Borsig L, Katopodis A G, Bowen B R, Berger E G
Institute of Physiology, University of Zürich, Switzerland.
Glycobiology. 1998 Mar;8(3):259-68. doi: 10.1093/glycob/8.3.259.
Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3-fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.
聚糖的外周α1,3-岩藻糖基化是由迄今克隆的五种不同的α1,3-岩藻糖基转移酶（Fuc-Ts）之一的作用引起的。Fuc-TVI是能够合成选择素配体的α1,3-岩藻糖基转移酶之一。人血浆中的主要α1,3-岩藻糖基转移酶活性由岩藻糖基转移酶VI的基因编码，该基因可能起源于肝细胞。虽然Fuc-TVI的序列、染色体定位和动力学特性是已知的，但由于缺乏特异性抗体，免疫细胞化学定位和转运研究一直无法进行。在这里，我们报告了一种对Fuc-TVI具有肽特异性的多克隆抗血清以及一种与Fuc-TIII和Fuc-TV交叉反应的针对纯化的可溶性重组Fuc-TVI的抗血清的开发和表征。两种抗血清都用于在稳定转染表达该酶全长形式的CHO细胞（CHO克隆61/11）中进行免疫检测。发现Fuc-TVI是高尔基体的驻留蛋白。此外，在培养基中发现超过30%的细胞相关和释放的酶活性。在代谢标记的CHO 61/11细胞中进行免疫沉淀后，分析了Fuc-TVI的成熟和释放。Fuc-TVI以47 kDa和43 kDa条带的两种形式出现，而分泌形式检测为43 kDa。如脉冲追踪实验所示，这两种不同的细胞内形式是通过翻译后修饰产生的。Fuc-TVI通过蛋白水解切割作为部分耐内切糖苷酶H的糖型释放到上清液中。
