(Investigative Ophthalmology and Visual Science. 2001;42:2164-2172.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Isolation and Characterization of Galectins in the Mammalian Retina
Fumiyuki Uehara1,
Norio Ohba1 and
Masayuki Ozawa2
1 From the Departments of Ophthalmology and
2 Biochemistry, Kagoshima University Faculty of Medicine, Japan.
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Abstract
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PURPOSE. Previous studies have suggested that galectins may be involved in
retinal adhesion and photoreceptor cell survival. To elucidate the
underlying mechanisms, the authors isolated retinal galectins,
determined their types and distributions, and investigated the validity
of the hypothesis, using rat models.
METHODS. An antibody was prepared against a bovine retinal lectin that was
isolated by use of a lactose-agarose column. cDNA of the lectin was
isolated by screening of a bovine retinal cDNA library, using the
antibody, and then was sequenced. The cDNAs of rat retinal galectins
were also isolated by means of polymerase chain reaction and used to
produce an antibody against recombinant galectin-3. Using the described
antibodies, the authors examined the distributions of galectins in
bovine and rat retinas, morphologic changes of rat retinas induced by
the antibodies, and distributional changes of galectins in
constant-light–exposed rat retinas.
RESULTS. The cDNAs of bovine galectin-1, rat galectin-1, and rat galectin-3 were
isolated. Galectin-1 was found in various regions, including the
retinal pigment epithelium, outer limiting membrane, and outer
plexiform layer in bovine and rat retinas. Galectin-3 was increasingly
detected in the cytoplasm of Müller cells after constant light
exposure after an increase in its transcript. Retinal detachment and
vacuolation of the outer plexiform layer were induced in rat eyes by
intravitreous injection of the anti-galectin-1 antibody.
CONCLUSIONS. Galectin-1 may be involved in adhesion of the photoreceptor and outer
plexiform layers by interacting with glycoconjugates with
ß-galactoside residues in the interphotoreceptor matrix and synaptic
cleft matrix. Galectin-3 may increase in Müller cells of a
degenerative rat retina, probably through endogenous
anti-apoptosis.
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Introduction
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Various kinds of glycoconjugates with ß-galactose
residues have been detected in the retina, including the
interphotoreceptor matrix (IPM), using galactose-binding plant
lectins.1
2
3
4
5
Glycoconjugates in the IPM play a number of
important roles in the interactions between photoreceptors, retinal
pigment epithelium (RPE) cells, and Müller cells, including
mediation of intercellular adhesion6
7
8
and transfer of
substances between these cells.8
9
10
We have observed that
the IPM around photoreceptors is lost when the retinal tissue is
incubated with an exogenous lectin in the presence of
D-galactose.11
This finding suggests that
galactose residues may be involved in retinal adhesion mediated by the
IPM. Galectin is an endogenous animal lectin that exhibits an affinity
for ß-galactoside sugars and promotes cell–matrix adhesion by
cross-linking cell surface and substrate
glycoconjugates.12
These properties indicate that galectin
may be a mediator of retinal adhesion.
In addition to cell–matrix adhesion, another function of galectin has
been reported. Galectin-3 inhibits apoptosis through the
bcl-213
or cysteine protease pathways.14
We
recently observed that intravitreous injection of an anti-galectin-3
antibody accelerates the rat photoreceptor degeneration due to constant
light, whereas that of galectin-3 inhibits the light
damage.15
These findings suggest that increased expression
of endogenous galectin-3 may represent an anti-apoptotic action in the
rat retina after constant light (CL) exposure.
An endogenous 16-kDa galactose-binding lectin has been isolated from
the chicken retina16
and shown to be distributed in the
apical villi of Müller cells and some other regions of the
retina.17
Although its molecular size is similar to that
of galectin-1, it has not yet been confirmed to be galectin-1 or
another type of galectin. In the present study, therefore, we isolated,
and determined the types and distributions of galectins in mammalian
retinas. Then, we examined whether intravitreous injection of an
anti-galectin antibody induces retinal detachment to confirm that
galectin is involved in retinal adhesion. We also investigated whether
the expression of galectin-3 increases in CL-exposed rat retinas, to
elucidate the mechanism underlying the acceleration of photoreceptor
degeneration by an intravitreous injection of an anti-galectin-3
antibody.
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Materials and Methods
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Purification of Bovine Retinal Lectins and Their Antibodies
Twelve bovine retinas were isolated and homogenized in 50 ml of
1.25% Triton X-100 in 0.01 M Tris-HCl buffer (pH 7.6), containing 0.15
M NaCl, 2 mM 2-mercaptoethanol, 2 mM EDTA, and 1 mM
phenylmethylsulfonyl fluoride (PMSF) (extraction buffer), followed by
centrifugation at 140,000g for 1 hour. The supernatant
(Triton X-100–extracted sample) was decanted and applied to a column
of lactose-agarose (column volume, 1 ml; Seikagaku Corp., Tokyo, Japan)
equilibrated with 0.1% Triton X-100 in 0.01 M-Tris-HCl buffer (pH
7.6), containing 0.15 M NaCl, 2 mM 2-mercaptoethanol, 2 mM EDTA, and 1
mM PMSF (equilibration buffer) at 4°C. After the column had been
washed with 20 ml equilibration buffer, the lactose binding lectins
bound to the lactose column were eluted with 4 ml equilibration buffer
containing 0.2 M lactose. Approximately 130 µg lectins was purified
from the 12 bovine retinas (10.8 mg/retina), as determined with a
protein assay (DC Protein Assay System; Bio-Rad,
Hercules, CA). Therefore, to obtain enough of the lectins for the
immunization of two rabbits and affinity purification of antibodies, we
purified 4.5 mg lectins from 440 bovine retinas by 10 repetitive cycles
of the procedure, the column volume being 4 ml each time. The lectins
were analyzed by Western blot analysis as described in the section,
Protein Electrophoresis and Western Blot Analysis, after incubation
with a biotinylated D-galactose probe (Seikagaku
Corp.). Rabbit antisera were raised against the lectins by immunizing
two New Zealand White rabbits, and then antibodies were affinity
purified on columns of the lectins coupled to an
NH2-crosslinked agarose (Affi-Gel 10;
Bio-Rad), according to a method previously described.18
Screening of a Bovine Retinal cDNA Expression Library
A cDNA library of bovine retinas (Uni-ZAP XR; Stratagene, La
Jolla, CA) was screened with an affinity-purified antibody against
bovine retinal lectins. Screening was performed according to the
instructions provided by the manufacturer. Seven positive plaques were
detected on the screening of approximately 1.0 x
106 phages in the library. The DNAs of the
positive clones were sequenced on both strands, using T3 and T7
universal primers (Stratagene), according to a terminator cycle
sequencing protocol (Prism Big Dye; PE-Applied Biosystems, Foster City,
CA).
Polymerase Chain Reaction
To determine the kinds of galectins that are expressed in the
rat retina, cDNA clones of rat retinal galectins were amplified by
means of polymerase chain reaction (PCR) from the rat retinal cDNAs in
the pAP3neo vector (mRNA source: pooled eyes from 100 Sprague-Dawley
outbred males, aged 10–11 weeks; Takara Shuzo, Shiga, Japan), by using
three pairs of oligonucleotides (5'-CGGGATCCTTCGCTTCAATCATGGCCTG-3' and
5'-GGGCTGGGGCTGGCTGGCTTCACTC-3' for galectin-1,19
5'-CGGGATCCAGGAAAATGGCAGACGGCTTC-3' and
5'-GGGGTACCTCATAACACACAGGGCAGTTC-3' for galectin-3,20
and
5'-CGAATTCCGACTCTCAAGATGGCCTATTG-3' and
5'-GATTAGATGGAACTTGGGATCTCTCTGC-3' for galectin-421
) as
PCR primers. PCR was performed with a kit (Advantage cDNA PCR; Clontech
Laboratories, Palo Alto, CA) according to the manufacturer’s
instructions. After thirty cycles (1 minute at 94°C and 3 minutes at
68°C of amplification), the products were isolated from agarose gels
for subcloning into a vector (Bluescript II SK[+]; Stratagene) using
PCR DNA (GFX; Amersham Pharmacia Biotech, Piscataway, NJ) and a gel
band purification kit (Amersham Pharmacia Biotech). After ligation, the
DNA was sequenced on both strands, using T3 and T7 universal primers,
according to the manufacturer’s protocol (PE Applied Biosystems).
