(Received for publication, October 31, 1996, and in revised form, December 9, 1996)
From the Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611
A 36-kDa -galactoside mammalian lectin
protein, designated as galectin-9, was isolated from mouse embryonic
kidney by using a degenerate primer polymerase chain reaction and
cloning strategy. Its deduced amino acid sequence had the
characteristic conserved sequence motif of galectins. Endogenous
galectin-9, extracted from liver and thymus, as well as recombinant
galectin-9 exhibited specific binding activity for the lactosyl group.
It had two distinct N- and C-terminal carbohydrate-binding domains
connected by a link peptide, with no homology to any other protein.
Galectin-9 had an alternate splicing isoform, exclusively expressed in
the small intestine with a 31-amino acid insertion between the
N-terminal domain and link peptide. Sequence homology analysis revealed
that the C-terminal carbohydrate-binding domain of mouse galectin-9 had
extensive similarity to that of monomeric rat galectin-5. The presence
of galectin-5 in the mouse could not be demonstrated by polymerase
chain reaction or by Northern or Southern blot genomic DNA analyses.
Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did
not reveal identical nucleotide sequences in the overlapping C-terminal
carbohydrate-binding domain, indicating that galectin-9 is not an
alternative splicing isoform of galectin-5. However, galectin-9 had a
sequence identical with that of its intestinal isoform in the
overlapping regions in both species. Southern blot genomic DNA
analyses, using the galectin-9 specific probe derived from the
N-terminal carbohydrate-binding domain, indicated the presence of
a novel gene encoding galectin-9 in both mice and rats. In contrast to
galectin-5, which is mainly expressed in erythrocytes, galectin-9 was
found to be widely distributed, i.e. in liver, small
intestine, thymus > kidney, spleen, lung, cardiac and skeletal
muscle > reticulocyte, brain. Collectively, these data indicate
that galectin-9 is a new member of the galectin gene family and has a
unique intestinal isoform.
There is growing evidence that specific carbohydrate moieties and
their putative binding proteins, i.e. lectins, play diverse roles in mammalian physiology and development and in various
pathological states (1). The mammalian lectins are classified into four categories, C-type lectins (including selectins), P-type lectins, pentraxins, and galectins; the latter are referred to as S-type or
S-Lac lectins (2, 3). Galectins are endowed with two essential
biochemical properties: 1) characteristic amino acid homologous
sequences; and 2) affinity for -galactoside sugars, i.e.
carbohydrate-binding domain. In addition, all the known galectins lack
a signal peptide, have a cytoplasmic localization, and are secreted as
soluble proteins by a nonclassical secretory pathway (4). Seven
mammalian galectins, i.e. galectins-1 (5), -2 (6), -3 (7),
-4 (8), -5 (9), -7 (10, 11), and -8 (12), have been cloned and
characterized. Structural analyses of various galectins indicate the
presence of homodimers of carbohydrate-binding domains in galectin-1
and galectin-2, a monomer of the carbohydrate-binding domain in
galectin-5, and a single polypeptide chain with two carbohydrate-binding domains joined by a link peptide in galectin-4 and
galectin-8. Galectin-3 has a carbohydrate-binding domain, a short
N-terminal segment, consisting of PGAYPG(X)1-4
repeats, and an intervening stretch of amino acids, enriched with
proline, glycine, and tyrosine (2, 3). Although the overall structure of galectins varies, each carbohydrate-binding domain is highly conserved and is encoded by three major exons (13, 14). Expression analyses have revealed that certain galectins display a restricted distribution, e.g. galectin-2 in hepatoma, galectin-4 in the
small intestine, galectin-5 in erythrocytes, and galectin-7 in
keratinocytes. Galectins with a broad tissue distribution include
galectin-1, expressed in cardiac, smooth, and skeletal muscles,
neurons, thymus, kidney, and placenta; galectin-3, present in blood
cells, intestine, kidney, and neurons; and galectin-8, expressed in
liver, kidney, cardiac muscle, lung, and brain (2, 3, 5 -12). Some of
these are believed to be involved in cell:cell or cell-matrix
interactions (2).
Such interactions between cell adhesion molecules and extracellular matrix glycoproteins, i.e. collagen, laminin, fibronectin, and proteoglycans, and their receptors (integrins) are highly relevant during embryonic development, including organogenesis of the kidney (15, 16). Conceivably, these interactions also involve the participation of other matrix- and plasmalemmal-bound macromolecules, such as galectins-1, -3, and -8, that are abundantly expressed in the developing metanephric kidney. In view of these considerations, studies were initiated to search for other developmentally regulated novel galectins that may be pertinent to the biology of cell-cell and cell-matrix interactions. In this communication, we report the identification and characterization of a new galectin, i.e. galectin-9, including its isoform and relationship with galectin-5.
