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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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多糖，有助于促进寄生虫的进入，例如，通过伪装成宿主衍生的物种，促进黏附，并导致细胞毒性。我们目前正在研究蓝氏贾第鞭毛虫的这种相互作用，这种相互作用会渗透到肠道并导致腹泻，内阿米巴是发展中国家阿米巴痢疾和肝脓肿的主要原因，全世界高达50%的人口(美国为1-3%)都携带这种细菌。阴道毛滴虫感染引发一种性传播疾病 会导致不孕不育和癌症。独特的多聚糖的结构决定代表 开发治疗药物的机会，这些药物将在不对宿主产生副作用的情况下干扰寄生虫。蓝氏贾第鞭毛虫(又称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N-乙酰半乳糖胺(GalNAc)均聚体的纤维和至少三种囊壁蛋白(CWPs)，CWPLRR由富含亮氨酸的重复序列(CWPLRR)和C末端保守的Cys富集区(CWPCRR)组成。我们的目标是解剖囊壁的结构，并确定它是如何 在抽搐过程中被打乱。我们的结果表明，CWP1的富含Leu的重复结构域是一种凝集素，它与GalNAc均聚物的卷曲纤维结合。在激活过程中，宿主和贾第虫蛋白酶似乎降解结合的CWP，暴露出GalNAc均聚物的纤维，这些纤维被阶段特定的糖水解酶消化。 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，它有一个由七种糖(Man5GlcNAc2)组成的截短的天冬氨酸连接的葡聚糖(N-Glycan)前体。我们发现，含有未修饰N-糖链的糖蛋白被抗逆转录病毒凝集素氰病毒素-N聚集并覆盖在溶组织棘球绦虫滋养体的表面，然后从大的胞内池中补充。氰化韦林-N共包裹Gal/GalNAc黏附凝集素，以及含有O-磷酸二酯连接的糖蛋白的糖蛋白，该糖蛋白可被抗蛋白磷酸多糖单抗识别。氰韦林-N抑制粘蛋白包裹的微球中的溶组织埃希氏菌滋养体的吞噬作用，这是阿米巴毒力的替代试验。我们富集了分泌物和膜蛋白，用于质谱学鉴定，发现了具有占据N-糖基的糖蛋白，包括Gal/GalNAc凝集素、蛋白酶和17种先前假设的蛋白。这些蛋白和之前假设的50种蛋白可能是疫苗的靶标，因为它们丰富而独特。 阴道毛滴虫表面有一种独特的脂磷脂多糖(LPG)，在对宿主细胞的黏附和细胞毒性方面发挥作用。我们正在研究修饰滴虫蛋白的天冬氨酸连接的(N-糖链)和O-磷酸二酯连接的(O-P-糖链)糖链。毛滴虫N-糖链是建立在含有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化韦林-N识别。O-P-葡聚糖形成一种线性聚合物，主要由葡萄糖组成，既不被淀粉酶也不被葡聚糖酶消化。植物凝集素刀豆蛋白A极大地丰富了毛滴虫分泌的糖蛋白和胰酶消化后的质谱学鉴定的膜糖蛋白。毛滴虫的“CON Aome”含有参与N-糖依赖的蛋白质折叠质量控制(钙网蛋白)和N-糖非依赖的蛋白质折叠质量控制(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。令人感兴趣的是，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的新靶点。综上所述，这些结果表明毛滴虫糖蛋白具有新的N-多糖和O-P-多糖，并包括许多先前假设的蛋白质。