Trafficking and localization studies of recombinant α1,3-fucosyltransferase VI stably expressed in CHO cells
Trafficking and localization studies of recombinant α1,3-fucosyltransferase VI stably expressed in CHO cells
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DOI：
10.1093/glycob/8.3.259
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发表时间：
1998-03-01
期刊：
影响因子：
4.3
通讯作者：
Berger, EG
中科院分区：
文献类型：
--
作者：
Borsig, L;Katopodis, AG;Berger, EG
目前已克隆的五种不同的α-1，3-岩藻糖基转移酶(Fuc-ts)作用于糖链的外周α1，3-岩藻糖化。FUC-TVI是一种能够合成选择素配体的α-1，3-岩藻糖基转移酶。人血浆中的主要α-1，3-岩藻糖基转移酶活性是由岩藻糖基转移酶VI基因编码的，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运输研究一直是不可能的。在这里，我们报道了一种针对Fuc-TVI的多肽特异性多克隆抗血清和一种针对纯化的可溶性重组Fuc-TVI与Fuc-TII和Fuc-TV交叉反应的抗血清的研制和鉴定。将这两种抗血清应用于稳定表达该酶全长形式的CHO细胞(CHO克隆61/11)的免疫检测。FUC-TVI是高尔基体的一种驻留蛋白。此外，超过30%的细胞相关和释放的酶活性在培养基中被发现。在代谢标记的CHO 61/11细胞中分析了Fuc-TVI的成熟和释放，免疫沉淀后，Fuc-TVI出现了47 kDa和43 kDa两种形式，而分泌形式被检测到为43 kDa。脉冲追逐实验表明，这两种不同的胞内形式是通过翻译后修饰而产生的。FUC-TVI通过蛋白水解酶裂解释放到上清液中，形成部分抗Endo-H的糖形式。
Peripheral alpha 1,3-fucosylation of glycans occurs by the action of either one of five different alpha 1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha 1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha 1,3-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.