Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum
Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum
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DOI：
10.1074/jbc.m011323200
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发表时间：
2001-04-06
影响因子：
4.8
通讯作者：
Kieliszewski, MJ
中科院分区：
文献类型：
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作者：
Shpak, E;Barbar, E;Kieliszewski, MJ
羟脯氨酸(Hyp)O-糖基化是植物细胞外基质中富含羟脯氨酸的糖蛋白(HRGP)超家族的特征。糖基化以两种方式发生：阿拉伯糖基化添加短阿拉伯糖苷(Hyp-阿拉伯糖苷)，而半乳糖化导致更大的阿拉伯半乳糖多糖(Hyp-Polychardes)的添加。我们假设，小肽基序的序列依赖的糖基化导致了糖模块。这些小的功能单位与其他重复的多肽模块一起定义了HRGP的性质。Hyp邻接性假说预测相邻Hyp残基的阿拉伯糖基化和聚集的非毗邻Hyp残基的半乳糖化。为了确定引导阿拉伯糖基化的最低水平的Hyp邻接性，我们设计了一系列编码重复(Ser-Pro(2))(N)、(Ser-Pro(3))(N)和(Ser-Pro(4))(N)的合成基因。将这些内源底物定向到内质网/高尔基体的信号序列，用于转化烟草细胞中的翻译后脯氨酸羟化和糖基化。融合糖蛋白中还含有绿色荧光蛋白，便于检测和分离。(Ser-Pro(2))(N)和(Ser-Hyp(4))(N)融合糖蛋白产生阿拉伯糖苷，但不产生多糖。基序(Ser-Pro(3))(N)是不完全羟化的，产生了连续/非连续的混合Hyp和相应的Hyparabinoside和Hyp-Polyps的混合物，这些结果加上糖基化和脱糖的(Ser-Pro(2))(N)、(Ser-Pro(3))(N)和(Ser-Pro(4))(N)模块的圆二色谱证实了Hyp的邻接性假说，并表明Hyp O-糖基化确实是序列驱动的。
Hydroxyproline (Hyp) O-glycosylation characterizes the hydroxyproline-rich glycoprotein (HRGP) superfamily of the plant extracellular matrix. Hyp glycosylation occurs in two modes: Arabinosylation adds short oligoarabinosides (Hyp-arabinosides) while galactosylation leads to the addition of larger arabinogalactan polysaccharides (Hyp-polysaccharides). We hypothesize that sequence-dependent glycosylation of small peptide motifs results in glycomodules. These small functional units in combination with other repetitive peptide modules define the properties of HRGPs. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered noncontiguous Hyp residues. To determine the minimum level of Hyp contiguity that directs arabinosylation, we designed a series of synthetic genes encoding repetitive (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n). A signal sequence targeted these endogenous substrates to the endoplasmic reticulum/Golgi for post-translational proline hydroxylation and glycosylation in transformed Nicotiana tabacum cells. The fusion glycoproteins also contained green fluorescence protein, facilitating their detection and isolation. The (Ser-Pro(2))(n) and (Ser-Hyp(4))(n) fusion glycoproteins yielded Hyp-arabinosides but no Hyp-polysaccharide. The motif (Ser-Pro(3))(n) was incompletely hydroxylated, yielding mixed contiguous/noncontiguous Hyp and a corresponding mixture of Hyparabinosides and Hyp-polysaccharides, These results plus circular dichroic spectra of the glycosylated and deglycosylated (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n) modules corroborate the Hyp contiguity hypothesis and indicate that Hyp O-glycosylation is indeed sequence-driven.