Purification and cDNA cloning of Xenopus liver galectins and their expression

Hiroki Shoji2, Nozomu Nishi2, Mitsuomi Hirashima3 and Takanori Nakamura1,2

2Department of Endocrinology, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan, and 3Department of Immunology and Immunopathology, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan

Received on August 4, 2001; revised on October 29, 2001; accepted on November 6, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We have characterized galectin family proteins in adult tissues of Xenopus laevis and purified 14-kDa and 36-kDa proteins from the liver. The liver galectins showed comparable hemagglutination activities to those of mammalian galectins. Furthermore, we isolated five galectin cDNAs from a Xenopus liver library. These cDNAs revealed that X. laevis galectins (xgalectins) form a family consisting of at least proto and tandem repeat types based on their domain structures, like the mammalian galectin family. Two proto-type xgalectins, -Ia and -Ib, exhibited a high sequence identity (91%) with each other at the amino acid level and were most similar (49–50% identity) to human galectin-1. From their sequence similarity and ubiquitous tissue distributions, xgalectins-Ia and -Ib both seemed to be Xenopus homologues of mammalian galectin-1. Three tandem repeat–type xgalectins were newly identified. Two of them, xgalectins-IIa and -IIIa, seemed to be homologous to human galectins-4 and -9, respectively, judging from their high sequence similarities (42–50% identity). However, xgalectin-IVa seemed to be a novel type. Distributions of mRNAs of xgalectins were analyzed by northern hybridization. In addition to adult tissues, either of three tandem repeat–type xgalectins were expressed in whole embryos. Moreover, amino acid sequence analysis of liver proteins indicated that xgalectins-Ia, -IIa, and -IIIa are produced as abundant galectins in the adult liver.

Key words: galectin-1/galectin-4/galectin-9/galectin family/Xenopus


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ß-Galactoside-binding lectins, galectins, make up a family of which large numbers are found in both vertebrates and invertebrates (Barondes et al., 1994a,b; Kasai and Hirabayashi, 1996Go; Cooper and Barondes, 1999Go). Although galectins are broadly distributed in various tissues of these animals, some members are ubiquitously expressed and others are expressed in a tissue-specific manner. Recently, structural studies on galectins have rapidly increased with the cDNA cloning method. Galectins are thought to play important roles in various biological systems, such as tissue organization, immunity, and development, but a functional analysis of whole members has yet to be performed.

In mammals, cDNAs of 12 members of the galectin family have been isolated and characterized. They are classified into three subgroups, the proto, chimera, and tandem repeat types, based on their domain structures (Kasai and Hirabayashi, 1996Go). A structural conserved domain, the carbohydrate-recognition domain (CRD), of the galectin family can specifically recognize the ß-galactoside-containing carbohydrate moieties of glycoconjugates and mediate some biological activities, such as the hemagglutination of animal erythrocytes. Proto-type galectins-1, -2, -5, -7, -10, and -11 contain one CRD, and chimera-type galectin-3 consists of one CRD and an N-terminal-elongating protein domain (Hirabayashi and Kasai, 1988Go; Jia and Wang, 1988Go; Couraud et al., 1989Go; Hirabayashi et al., 1989Go; Oda et al., 1991Go; Raz et al., 1991Go; Gitt et al., 1992Go, 1995; Ackerman et al., 1993Go; Madsen et al., 1995Go; Dunphy et al., 2000Go). Galectin-1 is the most extensively studied galectin in the proto-type subfamily (Perillo et al., 1998Go). It is ubiquitously expressed and has been proposed to mediate cell-to-cell and cell-to-matrix adhesion, to induce or inhibit cell proliferation, and to induce T cell apoptosis (Perillo et al., 1995Go). Galectin-1 also exhibits neurite outgrowth activity in vitro and plays a role in development of the mouse olfactory system (Puche et al., 1996Go; Horie et al., 1999Go). Chimera-type galectin-3 has been proposed to either trigger or inhibit cell adhesion, to participate in neutrophil and macrophage activation, and to protect T cells from apoptosis (Perillo et al., 1998Go). Both galectins-1 and -3 are associated with pre-mRNA splicing in cell lysates in vitro (Dagher et al., 1995Go; Vyakarnam et al., 1997Go). Five tandem repeat-type galectins, -4, -6, -8, -9, and -12, have been successively identified, but their functions are largely unknown (Rechreche et al., 1997Go; Su et al., 1996Go; Tureci et al., 1997Go; Wada and Kanwar, 1997Go; Gitt et al., 1998Go; Yang et al., 2001Go). Recently, Matsumoto and colleagues (Matsumoto et al., 1998Go; Matsushita et al., 2000Go) identified and characterized human galectin-9 as a potent eosinophil chemoattractant. Galectin-9 has also been proposed to be a component of the urate transporter in rat kidney (Leal-Pinto et al., 1997Go).

