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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 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。我们目前正在研究蓝氏贾第鞭毛虫（Giardia lamblia）的这种相互作用，蓝氏贾第鞭毛虫（Giardia lamblia）浸润肠道并导致腹泻，内阿米巴（Entamobea）是发展中国家阿米巴痢疾和肝脓肿的主要原因，内阿米巴占全球人口的50%（美国为1 - 3%）。阴道毛滴虫感染引发性传播疾病， 会导致不育和癌症独特聚糖的结构测定代表 为开发治疗药物提供了机会，这些药物将干扰寄生虫而不会对宿主产生副作用。蓝氏贾第鞭毛虫（Giardia lamblia，又称G. G.nov.囊肿（cyst）。贾第鞭毛虫囊壁含有独特的<$1，3-连接的N-乙酰半乳糖胺（GalNAc）均聚物和至少三种由富含亮氨酸重复序列（CWPLRR）和C-末端保守的富含半胱氨酸区域（CWPCRR）组成的囊壁蛋白（CWPs）的纤维。我们的目标是解剖囊肿壁的结构， 在脱囊过程中被破坏。我们的研究结果表明，富含亮氨酸重复结构域的CWP1是一种凝集素，结合到卷曲的纤维的GalNAc均聚物。在脱囊期间，宿主和贾第虫蛋白酶似乎降解结合的CWP，暴露由阶段特异性糖水解酶消化的GalNAc均聚物的原纤维。 溶组织内阿米巴（Entamoeba histolytica）是引起阿米巴痢疾和肝脓肿的原生生物，具有由七种糖（Man5GlcNAc 2）组成的截短的天冬酰胺连接聚糖（N-聚糖）前体。我们发现，具有未修饰的N-聚糖的糖蛋白在E.溶组织滋养体的抗逆转录病毒凝集素cyanovirin-N，然后从大的细胞内池补充。Cyanovirin-N共封端Gal/GalNAc粘附凝集素，以及含有由抗蛋白磷酸聚糖单克隆抗体识别的O-磷酸二酯连接聚糖的糖蛋白。Cyanovirin-N抑制E.粘蛋白包被珠的溶组织滋养体，阿米巴毒力的替代测定。我们富集了分泌蛋白和膜蛋白用于质谱鉴定，发现E.具有被占据的N-聚糖位点的溶组织糖蛋白，包括Gal/GalNAc凝集素、蛋白酶和17种先前假设的蛋白质。这些和50个先前假设的蛋白质可能是疫苗的靶点，因为它们丰富而独特。 阴道毛滴虫表面具有独特的脂磷酸聚糖（LPG），在对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天冬酰胺连接（N-聚糖）和O-磷酸二酯连接（O-P-聚糖）聚糖。毛滴虫N-聚糖构建在含有5个甘露糖残基的截短前体上，这些甘露糖残基可被抗逆转录病毒凝集素氰威蛋白-N识别。0-β-聚糖形成主要由葡萄糖组成的线性聚合物，其不被淀粉酶或葡聚糖酶消化。植物凝集素伴刀豆球蛋白A显著地富集毛滴虫分泌的和膜糖蛋白，这些糖蛋白在胰蛋白酶消化后通过质谱法鉴定。毛滴虫的"Con Aome"含有参与蛋白质折叠的N-聚糖依赖性质量控制（钙网蛋白）和蛋白质折叠的N-聚糖非依赖性质量控制（BiP）的ER蛋白。ConAome还含有溶酶体蛋白，包括酸性磷酸酶和半胱氨酸蛋白酶。非常感兴趣的是，ConAome由独特的跨膜蛋白和独特的分泌蛋白主导，这些蛋白可能是针对这种原生生物的疫苗接种的新靶标。总之，这些结果表明毛滴虫糖蛋白具有新的N-聚糖和0-P-聚糖，并且包括许多先前假设的蛋白质。