Shoji H et al. (2003), Characterization of the Xenopus galectin family...

XB-ART-5865

J Biol Chem 2003 Apr 04;27814:12285-93. doi: 10.1074/jbc.M209008200.

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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 .

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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.

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Species referenced: Xenopus laevis
Genes referenced: cga gal.1 lgals1.1 lgals3 lgals4.2 lgals9b odc1 tbl1x

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	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.