Reverse Transcription-Polymerase Chain Reaction
To compare the mRNA-expression of galectin-3 after CL exposure
(described in the Immunohistochemistry section), RT-PCR was performed
with ß-actin as an internal control. The cDNA for rat galectin-3 was
amplified by RT-PCR using the same oligonucleotides as described under
PCR, whereas that for ß-actin was amplified with a pair of
oligonucleotides, 5'-TTGTAACCAACTGGGACGATATGG-3' and
5'-GATCTTGATCTTCATGGTGCTAGG-3' (Clontech Laboratories). Because this
pair of ß-actin primers spans an intron, it seems to be a control for
genomic contamination as well. Total RNA (1 µg/sample for each set of
CL exposure conditions) was reverse transcribed using oligo dT primers
(Boehringer–Mannheim, Mannheim, Germany) and reverse transcriptase
(Superscript II; Life Technologies, Rockville, MD) according to the
manufacturer’s instructions. An aliquot of the same RT-product from
each sample (1/20 of the total volume) was used for PCR using the cDNA
PCR kit (Advantage; Clontech Laboratories) following the method used
for PCR. Five microliters of each PCR-product was electrophoresed on a
1% agarose gel. Photographs were taken under ultraviolet light, and
the intensities of the PCR bands were determined by measurement with
image analysis software (NIH Image; provided in the public domain by
the National Institutes of Health [NIH], Bethesda, MD, and available
at http://www.nb.nih.ncbi.gov).
Purification of an Anti-Rat Galectin-3 Antibody
The cDNA encoding rat retinal galectin-3 was cloned into plasmid
vectors (pMALc2; New England Biolabs, Beverly, MA, and pGEX-4T1;
Amersham Pharmacia Biotech) to express rat galectin-3 as fusion
proteins with maltose-binding protein (MBP) and glutathione
S-transferase (GST) in Escherichia coli BL21
cells, respectively. The fusion proteins were induced and
affinity-purified by methods previously described.15
22
Rabbit antiserum was raised against the MBP/galectin-3 fusion protein
in a New Zealand White rabbit, and the antibody against galectin-3 was
affinity purified on a column of the GST/galectin-3 fusion protein
coupled to Affi-Gel 10 (Bio-Rad), by using a method previously
described.15
18
22
Protein Electrophoresis and Western Blot Analysis
Protein electrophoresis and Western blot analysis were performed
essentially as previously described,18
22
using Laemmli’s
sample buffer.23
The blotted lectins eluted from a
lactose-agarose column were incubated with a biotinylated ß-galactose
probe (20 µg/ml PBS; Seikagaku Corp.) for 1 hour at room temperature.
The blotted proteins (10 µg/10 µl each) of bovine and rat retinas
(with or without CL exposure) were incubated with the anti-bovine
retinal lectin or anti-rat galectin-3 antibody (all the antibodies were
1:50 diluted with PBS) for 1 hour at room temperature. The bands of
galectins were then detected with biotinylated anti-rabbit IgG (Vector
Laboratories, Burlingame, CA; 1:100 diluted with PBS) and an ABC kit
(Vectastain Elite; Vector Laboratories), by a method previously
described.15
22
Color slides of the blotted proteins of
rat retinas were obtained and transferred to a computer by a scanner
(Coolscan; Nikon, Tokyo, Japan). The intensities of the protein bands
were determined by measurement with NIH Image on the computer screen
and compared.
Immunohistochemistry
According to the procedures described in previous
papers,18
22
tissue sections of bovine and rat eyes for
immunohistochemistry were prepared and reacted with anti-bovine retinal
lectin (galectin-1; described in the Results section) or anti-rat
galectin-3 antibodies (1:50 diluted with PBS) for 1 hour at room
temperature. In rats, we also examined the distribution of galectins in
the retina after CL exposure at an illuminance of 130 to 150
foot-candles for 1 day, 3 days, 1 week, or 2 weeks (at least four rats
for each experiment). The antibody-binding sites were visualized with
biotinylated anti-rabbit IgG (Vector Laboratories) and an ABC kit
(Vectastain Elite; Vector Laboratories), according to a previously
described method.22
Intravitreous Injection of the Antibodies
Albino Wistar rats, 2 to 3 months of age, were anesthetized and
2 µl anti-bovine retinal lectin antibody (galectin-1; 1:50 diluted
with PBS; five rats; described in the Results section), anti-rat
galectin-3 antibody (1:50 diluted with PBS; five rats), or PBS (five
rats) was injected into the vitreous by inserting a 32-gauge beveled
needle for 2 mm through the inferior temporal equator of the left eye
at an angle of approximately 45°. After the rats had been maintained
under cyclic lighting (12-hours on–12-hours off) conditions for 3 days
after the injection, they were killed with an overdose of carbon
dioxide, after which their eyes were enucleated and immersed in a
fixative comprising 2.5% glutaraldehyde and 2% paraformaldehyde. The
eyes were bisected along the vertical meridian and embedded in
paraffin, by using a procedure previously
described.15
18
22
The nasal halves were sectioned at 5
µm thickness and stained with hematoxylin-eosin. Sections along the
vertical meridian of the eyes at approximately 20 µm from the
bisected edge containing the entire retina were examined (one section
per eye). Photographs were made of each section and printed. The
horizontal lengths of the RPE, with or without retinal detachment, were
determined by measurement with a flexible rule. The degree of retinal
detachment was then determined by calculating the ratio of RPE length
to retinal detachment/total RPE-length.
