Glycobiology vol. 20 no. 1 pp. 13–23, 2010
Advance Access publication on September 7, 2009
Analysis of differential expression of glycosyltransferases in healing corneas
by glycogene microarrays
Chandrassegar Saravanan2,3, Zhiyi Cao3, Steven R Head4,
and Noorjahan Panjwani1,2,3
2Program in Cell, Molecular and Developmental Biology, Sackler School of
Graduate Biomedical Sciences;3Department of Ophthalmology and The New
England Eye Center, Tufts University School of Medicine, Boston, MA; and
4The Scripps Research Institute, La Jolla, CA, USA
Received on December 18, 2008; revised on August 9, 2009; accepted on
August 28, 2009
It is generally accepted that the glycans on the cell sur-
face 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 occuring during corneal wound clo-
sure remains unknown. In this study, GLYCOv2 glycogene
microarray technology was used for the first time to iden-
tify the differentially expressed glycosylation-related genes
in healing mouse corneas. Of ∼2000 glycogenes on the ar-
ray, 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, glycosyl-
transferases, β3GalT5, T-synthase, and GnTIVb, were all
found to be induced in the corneas in response to injury,
whereas, GnTIII and many sialyltransferases were down-
regulated. Interestingly, it appears that the glycan struc-
tures on glycoproteins and glycolipids, expressed in healing
corneas as a result of differential regulation of these glyco-
syltransferases, 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. Addition-
ally, many glycogenes including a proteoglycan, glypican-3,
1 were identified for the first time to be differentially reg-
ulated during corneal wound healing. Results of glycogene
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.
1To whom correspondence should be addressed: Tel: +1-617-636-6776; Fax:
+1-617-636-0348; e-mail: firstname.lastname@example.org
Wound re-epithelialization in many organs including cornea,
skin, and mucous membranes occurs by the coordinated mi-
gration of adjacent epithelial cells over the wound surface
(Singer and Clark 1999; Lu et al. 2001). Defects in the re-
epithelialization process, including impaired migration or fail-
fects and ulceration (Singer and Clark 1999; Ma and Dohlman
2002). It is generally accepted that glycans on the plasma mem-
brane of the corneal epithelium play a pivotal role in the events
that modulate re-epithelialization (Gipson and Anderson 1980;
Gipson et al. 1984; Panjwani et al. 1990; Panjwani, Zhao,
et al. 1995; Yang et al. 1996; Cao et al. 2001). It has long
been demonstrated that increased amounts of plant lectins, such
as concanavalin A (ConA) and wheat germ agglutinin (WGA),
bind to migrating epithelium as compared to normal nonmigrat-
ing epithelium (Gipson et al. 1983), and a number of glycopro-
compared to normal corneal epithelium (Panjwani et al. 1990;
et al. 1996; Saika et al. 2000). While the studies conducted thus
far using plant lectins and glycan-specific monoclonal antibod-
ies have recognized that thereare substantial changes inspecific
glycan structures during re-epithelialization of corneal wounds,
the global alteration in the structure and composition of glycans
during corneal wound closure remains unknown.
Among the many factors that regulate the glycosylation pat-
Alteration in the expression pattern of genes encoding glyco-
syltransferases has been shown to have enormous impact on
cell behavior, morphology, and functions (Ohtsubo and Marth
2006). For example, extensive studies aimed at characteriza-
tion of the role of glycosyltransferases in cancer cell migra-
tion have demonstrated that changes in expression of glycosyl-
transferases alter the glycan profiles and hence the functions of
many glycoproteins (reviewed in Gorelik et al. 2001; Lau and
Dennis 2008). Thus far, only a few studies have focused on the
role of glycosyltransferases in the repair of wounds. For ex-
ample, β1,4-galactosyltransferase-1, which synthesizes type 2
chain (Galβ1,4GlcNAc) on N-glycans and the core 2 branch in
O-glycans, has been shown to participate in skin wound healing
by recruiting leukocytes, and by promoting angiogenesis and
collagen deposition at the sites of wound (Mori et al. 2004;
Shen et al. 2008). To date, the expression profile of glycosyl-
transferases during re-epithelialization of corneal wounds has
croarray approach that detects the transcript levels of enzymes
regulating glycosylation, we report that many glycosyltrans-
ferases are differentially regulated during re-epithelialization
c ?The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: email@example.com
C Saravanan et al.
