A Focused Microarray Approach to Functional Glycomics: Transcriptional Regulation of the Glycome. Mus musculus

Glycosylation is the most common post-translational modification of proteins, yet genes relevant to the synthesis of glycan structure and function are incompletely represented and poorly annotated on the commercially available arrays. To fill the need for expression analysis of such genes we employed the Affymetrix technology to develop a focused and highly annotated glycogene chip representing human and murine glycogenes, including glycosyltranferases, nucleotide sugar transporters, glycosidases, proteoglycans and glycan-binding proteins. In this report the array has been used to generate glycogene expression profiles of nine murine tissues. Global analysis with a hierarchical clustering algorithm, reveals that expression profiles in immune tissues (thymus, spleen, lymph node and bone marrow) are more closely related, relative to those of non-immune tissues (kidney, liver, brain and testes). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N-and O-linked oligosaccharides are ubiquitously expressed, while glycosyltransferase that elaborate terminal structures are expressed in a highly tissue-specific manner, accounting for tissue and ultimately cell type-specific glycosylation. Comparison of gene expression profiles with MALDI-TOF profiling of N-linked oligosaccharides suggested that the alpha-1-3 fucosyltransferase IX, Fut9, is the enzyme responsible for terminal fucosylation in kidney and brain, a finding validated by analysis of Fut9 knockout mice. Two families of glycan-binding proteins, C-type lectins and siglecs, are predominately expressed in the immune tissues, consistent with their emerging functions in both innate and acquired immunity. The glycogene chip reported in this study is available to the scientific community through the Consortium for Functional Glycomics (http://www.functionalglycomics.org). Keywords: Glycogenes, Glycomics, Glycosyltransferase, Lectin, Glycosylation, Glycome, Microarray, Kidney, Fut9 Overall design: Total RNA was extracted from 9 different wild-type mouse tissues comprising of liver, brain, kidney, spleen, thymus, lymphnodes, testis, lung and femurs for bone marrow. 3 samples were collected from each tissue type, resulting in a total of 27 samples (9 sets of 3 replicates). Glycogene expression profiling was performed on each sample using the Affymetrix custom-designed Glycov1 array.