Total RNA was extracted from embryonic metanephroi of CD-1
mice at day 13 and day 17 of gestation by the guanidinium
isothiocyanate-CsCl centrifugation method (17), and
poly(A)+ RNA was isolated by utilizing a FastTrack 2.0 kit
(Invitrogen). First strand cDNA was synthesized by using Moloney
murine leukemia virus-reverse transcriptase (RT, Rnase H)
and oligo(dT)25d(G/C/A) as a primer (CLONTECH) (18, 19). Double-stranded cDNA was prepared by using a mixture of
Escherichia coli DNA polymerase I, RNase H, and E. coli DNA ligase (20, 21). Subsequently, an adaptor-ligated
double-stranded cDNA was generated by a Marathon cDNA
Amplification Kit (CLONTECH), which was used as template DNA for rapid
amplification of 5
-cDNA ends (RACE) (19, 22) by PCR (23, 24). A
degenerate antisense 18-mer oligonucleotide primer, corresponding to
the
-galactoside-binding conserved sequence HFNPRF, was designed
with the following nucleotide sequence:
5
-(GA)AA(GATC)C(GT)(GATC)GG(GA)TT(GA)AA(GA)TG-3
. 5
-RACE PCR was
performed using template DNA in a 50-µl reaction mixture, containing
5 µl of 10× buffer (500 mM Tris-HCl (pH 9.2), 160 mM (NH4)2SO4, 17.5 mM MgCl2), 200 µM concentrations
of each dNTP, 0.2 µM adaptor primer 1 (supplied in the
kit), 1 µM degenerate antisense primer, and 1 µl of
polymerase mix; the latter is a mixture of 14.3 µl of
Taq/Pwo (Boehringer Mannheim) and 5.7 µl of TaqStart
antibody (CLONTECH). The reaction was carried out for 35 thermal
cycles, each consisting of 1.5 min at 95 °C, 2 min at 37 °C, 3 min at 68 °C, and a final 10-min extension at 68 °C. PCR products
purified by agarose gel electrophoresis, and subcloned into
pCRTMII vector (Invitrogen). Plasmid DNAs from ~100
colonies were prepared, and the vector inserts were sequenced by the
dideoxy chain termination method using modified T7 DNA polymerase
(Sequenase, Amersham). A sequence homology search was performed using
the Network BLAST program.
A ZAP mouse newborn kidney
cDNA library was prepared and screened as detailed previously (25,
26). Briefly, about 0.5 × 106 phage recombinants were
plated, and nitrocellulose filter lifts of phage plaques were prepared.
The filters were hybridized with a
[
-32P]dCTP-random-radiolabeled 226-base pair (bp)
mouse galectin-9 cDNA fragment obtained by the degenerate PCR
cloning method described above. Positive plaques were isolated and
purified by dilutional secondary and tertiary screening. Phage
cDNAs were amplified, agarose gel purified, and ligated into
pBluescript phagemid KS(+), using XL1-Blue MRF
E. coli
(Stratagene). Single-stranded DNAs were prepared by VCSM13 helper phage
and sequenced. Hydropathic (27), sequence homology (28), and
protein structural analyses (29) were performed using GCG PACKAGE
8.0.1.
Total RNAs from various organs of adult mice and rats
were used for the synthesis of cDNAs (26). For the synthesis of
cDNA from fetal red blood cells, 13-day-old mouse embryos were bled into the culture medium. Pellets of fetal erythrocytes were prepared by
centrifugation and utilized for isolation of mRNA by the Micro-Fast Track mRNA isolation kit (Invitrogen). In addition, total RNAs from
spleen and reticulocytes of phenylhydrazine-induced anemic mice and
rats (30) were isolated to prepare the cDNA. PCR analyses were
performed on cDNAs of various organs by utilizing primers with the
following nucleotide sequences: for mouse, 5-GCATTGGTTCCCCTGAGA TAG-3
(MG9SE) and 5
-CGTTCCAGAGACCGGATCC-3
(MG9AS); and for rat, 5
-GCGTT
GGTTCTCCCAAGACAG-3
(RG5SE) and 5
-CCTAGGCCAGAGACCTTC-3
(RG5AS) (Fig.
