CYST WALL ENDOPROTEASES AND GLYCANS OF PARASITES

寄生虫的囊壁内切蛋白酶和聚糖

• 批准号: 8170905
• 负责人: John C. Samuelson
• 金额: $ 0.46万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170905
• 关键词: Alpha-mannosidase; Amebic colitis; Amylases; Anabolism; Anti-Retroviral Agents; Antibodies; Back; Binding; Biochemical; Carbohydrates; Chromatography; Complex; Computer Retrieval of Information on Scientific Projects Database; Concanavalin A; Cyanovirin-N; Cyst; Dextranase; Dextrans; Endoplasmic Reticulum; Entamoeba; Entamoeba histolytica; Funding; Future; Glucans; Glucose; Glucosyltransferase; Glucosyltransferases; Glycoproteins; Grant; Hydroxyl Radical; Infection; Institution; Investigation; Lectin; Link; Liver Abscess; Mannose; Mass Spectrum Analysis; Methods; Monoclonal Antibodies; Parasites; Polymers; Polysaccharides; Proteins; Quality Control; Research; Research Personnel; Resistance; Resolution; Resources; Rhamnose; Source; Structure; Surface; System; Techniques; Trichomonas; Trichomonas vaginalis; United States National Institutes of Health; Vertebral column; Xylose; arm; base; bone; dextran; immunogenic; inorganic phosphate; interest; lipophosphonoglycan; novel; phosphodiester; protein folding; research study; sugar nucleotide; vaccine candidate

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man(5)GlcNAc(2)), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man(5)GlcNAc(2), which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc(1)Man(5)GlcNAc(2), which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive alpha-mannosidase trims some N-glycans to biantennary Man(3)GlcNAc(2). Complex N-glycans of E. histolytica are made by the addition of alpha1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man(5)GlcNAc(2), which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc. Entamoeba histolytica trophozoites are covered by proteophosphoglycans (PPGs), which are the target of anti-carbohydrate monoclonal antibodies. These anti-PPG antibodies confer resistance to Entamoeba infections in passive transfer experiments. The carbohydrate portion of the PPG consists of a polymer of alpha-1,6-linked glucose, which is linked by a phosphate group to hydroxyls on Ser or Thr. However, the protein back-bone of the PPGs has not been characterized. Biochemical and mass spectrometric methods were used to understand the binding of the monoclonal antibody on the surface of the parasite, to identify proteic backbones and to characterize PPgs glycan portion, with the following conclusions. By showing that anti-amebic antibody also reacts with bacterial dextran, that enzymatic incubation with dextranase inhibits binding of the anti-dextran antibody to blots of Concanavalin A-enriched glycoproteins of Entamoeba, we proved that PPG glycans behave like bacterial dextran. Thanks to a TFA treatment/muti-dimesional chromatography based method we then identified 7 putative candidates for specific anti-amebic antibody. Structural investigations on the PPGs glycan component are still in progress by means of high resolution mass spectrometry. We are also interested in studying the glycoproteins of Trichomonas vaginalis, the only medically important parasite that is sexually transmitted. Trichomonas has a surface lipophosphoglycan (LPG) that contains rhamnose, xylose, GlcNAc, and Gal. Other glycans of Trichomonas that are the focus of the studies here are Asn-linked glycans (N-glycans) and O-phosphodiester-linked glycans (O-P-glycans). We have now shown that the unmodified N-glycan of Trichomonas that contains 5 mannose rather than 9 mannose is recognized by the anti-retroviral lectin cyanovirin-N. We used mass spectrometric techniques to identify complex N-glycans of Trichomonas and the glycoproteins that contain the N-glycans. We have identified a novel O-P-glycan of Trichomonas that is a polymer of glucose with a small amount of mannose. The Trichomonas O-P-glycan does not digest with dextranase or amylase and so may represent a novel structure. Future studies will determine the structures of the Trichomonas complex N-glycans and O-P-glycans and determine whether either or both are immunogenic, and so may be vaccine candidates.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 溶组织内阿米巴是引起阿米巴痢疾和肝脓肿的原生生物，其N-糖链引起人们的极大兴趣，原因有很多。溶组乳杆菌产生一种不寻常的截短的N-糖前体(Man(5)GlcNAc(2))，几乎没有核苷酸糖转运体，其表面被刀豆蛋白A所覆盖。未加工的人(5)GlcNAc(2)是最丰富的N-糖链，通过N-糖特异性的抗逆转录病毒凝集素氰病毒素-N聚集在组织链球菌胞外膜上。GLC(1)Man(5)GlcNAc(2)是由UDP-GLC：糖蛋白葡萄糖转移酶产生的，它是保守的N-糖依赖的内质网蛋白质折叠质量控制系统的一部分，也存在于成熟的N-糖中。苦马豆素敏感的α-甘露糖苷酶将一些N-糖链修剪成双天线的Man(3)GlcNAc(2)。溶组埃希菌的复杂N-糖链是通过在小分子寡聚糖的两个臂上添加α-1，2-连接的半乳糖而形成的，半乳糖残基由一个或多个糖链封端。总之，溶解组织杆菌的N-聚糖包括未加工的Man(5)GlcNAc(2)，它是氰基韦林-N的靶标，以及含有Gal和GLC的独特的复杂N-聚糖。 溶组织内阿米巴滋养体被蛋白磷酸多聚糖(PPG)覆盖，PPG是抗碳水化合物单抗的靶标。这些抗PPG抗体在被动转移实验中对内阿米巴感染具有抵抗力。PPG的碳水化合物部分由α-1，6-连接葡萄糖的聚合物组成，该聚合物通过磷酸基团连接到丝氨酸或苏氨酸上的羟基。然而，PPG的蛋白质骨架还没有被表征。利用生物化学和质谱学方法了解单抗与寄生虫表面的结合，鉴定蛋白质骨架并鉴定PPgs的多糖部分，得出以下结论。通过证明抗阿米巴抗体也能与细菌葡聚糖反应，酶与葡聚糖酶孵育能抑制抗葡聚糖抗体与内阿米巴富含刀豆蛋白A的糖蛋白斑点的结合，我们证明了PPG葡聚糖的行为类似于细菌葡聚糖。多亏了基于TFA处理/多维层析的方法，我们确定了7个可能的抗阿米巴抗体候选者。利用高分辨率质谱仪对PPG多糖成分的结构研究仍在进行中。 我们还对研究阴道毛滴虫的糖蛋白感兴趣，这是唯一一种通过性传播的医学上重要的寄生虫。毛滴虫的表面脂多糖(LPG)含有鼠李糖、木糖、GlcNAc和Gal。毛滴虫的其他糖链是天冬氨酸连接的糖链(N-糖链)和O-磷酸二酯连接的糖链(O-P-链链)。我们现在已经证明，毛滴虫的未修饰的N-糖链含有5个甘露糖而不是9个甘露糖，它是由抗逆转录病毒凝集素氰韦林-N识别的。我们使用质谱学技术来鉴定毛滴虫的复杂N-糖链和含有N-糖链的糖蛋白。我们已经鉴定出一种新的滴虫O-P-葡聚糖，它是葡萄糖和少量甘露糖的聚合物。毛滴虫的O-P-葡聚糖不被葡聚糖酶或淀粉酶消化，因此可能代表了一种新的结构。未来的研究将确定滴虫复合体N-聚糖和O-P-聚糖的结构，并确定其中一种或两者是否具有免疫原性，因此可能是疫苗候选。