All animal procedures conformed to the Guidelines of the Kagoshima
University Faculty of Medicine for Animal Experiments and the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
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Results
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Western Blot Analysis of Retinal Lectins
The lactose-binding lectins of bovine retinas, bound to a
lactose-agarose column, were detected with a biotinylated
ß-galactose probe as two protein bands comprising an intense band
corresponding to Mr 15 kDa and a faint band
corresponding to Mr 31 kDa (Fig. 1 , lane A). The polyclonal antibody produced against bovine retinal
lectins selectively bound to the Mr 15-kDa band
for a bovine retina sample (Fig. 1
, lane B). This antibody
cross-reacted with the Mr 15-kDa band for a rat
retina lysate as well (Fig. 1 , lane C). This finding implies that this
antibody was mainly produced against the major retinal lectin of
Mr 15 kDa and thus was considered to be effective
for the isolation of its cDNA through screening of a retinal cDNA
library. The polyclonal antibody against rat galectin-3 reacted with
the Mr 31-kDa band for a rat retina sample (Fig. 2)
. The intensity of this band increased with 3 days of CL exposure (Fig. 2
, lanes A–D; band-intensity, light–dark cycle: 1.00; 1 day of CL
exposure: 0.98; 3 days: 6.56; and 1 week: 10.84). In contrast, the
intensity of the band that reacted with the anti-bovine lectin antibody
did not increase after CL exposure (Fig. 2
, lanes E–H; band-intensity,
light–dark cycle: 1.00; 1 day of CL exposure: 1.07; 3 days: 1.03; and
1 week: 0.62).
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Figure 1. Western blot analysis of retinal lectins in bovine retinas. Lane
A: Lectin bands of 15 kDa and 31 kDa were detected with a
biotinylated ß-galactose probe for the bovine retinal eluate from a
lactose-agarose column. Lane B: A 15-kDa band was
selectively detected with a polyclonal antibody against the bovine
retinal lectin for a bovine retina sample. Lane C: A
15-kDa band was detected with this antibody in a rat retina sample as
well.
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Figure 2. Western blot analysis of galectin-3 and galectin-1 in rat retinas.
Lanes A–D: A 31-kDa band reacted with the
anti-galectin-3 antibody. Its intensity increased after CL exposure.
Lane A: 0 days of CL exposure;
lane B: 1 day; lane
C: 3 days; and lane D: 1
week. Lanes E–H: A 15-kDa
band was constantly detected with the anti-galectin-1 antibody
independently of the period of CL exposure. Lane
E: 0 days of CL exposure; lane
F: 1 day; lane G: 3 days;
and lane H: 1 week.
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cDNA and Predicted Amino Acid Sequence of Bovine Retinal Lectin
The cDNA encoding the bovine retinal lectin of
Mr 15 kDa was cloned from the cDNA library of
bovine retinas (Uni-ZAP XR; Stratagene). Each phagemid isolated was
found to contain a cDNA insert of approximately 0.7 kbp, by agarose gel
electrophoresis. The sequencing data for the cDNA indicated that the
isolated clone contained a common open reading frame coding for 135
amino acids, which form a protein of 14.5 kDa. A search of GenBank
(provided in the public domain by NIH, Bethesda, MD, and available at
http://www.ncbi.nlm.nih.gov) for similar nucleotide sequences revealed
that this sequence is homologous to that of bovine galectin-1
(accession number, X14330) isolated from other tissues (e.g., trachea
fibroblasts).24
The predicted amino acid sequence encoded
by the cDNA isolated in the present study was completely identical with
that of bovine galectin-1.24
Polymerase Chain Reaction
A DNA band corresponding to 450 bp was observed for
the PCR product, which was amplified using primers for rat
galectin-1, electrophoresed on an agarose gel, and stained with
ethidium bromide (Fig. 3
, lane A). After the DNA was ligated into a vector (Bluescript
II SK [+]; Stratagene) and sequenced, a search of GenBank revealed
that the predicted amino acid sequence encoded by this DNA is identical
with that of rat galectin-1 (accession number, M19036).19
A DNA band corresponding to 900 bp was observed for the electrophoresed
PCR product amplified by using the primers for rat galectin-3
(Fig. 3
, lane B). After ligation and sequencing, a search of GenBank
revealed that the nucleotide sequence encoded by this cDNA is identical
with that of galectin-3 from rat basophilic leukemia
cells20
(accession number, J02962) except for one
nucleotide (nt 59: G
A). No PCR-product was detected by agarose gel
electrophoresis of the sample amplified by using the PCR primers for
rat galectin-421
(Fig. 3
, lane C). These results confirmed
that the galectin-1 and galectin-3 transcripts are both expressed in
the rat retina but that a galectin-4 transcript is not.
