From the Departments of Endocrinology and
¶ Immunology and Immunopathology, Kagawa Medical University,
1750-1 Miki, Kagawa 761-0793, Japan and § Galpharma Company,
Limited, Kagawa 761-0301, Japan
Received for publication, September 4, 2002, and in revised form, January 21, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have isolated six novel galectin cDNAs
from a Xenopus laevis kidney cDNA
library. The newly identified X. laevis
galectins (xgalectins) comprise one proto type (xgalectin-Vb), one
chimera type (xgalectin-VIIa), and four tandem repeat types
(xgalectin-IIb, -IIIb, -VIa, and -VIIIa). Thus, together with those
mentioned in our previous work (Shoji, H., Nishi, N., Hirashima,
M., and Nakamura, T. (2002) Glycobiology 12, 163-172), the
12 xgalectins are classified into three types based on their domain
structures, as in mammals. The xgalectins whose counterparts in other
species have not been identified (xgalectin-IVa, -Vb, and -VIa) were
confirmed to possess lactose-binding activity by expression of their
recombinant forms. This shows that they truly function as animal
lectins. The protein purification study revealed that the major
xgalectins in kidney are xgalectin-Ib, -IIa, -IIb, -IIIa, and -VIIa.
The mRNAs of xgalectin-IIb, -IIIb, -Vb, and -VIa were localized to specific adult tissues, whereas those of xgalectin-VIIa and -VIIIa were
broadly distributed. The temporal expression patterns of the mRNAs
of the 12 xgalectins during embryogenesis were analyzed and categorized
into three groups: 1) mRNA observed to exist throughout embryogenesis, i.e. maternal mRNA also exists
(xgalectin-Ia, -IIa, -IIIa, -IIIb, -Va, -VIIa, and -VIIIa); 2) mRNA
observed from the gastrula stage (xgalectin-VIa); and 3) mRNA
observed from the tail bud or the tadpole stage (xgalectin-Ib, -IIb,
-IVa, and -Vb). The mRNA of the most abundant xgalectin in embryos,
xgalectin-VIIa, was localized to the surface layer of embryos, the
epidermis, the cement gland, and various placodes. Xgalectin-VIIa
protein was also observed to exist throughout embryogenesis by Western blot analysis with specific antiserum. These results show that the
expression of each member is spatiotemporally regulated from eggs to
adulthood, suggesting that galectins play multiple roles not only in
adults, but also in development.
Galectins comprise a family of animal lectins that bind to
Studies have been recently accumulating that indicate that the galectin
family play roles in the development of vertebrate embryos. The
expression of some members is temporally and spatially regulated during
embryogenesis (13-16); galectin-1 regulates the axonal growth of
neural cells in mouse and rat (17-19); a proto-type galectin of
chicken affects the differentiation of cultured mesonephroi (20); a
null mutant mouse of galectin-3 has subtle but significant defects in
bones and inflammatory responses (21-23), etc. Other evidence that
galectins might contribute to developmental regulation has been
obtained for Xenopus. Milos and co-workers (24, 25) have
extensively studied the endogenous Our purpose is to clarify the functions of the galectin family in
animal development using the Xenopus system. However, when we started this project, only one proto-type galectin had been identified in Xenopus (26). Therefore, in a previous work
(27), we reported the isolation and characterization of five novel
galectins from Xenopus liver, which seemed to be the most
suitable organ for identifying novel galectins. In this study, we chose
Xenopus kidneys to further identify novel galectins because
they contain large amounts of tandem repeat-type galectins, including
other protein(s) that are absent in the liver (27). We successfully identified six novel galectins, with all three types, i.e.
the proto, chimera, and tandem repeat types, being represented. We have
comprehensively analyzed the expression of all the xgalectins identified so far and have demonstrated that they are specifically regulated throughout, from eggs to adulthood.
Xenopus Tissues and Embryos--
Female frogs were used for the
protein purification study and expression analysis of adult tissues,
except for testis. To obtain embryos, mature eggs were collected after
injection of 500 IU of human chorionic gonadotropin
(GONATROPIN®, Teikokuzouki, Inc., Tokyo, Japan) into females.
In vitro fertilized eggs were dejellied with 3%
L-cysteine hydrochloride monohydrate (Nacalai Tesque, Inc.,
Kyoto, Japan) in Steinberg's solution and then cultured at 20 °C.
Albino embryos were used only for whole-mount in situ hybridization. Embryos were staged according to Nieuwkoop and Faber
(28).
cDNA Cloning of Xgalectins--
A full-length cDNA clone
(1080-bp insert) of xgalectin-IIb was isolated by screening a
Xenopus kidney Construction of Expression Vectors and Preparation of Recombinant
Proteins--
Recombinant xgalectin-IVa, -Vb, and -VIa proteins were
expressed as fusion proteins with glutathione S-transferase
(GST) using the GST fusion system (Amersham Biosciences). Each cDNA
encoding an open reading frame was inserted into pGEX-4T, and the
expression plasmids were introduced into Escherichia coli
BL21. Expression of fusion proteins was carried out as described in our
previous work (11). The extracted recombinant proteins were purified by
affinity chromatography on a lactosyl-agarose column (Seikagaku Co.,
Tokyo). The proteins bound to the lactosyl-agarose were eluted with
buffer containing 20 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1 mM dithiothreitol, and 200 mM lactose and then analyzed by SDS-PAGE. The proteins were
stained with Coomassie Brilliant Blue R-250.
Purification of Xgalectins and Amino Acid Sequence
Analysis--
Purification of xgalectins and amino acid sequence
analysis were performed as described in our previous work (27).
Briefly, Xenopus kidneys obtained from adult females were
homogenized in a 5-fold volume of 10 mM Tris-HCl (pH 7.2),
0.15 M NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 5 mM benzamidine HCl, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM
diisopropylphosphofluoridate, and the homogenate was centrifuged. 0.1 volume of 4 M NaCl (final concentration of ~0.5
M) and 0.01 volume of 1% CHAPS (Sigma) were added to the supernatant recovered, and then the mixture was further centrifuged. The resulting supernatant was directly applied to a lactosyl-agarose column (2 ml) and washed extensively with 10 mM Tris-HCl
(pH 7.2), 0.5 M NaCl, 1 mM EDTA, 0.2 mM dithiothreitol, and 0.01% CHAPS and then with the same
buffer, except the NaCl concentration was 0.15 M. Proteins
adsorbed to the affinity resin were eluted with buffer containing 200 mM lactose. All steps were performed at 4 °C. The
protein mixture eluted from the lactosyl-agarose resin was separated
into two fractions by anion-exchange chromatography on a Resource Q
column (1 ml; Amersham Biosciences). The flow-through (30-36-kDa proteins) and adsorbed (14-kDa proteins) fractions were
separately pooled to examine the amino acid sequence.
