Analysis of differential expression of glycosyltransferases in healing corneas by glycogene microarrays

Program in Cell, Molecular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA.
Glycobiology (Impact Factor: 3.15). 10/2009; 20(1):13-23. DOI: 10.1093/glycob/cwp133
Source: PubMed


It is generally accepted that the glycans on the cell surface and extracellular matrix proteins play a pivotal role in the events that mediate re-epithelialization of wounds. Yet, the global alteration in the structure and composition of glycans, specifically occurring during corneal wound closure remains unknown. In this study, GLYCOv2 glycogene microarray technology was used for the first time to identify the differentially expressed glycosylation-related genes in healing mouse corneas. Of approximately 2000 glycogenes on the array, the expression of 11 glycosytransferase and glycosidase enzymes was upregulated and that of 19 was downregulated more than 1.5-fold in healing corneas compared with the normal, uninjured corneas. Among them, notably, glycosyltransferases, beta3GalT5, T-synthase, and GnTIVb, were all found to be induced in the corneas in response to injury, whereas, GnTIII and many sialyltransferases were downregulated. Interestingly, it appears that the glycan structures on glycoproteins and glycolipids, expressed in healing corneas as a result of differential regulation of these glycosyltransferases, may serve as specific counter-receptors for galectin-3, a carbohydrate-binding protein, known to play a key role in re-epithelialization of corneal wounds. Additionally, many glycogenes including a proteoglycan, glypican-3, cell adhesion proteins dectin-1 and -2, and mincle, and mucin 1 were identified for the first time to be differentially regulated during corneal wound healing. Results of glycogene microarray data were confirmed by qRT-PCR and lectin blot analyses. The differentially expressed glycogenes identified in the present study have not previously been investigated in the context of wound healing and represent novel factors for investigating the role of carbohydrate-mediated recognition in corneal wound healing.

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    • "Several high-throughput microarray studies have been applied to investigate the functional roles of various glycogenes [11] [19] [20]. However, to the best of our knowledge, no study till date has utilized RNA-Seq data in the existing databases to specifically explore the role of glycogenes. "
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    ABSTRACT: Glycogenes regulate a wide array of biological processes in the development of organisms as well as different diseases such as cancer, primary open-angle glaucoma, and renal dysfunction. The objective of this study was to explore the role of differentially expressed glycogenes (DEGGs) in three major tissues such as brain, muscle, and liver using mouse RNA-seq data, and we identified 579, 501, and 442 DEGGs for brain versus liver (BvL579), brain versus muscle (BvM501), and liver versus muscle (LvM442) groups. DAVID functional analysis suggested inflammatory response, glycosaminoglycan metabolic process, and protein maturation as the enriched biological processes in BvL579, BvM501, and LvM442, respectively. These DEGGs were then used to construct three interaction networks by using GeneMANIA, from which we detected potential hub genes such as PEMT and HPXN (BvL579), IGF2 and NID2 (BvM501), and STAT6 and FLT1 (LvM442), having the highest degree. Additionally, our community analysis results suggest that the significance of immune system related processes in liver, glycosphingolipid metabolic processes in the development of brain, and the processes such as cell proliferation, adhesion, and growth are important for muscle development. Further studies are required to confirm the role of predicted hub genes as well as the significance of biological processes.
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    • "Such complexity makes it difficult to characterize protein glycosylation patterns on a proteomic scale. The major limitations of the current glycoproteomic studies include: (1) Searching for changes in glycan structure or glycosylation site occupancy of a single glycoprotein, rather than in a high-throughput manner on a large proteomic scale [16,20]; (2) Investigating glycosyltransferase or the general changing trends of glycan structure in biological samples, regardless of the glycan structure of each specific glycoprotein [21-23]; and (3) Analyzing the expression levels of glycoproteins with specific glycan structure, but not describing the glycosylation sites and glycosylation site occupancy [24-27]. In order to overcome these limitations, we developed an integrated strategy which can be used to quantitatively analyze the abundance of glycoproteins/glycopeptides in a high-throughput manner, as well as the glycan structure and sites of altered N-glycosylation. "
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    ABSTRACT: The complexity of protein glycosylation makes it difficult to characterize glycosylation patterns on a proteomic scale. In this study, we developed an integrated strategy for comparatively analyzing N-glycosylation/glycoproteins quantitatively from complex biological samples in a high-throughput manner. This strategy entailed separating and enriching glycopeptides/glycoproteins using lectin affinity chromatography, and then tandem labeling them with 18O/16O to generate a mass shift of 6 Da between the paired glycopeptides, and finally analyzing them with liquid chromatography-mass spectrometry (LC-MS) and the automatic quantitative method we developed based on Mascot Distiller. The accuracy and repeatability of this strategy were first verified using standard glycoproteins; linearity was maintained within a range of 1:10-10:1. The peptide concentration ratios obtained by the self-build quantitative method were similar to both the manually calculated and theoretical values, with a standard deviation (SD) of 0.023-0.186 for glycopeptides. The feasibility of the strategy was further confirmed with serum from hepatocellular carcinoma (HCC) patients and healthy individuals; the expression of 44 glycopeptides and 30 glycoproteins were significantly different between HCC patient and control serum. This strategy is accurate, repeatable, and efficient, and may be a useful tool for identification of disease-related N-glycosylation/glycoprotein changes.
    Full-text · Article · Jan 2014 · Proteome Science
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    • "(Kiessling and Splain 2010; Lepenies and Seeberger 2010; Li and Richards 2010; Seeberger 2009; Varki et al. 2009; Wu and Wong 2011) For example, different carbohydrate array methodologies have recently been designed and evaluated for high-throughput analysis of protein-carbohydrate interactions. (Horlacher and Seeberger 2008; Krishnamoorthy and Mahal 2009; Lee and Shin 2005; Oyelaran and Gildersleeve 2009; Park et al. 2008; Park and Shin 2007; Pei et al. 2007c; Tyagi et al. 2010; Wu et al. 2009) Glycan arrays have thus been used to identify proteins involved in cancer metastasis, (Hatakeyama et al. 2009) enzymes involved in wound healing, (Saravanan et al. 2010) and glycans modulating T cell death; (Earl et al. 2010) to evaluate blood serum glycan binding, (Huflejt et al. 2009) antibodies towards HIV, (Luallen et al. 2010) and antibodies for use in cancer treatment; (Huang et al. 2006; Nagre et al. 2010; Sawada et al. 2011) to evaluate the binding specificity of glycan-binding proteins and receptors; (Feinberg et al. 2010; Gout et al. 2010; Hoorelbeke et al. 2011; Horlacher et al. 2011; Pipirou et al. 2011; Porter et al. 2010; Singh et al. 2009) to investigate the binding specificities of disease causing bacteria, (Hu et al. 2011) viruses, (Krishnamoorthy et al. 2009; Neu et al. 2010; Nilsson et al. 2011) and fungi;(Chachadi et al. 2011) as well as for the in-depth investigation of avian and swine influenza viruses.(de Vries et al. 2011; Lao et al. 2011; Liao et al. 2010; Pappas et al. 2010; Stevens et al. 2010; Xu et al. 2010) Although such glycan array methodologies yield significant knowledge of glycan interactions, there are still obstacles to overcome in the production of universally valid array methodologies. "
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    ABSTRACT: The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.
    Full-text · Article · Jan 2012 · Biosensors & Bioelectronics
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