CYST WALL ENDOPROTEASES AND GLYCANS OF PARASITES

寄生虫的囊壁内切蛋白酶和聚糖

• 批准号: 8365537
• 负责人: John C. Samuelson
• 金额: $ 0.62万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365537
• 关键词: Acetylgalactosamine; Acid Phosphatase; Adherence; Adhesions; Adverse effects; Amebic colitis; Amylases; Anti-Retroviral Agents; Binding; Biological Assay; Biology; C-terminal; Cell surface; Cells; Concanavalin A; Cyanovirin-N; Cyst; Cysteine Protease; Dextranase; Diagnostic; Diarrhea; Digestion; Entamoeba histolytica; Exhibits; Funding; Gal-GalNAc; Giardia; Giardia lamblia; Glucose; Glycoproteins; Glycoside Hydrolases; Goals; Grant; Infection; Integral Membrane Protein; Intestines; Lectin; Link; Liver; Liver Abscess; Malignant Neoplasms; Mannose; Mass Spectrum Analysis; Medicine; Membrane Glycoproteins; Membrane Proteins; Monoclonal Antibodies; Mucins; National Center for Research Resources; PAWR protein; Parasites; Peptide Hydrolases; Phagocytosis; Pharmaceutical Preparations; Plant Lectins; Play; Polymers; Polysaccharides; Population; Principal Investigator; Proteins; Quality Control; Research; Research Infrastructure; Resources; Role; Sexually Transmitted Diseases; Site; Source; Staging; Sterility; Structure; Surface; Trichomonas; Trichomonas vaginalis; Trypsin; United States National Institutes of Health; Vaccination; Vaccines; Virulence; calreticulin; cost; cytotoxicity; excystation; interest; lipophosphonoglycan; lysosomal proteins; mannosyl(5)-N-acetyl(2)-glucose; novel; phosphodiester; protein folding; sugar; therapeutic development