The 14- and 30-36-kDa kidney xgalectins were treated with 1% SDS and
20% methanol and then directly dot-blotted onto polyvinylidene difluoride membranes. The blotted proteins were reduced and
S-pyridylethylated on the membranes by the method of
Iwamatsu and Yoshida-Kubomura (30). The denatured proteins
were digested with trypsin (0.6 µg/450 µl) at 25 °C for 18 h, and the fragments liberated from the membranes were purified on a
µBondasphere 5C18 column (Waters Associates) in a reverse-phased high
performance liquid chromatography system (Tohso Co., Tokyo). The
N-terminal 10 amino acids of each peptide were determined with an ABI
492 gas-phase sequencer (Applied Biosystems).
RT-PCR--
Total RNAs were extracted from adult tissues and
embryos at various stages using Isogen (Nippon GENE, Tokyo). RT-PCR was
performed using an RNA PCR kit (Applied Biosystems) and 1 µg of each
total RNA for each reaction. Synthesis of first-strand cDNA was
performed according to the manufacturer's protocols. The primers used
for PCRs were as follows: Ia, 5'-CACGGAGACACAAATAAGATC-3'
(sense) and 5'-GCCCCTGGAGTGCATTATGG-3' (antisense); Ib,
5'-GAAGGCGACACCAACAAAATA-3' (sense) and 5'-TTAATGAAGCGAGATAGCCT-3'
(antisense); IIa and IIb, 5'-TCAACAGCAGCAGCTTTGAG-3' (sense) and
5'-CCCTTGATTTGGGAACATGG-3' (antisense); IIIa and IIIb,
5'-AATACCATCACCGAATCCCAATAC-3' (sense) and
5'-TATGAAACCTTTTTGGGTTTGCAG-3' (antisense); IVa,
5'-CATTGTTTGGGGCATCTAT-3' (sense) and 5'-TCAGAATTGTACGTAGGATA-3'
(antisense); Va, 5'-GATAACCAACCTCAACCTGC-3' (sense) and
5'-CAGCCAATAGCATAACAGCAGA-3' (antisense); Vb, 5'-ATGGACATGCAGCCAGAT-3' (sense) and 5'-TTAATGAACCGAGATGGA-3' (antisense); VIa,
5'-GGCTTCAGGTGACAGGAGAC-3' (sense) and 5'-TCAGAAATGTACAAAGCA-3'
(antisense); VIIa, 5'-GAACCTCCTAAACCATCT-3' (sense) and
5'-TCAGACCATGGTAACATT-3' (antisense); VIIIa, 5'-ATGGCCCAAACAGGACTT-3' (sense) and 5'-CTACCAGATGCGCACATC-3' (antisense); elongation
factor-1 Northern Hybridization--
Poly(A)+ RNAs were
prepared from adult tissues. RNA (1 µg) from each sample was
fractionated and blotted onto a nylon membrane. cDNA fragments with
the following sequences were labeled with digoxigenin-11-dUTP (Roche
Molecular Biochemicals) by PCR and then used as probes: xgalectin-VIa,
nucleotides 693-1427; xgalectin-VIIa, nucleotides 1-1074;
xgalectin-VIIIa, nucleotides 96-1043; and EF-1 Whole-mount in Situ Hybridization--
Whole-mount in
situ hybridization was performed by the method described
previously (33). Digoxigenin-labeled complementary RNA probes were
synthesized with a digoxigenin RNA labeling kit (Roche Molecular
Biochemicals). A plasmid clone with a cDNA fragment (nucleotides
84-864) of xgalectin-VIIa was used as template DNA. A sense probe was
used as a negative control.
Preparation of Anti-xgalectin-VIIa Serum--
Preparation of
recombinant xgalectin-VIIa was performed as described (11). A plasmid
clone carrying the entire open reading frame was constructed on the
pGEX-4T-2 vector (Amersham Biosciences). The protein was expressed as a
fusion protein with GST. The purified recombinant protein was used to
immunize Japanese white rabbits as described (27).
Western Blot Analysis--
For late tadpole stage samples
(stages 57-59), whole protein was extracted from whole embryos or
epidermis removed from embryos by homogenization in a 5-fold volume of
80% methanol and 20% Tris-buffered saline (20 mM Tris-HCl
(pH 7.5) and 150 mM NaCl). The extracts were centrifuged,
and the precipitates were dissolved in the general sample buffer for
SDS-PAGE. Proteins derived from 1 mg of wet tissues were applied to
each lane. At first, the same protocol was used for younger embryos;
but in this case, the huge amount of vitellogenin disturbed the
SDS-PAGE. To overcome this, unfertilized eggs and embryos
(gastrula/early tadpoles) were homogenized in buffer comprising 20 mM Tris-HCl (pH 7.5), 100 mM lactose, 2 mM EDTA, and 5 mM benzamidine HCl and then
centrifuged. Under these conditions, most of the vitellogenin
precipitate and xgalectin-VIIa are recovered in the supernatant.
Proteins in supernatants were precipitated by adding a 9-fold volume of
methanol and then dissolved in the SDS-PAGE sample buffer. The protein
amount for unfertilized eggs and embryos (gastrula/early tadpoles)
subjected to SDS-PAGE was standardized as to the number of embryos, and
protein equivalent to 0.5 embryos was applied to each lane.
Western blotting was performed in the same way as described in our
previous work (27), except that anti-xgalectin-VIIa serum was used at a
dilution of 1:500.
cDNA Cloning of Six Novel Galectins from a X. laevis kidney
cDNA library--
We have isolated and named six novel xgalectin
cDNAs. As described in our previous report (27), a Roman
numeral and letter were assigned to each Xenopus galectin
not according to the number of mammalian galectins, but according to
the order of their discovery, because complete correspondence of the
members of the Xenopus and mammalian galectin families was impossible.
A novel proto type (xgalectin-Vb) was structurally most similar to
Xenopus skin 16-kDa galectin (78% amino acid identity) (Fig. 1A), which was
previously described by Marschal et al. (26). As these two
xgalectins are structurally similar and also exhibit similar expression
patterns, being abundant in adult skin (to be described below), we
propose the designations xgalectin-Va for the skin 16-kDa galectin and
xgalectin-Vb for the newly identified galectin. Mammalian counterparts
of xgalectin-Va and -Vb have not been identified.
One chimera-type galectin, xgalectin-VIIa, was cloned, and its complete
sequence was determined. In the course of this study, we found nine
Xenopus EST clones similar to galectin-3, a mammalian chimera type, and the full coding sequence was reconstructed by combining them. The amino acid sequence deduced from the consensus nucleotide sequence is identical to that of xgalectin-VIIa (Fig. 1B). Thus, the xgalectin-VIIa and EST clones must have
originated from the same gene. As the amino acid sequence of
xgalectin-VIIa is highly similar to that of mammalian galectin-3 (Table
I), xgalectin-VIIa seems to be a
Xenopus homolog of mammalian galectin-3.