Besides those from mammals, cDNAs of two proto- and one chimera-type galectin homologue from chicken (Ohyama et al., 1986Go; Sakakura et al., 1990Go; Nurminskaya and Linsenmayer, 1996Go), two proto-type galectins from conger eel (Ogawa et al., 1999Go), several proto-type galectins from toad (Ahmed et al., 1996Go) and clawed frog, and others were originally isolated and characterized. However, the tandem repeat–type subfamily has not been reported in amphibians. In the clawed frog, Xenopus laevis, two proto-type galectins, 14 kDa and 16 kDa, were purified from skin and cDNAs encoding the latter protein were isolated (Marschal et al., 1992Go). The skin 16-kDa galectin is localized in skin and muscle tissues, but its role is unclear.

Some reports have shown that the expression of galectin mRNAs was regulatory during embryogenesis in mammals (Colnot et al., 1997Go; Wada et al., 1997Go). Thus, the galectin family must be associated with embryogenesis. In Xenopus, a typical proposal that ß-galactoside-binding lectins may play important roles in the development of neural crest cells has been presented (Evanson and Milos, 1996Go; Milos et al., 1998Go). We are mostly interested in determining the function(s) of the galectin family in embryogenesis. Therefore we decided to analyze X. laevis as a model animal that has been widely used in developmental biology.

In this study, we first identified galectin proteins belonging to the proto and tandem repeat types from Xenopus adult tissues by affinity purification and then isolated five independent cDNAs encoding these galectins from a liver cDNA library. A Roman numeral and alphabet was assigned to each Xenopus galectin (xgalectin) independently of numbers of mammalin galectins but in the order of their discovery, because it was impossible to make complete correspondence based on the amino acid sequences between the members of Xenopus and mammalian galectin families.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of xgalectin proteins
Xgalectin proteins were successfully purified from skin after extraction with lactose-containing buffer according to Marschal et al. (1992)Go. In this study, this method was first applied for characterization of the galectin family members from other tissues of X. laevis, such as adult liver. However, it was not possible to obtain a satisfactory protein yield reproducibly with this method. Therefore, we homogenized the tissues in the buffer without lactose and immediately applied the resulting extracts onto an affinity column of lactosyl-agarose resin. As a result, we could reproducibly obtain galectin proteins from various tissues of Xenopus by means of the one-step affinity chromatography. As shown in Figure 1, 14-kDa and/or 16-kDa proteins were obtained from all the tissues examined on elution with lactose-containing buffer from a lactosyl-agarose column. Interestingly, 35–36-kDa proteins were also obtained from the extracts of the tissues except for skin, muscle, and testis. However, the proteins could not be eluted with the buffer containing sucrose instead of lactose from the lactosyl-agarose column. In Xenopus, the liver seemed to be an appropriate material for the purification of galectin proteins because it was the largest organ and contained large amounts of the galectins, especially 36-kDa molecules, which had not been found in Xenopus tissues before the present study. Then, the liver galectins were further fractionated by anion-exchange chromatography to characterize the xgalectin family members.



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Fig. 1. SDS–PAGE profile of galectin family proteins isolated from Xenopus adult tissues. The eluates (2–3 µg protein/lane) of Xenopus male or female adult tissue extracts from lactosyl-agarose columns were subjected to SDS–PAGE (12% acrylamide gel). The proteins were visualized by Coomassie brilliant blue–R250 staining. The tissues examined were as follows: a, liver; b, lung; c, heart; d, spleen; e, kidney; f, stomach; g, intestine; h, skin; i, muscle (thigh); j, ovary; k, testis. The molecular weights of the Xenopus galectin proteins are indicated on the left. The molecular weights of the protein markers were indicated on the right.

 
In this step, 36-kDa protein(s) and 14-kDa protein(s) were obtained in the pass-through and adsorbed fractions (fractions1 and 2), respectively, as shown in Figure 2. Furthermore, at least two protein bands corresponding to about 36 kDa were observed on longer-term electrophoresis on a 12% polyacrylamide gel. The hemagglutination activities of the two protein fractions were almost identical toward glutaraldehyde-fixed and trypsin-treated rabbit erythrocytes. The minimal protein concentration of these fractions required for the agglutination was estimated to be 0.25 µg/ml each. This activity was comparable to those of mammalian galectins, as previously reported (Caron et al., 1987Go). Thus, these proteins isolated from the adult liver seemed to be typical galectin family proteins.



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Fig. 2. Elution profile of Xenopus liver galectins from a Resource Q column. (A) The liver galectins isolated by affinity chromatography were further fractionated into pass-through and adsorbed fractions on a Resource Q column. The adsorbed proteins were eluted with a gradient of increasing NaCl concentration, from 0.05 M to 0.2 M. (B) SDS–PAGE profile of liver protein fractions (fractions 1 and 2) obtained on the chromatography. Apparent 36-kDa liver proteins (fraction 1) passed through the anion resin, and 14-kDa liver protein(s) were adsorbed to the column. About 36-kDa proteins were visualized as a few bands on the longer 12% SDS–PAGE (left side of B).

