Characterization of Endoplasmic Reticulum-Localized UDP-D-Galactose: Hydroxyproline O-Galactosyltransferase Using Synthetic Peptide Substrates in Arabidopsis
Characterization of Endoplasmic Reticulum-Localized UDP-D-Galactose: Hydroxyproline O-Galactosyltransferase Using Synthetic Peptide Substrates in Arabidopsis
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DOI：
10.1104/pp.109.146266
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发表时间：
2010-01-01
期刊：
影响因子：
7.4
通讯作者：
Jigami, Yoshifumi
中科院分区：
文献类型：
--
作者：
Oka, Takuji;Saito, Fumie;Jigami, Yoshifumi
我们的特点是肽基羟脯氨酸（Hyp）O-半乳糖基转移酶（HGT），这是阿拉伯半乳聚糖生物合成途径的起始酶。以化学合成的荧光肽为受体底物，以拟南芥T87细胞提取物为粗酶源，建立了HGT活性的体外测定方法。半乳糖残基转移到肽可以检测到高效液相色谱和基质辅助激光解吸电离飞行时间质谱分析。HGT需要锰的二价阳离子以获得最大活性，并消耗UDP-D-半乳糖作为糖供体。HGT的最适pH范围为pH7.0 ~ 8.0，最适温度为35 ℃。活性的有利底物似乎是含有两个交替的亚氨基酸残基（包括至少一个受体Hyp残基）的肽，尽管具有单个Hyp残基而没有任何其他亚氨基酸的肽也起底物的作用。蔗糖密度梯度离心的结果显示，HGT活性的细胞定位是相同的内质网标记物，如Sec 61和Bip，表明HGT主要定位于内质网。据我们所知，这是HGT的第一个表征，并且数据提供了阿拉伯半乳聚糖生物合成发生在蛋白质转运途径中的证据。
We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis (Arabidopsisthaliana) T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by high-performance liquid chromatography and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analyses. HGT required a divalent cation of manganese for maximal activity and consumed UDP-D-galactose as a sugar donor. HGT exhibited an optimal pH range of pH 7.0 to 8.0 and an optimal temperature of 35 degrees C. The favorable substrates for the activity seemed to be peptides containing two alternating imino acid residues including at least one acceptor Hyp residue, although a peptide with single Hyp residue without any other imino acids also functioned as a substrate. The results of sucrose density gradient centrifugation revealed that the cellular localization of HGT activity is identical to those of endoplasmic reticulum markers such as Sec61 and Bip, indicating that HGT is predominantly localized to the endoplasmic reticulum. To our knowledge, this is the first characterization of HGT, and the data provide evidence that arabinogalactan biosynthesis occurs in the protein transport pathway.