Four tandem repeat-type galectins, xgalectin-IIb, -IIIb, -VIa, and
-VIIIa, were newly identified. Two of them, xgalectin-IIb and -IIIb,
are structurally very similar to xgalectin-IIa and -IIIa, respectively
(Fig. 2, A and B),
which we described in a previous work (27). The amino acid sequences of
the CRDs in xgalectin-IIa and -IIb or in xgalectin-IIIa and -IIIb are
88-90% identical. The major structural difference between each pair
of isoforms is the length of the link peptides. The link peptide of
xgalectin-IIa is longer by 12 amino acids compared with that of
xgalectin-IIb, and the link peptide of xgalectin-IIIa is 35 amino acids
longer than that of xgalectin-IIIb (Fig. 2, A and B). Based on the sequence similarity, both xgalectin-IIa and
-IIb seem to be Xenopus homologs of mammalian galectin-4,
and both xgalectin-IIIa and -IIIb seem to be Xenopus
homologs of mammalian galectin-9 (Table I).
There are three Xenopus EST clones related to xgalectin-VIa,
and the amino acid sequence deduced from the consensus nucleotide sequence completely matches that of part of the N-terminal CRD of
xgalectin-VIa (Fig. 2C). Thus, the xgalectin-VIa and EST
clones must have originated from the same gene. A mammalian counterpart of xgalectin-VIa has not been identified. One EST clone related to
xgalectin-VIIIa was found. Although the partial amino acid sequence
encoded by the EST sequence is only 88% identical to that of
xgalectin-VIIIa, the entire nucleotide sequence of the EST clone
matches the cDNA sequence of xgalectin-VIIIa (96% identity), even
though most of the compared region corresponds to the 3'-noncoding sequence (Fig. 2D). Thus, the disagreement between the
sequences of xgalectin-VIIIa and the EST clone may be due to allelic
differences and/or sequence errors, and the xgalectin-VIIIa and EST
clones may have originated from the same gene. Xgalectin-VIIIa seems to
be a Xenopus homolog of mammalian galectin-8 because its
amino acid sequence is highly similar to that of mammalian galectin-8 (53% identical to human galectin-8) (Table I).
Lactose-binding Activity of Xgalectins Whose Counterparts in Other
Species Have Not Been Identified--
Of the 12 xgalectins we have
identified so far, there are four xgalectins whose counterparts in
other species have not been identified, xgalectin-IVa, -Va, -Vb, and
-VIa (Table I). As described above, xgalectin-Va has been well
characterized as skin 16-kDa galectin by Marschal et
al. (26) and has been shown to bind lactose with high affinity. To
clarify whether xgalectin-IVa, -Vb, and -VIa truly act as lectins, we
tested the lactose-binding activity of their recombinant forms.
Recombinant xgalectin-IVa, -Vb, and -VIa proteins were expressed as GST
fusion proteins and purified by affinity chromatography on a
lactosyl-agarose column. As shown in Fig.
3, recombinant xgalectin-IVa, -Vb, and
-VIa proteins were successfully recovered from the adsorbed fractions
of each E. coli extract on the lactosyl-agarose column.
Protein Purification Study--
To identify the major xgalectins
in kidney, we determined the amino acid sequences of tryptic peptides
of xgalectins purified from kidney. Xgalectins were purified by an
affinity chromatography method using lactosyl-agarose resin, and the
eluted proteins were further fractionated into 14-kDa protein-rich and
30-36-kDa protein-rich fractions by anion-exchange chromatography.
From the 14-kDa protein-rich fraction, only the sequence of
xgalectin-Ib or the common sequence of xgalectin-Ia and -Ib was
obtained, and no particular peptide for xgalectin-Ia was recovered
(Table II). From the 30-36-kDa protein-rich fraction, three sequences of xgalectin-IIa, four sequences
of xgalectin-IIb, two common sequences of xgalectin-IIa and -IIb, three
sequences of xgalectin-IIIa, and six sequences of xgalectin-VIIa were
identified (Table II). There was no particular peptide for
xgalectin-IIIb. Thus, as summarized in Table
III, the dominant xgalectins in kidney
are xgalectin-Ib, -IIa, -IIb, -IIIa, and -VIIa, and they are quite
different from those in liver, which we reported in a previous study
(27).
Distribution of mRNAs of Novel Xgalectins in Adult
Tissues--
Fig. 4 shows the expression
profiles of the newly identified xgalectin mRNAs in adult tissues.
Expression of the mRNAs of xgalectin-IIb, -IIIb, and -Vb was
analyzed by RT-PCR to distinctly detect the structurally similar
isoforms (Fig. 4A). To distinguish the mRNAs of
xgalectin-IIa and -IIb, we used a primer pair that recognizes the
sequences of both xgalectin-IIa and -IIb and that amplifies the coding
region, including link peptides. In this way, cDNAs derived from
xgalectin-IIa and -IIb could be detected as different sized DNA bands
because of the different lengths of the link peptides. A control
experiment involving this primer set is presented in Fig.
5, which shows that, with the same amount of template DNA, the two bands exhibited the same intensity. The distribution of xgalectin-IIb mRNA was tissue-specific, and the major tissues producing xgalectin-IIb mRNA were stomach, kidney, and testis, whereas xgalectin-IIa mRNA was abundant in liver, stomach, intestine, and kidney (Fig. 4A) (27). The mRNAs
of xgalectin-IIIa and -IIIb were distinctly detected by the same method
used for xgalectin-IIa and -IIb. The mRNA of xgalectin-IIIb was
detected only in intestine, kidney, and testis, whereas that of
xgalectin-IIIa was broadly distributed. The mRNAs of xgalectin-Va and -Vb were distinctly detected using a specific primer pair for each.
The control experiments in Fig. 5 show the specific amplification by
the primers used. The mRNAs of both xgalectin-Va and -Vb were
extremely abundant in skin, and their expression in other tissues was
quite low. The quantitative differences in the mRNAs of both
xgalectin-Va and -Vb between skin and other tissues were more
conspicuously observed by Northern blot analysis (data not shown).
Expression of the mRNAs of xgalectin-VIa, -VIIa, and -VIIIa was
analyzed by Northern hybridization (Fig. 4B). The mRNA
of xgalectin-VIa was detected only in the tissues directly in contact with the outside of the body, i.e. lung and skin. The
mRNA of xgalectin-VIIa was broadly distributed and was particularly
abundant in ovary, consistent with the fact that a large amount
of xgalectin-VIIa mRNA and protein exists in eggs, as described
below. The mRNA of xgalectin-VIIIa was also distributed broadly,
but uniformly, in tissues other than heart, stomach, and muscle.