3A). To further ensure the detection of expression of
galectin(s) in mouse reticulocytes, RACE PCR was also performed using
adaptor-ligated double-stranded cDNA and primer set adaptor primer
1 and MG9AS. The PCR products were gel purified, ligated into
pCRTMII cloning vector (Invitrogen), and sequenced as
described above.
Expression of Galectins by Northern Blot Analyses
Analyses
were performed on total RNA, isolated from various adult mouse and rat
tissues, as described previously (26). In brief, 30 µg of total RNA
of each organ were glyoxalated, subjected to 1% agarose gel
electrophoresis, and transferred to a nylon membrane (Pall Biosupport).
Under high stringency conditions, the membranes were hybridized with a
[-32P]dCTP-random-radiolabeled "mouse galectin-9
cDNA probe," generated by PCR using MG9S.E. and MG9AS primers
(see above, and see Figs. 3A and 4C). In
addition, separate membrane filters were hybridized with a radiolabeled
"mouse galectin-9-specific cDNA probe," generated by PCR using
sense (5
-TACTGGACCAATCCAAGGAGG-3
) and antisense (5
-AGTAGAGAACATCTGTCCAG-3
) primers (Figs. 3A and
4C). The latter cDNA probe specifically detects the
expression of galectin-9 since it spans only the N-terminal
carbohydrate-binding domain, which is absent in galectin-5; whereas the
mouse galectin-9 cDNA probe spans the N- as well as C-terminal
carbohydrate-binding domains and detects the presence of both
galectin-9 and galectin-5 since they share 80.9% nucleotide sequence
homology in the C-terminal carbohydrate-binding domain.
-Actin
(GenBankTM accession no. M62174[GenBank], ATCC) cDNA was used as
a control.
Southern Blot Analyses of Genomic DNA
To confirm the
presence or absence of galectin-9 and -5 in mouse and rat species,
genomic Southern blot analyses were performed as described previously
(31). Genomic DNAs from rat and mouse livers were isolated. Aliquots
(20 µg each) of genomic DNAs were digested with EcoRI,
XbaI, PstI, BamHI, and
HindIII restriction enzymes; subjected to agarose gel
electrophoresis; and transferred to nylon membranes. The transferred
DNAs were hybridized either with the
[-32P]-radiolabeled mouse galectin-9 probe (G9 probe),
or the galectin-9-specific cDNA probe (G9-specific probe) under
high stringency conditions.
Expression of
recombinant protein with C-terminal c-myc-(His)6
tag for mouse galectin-9 was carried out with pTrcHis2 vector (Invitrogen). As controls, mouse galectin-1 and -3 cDNAs,
containing entire coding segments, were generated by RT-PCR using mouse
newborn kidney cDNA. Primers were: 5-for galectin-1,
GCTTCAATCATGGCCTGT-3
and 5
-GGCTGGCTTCACTCAAAGGC-3
(32); and
for galectin-3, 5
-GCACAGAGAGCATACCCAGG-3
and 5
-CTTCTGGCTTAGATC
ATG-3
(33). PCR products of galectins-1 and -3 were subcloned into
pCRTMII vector and sequenced. cDNA inserts of these 3 mouse galectins were reamplified by using the following primer sets:
5
-GGGGGG
CATGGCCTGTGGTCTGGTC-3
and 5
-GGG
GGG
CTCAAAGGCCACGCACTT-3
for galectin-1;
5
-GGGGGG
AATGGC AGACAGCTTTTCG-3
and
5
-GGGGGG
GATCATGGCGTGGTTAGC-3
for galectin-3;
5
-GGGGGG
GATGGCTCTCTTCAGTGCC-3
and
5
-GGGGGG
TGTCTG CACGTGGGTCAG-3
for galectin-9.