 
cDNA cloning of the xgalectin family
Although liver galectin proteins obtained on the chromatographies seemed to make up a mixture of several proteins, further purification to isolate individual components has not been successful. Therefore, we attempted to isolate cDNAs of xgalectins expressed in the liver. Degenerate primers were designed according to the highly conserved sequence of mammalian galectins and used for reverse transcription polymerase chain reaction (RT-PCR)–based cloning of partial cDNA fragments from the liver mRNAs. As a result, four kinds of xgalectin cDNA fragments were isolated. Using these cDNA fragments as probes, a Xenopus liver cDNA library was screened, and two proto- and three tandem repeat-type xgalectin cDNAs were isolated. As shown in Figures 3 and 4, Xenopus liver galectins form a family consisting of a large number of members like the mammalian galectin family. The two proto-type xgalectins, which each contained one CRD, were designated as xgalectins-Ia and -Ib. Both of them consisted of 134 amino acids, and the molecular weights of xgalectins-Ia and -Ib were calculated to 15,297 and 15,206, respectively. The amino acid sequences of xgalectins-Ia and -Ib exhibited the highest similarity with each other (91% identity) and also exhibited high similarity with those of Bufo ovarian galectin (56%) and human galectin-1 (49%), but were not so similar to that of Xenopus skin 16-kDa galectin (40%) reported previously (Figure 3). Thus, both xgalectins-Ia and -Ib seemed to be Xenopus homologues of mammalian galectin-1.



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Fig. 3. Comparison of the amino acid sequences of proto-type Xenopus and other vertebrate galectins. The amino acid sequences of five members of the galectin family were compared as follows: xG-Ia, xgalectin-Ia (DDBJ accession number for nucleotide sequence AB056478); xG-Ib, xgalectin-Ib (DDBJ accession number for nucleotide sequence AB060969); bG-1, Bufo arenarum galectin-1 (Ahmed et al., 1996Go); hG-1, human galectin-1 (Hirabayashi et al., 1989Go); and xSkin, Xenopus skin 16-kDa galectin (Marschal et al., 1992Go). The amino acids identical to xgalectin-Ia are shaded. Asterisks indicate the positions of the six amino acids essential for ß-galactoside binding. The sequence identities (%) indicated on the right, were determined as based on that of xgalectin-Ia.

 


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Fig. 4. Comparison of the amino acid sequences of tandem repeat–type Xenopus and human galectins. The amino acid sequences of the xgalectin family were compared with those of human tandem repeat-type galectins-4 and -9, as follows: xG-IIa, xgalectin-IIa (DDBJ accession number for nucleotide sequence AB060970); xG-IIIa, xgalectin-IIIa (DDBJ accession number for nucleotide sequence AB060971); xG-IVa, xgalectin-IVa (DDBJ accession number for nucleotide sequence AB060972); hG-4, human galectin-4 (Rechreche et al., 1997Go); and hG-9, human galectin-9 (Tureci et al., 1997Go). The amino acids identical to xgalectin-IIa are shaded. Asterisks indicate the positions of the six amino acids essential for ß-galactoside binding. The positions of the replaced amino acids found in the xgalectin CRDs are indicated by arrowheads.

 
Three tandem repeat–type xgalectins, xgalectins-IIa, -IIIa, and -IVa, which contained two CRDs covalently linked through a unique link peptide, were obtained (Figure 4). Xgalectins-IIa, -IIIa, and -IVa consisted of 340, 343, and 332 amino acids, respectively, that is, they were larger than mammalian tandem repeat galectins. The molecular weights of xgalectins-IIa, -IIIa, and -IVa were calculated to be 37,232, 38,364, and 36,825, respectively. They exhibited low sequence identities of 33–34% at the amino acid level with one another. The amino acid sequences of xgalectins-IIa and -IIIa were most similar to those of human galectins-4 (50 %) and -9 (42 %), respectively, suggesting that they are Xenopus homologues of mammalian galectins-4 and -9, respectively (Figure 5). However, it was difficult to identify the mammalian counterpart of xgalectin-IVa, because xgalectin-IVa was low and equally similar to both of human galectins-4 and -9. The sequence identity of xgalectin-IVa was 38% and 35% with those of human galectins-4 and -9, respectively.



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Fig. 5. A schematic illustration showing the sequence similarities of the CRDs of tandem repeat–type Xenopus and human galectins. The amino acid sequences of the N- and C-terminal CRDs and linker peptides of xgalectins-IIa, -IIIa, and -IVa were compared with one another (A). The amino acid sequences of the N- and C-terminal CRDs and linker peptides of xgalectins-IIa, -IIIa, and -IVa were also compared with those of human galectins-4 and -9 (B, C, D), respectively. The sequence identities (%) of each CRD, linker peptide, and full-length were determined based on that of xgalectin-IIa (A, B), xgalectin-IIIa (C), and xgalectin-IVa (D), respectively. The identities of the full-length sequences are shown on the right.

