Regulation of glycan structures in animal tissues - Transcript profiling of glycan-related genes
Regulation of glycan structures in animal tissues - Transcript profiling of glycan-related genes
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DOI：
10.1074/jbc.m801964200
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发表时间：
2008-06-20
影响因子：
4.8
通讯作者：
Moremen, Kelley W.
中科院分区：
文献类型：
--
作者：
Nairn, Alison V.;York, William S.;Moremen, Kelley W.
共价连接到蛋白质和脂质上的聚糖结构在哺乳动物细胞中发挥着多种作用，包括分子或细胞水平上的蛋白质折叠、靶向、识别和粘附。调节细胞糖蛋白和糖脂上聚糖结构的丰度是一个复杂的过程，取决于许多因素。大多数聚糖调控模型假设，参与聚糖合成、修饰和分解代谢的酶的转录控制决定了聚糖的丰度和多样性。然而，很少有基础广泛的研究探讨了聚糖结构和编码相关生物合成和分解代谢酶的转录本之间的相关性。许多聚糖相关基因的转录本丰度较低，阻碍了与聚糖结构数据进行比较的广泛转录本分析。为了便于与聚糖结构数据进行比较并确定聚糖结构改变的分子基础，我们开发了一种中等通量定量实时逆转录酶-PCR平台，用于分析小鼠组织和细胞中编码聚糖相关酶和蛋白质的转录本。该方法采用了超过 700 个基因的综合列表，包括参与糖核苷酸生物合成、转运蛋白、聚糖延伸、修饰、识别、分解代谢的酶以及众多糖基化核心蛋白。与平行微阵列分析的比较表明，我们的定量实时逆转录酶 PCR 方法具有显着更高的灵敏度和动态范围，特别是对于许多低丰度聚糖相关酶。将基因和转录物水平映射到各自的生物合成途径步骤可以与聚糖结构数据进行比较，并为模型提供支持，其中许多但不是全部的聚糖丰度变化是由相应生物合成酶转录物表达的改变引起的。
Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of > 700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes.