©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Galectin-8
A NEW RAT LECTIN, RELATED TO GALECTIN-4 (*)

(Received for publication, October 17, 1994; and in revised form, December 7, 1994)

Yaron R. Hadari (1) Keren Paz (1) Roi Dekel (2) Tomislav Mestrovic (3) Domenico Accili (4) Yehiel Zick (1)(§)

From the  (1)Department of Chemical Immunology, the Weizmann Institute of Science, Rehovot 76100, Israel, the (2)Technion School of Medicine, Haifa 31096, Israel, the (3)University of Zagreb, School of Medicine, Salata 3b, Zagreb 41000, Croatia, and the (4)Diabetes Branch, National Institutes of Health, Bethesda, Maryland 20982

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A protein of 35 kDa which has the characteristic properties of galectins (S-type lectins) was cloned from rat liver cDNA expression library. Since names for galectins 1-7 were already assigned, this new protein was named galectin-8. Three lines of evidence demonstrate that galectin-8 is indeed a novel galectin: (i) its deduced amino acid sequence contains two domains with conserved motifs that are implicated in the carbohydrate binding of galectins, (ii) in vitro translation products of galectin-8 cDNA or bacterially expressed recombinant galectin-8 are biologically active and possess sugar binding and hemagglutination activity, and (iii) a protein of the expected size (34 kDa) that binds to lactosyl-Sepharose and reacts with galectin-8-specific antibodies is present in rat liver and comprises 0.025% of the total Triton X-100-soluble hepatic proteins. Overall, galectin-8 is structurally related (34% identity) to galectin-4, a soluble rat galectin with two carbohydrate-binding domains in the same polypeptide chain, joined by a link peptide. Nonetheless, several important features distinguish these two galectins: (i) Northern blot analysis revealed that, unlike galectin-4 that is confined to the intestine and stomach, galectin-8 is expressed in liver, kidney, cardiac muscle, lung, and brain; (ii) unlike galectin-4, but similar to galectins-1 and -2, galectin-8 contains 4 Cys residues; (iii) the link peptide of galectin-8 is unique and bears no similarity to any known protein; (iv) the N-terminal carbohydrate-binding region of galectin-8 contains a unique WG-E-I motif instead of the consensus WG-E-R/K motif implicated as playing an essential role in sugar-binding of all galectins. Together with galectin-4, galectin-8 therefore represents a subfamily of galectins consisting of a tandem repeat of structurally different carbohydrate recognition domains within a single polypeptide chain.


INTRODUCTION

Lectins are involved in a wide variety of cellular functions, many of which are related to their only common feature, the ability to bind carbohydrates specifically and reversibly and to agglutinate cells (reviewed in (1) and (2) ). Animal lectins are classified as C-lectins, which are Ca-dependent and are structurally related to the asialoglycoprotein receptor, and galectins, previously known as S-type lectins, which are thiol-dependent and specifically bind beta-galactoside residues. In mammals, four galectin types have been sequenced and characterized, and there is evidence for the existence of other relatives(3, 4) . All known members of this family lack a signal peptide(5) , are found in the cytosol, and are isolated as soluble proteins. However, there is evidence that some members are externalized by an atypical secretory mechanism(5, 6, 7) .

Galectins require fulfillment of two criteria: affinity for beta-galactosides and significant sequence similarity in the carbohydrate recognition domain (CRD)(^1)(8, 9) , the relevant amino acids residues of which have been determined by x-ray crystallography(10, 11) . Galectin-1 and -2 are homodimers, with subunit molecular mass of 14 kDa, that are not subjected to post-translational modifications(12, 13) . Galectin-1 is found in the extracellular matrix (14, 15) and has been shown to interact with laminin(16, 17) . The function of galectin-1 and -2 is not yet fully understood, although there is evidence that they might be involved in regulation of cell growth(18, 19) , cell adhesion(17) , cell transformation(20) , and embryogenesis(21) .

Larger galectins (galectin-3) (previously known as CBP-35, Mac-2, RL-29) do exist ( (22) and references therein). These are monomeric 29-35-kDa mosaic proteins, composed of an N-terminal half made of tandem repeats characteristic of the collagen gene superfamily and a C-terminal half homologous to galectin-1 and -2(22) . Galectin-3 also binds laminin (23) and is implicated as component of growth regulatory systems(24) , mediator of cell-cell and cell-matrix interactions(2, 25) , modulator of immune response(26) , marker of neoplastic transformation, and indicator for metastatic potential of melanoma cells(27) .

