Arabinogalactan proteins (AGPs) are extracellular proteoglycans with many O-linked glycan chains. Precursors to many AGPs contain a C-terminal signal for the addition of a glycosylphosphatidylinositol (GPI)-anchor, but the role of this modification has not been elucidated. NtAGP1, a tobacco precursor to AGP, comprises a signal peptide, an AGP-coding region, and a GPI-anchoring signal, and it is classified as a member of the classical AGP family. Using green fluorescent protein (GFP) and sweet potato sporamin (SPO) as tags and tobacco BY-2 cells as the host, we analysed the transport and modification of NtAGP1. The fusion protein of GFP or SPO and NtAGP1 expressed in BY-2 cells migrated as a large smear on SDS-polyacrylamide gel. A confocal microscopic analysis indicated that the GFP and NtAGP1 fusion protein localized to the plasma membrane (PM) and intracellular structures. Fractionation studies of microsomes indicated that most of the fusion protein of SPO and NtAGP1 (SPO-AGP) localized to the PM. In contrast, the expression of mutants without a GPI-anchoring signal yielded several forms. The largest forms migrating as large smears on the gel were secreted into the culture medium, whereas other forms were recovered in the endomembrane organelles. A comparison of the glycan structures of the microsomal SPO-AGP and the secreted mutant SPO-AGP without a GPI-anchoring signal using antibodies against AGP glycan epitopes indicated that the glycan structures of these proteins differ. These observations indicate that a GPI-anchoring signal is required for both the proper transport and glycosylation of the AGP precursor.

中文翻译：

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烟草 BY-2 细胞中经典阿拉伯半乳聚糖蛋白前体的正确转运和糖基化需要糖基磷脂酰肌醇锚定

阿拉伯半乳聚糖蛋白 (AGP) 是具有许多 O-连接聚糖链的细胞外蛋白聚糖。许多 AGP 的前体包含用于添加糖基磷脂酰肌醇 (GPI) 锚点的 C 端信号，但这种修饰的作用尚未阐明。NtAGP1 是 AGP 的烟草前体，包含一个信号肽、一个 AGP 编码区和一个 GPI 锚定信号，它被归类为经典 AGP 家族的成员。我们以绿色荧光蛋白 (GFP) 和甘薯孢菌素 (SPO) 为标签，以烟草 BY-2 细胞为宿主，分析了 NtAGP1 的转运和修饰。在 BY-2 细胞中表达的 GFP 或 SPO 与 NtAGP1 的融合蛋白在 SDS-聚丙烯酰胺凝胶上迁移为大涂片。共聚焦显微镜分析表明 GFP 和 NtAGP1 融合蛋白定位于质膜 (PM) 和细胞内结构。微粒体的分级分离研究表明，大部分 SPO 和 NtAGP1 (SPO-AGP) 的融合蛋白定位于 PM。相反，没有 GPI 锚定信号的突变体的表达产生了几种形式。作为凝胶上的大涂片迁移的最大形式被分泌到培养基中，而其他形式则在内膜细胞器中回收。使用针对 AGP 聚糖表位的抗体比较微粒体 SPO-AGP 和没有 GPI 锚定信号的分泌突变体 SPO-AGP 的聚糖结构表明这些蛋白质的聚糖结构不同。