BamHI and HindIII restriction sites were
also introduced, and they are underlined in each of the
primer sequences. The generated PCR products were digested with
BamHI and HindIII, gel purified, and ligated into
pTrcHis2A plasmid to prepare galectins-1 (pTrcHis2/G1), -3 (pTrcHis2/G3), and -9 (pTrcHis2/G9) constructs. The constructs
were transfected into the TOP10 bacterial host
(Invitrogen) and sequenced to ensure proper in-frame ligation and
Taq polymerase fidelity. The bacteria were allowed to grow
in 1 liter of LB media until an A600 of 0.6 was
achieved. Expression of fusion proteins was induced by the addition of
1 mM isopropyl-1-thio-
-D-galactopyranoside
and further cultured for 5 h. To isolate recombinant proteins,
bacterial pellets were prepared, and suspended in 100 ml of either
MEPBS buffer (9, 12) or Tris-dithiothreitol (DTT) buffer (34)
containing 1.25% Triton X-100, 10 mM benzamidine, 10 mM
-amino-n-caproic acid, and 2 mM phenylmethanesulfonyl fluoride. They were lysed by
sonication, and the lysate was centrifuged at 20,000 × g at 4 °C for 30 min. Supernatants were applied to a
10-ml lactosyl-Sepharose column (Sigma), unbound proteins were washed,
and fusion proteins were eluted with either MEPBS buffer or Tris-DTT
buffer containing 200 mM lactose. The elution buffers
containing
-mercaptoethanol or DTT were always freshly prepared
to prevent oxidation of galectins by air (34, 35). Fractions of 1 ml
were collected, and the protein concentration was determined by the
Bradford method, using a Bio-Rad protein assay kit (Bio-Rad
Laboratories). Purified recombinant proteins were suspended in a sample
buffer (4% SDS, 150 mM Tris-HCl (pH 6.8), 20% glycerol,
0.1% bromphenol blue, 1%
-mercaptoethanol) and subjected to 12.5%
SDS-polyacrylamide gel electrophoresis (PAGE) (36). Gel bands were
visualized after staining with Coomassie Blue.
To determine the molecular size of
endogenous mammalian galectin-9, immunoprecipitation experiments were
performed. Polyclonal antibody was raised by immunizing rabbits with a
synthetic peptide, KTQNFRPAHQAPMAQT. Its sequence stretches between 148 and 162 amino acid residues of the link peptide of mouse galectin-9
(Fig. 1B). An additional lysine residue at the N-terminal
was used for conjugation of the peptide to keyhole limpet hemocyanin.
For immunoprecipitation experiments, newborn mouse liver and thymus
were radiolabeled in vivo by an intraperitoneal injection of
[35S]methionine (0.05 mCi/g body weight). After 24 h, the organs were harvested, homogenized in Tris-DTT buffer containing
10 mM benzamidine, 2 mM phenylmethanesulfonyl
fluoride, 1% Triton X-100, 10 mM benzamidine, and 10 mM -amino-n-caproic acid and sonicated. The
homogenates were centrifuged at 10,000 × g for 30 min
at 4 °C, and the supernatants were applied to lactosyl-Sepharose
columns and eluted with 0.2 M lactose in Tris-DTT buffer.
Immunoprecipitation was performed by adding 10 µl of anti-mouse
galectin-9 antibody to 0.5 ml of eluate, containing ~0.5 × 10
6 dpm. The eluate-antibody mixture was gently swirled
in an orbital shaker for 18 h. Protein A-Sepharose 4B (Pharmacia)
was added to the antibody-galectin-9 complex and mixed for 2 h,
following which the pellets were prepared and washed four times with
Tris-DTT buffer. The immunoprecipitated complexes were dissolved in a
sample buffer and subjected to 12.5% SDS-PAGE under reducing
conditions. The gels were fixed in 10% acetic acid, treated with 1 M salicylic acid, and dried, and autoradiograms were
prepared. Preimmune serum was used as a control.
Enzyme-linked Immunosorbent Assays (ELISA)
To assess the
specificity of the anti-galectin-9 antibody, ELISA assays were
performed (37, 38). Wells of RIA/EIA titer plates (Costar) were coated
with 50 µl of synthetic peptide solution (100 µg/ml) in 20 mM NaHCO3, pH 9.0. The plates were allowed to dry overnight at 37 °C. 100 µl of ice-cold methanol were added to
each well the next day and allowed to evaporate for 2 h at 37 °C. To reduce nonspecific binding of the antibody, 200 µl of bovine serum albumin solution (5 mg/ml), prepared in phosphate-buffered saline (PBS), was added, and plates were left at 22 °C for 1 h. The wells were washed twice with PBS, and 0.5 mg of antibody (IgG fraction) in 50 µl of bovine serum albumin-PBS (100 µg/ml) solution was added to the first well. Log dilutions of the antibody were made in
successive wells, and incubation was carried out with gentle shaking
for 90 min. Wells were rewashed three times with PBS containing 0.05%
Tween 20. Then horseradish peroxidase-conjugated goat anti-rabbit IgG
(Cappel) diluted 1:1000 in bovine serum albumin-PBS solution was added,
and incubation was extended for 60 min. After another three washes with
Tween 20, 100 µl of a solution containing 9 parts
3,3,5,5
-tetramethylbenzidine and 1 part H2O2
(Bio-Rad Laboratories) was added. For color development, the plates
were left in the dark for 30 min. The colorimetric reaction was stopped by the addition of 100 µl of 0.3 M
H2SO4. Finally, readings at A490 were made and plotted against log dilutions
of the antibody.