Temporal Expression Patterns of mRNAs from the Xenopus Galectin
Family during Embryogenesis--
RT-PCR was performed to analyze the
temporal expression patterns of mRNAs from all members of the
Xenopus galectin family identified so far during
embryogenesis (Fig. 5). The mRNAs of sets of xgalectins (Ia/Ib,
IIa/IIb, IIIa/IIIb, and Va/Vb) were distinctly detected as shown in
control experiments. The expression patterns of the 12 xgalectins can
be categorized into three groups: 1) mRNA observed to exist
throughout embryogenesis, i.e. maternal mRNA also exists
(xgalectin-Ia, -IIa, -IIIa, -IIIb, -Va, -VIIa, and -VIIIa); 2) mRNA
observed from the gastrula stage (xgalectin-VIa); and 3) mRNA observed
from the tail bud or tadpole stage (xgalectin-Ib, -IIb, -IVa, and -Vb).
Among these members, xgalectin-VIIa seems to be the most abundant
xgalectin in embryos because it was sufficiently detected even with 25 PCR cycles and because it was remarkably detected by Northern
analysis, which did not allow comparison with other members (data not shown).
mRNA of Xgalectin-VIIa Is Localized to the Surface of
Embryos--
To clarify the distribution of the mRNA of
xgalectin-VIIa during embryogenesis, whole-mount in situ
hybridization was performed in the neurula to early tadpole stages.
Xgalectin-VIIa mRNA was localized to various placodes, the cement
gland, and the epidermis throughout the developmental stages (Fig.
6). At the early neurula stage, a
hybridization signal was observed in the neural fold region (which
would give rise to placodes such as the lens and otic placodes), in the
cement gland anlage, and in the epidermal ectoderm surrounding the
embryo, but it was absent in the neural plate. The negative region
disappeared with development because, during neurulation, the neural
ectoderm was missing from the surface layer of the embryo. At the tail
bud stage, the entire surface of embryos was positively stained, except
for the proctodeum. This expression pattern persisted at least to the
early tadpole stage.
Western Blot Analysis of Expression of Xgalectin-VIIa in
Embryos--
To confirm the protein expression of xgalectin-VIIa,
Western blot analysis was performed using a specific rabbit antiserum raised against recombinant xgalectin-VIIa. Xgalectin-VIIa protein existed from unfertilized eggs to the early tadpole stages at almost
comparable levels (Fig. 7). Furthermore,
we prepared an extract of epidermis removed from late tadpoles and
compared it with a whole-embryo extract. As a result, xgalectin-VIIa
was found to be significantly abundant in the epidermal extract.
Together with those mentioned in our previous work (27), 12 Xenopus laevis galectins have been identified.
Also, with the identification of chimera-type xgalectin-VIIa in this
study, three structurally different types have been found. As
summarized in Table IV, a galectin family
comparable to that in mammals exists in Xenopus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactoside-containing carbohydrate moieties of glycoconjugates (1,
2). Fourteen galectins have been isolated from mammals and are
classified into the proto, chimera, and tandem repeat types based on
their structures (3-8). Proto-type galectins contain one carbohydrate
recognition domain (CRD),1 a
structurally conserved domain that specifically recognizes a
-galactoside-containing carbohydrate; and chimera-type
galectins consist of one CRD and an N-terminal elongating protein
domain. Mammalian galectin-1 (proto type) and -3 (chimera type) are the most extensively studied galectins (9). They have been proposed to play
roles in tissue organization, development, immunity, and cancer growth
and metastasis by regulating such processes as cell adhesion and
apoptosis. Tandem repeat-type galectins contain two CRDs covalently
linked through a unique link peptide. Tandem repeat-type galectins are
less well understood, but they have been shown to be expressed in a
tissue-specific manner in adult animals as well as in mouse embryos.
Also, our recent studies revealed that they play roles in immunity,
e.g. the chemoattractant activity of galectin-9 for
eosinophils (10, 11) and activation of
neutrophils by galectin-8.2 Thus, the galectin
family seems to be significantly associated with development, immunity,
and tumorigenesis, but the details of their functional mechanisms
remain unclear.
-galactoside-binding lectins in
Xenopus embryos and have proposed that these lectins are
important for the development of neural crest cells, craniofacial tissue, and heart, etc. Although the identity of these
-galactoside-binding lectins has not yet been determined, they are
very likely to be members of the galectin family.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cDNA library (Stratagene) using a
cDNA fragment of xgalectin-IIa (nucleotides 1-786,
GenBankTM/EBI accession number AB060970) as a probe.
Partial cDNA clones of xgalectin-IIIb were isolated by screening
the kidney library using a cDNA fragment of xgalectin-IIIa
(nucleotides 26-452; accession number AB060971) as a probe. Because
all the clones lacked the 5'-region (even the clone with the longest
insert lacked nucleotides 1-77), 5'-RACE-PCR was performed with a
Marathon cDNA amplification kit (Clontech) and
kidney mRNA. A specific primer with the following sequence was
used: 5'-ATTCGGTGATGGTATTCCAAGTAA-3'. As a result, a cDNA fragment
of 422 bp covering the entire 5'-region of xgalectin-IIIb was obtained.
The full-length sequence (2000 bp) of xgalectin-IIIb was
reconstructed by combining the sequence of the 5'-RACE-PCR product and
that of the clone with the longest insert. A partial cDNA fragment
of xgalectin-Vb was isolated by degenerate oligonucleotide-based PCR
cloning as described (27). A sense primer (Gd4,
5'-CACTTYAAYCCNCGNTTY-3') (where Y = T or C and N = G or A, or T
or C) corresponding to highly conserved amino acids in members of the
galectin family, HNFPRF, and an antisense primer (Gd7,
5'-TTCTTTNCCRTCNGGNA-3') (where R = G or A) corresponding to the amino
acid sequence of Xenopus skin 16-kDa galectin/xgalectin-Va
(26), LPDGKE, were used for amplification from kidney mRNA. The
resultant cDNA fragment (183 bp) was used as a probe to screen the
kidney library, and a full-length clone (583 bp) was isolated. A
cDNA clone of xgalectin-VIa was isolated in the same way. First,
sense primer Gd4 and another antisense primer (Gd9,
5'-CATATGYTGNCCRTTNAC-3') corresponding to the amino acid
sequence of mouse galectin-9 (29), VNGQHM, were used, and a partial
cDNA fragment of xgalectin-VIa (177 bp) was isolated from mRNA
of whole embryos (tail bud stage). The resultant cDNA fragment was
used as a probe to screen the kidney library, and a full-length clone
(1427 bp) was isolated. Although the mRNA of xgalectin-VIa was
under the detectable level in kidney by Northern analysis, we observed
weak expression by reverse transcription (RT)-PCR analysis (data not
shown). To isolate xgalectin-VIIa and -VIIIa, we utilized expressed
sequence tag (EST) sequence data from the GenBankTM/EBI Data Bank
(accession numbers AW765695 for xgalectin-VIIa and AW782523 for
xgalectin-VIIIa). We generated specific primers to amplify partial
cDNAs (nucleotides 28-578 of AW765695 and 1-486 of AW782523) by
RT-PCR according to the sequence data. The cDNA fragments obtained
from kidney total RNA were used as probes to screen the kidney cDNA
library. Full-length clones of xgalectin-VIIa (1074 bp) and
xgalectin-VIIIa (1456 bp) were isolated. The details of the library
screening and DNA sequencing methods are given in our previous report
(27).