Fig. 1. Dendrogram showing similarities in gene expression profiles within
each of the three replicates of healing and normal corneas. Six expression
arrays (three healing and three normal) were hierarchically clustered with the
algorithm BRB ArrayTools 3.2.2 and displayed with TreeView. The individual
samples were clustered in branches of the dendrogram based on overall
similarity in patterns of gene expression. Note that all normal corneal samples
clustered together on one side of the dendrogram, whereas all three samples of
healing corneas clustered on the other side of the dendrogram.
of corneal wounds after transepithelial excimer laser kerate-
ctomy. Of particular interest is our finding that among the
differentially expressed glycosyltransferases are enzymes such
as β1,3-galactosyltransferase 5 (β3GalT5), mannoside acetyl-
glucosaminyltransferase IVb (GnTIVb), T-synthase, and sialyl-
transferases, whose differential expression is likely to result in
the expression of glycans on glycoproteins and glycolipids that
serve as high-affinity ligands for galectin-3, a β-galactoside-
binding protein, that is known to promote re-epithelialization of
corneal wounds (Cao, Said, et al. 2002).
Analysis of RNA yield and quality
The total RNA extracted from the normal and healing corneas
were run on an Agilent 2100 Bioanalyzer for assessment of
quality and purity. The average yield of total RNA was 12.5 ±
1.0 μg and 8.8 ± 0.8 μg per 10 normal and healing corneas, re-
spectively. The ribosomal RNA 28S/18S ratio ranged between
1.3 and 1.7 (supplementary Figure 1A). Supplementary Fig-
ure 1B shows representative electropherograms and gel-like
images, wherein the gel bands and graph peaks correspond to
18S and 28S dominant components with insignificant amount
of low-molecular-weight RNA. This suggests that RNA degra-
dation in the samples used was minimal and the quality of RNA
preparations was satisfactory for the microarray analysis.
Normal and healing corneas show distinct glycogene
Glycov2 array was hybridized with biotinylated cRNA probes
prepared from total RNA from normal and healing corneas, and
the RMA algorithm was used to obtain the expression signal
values. Similarity in overall gene expression profiles between
individual samples was assessed by an unsupervised hierarchial
clustering. As shown in Figure 1, all normal corneal samples
clustered together on one side of the dendrogram, whereas all
three samples of healing corneas clustered on the other side
of the dendrogram. This implies that the glycogene expression
pattern in healing corneas was distinct from that of the normal
Identification of differentially expressed glycosyltransferases
and glycosidases in healing corneas after excimer laser injury
that of 39 genes was downregulated more than 1.5-fold in heal-
ing compared with normal, uninjured corneas with parametric
P-value of <0.01. The differentially expressed genes were vi-
sualized as a heat map using Cluster and Tree View software
(Figure 2A). The color-based view also demonstrates that the
healing corneas show a distinct gene expression profile from
that of the normal, uninjured corneas. For clarity, these genes
are grouped according to their involvement in specific cellu-
lar processes or functions as determined using David software,
Entrez gene, and Gene Cards (Figure 2B and supplementary Ta-
ble 1). Of the 75 differentially expressed genes, 30 genes (40%)
are glycosyltransferases and glycosidases, 36% fall under the
growth factors and their receptors category, and 9% are cell
adhesion proteins. Of the remaining 15% of differentially ex-
pressed genes, 4% were core proteins of proteoglycans and the
rest (11%) are known to participate in various biological func-
tions and were categorized as miscellaneous molecules (Fig-
ure 3B and supplementary Table 1). IL-1β, transforming growth
factor-β1 (TGF-β1), amphiregulin, and intercellular adhesion
molecule 1 (ICAM1), which are known to be upregulated in
healing corneas (Planck et al. 1997; Sotozono et al. 1997; Chen
et al. 2000; Zieske et al. 2000; Cao, Wu, et al. 2002), were all
found to be induced in response to injury in the current study,
thereby attesting to the validity of the analysis. The expression
of many glycogenes including core proteins of proteoglycans,
serglycin, glypican-3, cell adhesion proteins mincle, dectin-1
and -2, and MUC1 were identified for the first time to be differ-
entially expressed in healing corneas (supplementary Table 1).
The differentially expressed glycogenes that regulate glyco-
sylation were further categorized according to their specific
function and are listed in Table I. Among the glycosyltrans-
ferase and glycosidase enzymes, the expression of 11 glyco-
genes was upregulated, whereas that of 19 was downregulated
in healing compared to normal corneas. In an earlier study, we
but not galectin-1, plays a role in re-epithelialization of corneal
wounds (Cao, Said, et al. 2002). It was, therefore, of interest to
assess whether injury-specific glycosyltransferases identified in
the current study have the potential to synthesize glycans that
serve as ligands of galectin-3. In this respect, most notably, the
expression of β3GalT5, GnTIVb, and T-synthase was upregu-
to synthesize high-affinity ligands for galectin-3 (Yoshida et al.
