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

Abstract

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.

Detection of Differentially Expressed Wound-Healing–Related Glycogenes in Galectin-3–Deficient Mice

“…Among the differentially expressed genes in healing Gal-3 −/− corneas, the largest group (41%) were glycosyltransferases and glycosidases that regulate glycosylation of proteins and lipids. Specifically, the expression of a galactosyltransferase, β 3-galactosyltransferase 5 ( β 3GalT5) that synthesizes Gal β 1,3GlcNAc (type I chain) to create lactosamine repeats on glycoproteins and glycolipids and thereby has the potential to synthesize ligands for Gal-3,16-18 was downregulated in healing Gal-3 −/− compared with Gal-3 +/+ corneas. The enzymes that were upregulated include polypeptide N -acetylgalactosaminyltransferases-3 and -7 (ppGalNAcTs-3 and -7) that initiate mucin-type O -glycosylation19 and N -aspartylglucosaminidase that removes N-glycans 20.…”

“…This is, however, not surprising. Large changes are expected if the groups being compared are dramatically different such as normal versus healing corneas 18. On the other hand, low changes may be expected if the groups being compared are related.…”

“…The biological role of β 1,3GalT5 includes type I chain elongation of core 2 and core 3 O-glycans, N-glycans,16,17 lactoceramides,31 and globosides32 and create lactosamine residues, which are known to have affinity for Gal-3 8,9. In the wild-type mice, β 1,3GalT5 is markedly upregulated (~12-fold) in healing mouse corneas compared with the normal, unwounded corneas 18. The downregulated expression of β 1,3GalT5 in healing Gal-3 −/− corneas suggests that the glycan ligands on glycoproteins and glycolipids specific for Gal-3 may be reduced in Gal-3 −/− corneas, which may be the reason for the absence of effects of exogenous Gal-3 in promoting wound closure in Gal-3 −/− mouse corneas.…”

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

“…tions of cell adhesion molecules, including cell adhesion and cancer metastasis (10). These structures on proteins, including growth factor receptors and integrins, are shown to enhance growth signaling in motile tumor cells.…”

Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry in aqueous solution

“…(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.…”