Galectin-4 was cloned from rat intestine(28) , and a homologous protein was cloned from nematode(29) . Galectin-4 is a monomer with molecular mass of 36 kDa. It contains tandem domains of 140 amino acids each, homologous to galectin-1 and -2, that are separated by a link region (28) . The function of galectin-4 is presently unknown. Here we describe the cloning of a cDNA encoding for a novel protein that we term galectin-8. Galectin-8 has the characteristic properties of other galectins(3, 4) , and it is structurally related (34% identity) to rat galectin-4.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes were purchased from Fermentas. Radiolabeled nucleotides and [S]methionine were from Amersham Corp. (Buckinghamshire, UK). All other reagents were from Sigma unless stated otherwise.

Antibodies

Antisera to insulin receptor substrate 1 (30) (anti-IRS-1) were raised in rabbits, according to standard procedures (31) , by injection of a peptide CYASINFQKQPEDRQ corresponding to the C-terminal 14 amino acids of rat liver IRS-1 (and an additional Cys residue at the N-terminal site). Antibodies were affinity-purified from the serum by adsorption onto a column of peptide coupled to Affi-Gel 10, elution with 100 mM HCl glycine, pH 2.7, and immediate neutralization. Antisera (denoted lp-lec8) against a peptide CQISKETIQKSGKLHL, corresponding to amino acids 168-182 in the link peptide of galectin-8 (and an additional Cys residue at the N-terminal site), were raised in rabbits by a similar procedure. Anti-GST antiserum was a kind gift from Y. Yarden (Weizmann Institute).

Screening of Rat Liver cDNA Expression Library

-Zap rat liver cDNA library in the -ZAP II vector (Stratagene, La Jolla, CA), was screened separately and in duplicate with affinity-purified anti-IRS-1 antibodies (see above). Screening was carried out according to the instruction manual provided by the manufacturer (picoBlue Immunoscreening Kit, Stratagene). Positive plaques were isolated by three repetitive cycles of the procedure. The ExAssist/SOLR system (Stratagene) was used to allow efficient excision of the Bluescript phagemid from the -ZAP vector, and SOLR cells containing positive clones were isolated. Initial DNA sequencing of one positive clone was carried on both strands, using T3 and T7 universal primers with Sequenase version 2.0, (U. S. Biochemical Corp., Cleveland, OH). Subsequent sequencing was carried out with internal primers designed as the sequencing progressed. All other manipulations of nucleic acids, such as restriction, ligations, transformation, gel electrophoresis, blotting, gel elution, radiolabeling, and preparation of buffers, were done using standard protocols(32) . Search of the GenBank revealed that the isolated clone is unique, and it bears no sequence similarity with IRS-1, or the peptide, against which the antibodies were raised. The reason why this clone was picked up by the antibodies remains unclear.

Northern Blot Analysis

RNA extraction was carried out as previously described(33) . Total RNA (30 µg) was electrophoresed, the gel was blotted onto nitrocellulose, and the blot was probed with labeled PCR product which was obtained by the following procedure. Two primers, 5`-CCCGACAATCCCCTATGTCAGTACC-3 and 5`-GCATGGCCAGGCCTGACAACA-3`, were used to amplify the entire cDNA coding sequence of galectin-8, using the cloned cDNA in Bluescript as a template. The PCR products were labeled with [alpha-P]ATP by random priming with DECAprime II DNA labeling kit (Ambion, Austin, TX). Hybridization was carried out at 42 °C in 50% formamide 5 times SSC, and washes were at 60 °C in 0.1 times SSC, 0.1% SDS.

Expression of Recombinant Galectin-8 in Escherichia coli

Expression of galectin-8 as a GST fusion protein (GST-galectin-8) was carried out as follows: two primers, 5`-GGGGGGGGATCCATGTTGTCCTTAAGCAAT-3` and T7, were used to amplify the entire cDNA insert of galectin-8, using the cloned cDNA in Bluescript as a template. The PCR products were digested with BamHI and EcoRI, gel-purified, and ligated into pGeX-2X expression plasmid (Pharmacia) in the TOP bacterial host (Invitrogen). For direct expression of (tag-free) r-galectin-8, a sense primer 5`-GGGGGGCATATGTTGTCCTTAAGCAAT-3` and an antisense primer 5`-GGGGGGGGATCCGCCATTTTGTATTTCCAG-3` were used to amplify the entire coding sequence of galectin-8, using the cloned cDNA in Bluescript as a template. (The EcoRI, NdeI, and BamH I restriction sites, respectively, in the above primers are underlined.) The PCR products were digested by NdeI and BamHI, gel-purified, and ligated into a pET-3a expression plasmid (Novagen) in the pLysS bacterial host. Sequencing of both expression plasmids was carried out to ensure proper, in-frame, ligation of the inserts.

