Abstract
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.
Highlights
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
Whereas protein-carbohydrate interactions are known to play critical roles in biological recognition events, very little is known about the global regulation of glycan biosynthesis and catabolism
Our goal in this study was to examine correlations of glycan structural data with detailed transcript profiles of enzymes and proteins involved in glycan synthesis, modification, recognition, and catabolism in an effort to test the hypothesis that glycan structures are predominantly regulated at the transcriptional level
Summary
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. Most of our understanding of the roles of cellular glycosylation in physiology and pathology comes from a combination of glycan structural analysis on specific glycoproteins, cell surfaces, or total tissue extracts in combination with years of study on the biochemistry and enzymology of glycan biosynthetic and degradative enzymes (10 –13) Despite this array of biochemical and genetic information, very little is known about the global regulation of glycan synthesis and degradation. Numerous factors can impact the efficiency and penetrance of individual glycosylation steps on protein and lipid acceptors, including enzyme accessibility to glycan modification sites, the abundance of the respective protein or lipid acceptors, availability of sugar-nucleotide precursors, and relative enzyme levels or relative localization of biosynthetic enzymes that can compete for the same glycan substrates Despite these complexities in glycan biosynthesis, several lines of evidence indicate that one of the major modes of regulating cellular glycosylation is transcriptional regulation of the enzymes involved in glycan synthesis and catabolism [14]. Transcript Profiling of Mouse Glycan-related Genes scriptional regulation is the major mechanism driving the structural alterations (14, 18 –24)
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