(EF-1
), 5'-GTCGCCCAACTGATAAGCCTCTCC-3' (sense) and
5'-TGCCTTCTTTTCCACTGCCTTGAT-3' (antisense); ornithine decarboxylase,
5'-GTCAATGATGGAGTGTATGGATC-3' (sense) and
5'-TCCATTCCGCTCTCCTGAGCAC-3' (antisense). The primers for
ornithine decarboxylase were prepared according to Yamada et
al. (31). The reaction mixtures were preincubated for 2 min at
94 °C, followed by thermal cycles of the following: 94 °C for 30 s, 60 °C for 15 s, and 72 °C for 1 min. For analysis
of adult tissues, reactions were repeated for 30 cycles; and for
embryos, the cycle numbers were as indicated in legend to Fig. 5.
Ornithine decarboxylase or EF-1
(GenBankTM/EBI accession
number M25504) was amplified as an internal control. All PCR products
were analyzed by 2% agarose gel electrophoresis and visualized by
ethidium bromide staining. Control experiments to show the specific
amplification of xgalectin-Ia, Ib, Va, and -Vb were performed using 1 ng of each plasmid clone as template DNA in each reaction. A control
experiment to show the simultaneous and equal amplification of
xgalectin-IIa and -IIb was performed by adding 0.1 pg of each plasmid
clone used as template DNA to the reaction mixture. A control
experiment for xgalectin-IIIa and -IIIb was performed in the same way.
Specific amplification with each primer pair was confirmed by a
reaction without reverse transcriptase and also by sequence analysis of
the PCR products (data not shown).
, nucleotides
758-1378 (GenBankTM/EBI accession number M25504).
Hybridization, washing, and detection were performed as described
previously (32).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
Schematic illustrations of the protein
structures of novel proto- and chimera-type xgalectins. A,
protein structure of xgalectin-Vb compared with that of xgalectin-Va;
B, protein structure of xgalectin-VIIa compared with that
of the protein predicted from the EST sequence. Nine EST sequences
related to xgalectin-VIIa were combined, and the amino acid sequence
was predicted from its consensus sequence. Amino acid numbers are
indicated. The identity of the entire amino acid sequence is indicated
for each comparison. The structure of skin 16-kDa galectin
(skin16K)/xgalectin-Va was described in Ref. 26. The
GenBankTM/EBI accession numbers for the cDNA sequences
of others are as follows: xgalectin-Vb, AB080018; xgalectin-VIIa,
AB080020; and ESTs, AW765695, AW766406, BF428260, BG021071,
BG163149, BG413616, BG552788, BG885816, and BI446289. N-ter,
N-terminal.
Relationship of Xenopus and mammalian galectins
View larger version (27K):
[in a new window]
Fig. 2.
Schematic illustrations of the protein
structures of novel tandem repeat-type xgalectins. A,
protein structure of xgalectin-IIb compared with that of xgalectin-IIa.
The amino acid sequences of link peptides are compared below.
B, protein structure of xgalectin-IIIb compared with that of
xgalectin-IIIa. The amino acid sequences of link peptides are compared
below. C, protein structure of xgalectin-VIa compared with
that of the protein predicted from the EST sequence. Three EST
sequences related to xgalectin-VIa were combined, and the amino acid
sequence was predicted from its consensus sequence. D,
protein structure and cDNA sequence of xgalectin-VIIIa compared with
those of the EST sequence. Amino acid numbers are indicated. The
identity of the amino acid sequence of each domain and the entire
sequence is indicated for each comparison. cDNA sequence identity is
also indicated in D. The structures of xgalectin-IIa and
-IIIa were described in Ref. 27. The GenBankTM/EBI
accession numbers for the cDNA sequences of the others are as
follows: xgalectin-IIb, AB080016; xgalectin-IIIb, AB080017;
xgalectin-VIa, AB080019; xgalectin-VIIIa, AB080021; EST clones
related to xgalectin-VIa, BG813931, BG813457, and BG513146; and
EST clone related to xgalectin-VIIIa, AW782523. N-CRD and
C-CRD, N- and C-terminal CRDs, respectively;
link, link peptide; ORF, open reading
frame.
View larger version (53K):
[in a new window]
Fig. 3.
Lactose-binding activities of recombinant
xgalectin-IVa, -Vb, and -VIa. Recombinant xgalectin-IVa -Vb, and
-VIa proteins were expressed as GST fusions in E. coli
carrying each expression plasmid and were collected by affinity
chromatography on lactosyl-agarose columns. The adsorbed fractions on
the lactosyl-agarose columns were analyzed by SDS-PAGE (12% acrylamide
gel). The protein bands were visualized by Coomassie Brilliant Blue
R-250 staining. Two minor bands observed in the GST/xgalectin
(xgal)-VIa column (~42.7 kDa) are degraded products of the
fusion protein. The calculated molecular masses of the fusion proteins
are as follows: GST/xgalectin-IVa, 62.8 kDa; GST/xgalectin-Vb, 41.4 kDa; and GST/xgalectin-VIa, 61.4 kDa.
Tryptic peptide sequences of xgalectins from kidney
Dominant xgalectins in liver and kidney
View larger version (64K):
[in a new window]
Fig. 4.
Distribution of novel xgalectin mRNAs in
adult tissues. A, to detect isoforms distinctly, the
expression of the mRNAs of xgalectin-IIa, -IIb, -IIIa, -IIIb, -Va,
and -Vb was analyzed by RT-PCR. The mRNAs of xgalectin-IIa and -IIb
were successfully detected as distinct bands after simultaneous
amplification of a part of each cDNA including the region coding
the link peptide; and the same was true for xgalectin-IIIa and -IIIb.
The mRNAs of xgalectin-Va and -Vb were separately amplified using a
specific primer set for each. The sizes of the cDNA bands are as
follows: xgalectin-IIa, 230 bp; xgalectin-IIb, 194 bp; xgalectin-IIIa,
381 bp; xgalectin-IIIb, 276 bp; xgalectin-Va, 536 bp; and xgalectin-Vb,
408 bp. The mRNA of EF-1 was amplified as an internal control.
B, the expression of mRNAs of xgalectin-VIa, VIIa, and
-VIIIa was analyzed by Northern hybridization. The size of each
mRNA is indicated on the right. A probe of EF-1
was used as an
internal control.
View larger version (59K):
[in a new window]
Fig. 5.