To express GST-galectin-8, bacteria were cultured in 0.5 liter of LB medium until the absorbance at 600 nm was 0.5. Expression of GST-galectin-8 was then induced with 5 mM isopropyl-1-thio-beta-D-galactopyranoside for 4 h. To isolate the recombinant protein, a bacterial pellet was isolated by centrifugation, resuspended in 30 ml of buffer I (phosphate-buffered saline containing 4 mM beta-mercaptoethanol, 2 mM EDTA, 10 µg/ml soybean trypsin inhibitor, 2 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5), and lysed by sonication. Debris were removed by centrifugation at 38,000 times g at 4 °C for 45 min, and 30 ml of the soluble extract were passed over 5 ml of lactosyl-Sepharose. Unbound proteins were eluted with buffer I, while the lectin was subsequently eluted with buffer I containing 100 mM lactose. A similar procedure was utilized to express r-galectin-8 in the pET-3a expression plasmid, save for the fact that the bacteria were centrifuged when the absorbance at 600 nm was 0.3, without addition of isopropyl-1-thio-beta-D-galactopyranoside. Recombinant galectin-8 was isolated under reducing conditions, since in their absence the protein underwent denaturation even when maintained at 4 °C.

In Vitro Translation of Galectin-8

For in vitro translation of galectin-8, the BamHI/EcoRI-digested PCR product, described above, was cloned into pcDNA I mammalian expression plasmid (Invitrogen). In vitro translation in the presence of [S]methionine was performed using the TNT in vitro translation kit (Promega) according to the manufacturer's instructions.

Immunoprecipitation

lp-lec8 antibodies were added to 60 µl of 50% protein A-Sepharose in 0.1 M Tris buffer, pH 8.5, and were incubated for 1 h at 4 °C. Bacterial cell extracts were prepared in buffer I. 500-µl extracts (0.8 mg) were incubated for 2 h with the antibody-protein A-Sepharose complex. Immunocomplexes were washed, suspended in sample buffer(34) , resolved on 10-12% SDS-PAGE(35) , and transferred to nitrocellulose for Western blotting.

Protein Electrophoresis and Blotting

Immunoblotting was carried out essentially as previously described(36) . The blotted proteins were incubated with lp-lec8 antibodies at 4 °C for 16 h and then were extensively washed. To detect antibody binding, a horseradish peroxidase-conjugated protein A ECL kit (Amersham Corp.) was used according to the manufacturer's instructions.

Purification of Galectin-8 from Rat Liver

Freshly isolated rat livers from male Wistar rats were homogenized in buffer I (1 g/5 ml) supplemented with 10 µg/ml aprotinin and 5 µg/ml leupeptin. The homogenate was centrifuged for 1 h at 4 °C at 100,000 times g, and 25 ml of the supernatant were passed over 5 ml of lactosyl-Sepharose, following the procedure described above. The eluted fractions were kept frozen at -20 °C. Since intact galectin-8 denatures upon freezing, the frozen fractions were thawed and centrifuged at 12,000 times g, for 15 min at 4 °C to precipitate, and thus concentrate, galectin-8. Supernatants and pellets were resuspended in sample buffer(34) , resolved by 12% SDS-PAGE, transferred to nitrocellulose, and Western immunoblotted with lp-lec8 antibodies. The amount of galectin-8 in rat liver was estimated using 100,000 times g supernatants that were prepared in buffer I in the presence of 1% Triton X-100.

