STRUCTURE AND CELL-SURFACE MATURATION OF THE ATTACHMENT GLYCOPROTEIN OF HUMAN RESPIRATORY SYNCYTIAL VIRUS IN A CELL-LINE DEFICIENT IN O GLYCOSYLATION
STRUCTURE AND CELL-SURFACE MATURATION OF THE ATTACHMENT GLYCOPROTEIN OF HUMAN RESPIRATORY SYNCYTIAL VIRUS IN A CELL-LINE DEFICIENT IN O GLYCOSYLATION
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DOI：
10.1128/jvi.63.11.4767-4776.1989
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发表时间：
1989-11-01
影响因子：
5.4
通讯作者：
BALL, LA
中科院分区：
文献类型：
--
作者：
WERTZ, GW;KRIEGER, M;BALL, LA
在蛋白O糖基化缺陷的突变中国仓鼠卵巢（CHO）细胞系ldID中检测了人呼吸道合胞病毒广泛O糖基化附着蛋白G的合成及其在细胞表面的表达。这些细胞与N-连接寡糖合成抑制剂联合使用，可用于建立不发生碳水化合物添加或仅发生N-连接或O-连接碳水化合物添加的条件。构建了G蛋白的重组牛痘病毒表达载体，该载体除了含有人呼吸道合胞病毒G基因外，还含有牛痘病毒基因组的一部分，该部分避开了CHO细胞中牛痘病毒的正常宿主范围限制。重组载体在突变型IdID和野生型CHO细胞中表达高水平的G蛋白。G蛋白的几种未成熟形式被鉴定为仅含有N-连接或O-连接的寡糖侧链。代谢脉冲追踪研究表明，G蛋白的成熟途径是从32千道尔顿（kDa）多肽的合成开始的，伴随着高甘露糖N-连接糖的共翻译连接，形成一个表观质量为45 kDa的中间体。该步骤之后是高尔基体相关的N-连接糖转化为复合物类型和O-连接寡糖的完成，以获得天然90-kDa形式的G。从45-kDa的N-连接的形式的成熟的90-kDa的形式只发生在O-连接的糖添加的存在下，确认O-连接的寡糖构成的成熟G蛋白的质量的显着比例。在没有O糖基化的情况下，观察到G承载半乳糖缺乏的截短的N-连接和完全成熟的N-连接寡糖的形式。测量了N-和O-连接的糖添加对G运输到细胞表面的影响。间接免疫荧光和流式细胞仪检测结果表明，G蛋白在O糖基化和N糖基化缺失的细胞表面均可表达。然而，细胞表面的G缺乏N-和O-连接的寡糖的表达受到严重抑制。
The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldID, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant IdID and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the nature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.