RT-PCR analysis of the temporal expression
patterns of Xenopus galectin family mRNAs during
embryogenesis. All members of the Xenopus galectin
family currently identified were analyzed. The mRNAs of
xgalectin-Ia and -Ib were separately amplified using a specific primer
set for each. As shown in the control lanes for xgalectin-Ia and -Ib,
specific amplification by each primer set was confirmed by reaction
with a plasmid clone of each cDNA used as template DNA. The same
was true for xgalectin-Va and -Vb. The mRNAs of xgalectin-IIa and
-IIb were successfully detected as distinct bands after simultaneous
amplification of a part of each cDNA including the region coding
the link peptide. The control lanes show that, upon using equal amounts
of plasmid clones of xgalectin-IIa and -IIb as template DNAs in a
reaction, equally stained PCR products were observed. The same was true
for xgalectin-IIIa and -IIIb. The stages are indicated at the top. The
sizes of the cDNA bands are as follows: xgalectin-Ia, 353 bp;
xgalectin-Ib, 404 bp; xgalectin-IIa, 230 bp; xgalectin-IIb, 194;
xgalectin-IIIa, 381 bp; xgalectin-IIIb, 276 bp; xgalectin-IVa,
452 bp; xgalectin-Va, 536 bp; xgalectin-Vb, 408 bp; xgalectin-VIa, 557 bp; xgalectin-VIIa, 444 bp; and xgalectin-VIIIa, 948 bp. The numbers of
PCR cycles are indicated on the right. Ornithine decarboxylase
(ODC) was amplified as an internal control.
View larger version (74K):
[in a new window]
Fig. 6.
Distribution of xgalectin-VIIa in embryos as
analyzed by whole-mount in situ hybridization.
A, early neurula stage (stage 15). The entire
epidermal ectoderm and neural fold (nf) are positive,
whereas the neural plate (np) is negative. B,
negative control for A, hybridized with a sense probe.
C, late neurula stage (stage 18). The negative region became
narrower with the progression of neurulation. D, neural tube
stage (stage 20). The negative region disappeared with closure of the
neural tube. E, tail bud stage (stage 26). The entire
surface of the embryo was positively stained, except for the proctodeum
(arrowhead). F, late tail bud stage (stage
33/34). The lower embryo is a negative control hybridized with a sense
probe. A-D are anterior views, and E and
F are lateral views. cg, cement gland;
cga, cement gland anlage; ev, eye vesicle.
View larger version (81K):
[in a new window]
Fig. 7.
Western blot analysis of protein expression
of xgalectin-VIIa. The stages are indicated at the top. Specific
antiserum raised against recombinant xgalectin-VIIa was used. For each
lane from unfertilized eggs to the early tadpole stage, protein
equivalent to 0.5 whole embryos was applied. For each lane of the late
tadpole stage, protein equivalent to 1 mg of wet tissue (whole embryos
or epidermis) was applied. Recombinant xgalectin-VIIa
(rVIIa; 10 ng) was applied to the last lane as an internal
control, and the xgalectin-VIIa bands were stained with an ECL system
(Amersham Biosciences). Xgalectin-VIIa protein constantly existed
throughout embryogenesis. Localization in the epidermis was
successfully detected in the late tadpole stage.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Classification of mammalian and Xenopus galectins according to the
domain structure
The three newly identified xgalectins, xgalectin-IIb, -IIIb, and -Vb, are structurally very similar to the known xgalectins, xgalectin-IIa, -IIIa, and -Va, respectively. However, they are not alleles from different individuals, but rather distinct genes. For the proto-type xgalectins, xgalectin-Va and -Vb, the similarity of the cDNA sequence in the 3'-noncoding region is quite low; and furthermore, the expression patterns of their mRNAs differ in early embryogenesis. For two sets of tandem repeat-type xgalectins, xgalectin-IIa and -IIb and xgalectin-IIIa and -IIIb, we obtained two lines of strong evidence. First, there is a significant difference in the lengths of the link peptides. Second, as shown in Figs. 4 and 5, their expression is differently regulated in both adult tissues and whole embryos.
There are several EST clones related to xgalectins identified in either the previous (27) or present study. Analysis of their cDNA sequences revealed that they originated from the xgalectin -VIa, -VIIa, or -VIIIa gene, and there are no apparent sequences of isoforms.
The relationship of Xenopus and mammalian galectins is summarized in Table I. Our proposal of a homologous relationship is based not only on structural similarities, but also on their expression patterns. Xgalectin-IIa and -IIb and mammalian galectin-4 are apparently abundant in the digestive tract, although the xgalectins have some other expression sites (15). The expression patterns of xgalectin-IIIa and -IIIb share common characteristics with that of the mammalian counterpart, galectin-9. Three isoforms of mammalian galectin-9, galectin-9S, -9M, and -9L (29, 34), are produced through alternative splicing of exons coding the link peptide, resulting in the production of proteins with link peptides of different lengths. The major isoform of galectin-9, galectin-9M, is broadly distributed in adult tissues; but another isoform, galectin-9L, has been shown to be localized in small intestine in mouse (29). Almost the same is true for xgalectin-IIIa and -IIIb, viz. xgalectin-IIIa is broadly distributed in adult tissues, whereas the major expression site of xgalectin-IIIb is restricted to intestine, although minor expression is observed in kidney and testis.
Other xgalectins for which mammalian counterparts have been identified (xgalectin-Ia, -Ib, -VIIa, and -VIIIa) are broadly distributed in adult tissues, and the same is true for their mammalian counterparts (galectin-1, -3, and -8) (9, 35, 36). On the other hand, mammalian counterparts of xgalectin-IVa, -Va, -Vb, and -VIa have not been identified. Xgalectin-Va and/or -Vb is the most abundant protein in skin (5% of the total protein) (26). In mammals, a proto-type galectin, galectin-7, is abundantly expressed in skin keratinocytes (12, 37, 38), but its amino acid sequence is dissimilar from that of either xgalectin-Va or -Vb. Thus, galectin(s) distributed in skin may have evolved independently in various animal species. Xgalectin-VIa exhibits relatively high structural similarity to mammalian galectin-4 (49% amino acid sequence identity to human galectin-4). However, their expression patterns are completely different. The mRNA of xgalectin-VIa is localized to skin and lung, whereas that of mammalian galectin-4 is localized to the digestive tract (15). Therefore, xgalectin-IIa and -IIb are more suitable as Xenopus homologs of galectin-4. No mammalian galectin that is localized to skin and lung has been identified.
Because homologous proteins for xgalectin-IVa, -Va, -Vb, and -VIa have
not been identified in any other species, not only in mammals, they
must be demonstrated to bind -galactoside to be categorized into the
galectin family. Xgalectin-Va has been shown to bind lactose by
Marschal et al. (26), and we have demonstrated the
lactose-binding activities of xgalectin-IVa, -Vb, and -VIa in this
study. Furthermore, we performed a preliminary analysis using antiserum
raised against the recombinant C-terminal CRD of xgalectin-VIa. As a
result, we detected native xgalectin-VIa protein in the lactose-binding
fraction of the adult lung extract, where the xgalectin-VIa mRNA
exists abundantly (data not shown). Therefore, all four galectins
identified only in Xenopus have been shown to truly act as
lectin proteins.