Assay of Lectin Activity

The biological activity of galectin-8 was assayed by measuring its ability to agglutinate formaldehyde-fixed, trypsin-treated rabbit erythrocyte. Rabbit erythrocytes were trypsin-treated according to Lis and Sharon(37) . Cells were incubated for 1 h at 37 °C with 0.1% trypsin in phosphate-buffered saline, washed five times in 10 volumes of 0.9% NaCl/packed ml of cells, and resuspended in 0.9% NaCl to yield an erythrocyte suspension with an absorbance of 1.5 at 620 nm. An aliquot (0.5 ml) of erythrocyte suspension was incubated for 45 in at room temperature with the lectin solution. Aliquots (0.2 ml) of the upper part of the tube were removed and mixed with 0.8 ml of phosphate-buffered saline, and the optical density at 620 nm was monitored.


RESULTS

Isolation of Galectin-8, a Novel Mammalian Galectin

A cDNA encoding for a new galectin, termed galectin-8, was cloned from a -Zap rat liver cDNA library (Fig. 1). The isolated clone contained an open reading frame (nucleotides 112-1068) with a potential initiation ATG codon at position 121. This open reading frame coded for 316 amino acids, which form a protein of about 35 kDa. The putative coding sequence was followed by a signal for translation termination (TAG) and 176 nucleotides of 3`-untranslated region. Search of the GenBank for similar nucleotide sequences revealed that this sequence is unique. Analysis of galectin-8 using alignment algorithms suggested the presence of two homologous domains 140 amino acids each, linked by a stretch of 32 amino acids (Fig. 2, top). Thirty-eight percent of the amino acids were identical between the first and second domains (Fig. 2, bottom). Both domains contained sequence motifs (e.g. H-NPR, WG-EE) that have been conserved among most CRDs of galectins analyzed so far(2, 38) . Structurally, galectin-8 resembles a 32-kDa beta-galactoside-binding protein from Caenorhabditis elegans(29) (CE-galectin), and rat galectin-4 (galectin-4) (28) that also contain two CRDs connected by a link peptide (Fig. 2). At the level of nucleic acids, galectin-8 is 50 and 45% homologous to galectin-4 and CE-galectin, respectively. At the level of amino acids, galectin-8 shares 34 and 31% identity, respectively, with the above proteins. No homology with any known protein was found in the region of the link peptide. Like other galectins, galectin-8 lacks classical signal sequence or transmembrane segment, but it contains three potential N-linked glycosylation (Asn-X-Ser/Thr) sites. Analysis of its predicted secondary structure (not shown), revealed that the N- and C-terminal domains of galectin-8 share a great degree of structural homology, as expected from their primary structure. Both domains are predicted to form several beta-sheets, structural features of other galectins(10, 11) .


Figure 1: cDNA sequence of galectin-8 and deduced protein sequence. The cDNA sequence of 1247 base pairs contains an open reading frame from 121 to 1069 base pairs, which encodes for a protein of 316 amino acids.




Figure 2: Galectin-8 encodes for a galectin with two homologous carbohydrate-binding regions. A schematic structure of galectin-8 is presented (top). Each box represents a putative carbohydrate-binding domain, linked by a 32-amino acid long peptide. Shown are invariant amino acids preserved in most galectins analyzed so far. The Arg residue, indispensable for sugar binding(9) , located at the C-terminal CRD, and its corresponding Ile residue, localized to the N-terminal CRD, are shown in bold. Amino acid sequences of different galectins are presented for comparison (bottom). These include human galectin-1 (Galec-1)(44) , human galectin-2 (Galec-2)(45) , the carbohydrate binding domain (amino acids 128-263) of rat galectin-3 (Galec-3)(26) , N-terminal (Galec-4-Nt) and C-terminal (Galec-4-Ct) halves of galectin-4(28) , N-terminal (CE-Nt) and C-terminal (CE-Ct) halves of a 32-kDa beta-galactoside-binding protein from C. elegans(29) ; N-terminal (Galec-8-Nt) and C-terminal (Galec-8-Ct) halves of galectin-8. Residues with shared identity are boxed. Residues with shared similarity are shaded.



Galectin-8 Is Widely Expressed

The expression of galectin-8 in different rat tissues was examined by Northern blots (Fig. 3). A single mRNA transcript of 3 kilobases hybridized with the galectin-8 PCR product probe. Unlike galectin-4, which is confined to intestine and stomach(28) , galectin-8 mRNA is highly expressed in lung and to a lower extent in liver, kidney, spleen, hind-limb, and cardiac muscle (Fig. 3, Table 1). Lower levels of expression were detected in brain, and almost no expression was found in whole rat embryos.


