From the Galectins are a family of carbohydrate-binding
proteins that share a conserved sequence and affinity for
Galectins (1, 2) are a family of proteins that have at least one
characteristic carbohydrate recognition domain
(CRD)1 of about 14 kDa with
affinity for Although the functions of galectins are not established, there is
evidence that the best studied, galectin-1 and galectin-3, play roles
in cell adhesion and signaling by cross-linking of glycoconjugate
ligands (2, 11). Galectin-4 has been found in the epithelium of the rat
(12-14), porcine (15), and human (16) alimentary tract, and may
sometimes be associated with adherens junctions (17).
In the course of cloning of galectin-4 cDNA from mouse colon, we
isolated an additional closely related cDNA. Since the nomenclature of galectins was being established at that time (1), we reserved the
name galectin-6 for the protein it encodes. Here, we provide the
sequence and demonstrate carbohydrate binding activity of this new
galectin. In addition, we have begun to address the biological functions of galectin-4 and galectin-6 by determining their
distribution in embryonic and adult mouse tissues.
General Information--
All materials, equipment, and
experimental conditions were identical to those described by Gitt
et al. (3, 18) unless stated otherwise.
Polymerase Chain Reaction (PCR)--
We used the following
conditions for all amplifications. We used Ampli-Taq
(Perkin-Elmer Cetus), 250 µM deoxynucleotides, 25 pmol of
each primer, and buffer provided by the enzyme manufacturer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin). We amplified
for 45 cycles, with each cycle consisting of a 40-s denaturation at
95 °C, a 1-min annealing at 60 °C, and a 1-5-min extension at
72 °C. Amplified fragments were visualized on ethidium
bromide-stained 1% agarose gels.
Isolation and Characterization of Galectin-4 and Galectin-6
cDNAs--
Total RNA was purified from mouse colon using RNAzol
(Tel-Test, Friendswood, TX), following the manufacturer's protocol.
Concentration was determined by absorbance at 260 nm. Ten micrograms of
the RNA was reverse-transcribed for 2 h at 42 °C using 200 units of Moloney murine leukemia virus reverse transcriptase (U. S. Biochemical Corp.) in a 20-µl solution containing 50 mM KCl, 20 mM Tris-HCl, pH 8.4, 2.5 mM MgCl2, 100 mg/ml nuclease-free bovine serum
albumin, 1.0 mM deoxynucleotide triphosphates, 20 units
RNasin (Promega, Madison, WI), and using 50 pmol of a
poly(dT)-containing primer (GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT). After
reverse transcription, the solution was heated for 5 min at 95 °C,
immediately cooled on ice, and diluted to 1 ml with 10 mM
Tris-HCl, pH 7.6, 1 mM EDTA. Ten µl of this solution was
used as a template in PCR with different gene-specific primer pairs as
described in Figs. 1 and 2. The amplified products were ligated
directly to plasmid pCR1000 or pCR3 (Invitrogen, San Diego, CA)
according to the manufacturer's protocol.
Expression of Galectin-6 Domain 2--
A construct encoding the
amino acid 161-301 fragment of galectin-6 was generated in the pET11d
vector (Novagen, Madison, WI) using the same primers as used for
expression of domain 2 of rat galectin-4 (12), and expressed in the
BL21 host. In brief, the cDNA clone pmG6-2 (Fig. 1), constructed
to provide a galectin-6-specific template encompassing all of domain 2, was used as template in a PCR amplification with primers rG4E and rG4K
(Fig. 2) and the product cloned into pCR3 (Invitrogen). Primer rG4E has
an engineered NcoI site encompassing the ATG encoding
Met-161, and rG4K has additional sequence generating a BamHI
site downstream of the stop codon. The plasmid DNA prepared from this
clone was digested with NcoI and BamHI, and the
insert was ligated into NcoI/BamHI-digested pET11d (Novagen, Madison WI). A clone containing the correct galectin-6 fragment was grown and induced by
isopropyl-1-thio- Northern Blots--
Total RNA was isolated from various mouse
tissues using RNAzol (Tel-Test Inc.). The amount of RNA in each sample
was estimated by absorbance at 260 nm. Samples were electrophoresed
along with size markers (Life Technologies, Inc.) on a 1.2% agarose
gel containing 6% formaldehyde and 20 mM MOPS and the gel
transferred to MagnaGraph nylon filters (Micron Separations, Inc.,
Westborough, MA) in 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate). Ultraviolet cross-linking, hybridization
buffer, and wash solutions were the same as described (19). For probes,
we used PCR-amplified cDNA containing all the coding sequence but
no untranslated sequence of rat galectin-4 (12), or, as a control,
human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA
amplified from a plasmid kindly provided by Dr. Alex Bulfone,
Department of Psychiatry, University of California, San Francisco).