1998; Glinsky et al. 2000; Zhou et al. 2000; Salvini et al. 2001;
trast, the expression of GnTIII and ST6GalI sialyltransferases,
which synthesize glycans that block galectin-3 binding to its
ligands (Patnaik et al. 2006; Zhuo et al. 2008), was downreg-
ulated in healing corneas. Based on these data, it appears that
the differential expression of the glycosyltransferases in healing
corneas leads to the upregulation of the high-affinity glycans for
galectin-3 on glycoproteins and glycolipids.
Glycosyltransferases in healing corneas
Fig. 2. (A) Heat map of 75 differentially expressed genes (change >1.5-fold; P < 0.01). All signals are compared to a median value, and fold change from the
median is visually represented by color assignment (see scale on the right side). Healing and normal corneas showed visibly distinct profiles of gene expression.
(B) Pie diagram showing the classification of differentially expressed genes based on molecular functions. Categories ascribed to genes were determined using
DAVID, Gene Cards, and Entrez gene. Note that 40% of the differentially expressed genes are glycosyltransferases and glycosidases.
C Saravanan et al.
Fig. 3. (A) Lectin blots demonstrating that alteration in transcript levels of glycosyltransferases in healing corneas is associated with corresponding changes in
glycan structures of glycoproteins. Protein extracts (5 μg) of normal (N) and healing (H) corneas were electrophoresed on 10% SDS–polyacrylamide gels; protein
blots of the gels were stained with Ponceau S to ensure equal loading of samples and were then probed with biotinylated E-PHA, DSL, and MAA that recognize
glycan products of GnTIII, GnTIV, and α2,3-sialyltransferases, respectively. Note that consistent with the increased mRNA levels of GnTIVb and
α2,3-sialyltransferases (ST3GalI and ST3GalIV) and the reduced mRNA level of GnTIII detected in the healing corneas by microarrays (Table I) and qRT-PCR
(Table II), the increased expression of DSL- and MAA-reactive glycoproteins and the reduced expression of E-PHA-reactive glycoproteins is detected in healing
corneas compared to normal, uninjured corneas. (B) The intensity of lectin-reactive bands from the lanes of normal and healing corneal samples from each lectin
blot was quantified by densitometry. A value of 1.0 was assigned to the intensity value of the lectin-reactive components of normal corneas. The intensity values of
the lectin-reactive components of healing corneas are expressed as a change in the intensity value with respect to normal corneas. E-PHA, Phaseolus vulgaris
erythroagglutinin; DSL, Datura Stromium lectin; MAA, Maakia Amurensis agglutinin.
qRT-PCR confirmation of differentially expressed genes
Gene-specific qRT-PCR was performed to confirm the differ-
ential expression of 11 selected genes. Of these 11 genes, we
chose 7 glycosyltransferases because they are likely to have an
known to play a role in corneal wound healing (Cao, Said, et
al. 2002). The expression level of a housekeeping gene, RPL19,
that was used as a reference gene in the current study, was sim-
ilar in both normal and healing corneas (fold change: 1.06).
For the most part, the qRT-PCR data were in agreement with
those obtained by array hybridization. For genes measured by
qRT-PCR, the mRNA levels were shown to change in the same
direction as the changes measured by the microarray detection
for 10 of the 11 genes tested (∼91%) (Table II). Consistent with
TIVb, β3GalT5, T-synthase, ST3GalI (representative gene from
the differentially expressed ST3 sialyltransferases), MUC1, and
IL-1β, and significant downregulation of GnTIII, ST6GalI (rep-
resentative gene from the differentially expressed ST6 sialyl-
transferases), ST8SiaIV (representative gene from the differ-
entially expressed ST8 sialyltransferases), and glypican-3, in
healing corneas by qRT-PCR. While modest downregulation of
the lumican gene was detected in healing corneas by microarray
analyses (1.6-fold), qRT-PCR analysis contradicted this result
(2.9-fold induction), thereby confirming data in an earlier pub-
lished report (Saika et al. 2000).