Figure 3: Northern blot analysis of RNA from rat tissues probed with galectin-8 cDNA. Top, 30 µg of total RNA from the indicated tissues was electrophoresed, blotted, and probed with labeled galectin-8 PCR product as described under ``Experimental Procedures.'' The migration of the 18 and 28 S rRNA are marked. Bottom, the same blot was stripped and reblotted with cDNA encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).





In Vitro Translated Galectin-8 Is Biologically Active

Galectin-8 cDNA was transcribed and translated in vitro using a TNT (Promega) kit. A S-labeled product of the expected size (34 kDa) was synthesized (Fig. 4). This in vitro translated product was indeed galectin-8 since it could be immunoprecipitated with lp-lec8 antibodies directed against a unique sequence within the link peptide of galectin-8 (Fig. 4). As predicted by its primary amino acid sequence, in vitro translated galectin-8 exhibited the key feature of galectins, namely, capacity to bind to a column of lactosyl-Sepharose in the presence of reducing agents and to be eluted with 0.1 M lactose (not shown).


Figure 4: Immunoprecipitation of in vitro translation product of galectin-8 by lp-lec8 antibodies. Fifty µl of the S-labeled galectin-8, expressed as in vitro translation product (see ``Experimental Procedures''), were immunoprecipitated by lp-lec8 antibodies as described under ``Experimental Procedures.'' Five µl of the total S-labeled galectin-8 (Total), 5 µl of the fraction not precipitated by the antibodies (Sup), and 50 µl of the immunoprecipitated fraction (IP) were subjected to 12% SDS-PAGE and autoradiography.



Recombinant Galectin-8, Expressed in Bacteria, Remains Soluble and Retains Lectin Biological Activity

To further characterize galectin-8, it was expressed in bacteria as a GST fusion protein. GST-galectin-8 remained bound to glutathione-Sepharose beads, and could be eluted with glutathione (not shown). GST-galectin-8 retained its sugar-binding capacity and could be purified by binding to lactosyl-Sepharose and elution with 0.1 M lactose (not shown). Routinely, 3 mg of GST-galectin-8 could be purified in such a way from 1 liter of bacterial extracts. Like other galectins, GST-galectin-8 also maintained hemagglutination activity. Half and maximal activities were obtained with 0.1 and 1 µg/ml GST-galectin-8, respectively.

In a different approach a (tag-free) r-galectin-8 was expressed employing a pET-3a expression plasmid (Novagen) in the pLysS bacterial host. Unlike intestinal recombinant galectin-4 that precipitates and cannot be extracted with buffers that preserve its lectin activity (28) , r-galectin-8 could be readily extracted from bacteria in a soluble form. r-galectin-8 was not subjected to major proteolytic cleavage, as it migrated at the expected size of 34 kDa. Most important, r-galectin-8 retained its sugar-binding activity, and 1.2 mg of protein/liter of bacteria were obtained following its purification over lactosyl-Sepharose column (Fig. 5).


Figure 5: Binding of tag-free r-galectin-8 to lactosyl-Sepharose. Tag-free r-galectin-8 was expressed in pLysS as described under ``Experimental Procedures.'' After centrifugation, 30 ml of the soluble bacterial proteins were purified over 5 ml of lactosyl-Sepharose. r-galectin-8 was eluted with 100 mM lactose in buffer I, and 1-ml fractions were collected. Ten µl of the total and effluent fractions and 50 µl from each elution fraction were resolved by 12% SDS-PAGE, transferred to nitrocellulose, and Western immunoblotted with lp-lec8 antibodies.



Endogenous Galectin-8 Is Present in Rat Liver

To demonstrate the presence of endogenous galectin-8 in rat liver, a cytosolic (100,000 times g supernatant) liver extract was prepared and applied to a column of lactosyl-Sepharose, and proteins retained specifically by the column were eluted with 0.1 M lactose. Advantage was taken of the fact that hepatic galectin-8 denatures and precipitates upon freezing. Fractions, eluted from the lactosyl-Sepharose column, were therefore frozen at -20 °C, thawed, and centrifuged to precipitate, and thus concentrate, the hepatic galectin-8. Staining with Coomassie Blue revealed that most hepatic proteins failed to interact with lactosyl-Sepharose and therefore remained in the flow-through fraction (Fig. 6, bottom). Immunoblotting with lp-lec8 antibodies (Fig. 6, top) revealed that, while hepatic galectin-8 could not be detected in total cytosolic liver extracts, a 36-kDa protein, with the expected size of galectin-8, remained bound to, and could be eluted from, the lactosyl-Sepharose column. Hepatic galectin-8 was readily detected in the pellets, but not in the supernatants of the (frozen and thawed) eluted fractions, indicating that indeed it denatures upon freezing. These results suggest that functionally active cytosolic galectin-8 is present in rat liver (Fig. 6).