 
Six amino acids were identified as essential residues for sugar binding of human galectin-1, and they are well conserved in most galectins (Hirabayashi and Kasai, 1991Go, 1994). The corresponding residues in xgalectins-Ia, -Ib, and -IIa completely matched the consensus sequence. However, one amino acid replacement in the N-terminal CRD of each of xgalectin-IIIa and C-terminal CRD of xgalectin-IVa was observed. Asn-83 of xgalectin-IIIa and Ala-248 of xgalectin-IVa replaced Arg/Lys and Asn, respectively, of the consensus sequence (Figure 4).

Distribution of mRNAs of the xgalectin family
Figure 6 shows the expression profiles of xgalectin mRNAs in adult tissues and whole embryos (tail bud stage) on northern hybridization. Using cDNA probes of either xgalectin-Ia or -Ib, ubiquitous expression patterns were observed for the adult tissues, and they were indistinguishable. Although we failed to distinctly detect hybridization signals of xgalectins-Ia and -Ib on northern analysis, because of the high sequence similarity, we observed ubiquitous expression of both their mRNAs by RT-PCR analysis using primer pairs specific for xgalectin-Ia or -Ib (data not shown). The mRNA of xgalectin-IIa was distributed in a tissue-specific manner. It was abundant in adult liver, stomach, intestine, and kidney and in whole embryos and was not at all or only slightly detected in the other tissues. The mRNA of xgalectin-IIIa was broadly distributed in the adult tissues except for muscle, being especially abundant in lung and liver. Xgalectin-IVa mRNA was also broadly expressed in the adult tissues. Expression of the xgalectin-IIIa and -IVa mRNAs was also weakly detected in the embryos. In skin, tandem repeat xgalectins have not identified on protein analysis, but expression of the mRNAs of xgalectins-IIIa and -IVa was observed.



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Fig. 6. Tissue distributions of xgalectin mRNAs. The expression of xgalectin mRNAs was examined by northern hybridization analysis. The distributions of xgalectin-Ia and/or Ib mRNAs were analyzed using a probe for xgalectin-Ib. The tissues examined were as follows: a, heart; b, lung; c, liver; d, stomach; e, intestine; f, kidney; g, muscle (thigh); h, skin; i, ovary; j, testis; k, whole embryos (tail bud stage). The EF-1{alpha} probe was used as a control.

 
Immunoreactivity of the xgalectin family
The immunoreactivities of the isolated xgalectins were characterized by western blot analysis using anti-Xenopus skin 14-kDa or 16-kDa galectin antisera (Figure 7). Antiserum against skin 14-kDa galectin reacted with the 14-kDa xgalectin protein(s) isolated from all the adult tissues examined. The anti-skin 16-kDa galectin antiserum, which reacted with both the skin 14-kDa and 16-kDa galectins, recognized the 16-kDa xgalectin band for muscle and skin and also for liver, lung, and kidney, in addition to the 14-kDa xgalectin(s) from the adult tissues. Thus although the 16-kDa protein band was only observed for muscle and skin on Coomassie blue staining, a small amount of the 16-kDa xgalectin protein was also produced in other adult tissues, that is, not only in muscle and skin. Neither the anti-skin 14-kDa nor 16-kDa galectin antiserum exhibited cross-reactivity with the tandem repeat xgalectins, supporting that proto-type xgalectins are structurally quite different from any of the tandem repeat–type xgalectins shown in Figures 3 and 4.



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Fig. 7. Western blot analysis of xgalectin proteins obtained from Xenopus tissues. The eluates (50–100 ng protein/lane) of Xenopus adult male or female tissue extracts from lactosyl-agarose columns were subjected to SDS–PAGE (12% acrylamide gel). After SDS–PAGE, the proteins were electrically blotted onto PVDF membranes. The immunoreactivity of each protein was examined using anti-Xenopus skin 14-kDa (A) or 16-kDa galectin (B) antiserum. The tissues examined were as follows: a, liver; b, lung; c, kidney; d, stomach; e, intestine; f, muscle (thigh); g, skin. The molecular weights of the protein markers are indicated on the right.

 
Identification of liver xgalectin proteins
To classify major xgalectin proteins isolated from adult liver, amino acid sequence analysis was performed with concentrated proto- (14-kDa protein) and tandem repeat–type (36-kDa protein) xgalectin fractions (fractions 1 and 2 in Figure 2). Both proteins were blotted onto polyvinylidene difluoride (PVDF) membranes, reduced, S-alkylated, and then digested with trypsin. The tryptic peptides liberated from the membranes were purified, as shown in Figure 8. Then, the N-terminal ten-amino-acid sequence of each peptide was analyzed (Table I). Ten tryptic peptides (14K-T1-10) were obtained from the 14-kDa xgalectin fraction. The N-terminal sequences of 14K-T1, -T2, and -T6 corresponded to that of xgalectin-Ia. 14K-T3 and -T4 were mixtures of two peptides derived from xgalectin-Ia. The N-terminal sequences of 14K-T9 and -T10 were common sequences in xgalectins-Ia and -Ib. The sequences of 14K-T5, -T7, and -T8 could not be determined. Thus no particular peptide for xgalectin-Ib was recovered. This indicated that the most abundant proto-type xgalectin in the liver is xgalectin-Ia. Eight tryptic peptides (36K-T1 to -T8) were obtained from the 36-kDa xgalectin fraction. The N-terminal sequences of 36K-T2 to -T4, and -T8 corresponded to that of xgalectin-IIIa. The sequence of 36K-T5 corresponded to that of xgalectin-IIa. Both 36K-T1 and -T7 were mixtures of two peptides derived from xgalectins-IIa and -IIIa. The sequence of 36K-T6 could not be determined. No peptide derived from xgalectin-IVa was detected. This indicated that among the three tandem repeat–type xgalectins predicted from cDNAs, xgalectins-IIa and -IIIa are produced abundantly in adult liver.



