, at least in part, for the more than 700 moles of hexose present in the SAcP per mole of protein and for the extreme resistance of this enzyme to host proteolytic-degradation . In addition, using GFP constructs of several different truncated surface membrane proteins, we defined a unique endosomal trafficking pathway in Leishmania which interfaces with the secretory pathway an pathway and functions to regulate protein over-expression of these organisms.