Bencúrová Monika, Hemmer Wolfgang, Focke-Tejkl Margarete, Wilson Iain B H, Altmann Friedrich
Glycobiology Division, Institute of Chemistry, University of Natural Resources and Applied Life Sciences (Universität für Bodenkultur), Muthgasse 18, A-1190, Vienna, Austria.
Glycobiology. 2004 May;14(5):457-66. doi: 10.1093/glycob/cwh058. Epub 2004 Mar 19.
Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing beta1,2-xylose and core alpha1,3-fucose, the latter also found in insect glycoproteins. To determine the relative contributions of these two sugar residues to antibody binding, we prepared an array of glycomodified forms of human apo-transferrin. Using core-alpha1, 3-fucosyltransferase (EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF), or neither (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the alpha1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in western blot, ELISA, and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.
植物的交叉反应性碳水化合物决定簇本质上是含有β1,2-木糖和核心α1,3-岩藻糖的N-聚糖混合物，后者也存在于昆虫糖蛋白中。为了确定这两种糖残基对抗体结合的相对贡献，我们制备了一系列糖基化修饰的人脱铁转铁蛋白。利用在毕赤酵母中重组表达的核心α1,3-岩藻糖基转移酶（EC 2.4.1.214）和β1,2-木糖基转移酶（EC 2.4.2.38）以及合适的糖苷酶，生成了仅连接岩藻糖（MMF）、仅连接木糖（MMX）、同时连接二者（MMXF）或均不连接（MM）到常见五糖核心的糖型。通过酶促去除α1,3-连接的甘露糖残基获得了其他糖型。这些转铁蛋白糖型用于确定蛋白质印迹法、酶联免疫吸附测定（ELISA）和抑制ELISA中抗体的结合特异性。兔抗辣根过氧化物酶血清与岩藻糖基化（MMF）和木糖基化（MMX）糖型均结合。抑制研究表明存在两个独立的、与两个表位之一反应的高度特异性群体。相比之下，单克隆抗体YZ1/2.23似乎识别一个更大的结构，包括岩藻糖基和木糖基残基。缺乏甘露糖的糖型对两种抗体的抑制作用较差。末端N-乙酰葡糖胺残基可阻止抗体结合。兔抗蜂毒血清仅与岩藻糖基化形式（MMF和MMXF）反应。对过敏患者血清的实验表明，糖基化修饰的人转铁蛋白，尤其是MMXF糖型，是检测针对交叉反应性碳水化合物决定簇抗体的合适试剂。在所研究的血清组中，几份血清含有高水平的岩藻糖反应性IgE，但只有少数血清显示与MMX-转铁蛋白有任何结合。