Both probes were labeled with 32P by random primer
polymerization (20). Hybridization and washing was done at 45 °C.
After hybridization with the galectin-4 probe and autoradiography, the
galectin-4 probe was removed by washing for 1 h at 65 °C in
50% formamide, 2 × SSC, followed by a 0.1 × SSC rinse at
room temperature. The blot was then incubated with the GAPDH probe
under the same conditions. Quantitation of bound radioactivity was done
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
In Situ Hybridization of Mouse Embryos--
Antisense
35S-cRNA probes were prepared by T7 polymerase
transcription of clones pmG4-1 and pmG6-1 (Fig. 1) as described
below. In situ hybridization was performed as described by
Wilkinson and Green (21), using mouse embryos starting at first day of gestation (0.5 day post coitum (dpc)).
RNase Protection Assay (RPA)--
For generation of antisense
galectin-4 and galectin-6 transcripts, DNA from clones pmG4-1 and
pmG6-1 (Fig. 1) was linearized with EcoRI and then
incubated with 200 µg/ml proteinase K for 30 min at 37 °C to
remove RNases, then phenol-extracted and ethanol-precipitated. For
sense transcription templates, pmG4-1 and pmG6-1 inserts were released by HindIII/EcoRI digestion and recloned
into similarly digested pBluescript SK+ (Stratagene, La Jolla, CA). DNA
of the resultant clones and a plasmid containing a fragment of human GAPDH cDNA were linearized with HindIII, and treated as
above with proteinase K and phenol. A pBluescript SK+-derived plasmid containing a fragment of human GAPDH cDNA was linearized with EcoRI. Galectin-4 and galectin-6 antisense transcripts were
generated with T7 RNA polymerase, and sense galectin-4 and galectin-6
transcripts and antisense GAPDH transcripts were generated with T3 RNA
polymerase, using the Maxiscript system (Ambion, Inc., Austin, TX) with
10 mM each of ATP, GTP, UTP, 0.5 mM CTP, and 75 µCi of [ Western Blot Analysis of Tissue Extracts--
Total soluble
proteins were extracted from mouse stomach, small intestine, and colon
using MEPBS supplemented with 300 mM lactose and 2 mM phenylmethanesulfonyl fluoride. After homogenization with an Omni mixer (10 s, medium strength), the extracts were centrifuged at 17,000 × g for 15 min to remove cell
debris and the supernatants were immediately boiled in the presence of
SDS and 2-mercaptoethanol and applied to a 12% polyacrylamide gel. After electrophoresis, the gel bands were transferred to nitrocellulose (Micron Separations, Inc.) as described previously (3). The blot was
blocked with 3% bovine serum albumin and then probed with a 1/2000
dilution of chicken antibodies raised against domain-I of rat
galectin-4 (prepared by HTI-Bioproducts Inc., Ramona, CA). The bound
antibodies were visualized using a 1/15,000 dilution of a
peroxidase-conjugated rabbit anti-chicken IgG (Sigma) and the ECL
system (Amersham Life Sciences) as peroxidase substrate.
Identification of Galectin-4 and Galectin-6--
To isolate mouse
galectin-4 cDNA, we amplified a portion of cDNA prepared from
mouse colon RNA by PCR using different pairs of primers designed on the
basis of the sequence of rat galectin-4. To our surprise, we obtained
two sets of clones, pmG6-1, and pmG4-1 and -2, respectively (Fig.
1), representing two non-identical but
very similar sequences resembling the rat galectin-4 cDNA reported
by Oda et al. (12). An additional cDNA fragment
(pmG6-2) was obtained using a primer designed to specifically
recognize only one of the two sequences, and genomic clones
( Center for Neurobiology and Psychiatry,
Department of Pharmaceutical
Chemistry, University of California, San Francisco, California
94143-0984 and the ¶ Institut Cochin de Genetique Moleculaire,
Unite INSERM 257, 24 rue de Faubourg Saint-Jaques,
75014 Paris, France
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
-galactosides. Some, such as galectin-1, are isolated as dimers and
have a single carbohydrate recognition domain (CRD) in each monomer,
whereas others, such as galectin-4, are isolated as monomers and have
two CRDs in a single polypeptide chain. In the course of studying mouse
colon mRNA for galectin-4, we detected a related mRNA that
encodes a new galectin that also has two CRDs in a single peptide
chain. The new galectin, galectin-6, lacks a 24-amino acid stretch in the link region between the two CRDs that is present in galectin-4. Otherwise, these two galectins have 83% amino acid identity.