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Many parasites exhibit cell-surface glycans that interact with the host, helping to facilitate the entry of the parasite, e.g., by masquerading as host-derived species, contributing to adhesion, and causing cytotoxicity. We are currently studying such interactions of Giardia lamblia which infiltrates the intestine and causes diarrhea, Entamobea, which is harbored by up to 50% of the population worldwide (1-3% in US), is the main cause of amebic dysentery and liver abcesses in the developing world. Trichomonas vaginalis infection initiates a sexually transmitted disease that can cause sterility and cancer. Structural determinations of unique glycans represent opportunities for development of therapeutic drugs that will interfere with the parasite without producing side effects in the host. The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique ¿1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. Our results showed that the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase. Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man5GlcNAc2). We showed that glycoproteins with unmodified N-glycans are aggregated and capped on the surfaceof E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. We enriched the secreted and membrane proteins for mass spectrometric identification, finding E. histolytica glycoproteins with occupied N-glycan sites including Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. These and 50 previously hypothetical proteins may be vaccine targets as they are abundant and unique. Trichomonas vaginalis has on its surface a unique lipophosphoglycan (LPG) that plays a role in adherence and cytotoxicity to host cells. We are investigating the Asn-linked (N-Glycans) and O-phosphodiester-linked (O-P-glycans) glycans that modify Trichomonas proteins. Trichomonas N-glycans are built on a truncated precursor containing 5 mannose residues that are recognized by the anti-retroviral lectin cyanovirin-N. O-P-glycans form a linear polymer predominantly composed of glucose that is not digested with either amylase or dextranase. The plant lectin Concanavalin A dramatically enriches Trichomonas secreted and membrane glycoproteins that were identified by mass spectrometry after trypsin digestion. The "Con Aome" of Trichomonas contains ER proteins involved in N-glycan-dependent quality control of protein folding (calreticulin) and N-glycan-independent quality control of protein folding (BiP). The ConAome also contains lysosomal proteins including acid phosphatase and cysteine proteinase. Of great interest, the ConAome is dominated by unique transmembrane proteins and unique secreted proteins that may be novel targets for vaccination against this protist. In summary, these results show that Trichomonas glycoproteins have novel N-glycans and O-P-glycans and include many previously hypothetical proteins.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 许多寄生虫表现出与宿主相互作用的细胞表面聚糖，有助于促进寄生虫的进入，例如，通过伪装成宿主来源的物种，促进粘附并引起细胞毒性。我们目前正在研究兰伯氏贾第鞭毛虫的相互作用，兰伯氏贾第鞭毛虫渗入肠道并引起腹泻，而肠虫则被全世界 50% 的人口携带（美国为 1-3%），是发展中国家阿米巴痢疾和肝脓肿的主要原因。阴道毛滴虫感染会引发一种性传播疾病， 可导致不育和癌症。独特聚糖的结构测定代表 开发干扰寄生虫而不会对宿主产生副作用的治疗药物的机会。兰氏贾第鞭毛虫（也称为肠贾第鞭毛虫或十二指肠贾第鞭毛虫）的感染和诊断阶段是包囊。贾第虫囊壁含有独特的 1,3 连接的 N-乙酰半乳糖胺 (GalNAc) 均聚物原纤维和至少三个由富含亮氨酸重复序列 (CWPLRR) 和 C 端保守富含半胱氨酸区域 (CWPCRR) 组成的囊壁蛋白 (CWP)。我们的目标是解剖囊肿壁的结构并确定它是如何的 排泄期间中断。我们的结果表明，CWP1 的富含亮氨酸的重复结构域是一种凝集素，可与 GalNAc 均聚物的卷曲原纤维结合。在排泄过程中，宿主和贾第鞭毛虫蛋白酶似乎会降解结合的 CWP，从而暴露出 GalNAc 均聚物的原纤维，并被阶段特异性糖水解酶消化。 溶组织内阿米巴是一种引起阿米巴痢疾和肝脓肿的原生生物，它具有由七种糖 (Man5GlcNAc2) 组成的截短 Asn 连接聚糖 (N-聚糖) 前体。我们发现，具有未修饰的 N-聚糖的糖蛋白通过抗逆转录病毒凝集素cyanovirin-N 在溶组织内阿米巴滋养体表面聚集并加帽，然后从大型细胞内池中补充。 Cyanovirin-N 包覆 Gal/GalNAc 粘附凝集素以及含有 O-磷酸二酯连接聚糖的糖蛋白，可被抗蛋白磷酸聚糖单克隆抗体识别。 Cyanovirin-N 抑制溶组织内阿米巴滋养体对粘蛋白包被珠子的吞噬作用，这是阿米巴毒力的替代测定。我们富集了分泌蛋白和膜蛋白进行质谱鉴定，发现溶组织内阿米巴糖蛋白具有占据的 N-聚糖位点，包括 Gal/GalNAc 凝集素、蛋白酶和 17 种先前假设的蛋白。这些以及之前假设的 50 种蛋白质可能是疫苗的靶标，因为它们丰富且独特。 阴道毛滴虫表面有一种独特的脂磷酸聚糖（LPG），在宿主细胞的粘附和细胞毒性中发挥作用。我们正在研究修饰毛滴虫蛋白的 Asn 连接（N-聚糖）和 O-磷酸二酯连接（O-P-聚糖）聚糖。滴虫 N-聚糖构建在含有 5 个甘露糖残基的截短前体上，这些残基可被抗逆转录病毒凝集素cyanovirin-N 识别。 O-P-聚糖形成主要由葡萄糖组成的线性聚合物，不被淀粉酶或葡聚糖酶消化。植物凝集素伴刀豆球蛋白 A 显着富集毛滴虫分泌的糖蛋白和膜糖蛋白，这些糖蛋白在胰蛋白酶消化后通过质谱分析进行鉴定。毛滴虫的“Con Aome”含有 ER 蛋白，参与 N-聚糖依赖性蛋白质折叠质量控制（钙网蛋白）和 N-聚糖依赖性蛋白质折叠质量控制 (BiP)。 ConAome 还含有溶酶体蛋白，包括酸性磷酸酶和半胱氨酸蛋白酶。令人感兴趣的是，ConAome 以独特的跨膜蛋白和独特的分泌蛋白为主，它们可能是针对这种原生生物疫苗接种的新靶标。总之，这些结果表明毛滴虫糖蛋白具有新的 N-聚糖和 O-P-聚糖，并且包括许多以前假设的蛋白质。