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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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-聚糖由于多种原因而引起人们极大的兴趣。E.溶组织菌产生一种不寻常的截短N-聚糖前体（Man（5）GlcNAc（2）），几乎没有核苷酸糖转运蛋白，并且表面被凝集素伴刀豆球蛋白A覆盖。本文采用生物化学和质谱方法研究了大肠杆菌N-聚糖的生物合成和最终N-聚糖的合成。组织溶解，得出以下结论。未加工Man（5）GlcNAc（2），这是最丰富的E.溶组织菌N-聚糖在大肠杆菌表面聚集成帽状。通过N-聚糖特异性抗逆转录病毒凝集素cyanovirin-N. Glc（1）Man（5）GlcNAc（2）也存在于成熟的N-聚糖中，它由UDP-Glc：糖蛋白葡糖基转移酶产生，是保守的N-聚糖依赖性内质网蛋白质折叠质量控制系统的一部分。苦马豆素敏感的α-甘露糖苷酶将一些N-聚糖修剪成双触角Man（3）GlcNAc（2）。E.通过将α 1，2-连接的Gal添加到小寡聚甘露糖聚糖的两个臂上来制备溶组织剂，并且Gal残基被一个或多个Glc封端。综上所述，E.溶组织菌N-聚糖包括未加工的Man（5）GlcNAc（2），它是氰威菌素-N的靶标，以及含有Gal和Glc的独特复杂N-聚糖。 溶组织内阿米巴滋养体被蛋白磷酸聚糖（PPG）覆盖，这是抗碳水化合物单克隆抗体的靶标。 这些抗PPG抗体在被动转移实验中赋予对内阿米巴感染的抗性。PPG的碳水化合物部分由α-1，6-连接葡萄糖的聚合物组成，其通过磷酸基团连接到Ser或Thr上的羟基。然而，PPG的蛋白质骨架尚未表征。采用生物化学和质谱方法了解单克隆抗体与寄生虫表面的结合，鉴定蛋白质骨架并表征PPgs聚糖部分，得出以下结论。通过显示抗阿米巴抗体也与细菌葡聚糖反应，与葡聚糖酶的酶促孵育抑制抗葡聚糖抗体与内阿米巴的富含伴刀豆球蛋白A的糖蛋白的印迹的结合，我们证明PPG聚糖的行为类似于细菌葡聚糖。由于TFA处理/多维色谱法为基础的方法，我们然后确定了7个推定的候选人的特异性抗阿米巴抗体。PPG聚糖组分的结构研究仍在通过高分辨率质谱法进行中。 我们也有兴趣研究阴道毛滴虫的糖蛋白，阴道毛滴虫是唯一医学上重要的性传播寄生虫。 毛滴虫具有含有鼠李糖、木糖、GlcNAc和Gal的表面脂磷酸聚糖（LPG）。 本研究关注的毛滴虫的其他聚糖是Asn连接聚糖（N-聚糖）和O-磷酸二酯连接聚糖（O-P-聚糖）。我们现在已经证明，毛滴虫的未修饰的N-聚糖含有5个甘露糖，而不是9个甘露糖，被抗逆转录病毒凝集素氰威蛋白-N识别。 我们使用质谱技术来鉴定毛滴虫的复合N-聚糖和含有N-聚糖的糖蛋白。我们已经鉴定了毛滴虫的一种新的O-β-聚糖，它是葡萄糖与少量甘露糖的聚合物。毛滴虫O-P-聚糖不被葡聚糖酶或淀粉酶消化，因此可能代表一种新结构。未来的研究将确定毛滴虫复合体N-聚糖和O-β-聚糖的结构，并确定其中一种或两种是否具有免疫原性，从而可能成为候选疫苗。