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Fig. 8. Purification of tryptic peptides prepared from Xenopus liver galectins. About 15 µg protein of a 14-kDa or 36-kDa xgalectin fraction prepared as shown in Figure 2 was dot-blotted onto a PVDF membrane. The blotted proteins were reduced and S-pyridylethylated, then digested with trypsin (1.3 µg/ml) at 25°C for 18 h. The digests were applied onto a C18 column (µ-Bondasphere 5 µmC18, 3.9 mm x 150 mm) and eluted with a gradient of acetonitrile in 0.1% TFA. The elution of the peptides was monitored as the absorption at 215 nm, and the main peptide peaks were collected manually. Ten peptide peaks (14K-T1 to -T10) from the 14-kDa xgalectin fraction and eight peptide peaks (36K-T1 to -T8) from the 36-kDa xgalectin fraction were recovered.

 

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Table I. N-Terminal amino acid sequences of tryptic peptides obtained from Xenopus liver galectins and their corresponding positions deduced from cDNA clones
 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Two types of galectin family proteins were identified in Xenopus adult tissues on one-step affinity chromatography on a lactosyl-agarose column. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis, it was predicted that xgalectins could be primarily classified into two groups, the 14–16-kDa and 35–36-kDa galectin subfamilies. These subfamilies were further characterized, using the liver galectins, to be comparable to mammalian proto- and tandem repeat–type galectins, because each subfamily had almost the same molecular weight as and comparable hemagglutination activity with one of the mammalian subfamilies. Moreover, the complete amino acid sequences of the Xenopus liver galectins were deduced from five cDNAs isolated from a liver cDNA library. As a result, it was proven that both proto- and tandem repeat–type galectins distinctly exist in X. laevis. Also, a typical signal sequence was defective in the N-terminus in these galectins, as in other animal galectins.

Two cDNAs encoding proto-type galectin subfamily proteins were cloned. The amino acid sequences of xgalectins-Ia and -Ib are most similar to each other (91% amino acid sequence identity), and they seem to be homologous with human galectin-1, with almost the same sequence similarity, 49% and 50% identity, respectively. Xgalectins-Ia and -Ib are not alleles from different individuals but rather distinct genes, because we have identifed not only xgalectin-Ia as the major proto-type galectin protein in liver but also xgalectin-Ib as the major protein in kidney (unpublished data). Three tandem repeat–type galectins of Xenopus liver were newly identified. Xgalectins-IIa and -IIIa seem to be homologous to human galectins-4 and -9, respectively, because of the high sequence similarity. However, it was difficult to clarify the mammalian counterpart of xgalectin-IVa, because xgalectin-IVa exhibited slightly low similarity with both human galectins-4 and -9. The sequence identities were 38% and 35% with galectins-4 and -9, respectively. Thus xgalectin-IVa seems to be a unique protein in Xenopus or a novel subtype in vertebrates. During the course of this study, we identified other cDNA fragments encoding structurally very similar proteins to xgalectins-IIa and -IIIa from a kidney cDNA library, which seemed to be isoforms (unpublished data). This suggests that there are isoforms for most of xgalectins, so we decided to assign letters, in addition to the Roman numerals, to all xgalectins.

One of the structural properties of the galectin family is that the essential amino acids involved in ß-galactoside binding are well conserved in their CRDs. Six amino acids have been identified as essential residues for sugar binding of human galectin-1, and they are well conserved in most galectins (Hirabayashi and Kasai, 1991Go, 1994). In the CRD of each of xgalectins-Ia, -Ib, and -IIa, all the six amino acids completely matched the consensus sequence, that is, His-45, Asn-47, Arg-49, Asn-62, Gln-72, and Arg-74 of human galectin-1. However, one amino acid replacement was observed in the N-terminal CRD of xgalectin-IIIa and the C-terminal CRD of xgalectin-IVa. Asn-83 of xgalectin-IIIa and Ala-248 of xgalectin-IVa replaced Arg-74 and Asn-47 of human galectin-1, respectively. These replacements might effect the sugar recognition specificities of xgalectins-IIIa and -IVa.

