Borsig L, Kleene R, Dinter A, Berger E G
Institute of Physiology, University of Zürich, Switzerland.
Eur J Cell Biol. 1996 May;70(1):42-53.
The fucosyltransferases constitute a family of glycosyltransferases incorporating fucose residues into glycoprotein or glycolipid glycans. They afford one of the possible termination steps of glycoconjugate biosynthesis creating the sialyl Lewisx or sialyl Lewisa determinant, which play an important role in cell-cell interaction. While cDNA, chromosomal localization and kinetic properties of a number of fucosyltransferases are known, immunocytochemical localization and trafficking studies have been delayed because of the lack of specific antibodies due to the pronounced homology of alpha 1, 3 fucolsyltransferases III, V and VI. Here we report development and characterization of monospecific polyclonal antibodies to alpha 1-3 fucosyltransferase V (FucT-V) and their application for immunodetection in transfected cells. Antisera against FucT-V were raised in two different ways: first by producing a fusion protein beta-galactosidase-FucT-V in Escherichia coli, and by synthesizing a peptide stretch specific for FucT-V. Polyclonal antisera were raised against each of both antigens and characterized by enzyme-linked immunosorbent assay, neutralization of activity, immunoblotting, immunofluorescence and immunoprecipitation of metabolically labeled COS cells, transiently transfected with cDNA encoding FucT-V. Both antibodies recognized only FucT-V. No cross-reactivity to FucT-III or FucT-VI was observed. FucT-V was localized mainly to the Golgi apparatus by colocalization with beta 1, 4-galactosyltransferase, and to the cell surface of COS, CHO and HeLa cells. Expression of FucT-V in COS cells revealed three enzyme forms of 58, 53 and 50 kDa, respectively. These size differences arose by post-translational modifications, as shown by pulse-chase experiments. Our results indicate that alpha 1-3 fucosyltransferase is a Golgi-associated enzyme and suggest its possible occurrence on the cell surface.
岩藻糖基转移酶构成了一类糖基转移酶家族，可将岩藻糖残基掺入糖蛋白或糖脂聚糖中。它们是糖缀合物生物合成中可能的终止步骤之一，可产生唾液酸化路易斯x或唾液酸化路易斯a决定簇，这些决定簇在细胞间相互作用中发挥重要作用。虽然许多岩藻糖基转移酶的cDNA、染色体定位和动力学特性已为人所知，但由于α1,3岩藻糖基转移酶III、V和VI具有显著的同源性，缺乏特异性抗体，免疫细胞化学定位和运输研究一直滞后。在此，我们报告了针对α1-3岩藻糖基转移酶V（FucT-V）的单特异性多克隆抗体的研制及其在转染细胞免疫检测中的应用。针对FucT-V的抗血清通过两种不同方式制备：第一种是在大肠杆菌中产生融合蛋白β-半乳糖苷酶-FucT-V，第二种是合成对FucT-V特异的肽段。针对这两种抗原分别制备了多克隆抗血清，并通过酶联免疫吸附测定、活性中和、免疫印迹、免疫荧光以及对用编码FucT-V的cDNA瞬时转染的代谢标记COS细胞进行免疫沉淀来进行表征。两种抗体仅识别FucT-V。未观察到与FucT-III或FucT-VI的交叉反应。通过与β1,4-半乳糖基转移酶共定位，FucT-V主要定位于高尔基体，并定位于COS、CHO和HeLa细胞的细胞表面。FucT-V在COS细胞中的表达分别显示出58 kDa、53 kDa和50 kDa的三种酶形式。脉冲追踪实验表明，这些大小差异是由翻译后修饰引起的。我们的结果表明，α1-3岩藻糖基转移酶是一种与高尔基体相关的酶，并提示其可能存在于细胞表面。