Alteration in transcript levels of glycosyltransferases
in healing corneas is associated with corresponding changes
in glycan structures of glycoproteins
We used lectin blot analysis to determine whether differential
expression in transcript levels lead to corresponding alterations
entially expressed glycosyltranferases identified in the current
study (Table I), we randomly chose three glycosyltransferases,
two genes (GnTIVb and ST3GalI) representing the upregulated
group, and one gene (GnTIII) representing the downregulated
group, as examples to assess the potential of differentially ex-
pressed transcripts to modulate the expression of correspond-
ing glycans. Plant lectins, E-PHA, DSL, and MAA, have been
Glycosyltransferases in healing corneas
Table I. Glycosyltransferases and glycosidases differentially expressed in healing compared to normal mouse corneasa
Fold differenceGene nameAccession number
(i) Galactosomanyl transferases
β-1,4-N-acetyl-galactosaminyl transferase 1 (β4GalNAcT1)
N-acetylgalactosaminyltransferase 4 (Galnt4)
N-acetylgalactosaminyltransferase 11 (Galnt11)
UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase,
polypeptide 5 (β3GalT5)
Core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta
2.3Mannoside acetylglucosaminyltransferase 4, isoenzyme B
Mannoside acetylglucosaminyltransferase 3 (GnTIII)
Phosphatidylinositol glycan anchor biosynthesis, class P (PIG-P)
UDP-glucose ceramide glucosyltransferase (Ugcg) D89866
ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GalI)
ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GalIV)
ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4
ST3 beta-galactoside alpha-2,3-sialyltransferase 5 (ST3GalV)
ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6
acetylgalactosaminide alpha-2,6-sialyltransferase 1
ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GalI)
C. Glycan degradation
aGenes are considered differentially expressed when there is a difference of >1.5-fold between the geometric mean signal of the healing group (n = 3) and the
normal group (n = 3). Results of genes represented in bold were confirmed by qRT-PCR.
Fucosyltransferase 11 (Fut11)AK014029
Exostoses (multiple) 1 (Ext-1)U78539
Heparan sulfate 3-O-sulfotransferase 1 (Hs3st1)
Galactose-3-O-sulfotransferase 1 (Gal3ST1)
Carbohydrate sulfotransferase 12 (Chst12)
Sulfatase 1 (Sulf 1)
Lysosomal beta A mannosidase (Manba)
Hexosaminidase A (Hexa)
Hyaluronoglucosaminidase 1 (Hyal1)
GM2 ganglioside activator protein (Gm2a)
Neuraminidase 3 (Neu3)
Iduronate 2-sulfatase (Ids)
Lysosomal-associated membrane protein 2 (Lamp2)
widely used to analyze the glycan products of GnTIII (bisecting
GlcNAc-branched N-glycans), GnTIV (β1,4-GlcNAc branched
N-glycans), and ST3 sialyltransferases (α2,3-sialic acids), re-
spectively (Wang and Cummings 1988; Ohtsubo et al. 2005;
Patnaik et al. 2006; Abbott et al. 2008). Lectin blot analyses
using these plant lectins showed that the expression level of
DSL- and MAA-reactive glycoproteins is upregulated, whereas
that of E-PHA-reactive glycoproteins is downregulated in heal-
ing corneas compared to the normal controls (Figure 3). These
data are consistent with the qRT-PCR data (Table II) showing
GnTIVb, and ST3GalI are increased, but that of GnTIII is de-
creased in healing corneas.
The overall goal of the present study was to identify differen-
tially expressed glycosyltransferases in healing corneas that
may enable us to predict wound healing-specific glycan struc-
tures and identify novel genes that are differentially expressed
in response to injury. A comparison of gene expression profiles
of normal and healing corneas revealed that healing corneas
have a unique glycogene expression pattern that defines them
as a group distinct from the normal, unwounded corneas (Fig-
ures 2 and 3). In general, the structural variations in glycans on
glycoproteins could alter their interaction with the endogenous
carbohydrate-binding proteins and this, in turn, could modulate
C Saravanan et al.
Table II. Comparison of differential expression data obtained by microarray
hybridization and qRT-PCRa
aFold change expressed after normalization to the housekeeping gene, RPL19.
the role of the lectins in cell adhesion and signal transduction
events required for cell motility (Ohtsubo and Marth 2006),
a key event in wound healing. In an early study, we have
established that a carbohydrate-binding protein, galectin-3,
et al. 2002). Specifically, we have demonstrated that (i) among
the members of the galectin family, galectin-3 is the most abun-
dantly expressed lectin in corneal epithelium (experiment ID:
glycomics/publicdata/microarray.jsp), (ii) migrating epithe-
lium of healing mouse corneas express elevated levels of
galectin-3 protein compared to nonmigrating epithelium
of normal corneas, (iii) exogenous galectin-3 stimulates
re-epithelialization of corneal wounds, and (iv) the rate of
re-epithelialization of corneal wounds is significantly slower in
galectin-3-deficient mice compared to the wild-type mice. We
have also demonstrated that while galectin-1 is expressed in
and there is no difference in corneal epithelial wound closure
rates between galectin-1-deficient and wild-type mice (Cao,
galectin-1 does not stimulate re-epithelialization of corneal
wounds. Thus, our specific goal was to identify injury-specific
glycosyltransferases, which are likely to synthesize glycans
that serve as ligands of galectin-3.