For competitive inhibition ELISA assay, 250 µg of recombinant galectin-9 were added in the first well of the peptide-coated titer plate as a competitive antigen along with 0.5 µg of the antibody in 50 µl of the PBS-bovine serum albumin solution. Log dilutions of the antigen were made in successive wells, while the antibody concentration was kept constant. Conditions for incubation with secondary antibody and colorimetric reactions were the same as described above. Readings at A490 were made and plotted against the log dilutions of the antigen.
Using degenerate oligonucleotide-based PCR
cloning, PCR products of ~250 bp in both the 13-day and 17-day
embryonic kidney were identified. The products were ligated into
pCRTMII vector, and ~100 clones were isolated and
sequenced. By homology search (National Center for Biotechnology
Information), 73 clones were identified as mouse galectin-1, while 28 clones had other unrelated cDNA sequences. Translation of one
additional clone with 226 bp indicated that it had a consensus sequence
of the galectin gene family. However, it did not show >50% overall
sequence homology to other known galectins. Using the 226-bp clone as a hybridization probe, preliminary Northern blot analyses of embryonic and neonatal mouse kidney tissues revealed a ~2.0-kilobase pair (kbp)
transcript. We then proceeded to screen the newborn kidney ZAP
cDNA library, using 226-bp clone as a hybridization probe. A
1418-bp clone (GenBankTM accession no. U55060[GenBank]) was isolated
and was designated as "galectin-9."
The
1418-bp clone had an open reading frame (35-1000) and a potential
initiation codon. The open reading frame encoded 322 amino acids with a
predicted size of ~36-kDa protein. Alignment sequence analyses of
this protein with known galectin family members revealed that it had 2 homologous (38.5%) N-terminal (147 amino acids) and 1 C-terminal (149 amino acids) carbohydrate-binding domain, connected by a 26-amino acid
link peptide (Figs. 1A, 1B, and
2). Both domains contained galectin sequence motifs, which are
conserved in all the known galectins (Fig. 2). Among the
known mammalian galectins, only galectin-4 and galectin-8 have two
carbohydrate-binding domains joined by a link peptide (Table
I). The link peptide of galectin-9 (mG9) had no homology
with those of the galectin-4 and -8 (Fig. 2, 148-173 amino acids).
Hydropathic analysis revealed that like other galectins, it lacks a
classical signal sequence and a transmembrane hydrophobic segment (Fig.
1C). By secondary structural analyses, both domains shared
characteristics similar with those of several sheets, a common
structural feature of galectins.
|
At the
amino acid level, the C-terminal domain of galectin-9 shared an
extensive homology (81.3%) with rat erythrocyte galectin-5 (9). Also
at the nucleotide level, its 5-untranslated segment, the 3
half of
the coding region and the 3
-untranslated regions had a 80.3% homology
with rat galectin-5 (Table I, Fig. 3A). To
ensure that the cloned galectin-9 is not an isoform of galectin-5, a
PCR cloning strategy was used. Primers were designed from 5
- and
3
-untranslated regions of galectin-9 cDNA (Fig. 3A,
MG9SE and MG9AS). RT-PCR analyses of various mouse tissue mRNAs
(heart, brain, liver, kidney, spleen, muscle, and thymus) yielded
~1.0-kbp products (Fig. 3B, panel a). In addition, a
~1.1-kbp product, exclusively expressed in the small intestine, was
observed (Fig. 3B, panel a). By sequence analysis, the
~1.1-kbp product revealed a 31-amino acid insertion between the
N-terminal domain and the link peptide (GenBankTM accession
no. U55061[GenBank]) and was designated as a mouse galectin-9 intestinal
isoform. Except for the 31-amino acid insertion, the intestinal isoform
had a 100% homology with mouse galectin-9. Since rat galectin-5 was
originally isolated from reticulocytes, RT-PCR was also carried out on
mouse cDNAs, prepared from phenylhydrazine-induced reticulocytes
and embryonic erythrocytes. Only ~1.0-kbp products were detected, and
the expected 0.5-kbp product, i.e. mouse homologue of rat
galectin-5, could not be amplified. RACE PCR was also performed using
47-bp adaptor-ligated mouse reticulocyte cDNA, and it also yielded
only one ~1.1-kbp product; the 0.5-kbp band of galectin-5 was not
detected.