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Figure 3. DNAs of rat galectins amplified by PCR. Lane A: A 450-bp
DNA-band was detected for the electrophoresed PCR product, amplified
using primers for rat galectin-1. Lane B:
A 900-bp DNA band was observed for the electrophoresed PCR product,
amplified using primers for rat galectin-3. Lane C:
No PCR-product was detected in the sample amplified using PCR
primers for rat galectin-4. DNA molecular weight marker is shown in the
extreme left and right lanes.
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Reverse Transcription–Polymerase Chain Reaction
DNA bands corresponding to galectin-3 (Fig. 4
, lanes A–D) and ß-actin (Fig. 4
, lanes E–H) were observed for the
RT-PCR products. The ratio of the intensity of the DNA bands
(galectin-3 and ß-actin), which was calculated by measurement with
NIH Image, was 0.75 after the light–dark cycle, 1.03 after 1 day
of CL exposure, 1.68 after 3 days, and 1.28 after 7 days. These
findings showed that the RT-PCR product for galectin-3 increased
between 0 and 3 days of CL exposure (Fig. 4
, lanes A–C), whereas
it decreased between 3 and 7 days (Fig. 4
, lanes C, D).
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Figure 4. DNAs of rat galectin-3 and ß-actin amplified by RT-PCR from RNAs of
CL-exposed rat retinas. Lanes A–D: A
900-bp DNA band was observed in the electrophoresed PCR product,
amplified using primers for rat galectin-3. Lane
A: light–dark cycle; lane
B: 1 day after CL exposure; lane
C: 3 days after; and lane
D: 1 week after. Lanes
E–H: A 764-bp DNA band was detected in the
electrophoresed PCR product, amplified using primers for rat ß-actin.
Lane E: light–dark cycle;
lane F: 1 day after CL exposure;
lane G: 3 days after; and lane
H: 1 week after.
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Immunohistochemical Distribution of Galectins in Bovine and Rat
Retinas
The sequencing data for the cDNA coding the bovine retinal lectin
of Mr 15 kDa, which was detected as a single band
with the anti-bovine retinal lectin antibody in Western blot analysis,
showed that this antibody can be used for the detection of galectin-1
in the retina, RPE cells, and choroid (Figs. 5A
5B
5C
5D)
, although we cannot absolutely rule out the presence of antibodies
against other minor lectins, which are not detectable on Western blot
analysis. The antibody prominently reacted with the apical halves of
photoreceptor inner segments (IS), the outer limiting membrane (OL),
the outer plexiform layer (OP), the inner plexiform layer (IP), the
nerve fiber layer (NF), and vessels in the bovine retina (Fig. 5A)
. The
antibody also bound to RPE cells, choroidal arteries, and veins (Fig. 5C)
. This labeling pattern was significantly inhibited by preincubation
of the antibody with the bovine retinal lectin (Figs. 5B
5D)
.
Pigmented choroidal fibroblasts were observed in both sections with and
without this preincubation (Figs. 5C 5D
; arrows).
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Figure 5. Immunohistochemistry of a bovine retina, RPE and choroid
using the anti-galectin-1 antibody. (A, B)
Retina; (C, D) RPE and choroid. (A)
The apical halves of photoreceptor IS, the OL membrane, the OP layer,
the IP layer, the NF layer, and vessels were labeled. (C)
The RPE, almost the whole thickness; choroidal arteries; and veins were
labeled with the antibody. (B, D) Weak labeling
was observed after preincubation of the antibody with the bovine
retinal lectin. (C, D) Pigmented choroidal
fibroblasts (arrows) were observed in both sections, with
and without preincubation. AR, choroidal artery; VE, choroidal vein;
PE, pigment epithelium. Scale bar, 30 µm.