A major finding of the present study is that the expres-
sion of Mgat3 that encodes N-acetylglucosaminyltransferase-
III (GnTIII) was downregulated. GnTIII introduces a bisecting
β1,4GlcNAc to the β4-linked core mannose residues of
of studies have reported that the presence of bisecting GlcNAc
sugars on N-glycans is accompanied with a reciprocal change
in the expression of β1,6GlcNAc-branched N-glycans, which
are synthesized by N-acetylglucosaminyltransferase-V (GnTV)
(Isaji et al. 2004; Zhao et al. 2006; Kariya et al. 2008; Pinho et
al. 2009). It is thought that GnTIII dominantly competes with
GnTV for the modification of the same protein, and, therefore,
leads to the inhibition of the function of GnTV (Isaji et al.
2004; Zhao et al. 2006). These findings in conjunction with the
studies reporting that β1,6GlcNAc-branched N-glycans serve as
substrates for the synthesis of poly N-lactosamines, the high-
affinity counter-receptors for galectin-3 (Demetriou et al. 2001;
downregulation of Mgat3 gene expression in healing corneas
observed in the current study may result in the increased addi-
tion of poly N-lactosamine residues on N-glycans to synthesize
high-affinity counter-receptors for galectin-3 that, as described
above, plays a role in re-epithelialization of corneal wounds.
Indeed, Patnaik et al. (2006) have demonstrated that the binding
of galectin-3 to Lec10 CHO cells, which overexpresses GnTIII,
was reduced compared to that of wild-type CHO cells (Patnaik
et al. 2006).
Another key finding of the current study is that the genes
encoding the glycosyltransferases, GnTIVb, β1,3GalT5, and T-
synthase were all upregulated in healing corneas. The GnTIVb
enzyme generates distinct β1,4GlcNAc branches on α3-linked
core mannose of N-glycans (Figure 4A) that also serve as a sub-
strate for polylactosamine glycans (Yoshida et al. 1998). The
glycosyltransferase, β1,3GalT5 generates type I lactosamine
chain (Galβ1,3GlcNAc) elongation of core 2 and core 3 O-
glycans, N-glycans (Salvini et al. 2001; Holgersson and Lofling
2006), lactoceramides (Amado et al. 1999), and globosides
(Zhou et al. 2000). Since galectin-3 binds to glycans con-
taining Galβ1,3GlcNAc disaccharides (Sato and Hughes 1992;
Hirabayashi et al. 2002; Brewer 2004), the upregulated expres-
ligands may be synthesized during healing of corneal wounds.
T-Synthase generates the Thomsen–Friedenreich (TF) antigen
(Galβ1,3GalNAc, i.e.,core1dissacharide) andinitiatesthesyn-
thesis of core 1-derived O-glycans (Figure 4B) (Van den Steen
et al. 1998). The core 1 dissacharide is a substrate for a number
of glycosyltransferases including ST6GalNAcI that synthesize
the sialylated TF antigen (Takashima 2008). However, the ex-
pression level of ST6GalNAcI was downregulated in healing
corneas (see below). Clearly, the enhanced expression of T-
synthase coupled with downregulated ST6GalNAcI sialyltrans-
ferase (discussed below) may produce more unsubstituted TF
antigen in healing corneas. The unsubsituted TF antigen is also
a known ligand for galectin-3, and it has been shown that the
interaction between galectin-3 and unsubstituted TF antigen on
the cell surface mediates the homotypic aggregation of breast
carcinoma cells and adhesion of breast carcinoma cells to en-
dothelium for metastasis (Glinsky et al. 2003; Khaldoyanidi
et al. 2003). Thus, the increased unsubstituted TF antigen in
cell–matrix interactions. In this respect, it is noteworthy that in
the current study, the expression of MUC1, a transmembrane
O-glycosylated protein and a major carrier for the TF antigen
(Yu et al. 2007), was upregulated in healing corneas. In ep-
ithelial cancer cells, elevated expression of the MUC1 protein
is associated with an increased expression of the unsubstituted
TF antigen, and it has been shown that galectin-3 interactions
with MUC1 through the unsubstituted TF antigen facilitates the
adhesion of carcinoma cells to endothelium (McGuckin et al.
1995; Yu et al. 2007).
We found that sialyltransferases, which add sialic acids
to terminal sugars either by α2,6- and α2,8-linkages, includ-
ing ST6GalI (N-acetyllactosaminide α2,6 sialyltransferase),
ST6GalNAcI (sialyl-Tn, and mono- and di-sialyl T-synthase),
ST8SiaIV (polysialyltransferase), and ST8SiaVI (sialylates O-
glycans) (Takashima 2008), are largely downregulated in heal-
ing corneas. Recent studies have demonstrated that sialylation
distinctively modulates the recognition of cell surface glycans
and biological signaling by different galectins (Amano et al.