Expression of both galectin-4 and galectin-6 is confined to the
epithelial cells of the embryonic and adult gastrointestinal tract.
Galectin-4 is expressed at about equal levels in colon and small
intestine but much less in stomach, whereas galectin-6 is expressed at
about equal levels throughout the gastrointestinal tract.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-galactosides. In mammals, nine galectins have been
reported: galectins 1-4 (reviewed in Ref. 2), galectin-5 (3),
galectin-7 (4, 5), galectin-8 (6, 7), galectin-9 (8, 9), and
galectin-10 (10, 11). These are often expressed as abundant soluble
cytosolic proteins with 14-36 kDa subunits containing one
(galectins-1, -2,-3 -5, -7, and -10) or two (galectins-4, -8, and -9)
CRDs.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-D-galactopyranoside according to the
manufacturer's protocol. A pET11d clone with no insert was also grown
and induced as a negative control. Lysates were prepared by sonication
in MEPBS (58 mM Na2HPO4, 18 mM KH2PO4, 75 mM NaCl,
2 mM EDTA, 4 mM 2-mercaptoethanol) supplemented
with 2.5% Triton X-100 and 2 mM phenylmethanesulfonyl
fluoride, and
-galactoside-binding proteins purified by affinity
chromatography on lactosyl-Sepharose as described by Gitt et
al. (18). Western blot conditions are described below.
-32P]CTP per transcription. The transcripts
were purified on 5% polyacrylamide gels and eluted by diffusion into
0.5 M ammonium acetate, 1 mM EDTA, 0.1% SDS.
The RNase protection assay was done using an Ambion RNase protection
assay kit II. In brief, the antisense transcripts were hybridized
overnight at 42 °C with different amounts of total tissue RNA plus
yeast RNA carrier. In control experiments, tissue RNA was replaced by
in vitro synthesized sense transcripts or omitted entirely.
After hybridization, the mixtures were digested with 1 unit/ml RNase A + 200 units/ml RNase T1. The products were concentrated by
precipitation and then electrophoresed on an 8 M urea 5%
polyacrylamide gel. The gel was autoradiographed with intensifying
screens after drying. Densitometric quantitation was done with the
PhosphorImager system. Data were corrected for background and the
different sizes of protected bands, and then normalized relative to
GAPDH mRNA in each sample, as detected with GAPDH-specific probes
in either a Northern blot (Fig. 5) or in an RNase protection assay
(data not shown). Since dilutions of tissue RNAs resulted in
corresponding decreases in amount of protected probe for both
galectin-4 and galectin-6, the assay is RNA-limiting under the
conditions employed. Molecular weight markers were prepared by
incubation of HindIII-cut
DNA (Life Technologies, Inc.)
with 0.08 pmol/µl [
-32P]dCTP, a mixture of 80 µM each dATP, dGTP, dTTP, and Klenow fragment of DNA
polymerase I. In addition, a sequencing ladder was run in an adjacent
lane.
RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
Lgals6 and pLgals4-1, Fig. 1) that included
either sequence (22) were also obtained. The composite sequences are
given in Fig. 2, and the encoded amino
acid sequences are given in Fig. 3 and
compared with other bi-CRD galectins.
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Fig. 1.
Schematic of the isolated galectin-4 and
galectin-6 clones. The composite cDNAs are represented by
bars with sequence encoding carbohydrate binding domains
filled. Clones isolated by PCR are shown as
lines, and the relevant part of a genomic clone
(
Lgals 6; Ref. 22) as a line with
arrowheads. The PCR cDNA clones (with corresponding
primer pairs as given in Fig. 2) were as follows: pmG6-1 (rG4B and
rG4F), pmG6-2 (mG6R and rG4K), pmG4-1 (rG4B and rG4G), and pmG4-2
(rG4B and rG4K). The genomic clones
Lgals6, which
contains the whole galectin-6 gene (Lgals6), and
pLgals4-1, which contains a fragment of the galectin-4 gene (Lgals4), are described in the accompanying paper (22). All regions of overlap between the clones, whether cDNA or genomic, were identical in sequence except for a difference of three bases between the Lgals6 clone and the galectin-6 cDNAs,
presumably due to the origin of the DNAs in different strains.