The expression of mRNAs and proteins of xgalectin-Ia and/or -Ib was observed in all the adult tissues examined. This suggests that proto-type galectins are essential for fundamental function(s) of multicellular organisms, such as cell–cell contact or tissue organization. However, their mRNAs are expressed at fairly detectable levels in whole embryos. Xenopus skin 16-kDa galectin has also been shown to be absent in embryonic whole tissue (Marschal et al., 1994Go). Unidentified proto-type xgalectin(s) may play its role in embryos. Another explanation is that maternal proteins may play its role, instead of zygotic proto-type xgalectins. Although mRNAs of tandem repeat–type xgalectins (that is, xgalectins-IIIa and -IVa) are broadly distributed in the adult tissues, like proto-type xgalectins, mRNA of xgalectin-IIa is primarily expressed in adult liver, kidney, and digestive tract and in whole embryos. The restricted expression pattern of xgalectin-IIa is comparable to that of mammalian galectin-4, which is abundantly expressed in digestive tract (Rechreche et al., 1997Go; Gitt et al., 1998Go). This indicates that both xgalectin-IIa and mammalian galectin-4 may have a common function despite the species difference.

To identify the major xgalectin molecules translated in the Xenopus liver, liver xgalectin proteins were fractionated into the proto and tandem repeat types, and then the amino acid sequences of the resulting peptides derived from each fraction were analyzed. As shown in Table I, the abundant proto and tandem repeat types were xgalectin-Ia, and xgalectins-IIa and -IIIa, respectively. Neither xgalectin-Ib nor -IVa was detectable among the liver proteins on this sequence analysis. As the recombinant xgalectins-Ib and -IVa prepared from Escherichia coli cells could bind to a lactosyl-agarose column (data not shown), it seemed that they must not be produced abundantly in adult liver. These results indicate that xgalectins-Ia, -IIa, and -IIIa are major galectins expressed in adult liver. They also indicate that expressions of xgalectin isoforms are distinctly regulated and that each isoform may play a specific role in individual tissues.

Marschal et al. (1992) isolated two proto-type galectins, 14-kDa and 16-kDa galectins, from Xenopus skin and determined the complete structure of the 16-kDa galectin by cDNA cloning. Xenopus skin 16-kDa galectin is the most abundant protein in skin (5% of the total proteins) and is also expressed in muscle tissues. The amino acid sequence of skin 16-kDa galectin is unique and not so similar to those of any mammalian proto-type galectins. In mammals, galectin-7 is abundantly expressed in keratinocytes of the skin but does not exhibit high sequence similarity to Xenopus skin 16-kDa galectin. Mammalian galectin-7 is also a unique proto-type galectin in structural characteristics and tissue distribution. Thus, galectin(s) distributed in the skin may have evolved independently in various animal species.

Rabbit antisera against Xenopus skin 14-kDa and 16-kDa galectins were prepared, and the tissue distribution of xgalectin proteins was examined using these antisera. On western blot analysis, the 14-kDa protein(s) isolated from adult tissues including liver reacted with anti-skin 14-kDa galectin antiserum. On the other hand, although no 16-kDa protein bands were observed for the other tissues, except for muscle and skin on Coomassie blue staining, a 16-kDa protein band was detectable for liver, lung, and kidney with the anti-skin 16-kDa galectin antiserum. A small amount of a proto-type xgalectin, such as skin 16-kDa galectin or other related xgalectin(s), might be produced in the adult liver.

Recently, we identified 36-kDa galectins in the livers of chicken and fish (unpublished data), and Inagawa et al. (2001)Go have reported cDNA cloning of galectin-9 from rainbow trout. Thus tandem repeat–type galectins are extensively distributed in vertebrates and must play significant roles in various biological systems, such as cell growth and development. Another possible function of tandem repeat–type xgalectins in the liver is as carrier proteins for cellular metabolites, such as urate. The liver is the major organ for purine metabolism, and rat galectin-9 has been proposed to act as a urate transporter in the kidney (Leal-Pinto et al., 1997Go). However, the liver xgalectins exhibited no affinity to xanthine-Sepharose (data not shown), which was used for the purification of rat renal galectin-9. Hirabayashi et al. (1992)Go originally isolated tandem repeat–type galectins from a nematode, Caenorhabditis elegans; after that, completion of the genome project on C. elegans revealed the existence of at least 11 candidate proto- and tandem repeat–type galectins in C. elegans. The analysis of functional differences between proto- and tandem repeat–type galectins has progressed. Although neither the physiological function nor the specificity of sugar binding of the xgalectin family was addressed in the present study, it may be an useful tool for analysis on the roles of galectins in the embryogenesis, because either of three tandem repeat–type xgalectins were expressed in whole embryos. Further study is required to obtain a clue for elucidation of the functions of xgalectin family in X. laevis development.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of galectins from Xenopus tissues
Xenopus tissues obtained from adult males and females were each homogenized in a fivefold volume of 10 mM Tris–HCl (pH 7.2), 0.15 M NaCl, 1 mM ethylenediamine tetra-acetic acid (EDTA), 1 mM dithiothreitol (DTT), 5 mM benzamidine-HCl, 1 mM phenymethylsulfonyl fluoride (PMSF), and 1 mM diisopropylphosphofluoridate (DFP) with a Polytron homogenizer. The homogenates were centrifuged at 25,000 rpm for 30 min with a Hitachi Ultra centrifuge 55P-72. A one-tenth volume of 4 M NaCl (final concentration, about 0.5 M) and a 1/100 volume of 1% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS) (Sigma) were added to the supernatants recovered, and the mixtures were further centrifuged at 25,000 rpm for 30 min. The resulting supernatants were directly applied to a lactosyl-agarose column (2 ml; Seikagaku, Tokyo) and washed extensively with 10 mM Tris–HCl (pH 7.2), 0.5 M NaCl, 1 mM EDTA, 0.2 mM DTT, and 0.01% CHAPS, and then 10 mM Tris–HCl (pH 7.2), 0.15 M NaCl, 1 mM EDTA, 0.2 mM DTT, and 0.01% CHAPS. Proteins adsorbed to the affinity resin were eluted with 10 mM Tris–HCl (pH 7.2), 0.15 M NaCl, 1 mM EDTA, 0.2 mM DTT, and 0.01% CHAPS containing 200 mM lactose. All the steps were performed at 4°C.

