STRUCTURAL ANALYSIS OF GLYCOLIPID ANTIGENS OF LEISHMANIA PIFANOI AMASTIGOTES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。利什曼原虫属的原生动物寄生虫在人类身上引起许多疾病。这种寄生虫经历了一个复杂的生命周期，在昆虫媒介(前鞭毛体)和它们的哺乳动物宿主(无鞭毛体)之间交替。在小鼠模型中，接种皮氏利什曼原虫无鞭毛体的蛋白糖脂复合体(P-8)可部分或完全预防这种微生物的感染。因此，P-8复合体是一种潜在的利什曼病候选疫苗，而对皮氏利什曼原虫无鞭毛体糖脂抗原的结构表征是疫苗研制中的必要步骤。抗P-8复合体的单抗识别四个相关的糖脂成分，但活性组分中糖脂的详细结构仍有待确定。根据初步结果，糖脂有望属于糖肌醇-磷脂或脂磷脂前体之一。单糖组成分析证实了每个组分中都存在碳水化合物成分。MALDI FTMS为糖脂的结构分析提供了许多优点，例如保留在TLC板表面的样品的直接电离和MALDI产生的离子的多级解离能力。先前获得的P8样品作为未分离混合物、部分纯化组分和TLC上分离的斑点的MALDI FTMS和MS/MS谱与糖脂的存在一致，糖脂的碳水化合物部分具有不同的长度。到目前为止，所有的结构都与之前报道的类型相匹配。ESI-MS已经鉴定了一些简单的脂类，MALDI-TOF MS已经得到了以前表征的m/z值，但不是新的。进一步的提纯，再加上我们TLC MALDI FTMS能力的新改进，以及一个新建立的脂类LC MS系统，应该有助于获得完整的结构信息，从而能够识别活性馏分中的次要成分。

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. Protozoan parasites of the genus Leishmania cause a number of diseases in humans. The parasite undergoes a complex life cycle, alternating between an insect vector (promastigotes) and their mammalian hosts (amastigotes). In a mouse model, vaccination with proteoglycolipid complex (P-8) of Leishmania pifanoi amastigotes provides partial to complete protection against infection by this organism. Therefore the P-8 complex represents a potential vaccine candidate for leishmaniasis and structural characterization of the glycolipid antigens of Leishmania pifanoi amastigotes is a necessary step in the vaccine development. Monoclonal antibodies to the P-8 complex recognize four related glycolipid components, but detailed structures of the glycolipids in the active fractions remain to be determined. Based on the preliminary results, the glycolipids are expected to belong to one of the classes of either glycoinositol-phospholipids or lipophosphoglycan precursors. Monosaccharide composition analysis confirms the presence of carbohydrate components in each fraction. MALDI FTMS offers a number of advantages for structural analysis of glycolipids such as direct ionization of samples retained on the surface of TLC plates and capability for multistage dissociation of MALDI-generated ions. Previous MALDI FTMS and MS/MS spectra obtained for the P8 samples as unseparated mixtures, as partly purified components and as spots separated on TLC, are consistent with the presence of glycolipids with carbohydrate moieties of varying lengths. So far, all the structures match types previously reported. ESI-MS has led to the identification of a few simple lipids, and MALDI-TOF MS has yielded previously-characterized m/z values, but not novel ones. Further purification combined with new improvements of our TLC MALDI FTMS capabilities, and a newly set up system for lipid LCMS should help in the acquisition of full structural information that will enable identification of minor components in the active fractions.