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Characterization of the Xenopus galectin family. Three structurally different types as in mammals and regulated expression during embryogenesis.
Shoji H
,
Nishi N
,
Hirashima M
,
Nakamura T
.
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.
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; xgalectinVIIa,
AB080020; and ESTs, AW765695, AW766406, BF428260,
BG021071, BG163149, BG413616, BG552788, BG885816, and
BI446289. N-ter, N-terminal.
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 xgalectinVIa
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. NCRD
and C-CRD, N- and C-terminal
CRDs, respectively; link, link peptide;
ORF, open reading frame.
FIG. 3. Lactose-binding activities of recombinant xgalectinIVa,
-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 lactosylagarose
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.
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.
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 xgalectinIIa
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; xgalectinIb,
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.
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
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.