The protein purification study revealed that the dominant xgalectins in kidney are xgalectin-Ib, -IIa, -IIb, -IIIa, and -VIIa, which are quite different from those in liver, which we reported in a previous study (27). The point we should emphasize here is that the dominant proto-type galectin is different in liver and kidney, i.e. xgalectin-Ia in liver, but xgalectin-Ib in kidney. This shows that even though xgalectin-Ia and -Ib are structurally very similar (91% amino acid sequence identity and 86% identity in cDNA sequence, including noncoding regions), their expression is differentially regulated, suggesting that they have distinct roles in each organ.
The distribution of the newly identified xgalectin mRNAs in adult tissues was determined by RT-PCR and Northern hybridization. Here we found other paradigms of isoform pairs whose structures are homologous, but whose expression is differentially regulated, i.e. xgalectin-IIa and -IIb and xgalectin-IIIa and -IIIb. Xgalectin-IIb and -IIIb might have specific functions in the digestive tract; and to perform these functions, shorter link peptides than those in xgalectin-IIa and -IIIa, respectively, might be required. The mRNA of xgalectin-VIa was localized to skin and lung. Xenopus skin and lung are rich in mucosa and are in direct contact with the outside of the body. Another tissue with the same characteristics, i.e. mucosa-rich and in contact with the outside, is the digestive tract. From this point of view, many xgalectins are abundant and/or localized to tissues that are mucosa-rich and in contact with the outside. This suggests that xgalectins play roles in immunity, especially in innate immunity concerned with the function of the mucosa.
Comprehensive analysis was performed on the temporal expression patterns of the Xenopus galectin family during embryogenesis. As a result, we can categorize the 12 members into three groups: 1) mRNA observed to exist throughout embryogenesis, i.e. maternal mRNA also exists (xgalectin-Ia, -IIa, -IIIa, -IIIb, -Va, -VIIa, and -VIIIa); 2) mRNA observed from the gastrula stage (xgalectin-VIa); and 3) mRNA observed from the tail bud or tadpole stage (xgalectin-Ib, -IIb, -IVa, and -Vb). This shows that most xgalectins exist in embryos and that their expression is specifically regulated not only in adult tissues, but also in embryos. Xgalectins whose mRNAs exist as maternal mRNAs may be required for the maintenance of unfertilized eggs and/or in early development until zygotic gene expression is initiated at the mid-blastula stage (mid-blastula transition). Furthermore, expression of these xgalectins persists even after the mid-blastula transition. Therefore, they seem to play roles throughout development. The expression of xgalectin-VIa, whose mRNA begins to be detectable at the gastrula stage, might be initiated at the mid-blastula transition, although more precise analysis is required. According to our preliminary results, xgalectin-VIa is highly expressed in the cement gland throughout its formation (data not shown). These facts suggest that xgalectin-VIa plays a role in early development, especially in the transient formation of the cement gland. Xgalectins whose expression starts at the tadpole stage may play roles in the organization of various tissues because the formation of various organs is actively underway at this stage.
Among these xgalectins, we chose a chimera type, xgalectin-VIIa, as the first to be analyzed precisely in early embryos because it was judged to be the most abundant in embryos. Furthermore, it is valuable to examine its expression pattern to estimate the common role(s) of chimera-type galectins in vertebrate development because mammalian galectin-3 is the only chimera type that has been studied extensively in mammalian development and immune systems. Whole-mount in situ hybridization was performed and revealed that the mRNA of xgalectin-VIIa is localized to surface tissues of embryos, the epidermis, placodes, and the cement gland. Furthermore, Western blot analysis revealed that xgalectin-VIIa protein exists throughout embryogenesis from unfertilized eggs and is still abundant in the epidermis of late stage tadpoles. These results suggest that xgalectin-VIIa plays a role in the organization of tissues expressing xgalectin-VIIa and/or in embryonic self-defense. Murine galectin-3, a homolog of xgalectin-VIIa, has also been shown to be expressed in the epidermis of embryos, but at rather later stages, from 13.5 days postcoitus. In Xenopus, xgalectin-VIIa is expressed in epidermal cells at least at the neurula stage, suggesting that Xenopus requires galectin expression for organization of the embryonic epidermis at an earlier stage than mammals. Another suggestion is that galectins play roles in embryonic immunity and that Xenopus must establish an immune system at an earlier stage than mammals because Xenopus is oviparous, whereas mammals are viviparous. The expression of murine galectin-3 was also observed in the notochord and cartilage primordia. However, we could not find significant hybridization signals in tissues other than the epidermis, placodes, and the cement gland, even upon sectional analysis of whole-mount in situ hybridization specimens. In Xenopus, the roles of galectins in the notochord and cartilage primordia may be compensated for by some other members, including unidentified ones.
We are currently performing comprehensive analysis of the expression of
other xgalectin mRNAs in early embryos by in situ hybridization and screening of the target molecule(s) of xgalectins. Identification of the targets of xgalectins in each tissue is essential
for elucidating their functions. We recently isolated candidate
40-46-kDa protein(s) for xgalectin-VIIa from
embryos.3 Information on the
structure and gene expression of this candidate molecule will be useful
in determining the role of xgalectin-VIIa in Xenopus embryogenesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Asashima, C. Yokota, and S. Takahashi (Graduate School of Arts and Sciences, University of Tokyo) for technical advice on whole-mount in situ hybridization and helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant-in-aid for Scientific Research in Priority Areas 13226084 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.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.
To whom correspondence should be addressed: Dept. of
Endocrinology, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Tel.: 81-87-891-2106; Fax: 81-87-891-2108; E-mail: tnaka@kms.ac.jp.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M209008200
2 N. Nishi, H. Shoji, M. Seki, A. Itoh, H. Miyanaka, K. Yuube, M. Hirashima, and T. Nakamura, manuscript in preparation.
3 H. Shoji, N. Nishi, and T. Nakamura, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CRD, carbohydrate
recognition domain;
xgalectin, X. laevis galectin;
RACE, rapid amplification of cDNA ends;
RT, reverse transcription;
EST, expressed sequence tag;
GST, glutathione S-transferase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-2-propanesulfonic acid;
EF-1, elongation factor-1
.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Barondes, S. H.,
Cooper, D. N.,
Gitt, M. A.,
and Leffler, H.
(1994)
J. Biol. Chem.
269,
20807-20810 |
2. | Barondes, S. H., Castronovo, V., Cooper, D. N., Cummings, R. D., Drickamer, K., Feizi, T., Gitt, M. A., Hirabayashi, J., Hughes, C., Kasai, K., et al.. (1994) Cell 76, 597-598[Medline] [Order article via Infotrieve] |
3. | Kasai, K., and Hirabayashi, J. (1996) J. Biochem. (Tokyo) 119, 1-8[Abstract] |
4. |
Cooper, D. N.,
and Barondes, S. H.
(1999)
Glycobiology
9,
979-984 |
5. |
Dunphy, J. L.,
Balic, A.,
Barcham, G. J.,
Horvath, A. J.,
Nash, A. D.,
and Meeusen, E. N.
