Zhao Zhan Dong, Tan Li, Showalter Allan Marshall, Lamport Derek Thomas Anthony, Kieliszewski Marcia Jane
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45710, USA.
Plant J. 2002 Aug;31(4):431-44. doi: 10.1046/j.1365-313x.2002.01365.x.
Functional analysis of the hyperglycosylated arabinogalactan-proteins (AGPs) attempts to relate biological roles to the molecular properties that result largely from O-Hyp glycosylation putatively coded by the primary sequence. The Hyp contiguity hypothesis predicts contiguous Hyp residues as attachment sites for arabino-oligosaccharides (arabinosides) and clustered, non-contiguous Hyp residues as arabinogalactan polysaccharide sites. Although earlier tests of naturally occurring hydroxyproline-rich glycoproteins (HRGPs) and HRGPs designed by synthetic genes were consistent with a sequence-driven code, the predictive value of the hypothesis starting from the DNA sequences of known AGPs remained untested due to difficulties in purifying a single AGP for analysis. However, expression in tobacco (Nicotiana tabacum) of the major tomato (Lycopersicon esculentum) AGP, LeAGP-1, as an enhanced green fluorescent protein fusion glycoprotein (EGFP)-LeAGP-1, increased its hydrophobicity sufficiently for chromatographic purification from other closely related endogenous AGPs. We also designed and purified two variants of LeAGP-1 for future functional analysis: one lacking the putative glycosylphosphatidylinositol (GPI)-anchor signal sequence; the other lacking a 12-residue internal lysine-rich region. Fluorescence microscopy of plasmolysed cells confirmed the location of LeAGP-1 at the plasma membrane outer surface and in Hechtian threads. Hyp glycoside profiles of the fusion glycoproteins gave ratios of Hyp-polysaccharides to Hyp-arabinosides plus non-glycosylated Hyp consistent with those predicted from DNA sequences by the Hyp contiguity hypothesis. These results demonstrate a route to the purification of AGPs and the use of the Hyp contiguity hypothesis for predicting the Hyp O-glycosylation profile of an HRGP from its DNA sequence.
对高糖基化阿拉伯半乳聚糖蛋白（AGP）进行功能分析，旨在将生物学功能与分子特性联系起来，这些分子特性很大程度上源于由一级序列推测编码的O-羟脯氨酸糖基化。羟脯氨酸连续性假说是指连续的羟脯氨酸残基作为阿拉伯寡糖（阿拉伯糖苷）的附着位点，而簇状、不连续的羟脯氨酸残基则作为阿拉伯半乳聚糖多糖位点。尽管早期对天然富含羟脯氨酸的糖蛋白（HRGP）以及由合成基因设计的HRGP进行的测试与序列驱动编码一致，但由于难以纯化单个AGP进行分析，从已知AGP的DNA序列出发，该假说的预测价值仍未得到验证。然而，通过在烟草（Nicotiana tabacum）中表达主要的番茄（Lycopersicon esculentum）AGP，即LeAGP-1，作为增强型绿色荧光蛋白融合糖蛋白（EGFP）-LeAGP-1，其疏水性增加到足以从其他密切相关的内源性AGP中通过色谱法进行纯化。我们还设计并纯化了LeAGP-1的两个变体用于未来的功能分析：一个缺少假定的糖基磷脂酰肌醇（GPI）锚定信号序列；另一个缺少一个12个残基的富含赖氨酸的内部区域。质壁分离细胞的荧光显微镜检查证实了LeAGP-1位于质膜外表面和赫氏线中。融合糖蛋白的羟脯氨酸糖苷谱给出了羟脯氨酸多糖与羟脯氨酸阿拉伯糖苷加非糖基化羟脯氨酸的比率，与羟脯氨酸连续性假说从DNA序列预测的比率一致。这些结果证明了一种纯化AGP的途径，以及利用羟脯氨酸连续性假说从HRGP的DNA序列预测其羟脯氨酸O-糖基化谱的方法。