Since RT-PCR and 5-RACE PCR failed to document the presence of
galectin-5 in mouse, we then selected the primer set RG5SE and RG5AS
from rat galectin-5 cDNA (GenBankTM accession no.
L36862[GenBank]), which corresponds to mouse MG9SE and MG9AS by a sequence
alignment program (Fig. 3A). With these rat primers, RT-PCRs
were performed on cDNAs from various adult rat tissues, including
reticulocytes. Two bands, i.e. ~1.0 and ~0.5 kDa, were
observed in several tissues, and another ~1.1-kbp band was confined
to the small intestine only (Fig. 3B, panel b). Analysis of
the ~0.5-kbp product revealed that it had a nucleotide sequence
identical with that of rat galectin-5 (30-472 bp,
GenBankTM accession no. L36862[GenBank]), while the ~1.0-kbp
product (GenBankTM accession no. U59462[GenBank]) had a 88.9%
homology to mouse galectin-9. Thus, the ~1.0-kbp product was regarded
as the rat homologue of mouse galectin-9. Sequence analysis of the
~1.1-kbp product (GenBankTM accession no. U72741[GenBank]) showed
that it is an isoform of rat galectin-9 since it had a 32-amino acid
insertion between the N-terminal carbohydrate domain and the link
peptide, as in the mouse galectin-9 intestinal isoform. These two rat
PCR products, the 0.5- and 1.0-kbp rat galectins-5 and -9 shared a
93.6% homology in the 443-bp overlapping region of C-terminal
carbohydrate-binding domain (Fig. 3C), while the rat
galectin-9 and its intestinal isoform had a 100% identity in the
974-bp overlapping region, spanning the N- and C-terminal
carbohydrate-binding domains. These results indicate that the newly
cloned galectin-9 is not an alternate splicing isoform of galectin-5,
that it has a long intestinal isoform in both rats and mice, and that
galectin-5 is not present in the mouse.
Using the galectin-9 (G9) cDNA probe (Fig. 4C), a ~2-kbp single transcript was observed in various mouse tissues (Fig. 4A). Smaller mRNA transcripts, corresponding to putative mouse galectin-5, were not detected. mRNA expression of galectin-9 in various mouse organs was as follows: liver, small intestine, thymus > kidney, spleen, lung, skeletal muscle, heart > reticulocyte, brain (Fig. 4A, upper panel). In rat tissues, the G9 cDNA probe hybridized with transcripts of ~2 and ~~1.5 kbp, corresponding to galectin-9 and galectin-5, respectively (Fig. 4B, upper panel). The 1.5-kbp mRNA transcripts of rat galectin-5 were abundantly expressed in reticulocytes and spleen. Although, mRNA expression of galectin-9 (~2 kbp) in various rat tissues was similar to that in the mouse, its expression was relatively low in the thymus and substantially lower in kidney and skeletal muscle (Fig. 4B, upper panel). By using the G9-specific probe, only ~2-kbp transcripts were observed, and smaller transcripts (~1.5 kbp) were not detected in various rat tissues (Fig. 4B, middle panel).
Southern blot analyses, using the G9 cDNA probe, revealed a single
major band in various restriction enzyme digests of mouse genomic DNA
(Fig. 5A, left panel). Identical
results were obtained for mouse genomic DNA digests in blots hybridized
with the G9-specific cDNA probe (Fig. 5A, right panel),
supporting the presence of a gene encoding galectin-9 only. Rat genomic
DNA digests revealed multiple bands when the G9-specific cDNA probe
was used for Southern blot hybridization analyses (Fig. 5A, right
panel); while hybridization with the G9 cDNA probe revealed a
few additional bands in various restriction enzyme digests (Fig.
5B, left panel), suggesting the presence of galectin-9 and
-5 genes in rat genomic DNA.
Recombinant Galectin-9 Exhibits Lactose-binding Activity
Preliminary experiments revealed a
time-dependent increasing
isopropyl-1-thio--D-galactopyranoside-inducible
expression of recombinant galectins, which was maximal at 5 h in
total E. coli lysates, prepared in the presence of DTT or
-mercaptoethanol. SDS-PAGE of the eluates of bacterial lysates from
lactosyl-Sepharose column revealed ~39-kDa, ~37-kDa, and ~17-kDa
bands of rat galectins-9, -3, and -1, respectively (Fig.
