Robijn Marjolein L M, Koeleman Carolien A M, Wuhrer Manfred, Royle Louise, Geyer Rudolf, Dwek Raymond A, Rudd Pauline M, Deelder André M, Hokke Cornelis H
Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Centre, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Mol Biochem Parasitol. 2007 Feb;151(2):148-61. doi: 10.1016/j.molbiopara.2006.10.019. Epub 2006 Nov 27.
The eggs of Schistosoma mansoni express a plethora of glycoconjugate antigens. A specific subset of these antigens can be detected in the serum or urine of infected individuals by a diagnostic sandwich ELISA using the anti-carbohydrate monoclonal antibody (mAb) 114-4D12-A [Nourel Din MS, Nibbeling R, Rotmans JP, Polderman AM, Krijger FW, Deelder AM. Quantitative determination of circulating soluble egg antigen in urine and serum of Schistosoma mansoni-infected individuals using a combined two-site enzyme-linked immunosorbent assay. Am J Trop Med Hyg 1994;50:585-94]. We used affinity chromatography to isolate the 114-4D12-binding glycoprotein subset from soluble egg antigens (SEA) of S. mansoni. SEA and the isolated SEA-subset (SEA-4D12) were subjected to reductive beta-elimination and hydrazinolysis to release intact glycans and glycan fragments, respectively, from the protein backbones. The released glycans were characterised by matrix-assisted laser-desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS), liquid-chromatography (LC)-MS and gas chromatography (GC)-MS linkage analysis. Glycans released by reductive beta-elimination from SEA-4D12 were larger and more heavily fucosylated than glycans released from SEA. Most SEA-4D12 glycans contained a branched O-glycan core structure carrying up to 4 N-acetylhexosamines per chain which were substituted with maximum 12 fucose residues. Hydrazinolysis of SEA-4D12 resulted in the release of fucosylated antenna fragments. After 2-aminobenzamide (2AB)-labelling these fragments were subjected to 114-4D12-affinity purification. Normal phase (NP)-LC analysis of the flow-through and retained fractions indicated that the Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1- element forms the epitope of mAb 114-4D12. Most O-glycans from SEA-4D12 contain this structural element. Epitope-bearing N-glycans have not been found. In terms of abundance in total SEA, only a minority of all glycans possesses the epitope. This multifucosylated motif has so far only been found in schistosomes, providing a structural basis for the high specificity of the diagnostic antibody.
曼氏血吸虫卵表达大量糖缀合物抗原。通过使用抗碳水化合物单克隆抗体（mAb）114 - 4D12 - A的诊断夹心酶联免疫吸附测定（ELISA），可以在感染个体的血清或尿液中检测到这些抗原的一个特定子集[努雷尔·丁·M·S，尼布林·R，罗特曼斯·J·P，波尔德曼·A·M，克里杰·F·W，德尔德·A·M。使用联合双位点酶联免疫吸附测定法定量测定曼氏血吸虫感染个体尿液和血清中循环可溶性虫卵抗原。《美国热带医学与卫生杂志》1994年；50：585 - 94]。我们使用亲和色谱法从曼氏血吸虫的可溶性虫卵抗原（SEA）中分离出与114 - 4D12结合的糖蛋白子集。SEA和分离出的SEA子集（SEA - 4D12）分别进行还原性β - 消除和肼解，以从蛋白质主链上释放完整的聚糖和聚糖片段。通过基质辅助激光解吸电离飞行时间（MALDI - TOF）质谱（MS）、液相色谱（LC） - MS和气相色谱（GC） - MS联用分析对释放的聚糖进行表征。从SEA - 4D12通过还原性β - 消除释放的聚糖比从SEA释放的聚糖更大且岩藻糖基化程度更高。大多数SEA - 4D12聚糖含有一个分支的O - 聚糖核心结构，每条链最多携带4个N - 乙酰己糖胺，其被最多12个岩藻糖残基取代。SEA - 4D12的肼解导致岩藻糖基化天线片段的释放。用2 - 氨基苯甲酰胺（2AB）标记后，这些片段进行114 - 4D12亲和纯化。对流出物和保留部分的正相（NP） - LC分析表明，Fucα1 - 2Fucα1 - 3GalNAcbeta1 - 4（Fucα1 - 2Fucα1 - 3）GlcNAcbeta1 - 元件构成mAb 114 - 4D12的表位。SEA - 4D12的大多数O - 聚糖含有这种结构元件。尚未发现带有表位的N - 聚糖。就SEA总量中的丰度而言，所有聚糖中只有少数具有该表位。这种多岩藻糖基化基序迄今为止仅在血吸虫中发现，为诊断抗体的高特异性提供了结构基础。