Glycosyltransferases in healing corneas
Fig. 4. Schematic diagrams showing biosynthesis of N- and O-glycan structures by enzymes discussed in the paper. (A) The specific activity of GnT enzymes in
the N-glycan biosynthesis. Note that GnTIII adds bisecting GlcNAc to the core mannose, whereas, GnTIVb and GnTV add branching GlcNAc residues on
N-glycans and are then extended by galactosyltransferases and/or sialyltransferases. The synthesis of bisecting GlcNAc on N-glycans blocks the activity of GnTV
thereby reducing the available substrate for the synthesis of high-affinity glycan ligands of galectin-3 (Isaji et al. 2004; Patnaik et al. 2006; Zhao et al. 2006). (B) A
schematic diagram showing the biosynthesis of core 1 structures of mucin-type O-glycans. N-Acetylgalactosaminyltransferases (GalNAcTs) initiate
O-glycosylation to which core 1 galactosyltransferase (T-synthase) adds galactose to produce T-antigen. Galectin-3 has been shown to have high affinity toward
unsubsituted T-antigen, but not to the sialylated T-antigens (Glinsky et al. 2003; Khaldoyanidi et al. 2003).
2003; Toscano et al. 2007; Stowell et al. 2008a; Zhuo et al.
2008). Of particular relevance to the current study are the
reports demonstrating that the presence of α2,6-linked sialic
acids in the glycans substantially reduce their ability to inter-
act with galectin-3 (Hirabayashi et al. 2002; Brewer 2004) and
that ST6GalI-mediated α2,6-sialylation of β1 integrins prevents
galectin-3-induced apoptosis of colon tumor cells (Zhuo et al.
2008). In a different study, it was shown that the removal of
α2,8-sialic acids by neuraminidase primes the neutrophils for
stimulation by galectin-3 (Almkvist et al. 2004). Collectively,
these observations lead us to propose that downregulation of
ST6GalI, ST6GalNAcI, ST8SiaIV, and ST8SiaVI sialyltrans-
ferases that synthesize α2,6- and α2,8-sialic acids may assist in
the enhanced interaction of galectin-3 withitscounter-receptors
during re-epithelialization of corneal wounds. It is notewor-
thy that the expression of ST3 sialyltransferases (ST3GalI and
ST3GalIV) that synthesize α2,3-linked sialic acids was upreg-
ulated in healing corneas (Takashima 2008). Coincidentally, it
has been demonstrated that while galectin-3 does not prefer
α2,6-sialylated glycans, it binds well to α2,3-sialylated glycans
(Hirabayashi et al. 2002; Brewer 2004).
2004; Friedrichs et al. 2007; Stowell et al. 2007, Stowell et al.
2008a; Paclik et al. 2008) as well as unique (Fukumori et al.
2003; Sturm et al. 2004; Hernandez et al. 2006; Stillman et al.
2006; Lu et al. 2007; Stowell et al. 2008a, 2008b; Diskin et al.
2009) saccharide binding specificity. It is therefore, reasonable
to expect that injury-specific glycosyltransferases discussed
sion of counter-receptors of other members of galectin family
besides galectin-3. Another galectin that has been implicated in
the process of corneal wound healing is galectin-7 (Cao, Said,
and -7 share counter-receptors remains to be determined. Our
early study demonstrating that galectin-7, but not galectin-3,
accelerated re-epithelialization of wounds in Gal3−/−corneas
(Cao, Said, et al. 2002) in conjunction with the findings that
unlike galectin-3, galectin-7 does not show affinity to the
unsubstituted TF antigen (http://www.functionalglycomics.org/
=DATsideMenu=noobjId=1000348) may suggest that the two
lectins may bind to distinct cell surface and/or ECM counter-
receptors. However, based on the glycan array, thermodynamic
binding (Brewer 2004), and frontal affinity chromatographic
(Hirabayashi et al. 2002) studies showing that like galectin-3,
galectin-7 also binds to Galβ1, 3GlcNAc, internal lactosamine,
α2,3-sialylated glycans but not α2,6-sialylated glycans, it is
reasonable to expect that the two lectins share at least some
Although, the focus of the current study was glycosyltrans-
ferases, we report here for the first time that a number of
other genes encoding core proteins of proteoglycans and cell
adhesion proteins were also differentially expressed in heal-
ing corneas. These genes include mincle, dectin-1, dectin-
2, glypican-3, and serglycin (supplementary Table 1). These
C Saravanan et al.
differentially expressed genes have not previously been investi-
gated in the context of wound healing and represent the novel
The current functional genomics approach is the first study
aimed at identifying alterations in the expression of enzymes
regulating glycosylation during corneal wound healing. In sum-
that corneal wound healing response is characterized by the dif-
ferential expression of a number of glycosyltransferases. The
glycans produced by these differentially regulated enzymes
in healing corneas are likely to render the glycoproteins and
glycolipids as high-affinity counter-receptors for galectin-3, a
carbohydrate binding protein, known to play a key role in re-
epithelialization of corneal wounds.