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In the rat retina, almost the same antibody-binding pattern was
observed as in the bovine retina, although the binding to regions of
the inner retina was relatively weak (Fig. 6A)
. This labeling pattern did not change after CL exposure (Fig. 6B : 1
day of CL exposure, and Fig 6C
: 1 week), and was significantly
inhibited by preincubation of the antibody with the bovine retinal
lectin (Fig. 6D
: 1 week of CL exposure).
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Figure 6. Immunohistochemistry of rat retinas using the
anti-galectin-1 antibody. (A–C) The apical
halves of photoreceptor IS, the OL membrane, the OP layer, and vessels
intensely reacted with the antibody. The IP and NF layers were weakly
labeled. This reaction pattern remained unchanged, although the
thickness of the ON layer gradually decreased after CL exposure
(A: 0 days of CL exposure; B: 1 day;
C: 1 week). (D) A small reaction was observed
after preincubation of the antibody with the bovine retinal lectin (1
week of CL exposure). Scale bar, 30 µm.
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When rats were maintained with a light–dark cycle, galectin-3 was
faintly detected in the OL, OP, IP, ganglion cell layer (GC), NF, and
inner limiting membrane (IL; Fig. 7A
). After 1 day of CL exposure, the cytoplasm of certain cells with
projections in the inner nuclear layer (IN) and strands in the outer
nuclear layer (ON) reacted with the antibody, in addition to the
increased labeling in the OL, OP, IP, GC, NF, and IL (Fig. 7B)
. After 3
days of CL exposure, the immunoreactivity detected was increased,
especially in the ON and IN (Fig. 7C)
. The reaction products appeared
as strands extending from the IL to the OL across the neural retina
with side branches through the OP. This staining pattern closely
matched the distribution of Müller cells and their
processes.25
After 1 and 2 weeks of CL exposure, this
labeling pattern remained unchanged although the thickness of the ON
had gradually decreased (Fig. 7D
: 1 week of CL exposure, and Fig 7E
: 2
weeks). This immunoreactivity to galectin-3 was significantly inhibited
by preincubation of the antibody with the GST/galectin-3 fusion
protein, suggesting that this reaction was specifically induced by the
antibody (Fig. 7F
: 1 week of CL exposure).
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Figure 7. Immunohistochemistry of rat retinas using the
anti-galectin-3 antibody. (A–E) The labeling of
Müller cells increased with CL exposure (A: 0 days of
CL exposure; B: 1 day; C: 3 days; D: 1
week; E: 2 weeks). (F) This immunoreactivity to
galectin-3 was inhibited by preincubation of the antibody with the
GST-galectin-3 fusion protein (1 week of CL exposure).
Arrows, OP layer; OL, outer limiting membrane; ON, outer
nuclear layer; IN, inner nuclear layer; IP, inner plexiform layer; GC,
ganglion cell layer; IL, inner limiting membrane. Scale bar, 30 µm.
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Anti-galectin Antibody–Induced Changes in the Rat Retina
Retinal detachment and vacuolation of the OP were observed in all
five rat eyes at 3 days after vitreous injection of the anti-galectin-1
antibody, whereas the morphology of the detached photoreceptor outer
(OS) and inner segments (IS) was well organized (Figs. 8A
9A)
. In the case of anti-galectin-3 antibody injection, little morphologic
change was observed at 3 days after injection in most regions of the
eye (Figs. 8B
9B)
, although retinal regions with torn photoreceptor
outer segments were occasionally detected (Fig. 9D)
. In these regions,
because residual fragments of the OS were attached to the RPE (Fig. 9D)
, we did not include them with those with retinal detachment. In the
case of PBS injection, little morphologic change was observed as well
(Figs. 8C 9C)
. After PBS injection in one eye, vitreous and subretinal
hemorrhages were observed as a severe complication. In this retina,
disorganized photoreceptor OS and IS were detected, whereas no
morphologic change was observed in the OP (Figs. 9E
9F)
. Although
significant differences were obvious between anti-galectin-1–injected
eyes and either anti-galectin-3 antibody– or PBS-injected eyes, we
compared the ratio of RPE length with retinal detachment/total RPE
length (RD ratio: mean ± SD) in those eyes to ascertain the
retinal detachment–inducing effect of the anti-galectin-1 antibody.