Purification of Xenopus liver galectins
The liver protein mixtures eluted from the lactosyl-agarose resin were separated into two fractions by anion-exchange chromatography. Briefly, the crude liver galectins were extensively dialyzed against 10 mM Tris–HCl (pH 8.0), 0.05 M NaCl, 1 mM EDTA, 0.2 mM DTT, and 0.01% CHAPS for 24 h at 4°C. After dialysis, the galectin mix was applied to a Resource Q column (1 ml; Amersham Pharmacia Biotech) and then fractionated with a linear gradient of NaCl from 0.05 M to 0.2 M. The pass-through fraction and adsorbed fraction were separately pooled to examine the hemagglutination activity and the amino acid sequence.

Hemagglutination assay
Hemagglutination activities of Xenopus liver galectins were measured using glutaraldehyde-fixed and trypsin-treated rabbit red blood cells as described elsewhere (Nowak et al., 1976Go). The minimal concentrations of the galectins required for the induction of hemagglutination were determined in a U-shaped 96-well plate by visual inspection. Protein concentration was spectrophotometrically determined under the assumption that the absorption at 280 nm for a 1% (w/v) solution is 10.

Degenerate oligonucleotide-based PCR cloning of cDNA fragments of xgalectins
Total RNA was extracted from Xenopus liver and poly(A)+ RNA was purified using a PolyATtract System (Promega). Double-stranded cDNA was synthesized using a Marathon cDNA Amplification Kit (Clontech). A degenerate sense (Gd4) and three degenerate anti-sense primers (Gd5, 7, and 9) were synthesized according to the conserved amino acid sequence of the CRD. Gd4 corresponds to a highly conserved amino acid sequence in most members of the galectin family, HNFPRF. Gd5 and Gd9 correspond to the amino acid sequences of mouse galectin-9, FKVMVN and VNGQHM, respectively. Gd7 corresponds to the amino acid sequence of Xenopus skin 16-kDa galectin, LPDGKE, which is a conserved region in proto-type galectins. The nucleotide sequences of the primers were as follows:

• Gd4: 5'-CA(TC)TT(TC)AA(TC)CC(GATC)CG(GATC)TT(TC)-3',

• Gd5: 5'-GTT(GC)ACCAT(GATC)AC(TC)TT(AG)AA-3',

• Gd7: 5'-TTCTTT(GATC)CC(AG)TC(GATC)GG(GATC)A-3', and

• Gd9: 5'-CATATG(CT)TG(GATC)CC(AG)TT(GATC)AC-3'.

PCR was performed with an Advantage cDNA PCR Kit (Clontech). Reaction mixtures were prepared by adding a pair of degenerated primers, each at the concentration of 1.6 µM. Reaction mixtures were preincubated for 2 min at 94°C, followed by 30 or 35 thermal cycles of the following: 94°C for 30 s, 40°C for 30 s, 68°C for 2 min. The Gd4 sense primer was used for all the reactions. Using Gd7 as the anti-sense primer, a 186-bp cDNA fragment of xgalectin-Ia was amplified. Using Gd9 as the anti-sense primer, 180-bp and 177-bp cDNA fragments of xgalectin-IIa and -IVa, respectively, were amplified. Using Gd5 as the anti-sense primer, a 168-bp cDNA fragment of xgalectin-IIIa was amplified. The isolated cDNA fragments were used as probes for cDNA library screening.

Screening of a cDNA library, isolation of full-length cDNAs, and nucleotide sequencing
A Xenopus liver Lambda cDNA Library (Stratagene) was screened using cDNA probes labeled with digoxigenin-11-dUTP (Roche Molecular Biochemicals). Labeling of the probes and detection of hybridization signals were performed as described previously (Nishi et al., 1996Go). Full-length clones for xgalectins-Ia, -IIa, and -IVa and only a partial clone (with the 5'-region deleted) for xgalectin-IIIa were obtained through the screening. A full-length clone for xgalectin-Ib was isolated through the screening using a cDNA fragment of xgalectin-Ia as a probe.

