A focused microarray approach to functional glycomics: transcriptional regulation of the glycome

Abstract

Glycosylation is the most common posttranslational modification of proteins, yet genes relevant to the synthesis of glycan structures 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 glycosyltransferases, 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 [THY], spleen [SPL], lymph node, and bone marrow [BM]) are more closely related, relative to those of nonimmune tissues (kidney [KID], liver [LIV], brain [BRN], and testes [TES]). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N- and O-linked oligosaccharides are ubiquitously expressed, whereas glycosyltransferases 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 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) profiling of N-linked oligosaccharides suggested that the alpha1-3 fucosyltransferase 9, Fut9, is the enzyme responsible for terminal fucosylation in KID and BRN, 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 (CFG) (http://www.functionalglycomics.org).

Glycan-related gene expression signatures in human metastatic hepatocellular carcinoma cells

“…Although there are various technologies that are capable of decoding glycan structure, such as mass spectrometry, affinity chromatography technology, nuclear magnetic resonance and carbohydrate microarray, none of them have enabled systematic analysis of the altered carbohydrate structure. The microarray strategies, including lectin, glycan and glycogene arrays, represent a set of essential tools to speed up the development of functional glycomics (7). Exploring gene expression is experimentally more tractable than comprehensively characterizing glycan structures associated with metastatic tendency.…”

“…Exploring gene expression is experimentally more tractable than comprehensively characterizing glycan structures associated with metastatic tendency. The expression alterations in glycogenes have previously been examined using focused microarrays or PCR arrays designed to specifically measure the relevant transcripts (7,8). In this study, we demonstrate the use of glycan gene expression profiling and membrane glycoprotein lectin array assay to capture the metastasis-related information of HCC.…”

Diversity in cell surface sialic acid presentations: implications for biology and disease

“…Tools for In Situ Detection of Sialic Acid Types and Linkages, Using Glycan-Recognizing Probes Recent efforts at 'glycomics' using mass spectrometry are defining the array of terminal glycan structures present in tissues of model organisms such as the mouse. 42 However, such methods extract entire tissue samples, and do not take into account the diversity of cell types within a given tissue or organ-and can even miss major glycans on minor cell types. This approach to glycomics must thus be complemented with probing of tissue sections using natural and recombinant glycan-recognizing probes (GRPs) capable of detecting specific glycan structures in situ.…”

Activation of Murine CD4+ and CD8+ T Lymphocytes Leads to Dramatic Remodeling ofN-Linked Glycans

“…In principle, such analysis can now be conducted by high throughput methods for assessment of glycan structure and expression of the glycosyltransferase genes responsible for their synthesis (29). To this end, we have investigated changes in glycosylation of N-linked glycans of resting and activated murine T and B lymphocytes using MALDI-TOF mass spectrometry (MS) profiling for assessment of glycan structure, and a custom gene microarray based on Affymetrix technology, developed and annotated by the Consortium for Functional Glycomics (͗www.functionalgly comics.org͘) for evaluating changes in expression of glycosyltransferase genes.…”

“…Analysis of gene expression was conducted using a custom gene microarray (GLYCOv1 chip) produced by Affymetrix for the Consortium for Functional Glycomics (͗www.functionalglycomics.org͘), and containing probe sets for 122 glycosyl-and sulfotransferases (29). Three independent experiments were performed for each activation condition of each lymphocyte type (CD4, CD8, and B lymphocytes), resulting in triplicate RNA samples per condition.…”