(2000)
J. Biol. Chem.
275,
32106-32113 |
6. |
Yang, R. Y.,
Hsu, D. K., Yu, L.,
Ni, J.,
and Liu, F. T.
(2001)
J. Biol. Chem.
276,
20252-20260 |
7. |
Visegrady, B.,
Than, N. G.,
Kilar, F.,
Sumegi, B.,
Than, G. N.,
and Bohn, H.
(2001)
Protein Eng.
14,
875-880 |
8. |
Dunphy, J. L.,
Barcham, G. J.,
Bischof, R. J.,
Young, A. R.,
Nash, A.,
and Meeusen, E. N.
(2002)
J. Biol. Chem.
277,
14916-14924 |
9. | Perillo, N. L., Marcus, M. E., and Baum, L. G. (1998) J. Mol. Med. 76, 402-412[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Matsumoto, R.,
Matsumoto, H.,
Seki, M.,
Hata, M.,
Asano, Y.,
Kanegasaki, S.,
Stevens, R. L.,
and Hirashima, M.
(1998)
J. Biol. Chem.
273,
16976-16984 |
11. |
Matsushita, N.,
Nishi, N.,
Seki, M.,
Matsumoto, R.,
Kuwabara, I.,
Liu, F.,
Hata, Y.,
Nakamura, T.,
and Hirashima, M.
(2000)
J. Biol. Chem.
275,
8355-8360 |
12. | Sato, M., Nishi, N., Shoji, H., Kumagai, M., Imaizumi, T., Hata, Y., Hirashima, M., Suzuki, S., and Nakamura, T. (2002) J. Biochem. (Tokyo) 131, 255-260[Abstract] |
13. | Poirier, F., Timmons, P. M., Chan, C. T., Guenet, J. L., and Rigby, P. W. (1992) Development 115, 143-155[Abstract] |
14. | Fowlis, D., Colnot, C., Ripoche, M. A., and Poirier, F. (1995) Dev. Dyn. 203, 241-251[Medline] [Order article via Infotrieve] |
15. |
Gitt, M. A.,
Colnot, C.,
Poirier, F.,
Nani, K. J.,
Barondes, S. H.,
and Leffler, H.
(1998)
J. Biol. Chem.
273,
2954-2960 |
16. |
Wada, J.,
Ota, K.,
Kumar, A.,
Wallner, E. I.,
and Kanwar, Y. S.
(1997)
J. Clin. Invest.
99,
2452-2461 |
17. | Puche, A. C., Poirier, F., Hair, M., Bartlett, P. F., and Key, B. (1996) Dev. Biol. 179, 274-287[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Horie, H.,
Inagaki, Y.,
Sohma, Y.,
Nozawa, R.,
Okawa, K.,
Hasegawa, M.,
Muramatsu, N.,
Kawano, H.,
Horie, M.,
Koyama, H.,
Sakai, I.,
Takeshita, K.,
Kowada, Y.,
Takano, M.,
and Kadoya, T.
(1999)
J. Neurosci.
19,
9964-9974 |
19. |
Inagaki, Y.,
Sohma, Y.,
Horie, H.,
Nozawa, R.,
and Kadoya, T.
(2000)
Eur. J. Biochem.
267,
2955-2964 |
20. | Murphy, K. M., and Zalik, S. E. (1999) Dev. Dyn. 215, 248-263[CrossRef][Medline] [Order article via Infotrieve] |
21. | Colnot, C., Sidhu, S. S., Balmain, N., and Poirier, F. (2001) Dev. Biol. 229, 203-214[CrossRef][Medline] [Order article via Infotrieve] |
22. | Colnot, C., Ripoche, M. A., Milon, G., Montagutelli, X., Crocker, P. R., and Poirier, F. (1998) Immunology 94, 290-296[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Hsu, D. K.,
Yang, R. Y.,
Pan, Z., Yu, L.,
Salomon, D. R.,
Fung-Leung, W. P.,
and Liu, F. T.
(2000)
Am. J. Pathol.
156,
1073-1083 |
24. | Evanson, J. E., and Milos, N. C. (1996) J. Craniofac. Genet. Dev. Biol. 16, 74-93[Medline] [Order article via Infotrieve] |
25. | Frunchak, Y. N., Martha, G. N., McFadden, K. D., and Milos, N. C. (1993) Anat. Embryol. 187, 299-316[Medline] [Order article via Infotrieve] |
26. |
Marschal, P.,
Herrmann, J.,
Leffler, H.,
Barondes, S. H.,
and Cooper, D. N.
(1992)
J. Biol. Chem.
267,
12942-12949 |
27. |
Shoji, H.,
Nishi, N.,
Hirashima, M.,
and Nakamura, T.
(2002)
Glycobiology
12,
163-172 |
28. | Nieuwkoop, P. D., and Faber, J. (1967) Normal Table of Xenopus laevis (Daudin) , North-Holland Publishing Co., Amsterdam |
29. |
Wada, J.,
and Kanwar, Y. S.
(1997)
J. Biol. Chem.
272,
6078-6086 |
30. | Iwamatsu, A., and Yoshida-Kubomura, N. (1996) J. Biochem. (Tokyo) 120, 29-34[Abstract] |
31. | Yamada, K., Takabatake, Y., Takabatake, T., and Takeshima, K. (1999) Dev. Biol. 214, 318-330[CrossRef][Medline] [Order article via Infotrieve] |
32. | Nishi, N., Oya, H., Matsumoto, K., Nakamura, T., Miyanaka, H., and Wada, F. (1996) Prostate 28, 139-152[CrossRef][Medline] [Order article via Infotrieve] |
33. | Okabayashi, K., Shoji, H., Onuma, Y., Nakamura, T., Nose, K., Sugino, H., and Asashima, M. (1999) Biochem. Biophys. Res. Commun. 254, 42-48[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Sato, M.,
Nishi, N.,
Shoji, H.,
Seki, M.,
Hashidate, T.,
Hirabayashi, J.,
Kasai, K.,
Hata, Y.,
Suzuki, S.,
Hirashima, M.,
and Nakamura, T.
(2002)
Glycobiology
12,
191-197 |
35. |
Couraud, P. O.,
Casentini-Borocz, D.,
Bringman, T. S.,
Griffith, J.,
McGrogan, M.,
and Nedwin, G. E.
(1989)
J. Biol. Chem.
264,
1310-1316 |
36. | Gopalkrishnan, R. V., Roberts, T., Tuli, S., Kang, D., Christiansen, K. A., and Fisher, P. B. (2000) Oncogene 19, 4405-4416[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Madsen, P.,
Rasmussen, H. H.,
Flint, T.,
Gromov, P.,
Kruse, T. A.,
Honore, B.,
Vorum, H.,
and Celis, J. E.
(1995)
J. Biol. Chem.
270,
5823-5829 |
38. | Magnaldo, T., Fowlis, D., and Darmon, M. (1998) Differentiation 63, 159-168[CrossRef][Medline] [Order article via Infotrieve] |