To obtain the 5'-region of xgalectin-IIIa from the liver, 5'- rapid amplification of cDNA ends (RACE) PCR was performed with a Marathon cDNA Amplification Kit (Clontech) and mRNAs of the liver. A specific primer with the following sequence was used: 5'-GTTAAGGTAATGCATCCACC-3'. As a result, a cDNA fragment of 452 bp covering the entire 5'-region of xgalectin-IIIa was obtained. The full-length sequence of xgalectin-IIIa was reconstructed by combining the sequence of the 5'-RACE PCR product and that of a clone with the longest insert. Although we failed to obtain a full-length cDNA clone of xgalectin-IIIa from the liver library, one was obtained from a Xenopus kidney cDNA library (unpublished data), and the sequence was identical to that of cDNAs from the liver. This supports the reliability of the 5'-RACE PCR product from the liver.

Both strands of degenerate PCR products, inserts of cDNA clones obtained from the library, and 5'-RACE products were analyzed with a DNA sequencer Model 377 (PE Biosystems).

Northern hybridization
Poly(A)+ RNAs were prepared from adult tissues and whole embryos (tail bud stage). 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 by PCR, and then used as probes: xgalectin-Ia, nucleotides 14–418; xgalectin-Ib, nucleotides 23–427; xgalectin-IIa, nucleotides 3–1025; xgalectin-IIIa, nucleotides 436–1164; xgalectin-IVa, nucleotides 38–1036; and EF-1{alpha}, nucleotides 758–1378 (Genbank accession number M25504). Hybridization, washing, and detection were performed as described elsewhere (Nishi et al., 1996Go).

Preparation of rabbit antisera against Xenopus skin galectins and western blotting
Two Xenopus proto-type galectins, 14-kDa and 16-kDa galectins, were purified from skin by the method of Marschal et al. (1992)Go and used for the preparation of anti-serum. Rabbit anti-14-kDa galectin and anti-16-kDa galectin sera were prepared by a general method. Rabbits were injected with 100–200 µg of 14-kDa or 16-kDa galectin mixed with Freund’s complete adjuvant. The rabbits were boosted three times with the same antigen every 2 weeks.

The specificities of the antisera were examined by western blot analysis. Anti-14-kDa galectin antiserum recognized the Xenopus skin 14-kDa galectin but not the 16-kDa galectin. Anti-16-kDa galectin antiserum reacted with both the skin galectins. However, neither of the antisera cross-reacted with partially purified rat skin and liver galectins (data not shown). Xenopus galectins (xgalectin family) purified from adult tissues were analyzed using the above antisera. The xgalectin proteins (100–200 ng) were subjected to 12% SDS–PAGE under reducing conditions. The proteins were electrically transferred to PVDF membranes (Immobilon; Millipore), and then the membranes were blocked with 5% nonfat milk in 20 mM Tris–HCl (pH 7.2), 0.15 M NaCl (Tris buffered saline; TBS) for 1 h. Then, the membranes were incubated with the anti-14-kDa galectin or anti-16-kDa galectin antiserum (1:200) in TBS containing 1% milk for 12 h and washed with TBS-1% milk three times for 10 min each. After incubation with peroxidase-conjugated anti-rabbit IgG donkey antibodies (1:1000; Amersham Pharmacia Biotech.) in TBS-1% milk for 1 h, the membranes were washed twice with TBS-1% milk and then twice with TBS for 10 min each. The secondary antibody was detected by mean of a chemiluminescence method using an electrochemiluminescence kit (Amersham Pharmacia Biotech.).

Amino acid sequence analysis of tryptic peptide fragments obtained from the liver xgalectins
The 14-kDa and 36-kDa liver xgalectins were treated with 1% SDS and 20% methanol, then directly dot-blotted onto PVDF membranes. The blotted proteins were stained with Coomassie brilliant blue R-250, cut out, and then reduced and S-pyridylethylated on the membranes by the method of Iwamatsu and Yoshida-Kubomura (1996)Go. 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 with a reverse-phased high-performance liquid chromatography system (Tohso, Tokyo). The mixture of tryptic fragments was applied onto a 5C18 column (µBondasphere 5C18; Waters) and then separated with an acetonitrile gradient prepared with 0.1% trifluoracetic acid (TFA) and 0.1% TFA containing 80% acetonitrile. The N-terminal ten amino acids of each peptide were determined with a gas-phase sequencer ABI 492 (Applied Biosystems).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We wish to thank Ms. N. Yoshiga for expert technical assistance in the amino acid sequence analysis. This work was supported by grants from the Ministry of Science and Culture of Japan.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CHAPS, 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid; CRD, carbohydrate recognition domain; DFP, diisopropylphosphofluoridate; DTT, dithiothreitol; EDTA, ethylenediamine tetra-acetic acid; PMSF, phenymethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris buffered saline; TFA, trifluoroacetic acid; xgalectin, Xenopus laevis galectin.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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