The ratio was 0.82 ± 0.15 after anti-galectin-1 antibody
injection (n = 5), 0.16 ± 0.10 after anti-galectin-3
antibody injection (n = 5), and 0.18 ± 0.10 after PBS
injection (n = 5). These results showed that the RD ratio
after anti-galectin-1 antibody injection was greater than that after
the injection of either the anti-galectin-3 antibody or PBS
(P < 0.05).
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Figure 8. Hematoxylin-eosin staining of rat retinas after intravitreous injection
of anti-galectin antibodies or PBS. (A) Retinal detachment
was observed after vitreous injection of an anti-galectin-1 antibody.
(B, C) Little morphologic change was observed
after injection of an anti-galectin-3 antibody (B) or PBS
(C). Scale bar, 200 µm.
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Figure 9. Hematoxylin-eosin staining of rat retinas after intravitreous injection
of anti-galectin antibodies or PBS. (A) Retinal detachment
and vacuolation of the OP layer were observed after vitreous injection
of the anti-galectin-1 antibody. (B, C) Little
morphologic change was observed after injection of the anti-galectin-3
antibody (B) or PBS (C). (D) Even in
the anti-galectin-3 injected retinal region with torn photoreceptor OS,
no morphologic change was observed in the OP. (E,
F) Even in a PBS-injected eye with hemorrhage, and
disorganized photoreceptor OS and IS segments, no morphologic change
was observed in the OP. OS, outer segments; IS, inner segments; ON,
outer nuclear layer; arrows, OP; IN, inner nuclear layer;
PE, pigment epithelium. Scale bars, (A–D,
F) 20 µm; (E) 80 µm.
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Discussion
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Exogenous lectins, which recognize specific sugar sequences, have
been used for the detection and isolation of glycoconjugates in the
retina.1
2
3
4
5
11
Several kinds of endogenous animal lectins
have been isolated and characterized.12
19
20
21
26
Most
animal lectins can be classified into four distinct families: C-type
lectins including selectins, P-type lectins, pentraxins, and
galectins.12
Some of these are believed to be involved in
cell–cell or cell–matrix interactions, including cell
adhesion.12
26
Galectins, previously known as S-type
lectins, specifically bind to ß-galactoside residues and share a
carbohydrate-binding domain.12
26
They are found in many
animal species, and nine galectins have been isolated so
far.27
Expression analyses have revealed that galectins-2,
-5, and -7 exhibit a restricted distribution, whereas galectins-1, -3,
-8, and -9 exhibit a broad tissue distribution.26
27
Galectin-1 has been reported to be abundant in the cardiac, smooth, and
skeletal muscles; motor and sensory neurons; thymus; kidneys; and
placenta. In the nervous system, galectin-1 expression has been
observed in the brain, cochlea, and olfactory system.28
The present study revealed that the cDNA of galectin-1 can be isolated
either by screening of a retinal cDNA library using an antibody or by
PCR using primers for galectin-1. The present immunohistochemical study
also confirmed that galectin-1 is distributed in bovine and rat
retinas, and this finding indicates that the retina should be added to
the list of sites of galectin-1 expression in neural tissues.
The present Western blot analysis and cDNA sequencing showed that
the lactose-binding lectins eluted from a lactose column consisted of
two proteins: galectin-1 and a protein of Mr 31
kDa. We did not try to isolate the cDNA encoding the lectin of
Mr 31 kDa by increasing the screened number of
phages in the cDNA library, because the anti-bovine retinal lectin
antibody did not bind to the Mr 31-kDa band in
Western blot analysis, and the amount of this lectin in the retina
appeared to be much smaller than that of galectin-1 in both Western
blot analysis and immunohistochemistry. Therefore, the cDNA for the
31-kDa lectin could not be isolated by the cDNA library screening
method. Previous studies showed that the molecular size of galectin-3
is between 26.2 kDa and 30.3 kDa,20
whereas that
of galectin-4 is 36.3 kDa.21
The molecular weights of both
galectins are thus close to 31 kDa. The present study involving PCR
revealed that the cDNA for galectin-3 was amplified, whereas that for
galectin-4 was not. The present immunohistochemical study also showed
that galectin-3 is distributed in the retina. These findings suggest
that the lactose-binding lectin other than galectin-1 in the retina is
galectin-3.