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Fig. 2.
Composite sequences of mouse galectin-4 and
galectin-6 cDNAs (mG4 and mG6) compared with rat galectin-4 cDNA
(rG4). The mouse sequences were derived from a composite of
cDNA and genomic DNA clones (Fig. 1), and the rat sequence is from
Oda et al. (12). In the mouse sequences, residues identical
to the corresponding rat galectin-4 residue are shown as dots.
Dashes represent gaps introduced for alignment.
Numbers give the last residue on the line, with the first
translated nucleotide as 1 and assuming that the corresponding
translational initiation site is conserved in mG4. Locations,
orientations, and names of oligonucleotides used as primers are
indicated by arrows underneath the relevant sequence. The
oligonucleotides labeled with an asterisk had
additional "anchor sequence" or other modifications and are
described in full in Table II of Ref. 12, where primer 10 = rG4D,
11 = rG4E, 4 = rG4H, and 12 = rG4L.
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Fig. 3.
Comparison of deduced amino acid sequences of
mouse galectin-6 (mG6) with mouse galectin-4 (mG4) and other bi-CRD
galectins. Dashes represent gaps introduced for alignment.
Residues identical to the corresponding galectin-6 residues are
indicated by dots. Filled bars over the sequences
indicate the regions that are part of the tightly folded carbohydrate
binding domains, and open bars indicate other regions.
Asterisks below the sequences denote the position of
residues within each carbohydrate binding domain that interact with
carbohydrate (23, 24). Published sequences presented are rat galectin-4
(rG4; Ref. 12), pig putative galectin-4 (pG4; Ref. 15), rat galectin-8 (rG8; Ref. 6), mouse galectin-9 (mG9; Ref. 9), and the galectin-4 homologs from
Caenorhabditis elegans (C.el.; Ref. 29) and
O. volvulus (O.vo.; Ref. 30). The insertion of 31 amino acids at the beginning of the link region in a second form of
galectin-9 attributed to alternative splicing (9) is indicated.
Structures of Galectin-4 and Galectin-6--
In the structures
determined for galectin-1 (23), and galectin-2 (24) by x-ray
crystallography, the CRD consists of a tightly folded
"-sandwich" of about 130 amino acids. The homologous sequences
that are predicted to form these domains in galectin-4 and galectin-6
are indicated by a black bar in Fig. 3. Hence, both
galectin-4 and galectin-6 have two CRDs in tandem, each containing all
the conserved amino acid residues involved in
-galactoside binding
in other galectins (asterisked residues in Fig. 3).
-Galactoside Binding Activity of Galectin-6--
Although
galectin-6 has consensus sequences that predict
-galactoside
binding, we sought to verify this directly to prove that it is indeed a
galectin (1). Lacking a complete cDNA, we tested for carbohydrate
binding activity by expressing the C-terminal putative CRD of
galectin-6 (amino acids 161-301) in Escherichia coli. After
solubilization, the protein product bound to lactosyl-Sepharose and
could be eluted with lactose, proving that it has galactoside binding
activity. The affinity-purified protein has an apparent molecular
weight of 17,000 on SDS-polyacrylamide gels, consistent with the value
predicted based on its sequence (15,861), and cross-reacts weakly with
anti-galectin-4 antibodies on a Western blot (data not shown).
Expression of Galectin-4 and Galectin-6 in the Adult Gastrointestinal Tract-- To examine the tissue distribution of galectin-4 and galectin-6 expression, we first used a RPA assay, which permitted the selective detection of each galectin. As probes, we used the antisense transcripts of clones pmG4-1 and pmG6-1 for galectins-4 and -6, respectively (Fig. 1). Since both these probes span the link region, we predicted that they should readily distinguish galectin-4 from galectin-6 in this type of assay, i.e. when RNA and a heterologous probe anneal, the link region will represent a large region of single stranded uncomplementary RNA that is a substrate for the RNases.