Material and methods
Sample collection and RNA preparation
Three groups (10 animals/group) of 6- to 8-week old mice
(C57BL/6 and 129 mixed genetic backgrounds) were used. All
procedures were approved by the Institutional Animal Care and
Use Committee (IACUC) of Tufts University and were per-
formed in accordance with the Association for Research in Vi-
sion and Ophthalmology Resolution on the Use of Animals in
tutes of Health Guide for the Care and Use of Laboratory Ani-
of 1.25% avertin (0.2 mL/10 g body weight) (Aldrich Chemi-
cal Co., Milwaukee, WI) and proparacaine eye drops (Alcain,
Alcon Labs, Inc., Fort Worth, TX) were applied to the cornea
as a topical anesthetic. Transepithelial excimer laser ablations
(2 mm optical zone; 42 to 44 micron ablation depth, photother-
apeutic keratectomy mode) were performed on the right eye of
each animal using Summit Apex Plus Excimer Laser (Waltham,
MA). The left eye of each animal was used as normal, unin-
jured cornea. After surgery, all animals received buprenorphine
(intramuscular, 0.2 mL of 0.3 mg/mL Buprenex, Reckitt and
Colman Pharmaceuticals, Inc., Richmond, VA) as a painkiller.
Antibiotic ointment (Vetropolycin, Pharmaderm, Melville, NY)
was applied, and the corneas were allowed to partially heal in
vivo for 18–22 h. At the end of the healing period, animals
were euthanized, and the corneas of both eyes were excised and
immediately placed in liquid nitrogen until use.
Total RNA from corneas was extracted using RNeasy mini
kit according to the manufacturer’s recommendations (Qiagen,
Chatsworth, CA). Briefly, the frozen corneas were crushed, sus-
pended in the guanidine thoiocyanate buffer (Buffer RLT), and
further homogenized using a QIAshredder column. The eluent
was loaded onto a silica gel base column and the bound RNA
were analyzed using the Bioanalyzer 2100 with RNA Pico Lab
Chips (Agilent, Waldbronn, Germany).
The glycogene microarray, GLYCOv2, is an oligonucleotide
microarray, custom designed by Affymetrix (Santa Clara, CA)
for the Consortium for Functional Glycomics at the Scripps
Institute, La Jolla, CA. The array contains approximately
2000 mouse and human glycogenes including glycosyltrans-
ferases, glycosidases, enzymes involved in nucleotide-sugar
synthesis and transport, proteoglycans, and glycan binding
proteins. A complete list of probe sets and annotation for
the GLYCOv2 oligonucleotide array is available at (http://
resourcecoree.shtml). In this array, transcripts are detected by
three identical probe sets for each glycogene. Each probe set
consisted of 11 probe pairs, with each probe pair made of one
25-bp perfect match oligonucleotide that matches the sequence
of the targeted transcript, and one 25-bp oligonucleotide
designed with a mismatch at the center position.
Hybridization and data analysis
To prepare hybridization probes, total RNA (100 ηg) from each
sample was amplified, and cDNA was synthesized according to
a modified Baugh/Harvard protocol (http://www.scripps.edu/
transcribed in vitro in the presence of biotin-labeled ribonu-
cleotides, and the labeled cRNA was hybrized to GLYCOv2
microarrays, and scanned using Affymetrix Scanner 3000
(www.affymetrix.com). Quantitation of expression signal val-
ues, quantile normalization, and background subtraction were
performed as described earlier (Diskin et al. 2006). The gene
and normal corneas) were then analyzed by hierarchical cluster-
ing using BRB ArrayTools 3.2.2 (http://linus.nci.nih.gov/BRB-
The differential expression of genes in healing compared to
normal, uninjured corneas was analyzed as described by Diskin
et al. (2006). Briefly, the transformed expression values for the
replicated probe sets were averaged to get a single expression
value for each probe set on each array. Then, statistically
significant changes in gene expression were identified using
BRB ArrayTools 3.2.2 software. The class comparison test was
conducted using a univariate alpha level cutoff of 0.001 and a
multivariate permutation-based false discovery rate calculation.