The chicken 16-kDa lectin that was previously isolated16
is considered to be galectin-1, based on the molecular weight
similarity. However, the distribution of the 16-kDa lectin in the
chicken retina17
is partially different from that of
galectin-1 in the mammalian retinas observed in the present study. The
chicken galectin has been reported to be distributed in Müller
cells, including their apical villi, and in the OL membrane and cone
IS.17
The distribution of galactin-1 in the OL membrane
and cone IS observed in the present study was the same as that of the
chicken galectin. However, the other labeling patterns were different
between the chicken galectin and galectin-1. The chicken galectin has
been detected in the cytoplasmic and nuclear compartments of
Müller cells throughout the different retinal
layers.17
In the present study, galectin-3 was more
markedly detected in Müller cells throughout the retina than was
galectin-1 in the CL-exposed retinas. In this respect, the chicken
galectin is very similar to galectin-3. It is possible that this
inconsistency is partially due to the species difference between
mammals and birds.
Galectin-1 was isolated as a homodimer with a subunit molecular weight
of approximately 14.5 kDa.12
21
In its active form,
galectin-1 exists as a dimer, which enables it to cross-link ligands on
apposing cell surfaces or to facilitate the adherence of cells to the
extracellular matrix.12
The principal interaction of the
binding site of galectin-1 is with terminal
N-acetyllactosamine residues.12
Laminin,
lysosome-associated membrane glycoproteins, and antigen CD45, which
contain polylactosamine chains, have been shown to be good ligands for
galectin-1.12
We preliminarily found that the mucinlike
glycoprotein associated with photoreceptor cells (MLGAPC), which has
N-acetyllactosamine residues,22
also binds to
galectin-1 in the retina, on affinity chromatography on an immobilized
galectin-1 column. The previous immunohistochemical study showed that
MLGAPC is distributed in the photoreceptor layer and OP.22
The present study showed that this region, positive for galectin-1,
includes the OL, IS, RPE, and OP. These observations suggest that
galectin-1 may interact with MLGAPC in the photoreceptor layer and OP.
However, the origin of galectin-1 in these regions, unlike galectin-3,
cannot be restricted to Müller cells, because galectin-1 was
detected in various regions including the IS, RPE, and vessels in the
present study.
In connection with this surmised phenomenon, the histologic changes of
the rat retina induced by an injection of an anti-galectin-1 antibody
were notable. When a fixative containing glutaraldehyde is used for the
fixation of rat eyes, retinal detachment does not usually occur during
the preparation of tissue sections. The retinal detachment observed in
the sections of all five rat eyes after the injection of the
anti-galectin-1 antibody was thought to have been induced by the weak
adhesion between the RPE and neural retina. In addition to retinal
detachment, an intravitreous injection of the antibody against
galectin-1 produced vacuolation of the OP. This type of change was not
induced by an injection of either the anti-galectin-3 antibody or PBS,
even in severely injured cases with vitreous and subretinal hemorrhage.
Therefore, this morphologic change of the OP was also thought to have
been induced by the anti-galectin-1 antibody. This finding is notable
in relation to the common distribution of galectin-1 and MLGAPC
described earlier. Not only in the photoreceptor layer but also in the
OP, galectin-1 and MLGAPC may be involved in the mediation of
interphotoreceptor and synaptic cleft adhesion.
The reason that the anti-galectin-3 antibody did not induce retinal
detachment is that only a small amount of galectin-3 was expressed in
the retina under a normal light–dark cycle. Galectin-3 may play a
physiological role other than retinal adhesion in the retina. The
present study revealed that the expression of galectin-3 increased in
the Müller cells of CL-exposed retinas. The present findings also
show that an increase in the galectin-3 transcript preceded that in the
galectin-3 protein, implying that galectin-3 expression is upregulated
in Müller cells in response to CL exposure. These findings are
consistent with the acceleration of rat photoreceptor degeneration due
to CL exposure with the anti-galectin-3 antibody and the inhibition of
light damage by galectin-3 observed in the previous
study.15
These findings suggest that the increased
expression of endogenous galectin-3 observed in the present study may
represent an anti-apoptotic action in CL-exposed rat retinas, as in
other tissues including T-cells.13
14
|
Footnotes
|
|---|
Supported by Grant-in-Aid for Scientific Research (C) from the Japanese
Ministry of Education, Science, Sports and Culture (13671844).
Submitted for publication January 9, 2001; revised May 11, 2001;
accepted May 31, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Fumiyuki Uehara, Department of Ophthalmology,
Kagoshima University Faculty of Medicine, 8-35-1 Sakuragaoka,
Kagoshima-shi 890-8520, Japan.
fuehara{at}med5.kufm.kagoshima-u.ac.jp
|
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Top
Abstract
Introduction