The undigested probes had the expected length (425 and 600 nt, respectively; Fig. 4A, lanes a and e), and no fragments were protected with RNA from yeast (lanes c and g). When the galectin-6 probe was mixed with small intestinal RNA and digested with RNases, the size of the largest protected fragment was 335 nt (Fig. 4A, lane b), which is equal to the length of the galectin-6-specific sequence in this probe (Fig. 4B). Similarly, with the galectin-4 probe, a 465-nt band was protected (Fig. 4A, lane f), equal to the length of the mouse galectin-4-specific sequence of this probe. In this case, the 42-nt sequence from the 3
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Expression of Galectin-4 or Galectin-6 during Embryogenesis-- To examine the tissue origin of galectins-4 and -6 further, we examined their expression in mouse embryos of different age by in situ hybridization (Fig. 6) with antisense RNA probes transcribed from clones pmG4-1 and pmG6-1 (Fig. 1). As expected, the two probes gave identical signals, each probably reacting with both galectin-4 and galectin-6 mRNAs. Hence, the observed signal represents galectin-4 or galectin-6 or a mixture of the two.
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Expression of Galectin-4 and Galectin-6 Proteins-- To examine the expression of galectin-4 and galectin-6 at the protein level, we probed Western blots of tissue extracts with an antiserum raised against the N-terminal CRDs of rat galectin-4. This antiserum detected a band migrating as about 36 kDa in small intestine and colon but not stomach (Fig. 7). The mobility of this band is the same as for recombinant rat galectin-4, and hence likely to be mouse galectin-4. Its detection in small intestine and colon, but not stomach, is consistent with the results of the RPA.
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Functional Correlates-- The organization of galectins-4 and -6 and other bi-CRD galectins such as galectins-8 and -9 suggests that they function by cross-linking ligands for each of the CRDs. In a simple model, this functional activity would then depend on the specificity of each of the two carbohydrate binding sites, as determined by the structure of the CRDs, and the distance and relative orientation of the two CRDs, as determined by the structure of the link region between the CRDs. The latter property is the most likely functional difference between galectin-4 and galectin-6 since they differ mainly in the length of their link regions. Interestingly, galectin-9 also occurs in two forms differing only in their link regions. The form specifically expressed in intestine has a longer link region, while the form expressed in other tissues has a shorter link region (Ref. 9; Fig. 3). This provides further evidence that expression of different link regions may be functionally significant whether by their effects on CRD spacing, as suggested above, or by other effects. Nevertheless, it is also possible that galectins-4 and -6 are functionally redundant.
Whether or not galectins-4 and -6 are functionally distinct, their prominent expression in gastrointestinal epithelial cells suggests that they function in these cells. Prior to day E12.5, when neither galectin-4 nor galectin-6 are expressed, the intestine consists of a multilayered endodermal epithelium surrounded by mesenchyme. Around day E13.5, when galectin-4 or galectin-6 expression starts, there is a wave of morphological change in the intestine, starting in the ileum and extending both proximally and distally as the epithelium differentiates to a single-layered epithelium surrounded by the developing muscles and connective tissue of the gut. The formation of villi and various cell-cell junctions also occur at this time (28). Therefore, major changes of cell-cell interactions are taking place in the intestinal epithelium between days 12.5 and 13.5 of development. Since porcine galectin-4 (15) was originally isolated as an adherens junction-associated protein (17), it is possible that galectin-4 or galectin-6 is involved in these processes. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Gail Martin for the 129/SV genomic DNA, Dr. Alex Bulfone for the plasmid containing the GAPDH cDNA, and Dr. Margaret Huflejt for stimulating discussions.
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FOOTNOTES |
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* This work was supported by grants from the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco-Related Disease Research Program of the University of California (to H. L.), by National Institutes of Health Grant HL38627 (to S. H. B.), and by a grant from the Philippe Foundation (to F. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF026794 and AF026795.
§ Present address: United States Department of Agriculture, Agricultural Research Station, Western Regional Research Center-CIU, Albany, CA 94710.
** To whom correspondence should be addressed. Present address: Inst. of Medical Microbiology, Dept. of Clinical Immunology, Sölvegatan 23, S 22362 Lund, Sweden. Tel.: 46-46-173274; Fax: 46-46-137468; E-mail: hakon.leffler{at}mmb.lu.se.
1 The abbreviations used are: CRD, carbohydrate recognition domain; PCR, polymerase chain reaction; kb, kilobase pair(s); nt, nucleotide(s); RPA, RNase protection assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; dpc, day(s) post coitum; MOPS, 3-(N-morpholino)propanesulfonic acid.
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REFERENCES |
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