The predicted proportion of false discoveries was preset at 10%
and a false discovery rate calculation was set at a confidence
deposited in the Consortium of Functional Glycomics database
The differentially expressed genes were visualized using
Cluster and Tree View software for heat map creation (Eisen
Lab, UC Berkeley; http://rana.lbl.gov/EisenSoftware.htm). For
this, each gene was compared to median unlogged RMA signal
intensity values using normal and healing arrays in the study.
Differences from the median were represented in varying inten-
sitiesof green (decreased fold-change compared tomedian) and
red (increased fold-change compared to median).
The Database for Annotation, Visualization and Inte-
grated Discovery (DAVID) software (Diskin et al. 2006),
(http://www.ncbi.nlm.nih.gov/entrez) were used to gain insight
into the biological functions of differentially expressed genes.
resultant cDNAs were
and Entrez gene
Quantitative real-time RT-PCR
Total RNA (300 ηg) was reversed transcribed using the High
Capacity kit (Applied Biosystems (ABI), Foster City, CA)
according to manufacturer’s instructions. Real-time PCR was
Glycosyltransferases in healing corneas
performed (Mx4000 real-time PCR machine, Stratagene, La
Jolla, CA) in triplicates using 5 μL of cDNA (derived from
15 ηg total RNA), TaqMan MGB probes, primers specific
for the selected genes, and TaqMan Universal PCR master
mix (ABI). Reactions performed in the absence of template
served as negative controls. The ABI primer sets used in-
cluded: ribosomal protein L19 (RPL19) (Mm020601633_g1),
(Mm00447798_m1), GnTIVb (Mm00521482_m1), β3GalT5
I (ST3GalI) (Mm00501493_m1),
(Mm00500510_m1), mucin 1 (MUC1) (Mm00449604_m1),
and glypican-3 (Mm00516722_m1). For amplification, Ampli-
taq Gold DNA polymerase was activated (95◦C for 10 min) and
the reactions were subjected to 50 cycles involving denaturation
(95◦C for 15 s) and annealing plus extension (60◦C for 1 min).
data analysis was performed using Mx4000 software version
2 (Stratagene). The FAM fluorescent signals were measured
against the ROX (internal reference dye) signal to normalize the
non-PCR-related fluctuations; amplification plots showing the
increase in FAM fluorescence with each cycle of PCR (?Rn)
were generated for all samples, and the threshold cycle values
(Ct) were calculated from the amplification plots. The Ct value
represents the cycle number at which the fluorescence was
detectable above an arbitrary threshold, based on the variability
of the baseline data during the first 15 cycles. All Ct values
were obtained in the exponential phase. Quantification data of
each gene were normalized to the expression of a housekeeping
gene, RPL19. A value of 1.0 was assigned to the expression
level of each gene in the normal, uninjured corneas. The values
for healing corneas were expressed as a change in expression
levels with respect to normal corneas.
Lectin blot analysis
Corneal wounds (2 mm) were produced on the right eye of four
mice by transepithelial excimer laser ablations. The wounds
were allowed to partially heal in vivo for 18–22 h, and the
corneas were excised as described above. The corneas from
left eyes served as normal controls. Protein extracts (5 μg)
of normal and healing corneas prepared in the radioimmuno-
precipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 8.0),
150 mM NaCl, 0.1% Nonidet P-40, and 0.5% deoxycholic acid)
were electrophoresed on 10% SDS–polyacrylamide gels and
transferred to nitrocellulose membranes. The protein blots of
the gels were stained with Ponceau S (Sigma, MO) to en-
sure equal loading of samples and were then probed with var-
ious biotinylated plant lectins (1 μg/mL) including Phaseolus
vulgaris erythroagglutinin (E-PHA), Datura Stromonium lectin
(DSL), and Maakia Amurensis agglutinin (MAA) (Vector Labs,
Burlingame, CA). The lectin-reactive components were then vi-
sualized using streptavidin-HRP (ABC kit, Vector Labs) and
a chemiluminescence detection system (PerkinElmer Life Sci-
ences, Waltham, MA). Films were scanned and densitometric
analysis was preformed using ImageJ.
Supplementary data for this article is available online at
National Eye Institute (EY007088 to N.P.), New England
Corneal Transplant Fund, Mass Lions Eye Research fund, and
a challenge grant from Research to Prevent Blindness.
The gene microarray analysis was conducted by the Gene Mi-
croarray (E) Core of the Consortium for Functional Glycomics
funded by the National Institute of General Medical Sciences
Conflict of interest statement
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