6A, lanes 3-8).
Immunoprecipitation and ELISA Assays
Homogenates of
[35S]methionine-labeled liver and thymus were applied to
a lactosyl-Sepharose column, followed by immunoprecipitation of the
eluted fractions with anti-galectin-9 antibody. SDS-PAGE autoradiograms
of the immunoprecipitates revealed a ~36-kDa band, indicating that
endogenous galectin-9 also has lactose-binding activity and a
comparable molecular weight (Fig. 6B, lanes 1 and 2). No discernible bands were observed when
immunoprecipitation was performed with preimmune serum (Fig. 6B,
lanes 3 and 4). Specificity of the antibody was also
assessed by ELISA assay in which a fixed amount of antigen,
i.e. synthetic peptide, and serial log dilutions of the
antibody were used. With increasing dilutions of the antibody, a
proportional decrease in A490 readings was
observed (Fig. 7A). To confirm the
specificity of the antibody, a competitive inhibition ELISA assay was
performed. A fixed amount of diluted antibody (1:000) along with serial
log dilutions of the competitive antigen, i.e. recombinant
galectin-9, were added into the wells of the titer plate coated with
the synthetic peptide. With increasing dilutions of the competitive
antigen, a proportional increase in A490
readings was observed (Fig. 7B), documenting the
specificity of anti-galectin-9 antibody.
In the present study, we have described a novel galectin, galectin-9, isolated from the embryonic kidney cDNA, using the degenerate oligonucleotide-based RACE-PCR cloning strategy. Although previous studies used traditional lactosyl-Sepharose column protein purification to identify galectins, the successful isolation of galectin-9 indicates that degenerate oligonucleotide-based RACE-PCR strategy is yet another useful method by which one can search for new galectins in various tissues. The biochemical characteristics of galectin-9 fulfill the criteria for its inclusion as a new member of the galectin family (2, 3). They include the following: 1) its deduced amino acid sequence which indicates two domains consisting of characteristic conserved sequence motifs that are implicated in binding to specific saccharides; and 2) the recombinant fusion protein (recombinant galectin-9) exhibits specific binding affinity for lactosyl groups. In addition, an endogenous protein, with an expected size of ~36 kDa, binds to lactosyl-Sepharose columns and can be immunoprecipitated by specific antibody, directed against a unique sequence of galectin-9 link peptide.
A feature common to galectin-9 and other galectins is that it lacks a classical signal sequence and a transmembrane hydrophobic segment. Thus, like other galectins that have a cytosolic distribution and are secreted as a soluble proteins by a nonclassical secretory mechanism (4), galectin-9 could be externalized with the aid of a carrier protein. Such a mechanism has been shown for other cytoplasmic proteins lacking a signal peptide, such as thymosin, interleukin-1, and fibroblast growth factor (39, 40).
Structural analyses of galectin-9 revealed some additional unique features. It consists of two distinct carbohydrate-binding domains connected by a link peptide. Among the known galectin family members, only galectin-4 (8), galectin-8 (12), and the Caenorhabditis elegans homologue (41) have similar dimeric structures. Other galectins, including galectin-1 (5) and galectin-2 (6), contain only one carbohydrate-binding domain but can function as homodimers to facilitate aggregation or agglutination of cell surface-bound glycoconjugates via noncovalent associations (3). Although galectins with two carbohydrate-binding domains may have similar agglutinating and aggregating potential, their properties could be influenced by the size and amino acid sequence of the link peptide affecting overall molecular conformation. Moreover, since the amino acid sequences of link peptides differ from one another, it raises the possibility that these may modify biological activities of various dimeric galectins in a given tissue. One of the unique features of galectin-9 is its alternate splicing isoform exclusively expressed in the small intestine. This isoform has a 31- and 32-amino acid insertion in mouse and rat, respectively. Since this insertion has no homology with the carbohydrate-binding domain sequences, it can be regarded as an extension of the link peptide. Such a long link peptide, with a stretch of 57-58 amino acids, may influence the macromolecular conformation of galectin-9 which may be necessary for certain yet to be defined functions related to the biology of intestinal epithelium. Such an extended version of the link peptide or the isoforms have not been reported in other dimeric galectins.
Analysis of the C-terminal domain of mouse galectin-9 revealed
substantial sequence homology (81.3%) with rat galectin-5, while the
N-terminal had a 23-48% amino acid sequence homology with other
galectins. At the nucleotide level, the coding region of the C-terminal
domain and the 5- and 3
-untranslated regions had a 80.9% similarity
to rat galectin-5 cDNA. Galectin-5 is a monomeric form of rat
galectin, which is mainly expressed in the erythrocytes (9). Its
genetic relationship with galectin-9 is not clear; i.e. are
they alternate splicing isoforms, or are they derived from separate
genes? Initially, we speculated that galectin-9 might be a novel
isoform of galectin-5 because of its segmental sequence homology.
Therefore, attempts were made to isolate putative mouse galectin-5 from
various tissues by PCR cloning methods, using the primers derived from
sequences of flanking the 5
- and 3
-untranslated regions of mouse
galectin-9. The ~0.5 kbp PCR product, corresponding in size to
galectin-5, could not be amplified in mouse cDNA, instead, we
identified galectin-9 and an intestinal alternate splicing isoform of
galectin-9. In contrast, in cDNAs of various rat tissues, three PCR
products, corresponding to galectin-5, galectin-9, and the intestinal
isoform of galectin-9, were amplified when primer sequences derived
from the 5
- and 3
-untranslated regions of the reported rat galectin-5 cDNA were used. However, a comparison of the cDNA sequences of rat galectin-5 with those of galectin-9 did not show identical nucleotide sequences in the overlapping regions; thus, galectin-9 is
not an alternate splicing form of galectin-5. The existence of a long
intestinal isoform in rat as well as in the mouse, with nucleotide
sequences identical with that of galectin-9 in the respective species,
further supports the notion that galectin-9 is indeed a novel member of
the galectin gene family.
To further characterize the relationship between the galectin-5 and galectin-9, Northern blot and genomic Southern blot analyses were performed. By Northern blot analyses, galectin-9 mRNA transcripts were found in various tissues with a wide distribution in both rats and mice. However, galectin-5 mRNA transcripts were detected largely in rat reticulocytes and spleen. Galectin-5 mRNA transcripts were absent in mouse reticulocytes as well as in other mouse tissues. By using the galectin-9 (G9)-specific cDNA probe, genomic Southern blot analyses affirmed the existence of a unique gene encoding galectin-9 protein both in rat and mouse. In mouse genomic Southern blot analyses, a single band was detected in various restriction enzyme digests of the genomic DNA after hybridization with G9 specific or G9 cDNA probes; thus, the existence of the galectin-5 gene in the mouse is doubtful. Certainly, in our hands, attempts to elucidate the presence of mouse galectin-5 by RT-PCR, prime] RACE PCR, Northern blot, or Southern blot genomic DNA analyses were unsuccessful.
By a recent homology search through the GenBankTM, we have
also found that Homo sapiens RCC313 mRNA (Z49107[GenBank])
(42)2 has a 70% homology with mouse
galectin-9 and thus regard it as the human homologue of galectin-9.
This cDNA was isolated by immunoscreening of a human Hodgkin's
lymphoma ZAP library with autologous patient serum and was found to
be distributed in lymphoid tissues (42). Since, in both mice and rats,
galectin-9 is expressed in lymphoid tissues, such as in the thymus and
spleen, its potential role in the biology of the immune system would be
anticipated, although this galectin is originally isolated from
embryonic kidney. In addition, a 17.5-kDa galectin with a high amino
acid sequence similarity to galectin-5 has been reported in the rat
kidney (43). This galectin may be galectin-5 or a proteolytic fragment
of galectin-9.
In summary, although the C-terminal carbohydrate domain of galectin-9 shares extensive homology with galectin-5, there are distinct differences between the two, such as different tissue distributions and carbohydrate-binding domains. Finally, like certain other galectins (2), galectin-9 seems to be developmentally regulated in various embryonic tissues,3 including the kidney, the organogenesis of which is heavily influenced by cell-cell and cell-matrix interactions (15, 16). Since galectins are believed to be involved in cell-matrix interactions (44-49) and are developmentally regulated, it would be of a great interest to investigate their relevance in various embryological processes regulating cell growth and differentiation (32, 50).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U55060[GenBank] (mouse galectin-9), U55061[GenBank] (mouse galectin-9 intestinal isoform), U59462[GenBank] (rat galectin-9), and U72741[GenBank] (rat galectin-9 intestinal isoform).
We are grateful to Dr. Samuel H. Barondes and his colleagues for valuable suggestions during the course of this investigation and for suggesting the inclusion of galectin-9 as a new member of the galectin gene family. We are also thankful to Drs. D. G. Scarpelli, E. I. Wallner, and K. Alveraz for carefully editing the manuscript.