Figure 6: Binding of rat hepatic galectin-8 to lactosyl-Sepharose. Five g of rat liver were homogenized in buffer I as described under ``Experimental Procedures,'' and cytosolic extracts (25 ml) were applied over 5 ml of lactosyl-Sepharose. After extensive washing the bound proteins were eluted with 100 mM lactose in buffer I. One-ml fractions were collected and frozen for a period of 16 h in -20 °C. Eluted fractions were thawed and centrifuged for 15 min at 12,000 times g, and the pellets were resuspended in 50 µl of sample buffer(34) . Ten µg of protein of total (A) and effluent (B) fractions as well as 50 µl of the supernatant (C) and resuspended pellet (D) of the eluted fractions (nos. 3-5) were resolved by 12% SDS-PAGE, transferred to nitrocellulose, and Western immunoblotted with lp-lec8 antibodies (top) or subjected to Coomassie staining (bottom).



To estimate the amounts of galectin-8 in rat liver, Triton X-100-soluble liver extracts were prepared and resolved by means of SDS-PAGE. Known amounts of recombinant galectin-8 were run in parallel. All samples were then subjected to Western immunoblotting, using anti galectin-8 antibodies. Assuming that the immunoreactivity of r-galectin-8 and the endogenous hepatic protein are comparable, we calculated that 25 ng of galectin-8 are present in 100 µg of Triton X-100-soluble liver extracts. These findings suggest that galectin-8 comprises 0.025% of total Triton X-100-soluble hepatic proteins.


DISCUSSION

In the present study we describe a novel, widely expressed protein with key features of mammalian galectins. Since the names galectins 1-7 were already assigned(3, 4) , we termed this new protein galectin-8. Three lines of evidence support the notion that galectin-8 has the characteristics of other mammalian galectins: (i) its deduced amino acid sequence contains two domains with conserved motifs that were implicated in carbohydrate binding of galectins; (ii) in vitro translation products of galectin-8 cDNA or recombinant galectin-8 retain biological activity, they specifically bind to a column of lactosyl-Sepharose, and possess hemagglutination activity; and (iii) a protein of the expected size (34 kDa), that binds to lactosyl-Sepharose and reacts with antibodies directed against a unique sequence of galectin-8, is present in rat liver.

Galectin-8 was cloned when a -ZAP rat liver cDNA library was screened with affinity-purified antibodies directed against a 14-amino acid peptide located at the C-terminal end of the IRS-1(39) . Since galectin-8 bears no sequence similarity either to IRS-1, or to the peptide used as immunogen, we suspect that the reactivity toward IRS-1 antibodies could be due to a false positive reaction. This conclusion is supported by the fact that the anti-peptide antibodies used for screening failed to react with purified recombinant galectin-8 either by means of immunoprecipitation, or immunoblotting. (^2)

The primary structure of galectin-8 resembles that of galectin-4, namely, two homologous (38% identity) carbohydrate-binding regions (CRDs) linked by a short 30-amino acid linking peptide. This unique architecture is shared so far only by two galectins: rat galectin-4 (28) and its C. elegans homologue(29) . Other galectin types that contain a single CRD exist and function as noncovalent dimers, which provides them with the potential to aggregate or agglutinate glycoconjugates(37) . Since galectin-4 exists as a monomer(28) , it remains to be determined whether galectin-8 shares a similar property. Hepatic galectin-8 (Fig. 6) has a similar mobility on SDS-PAGE as its recombinant counterpart (Fig. 5). This suggests, although not proves, that hepatic galectin-8 is neither heavily glycosylated, nor it is subjected to extensive post-translational modifications (e.g. phosphorylation).

Although galectin-8 contains two putative CRDs, potential differences in sugar binding between the domains is predicted from a critical difference in their sequence (WG-E-Iversus WG-E-R at the N- and C-terminal CRDs of galectin-8, respectively (cf. Fig. 2)). The (bold) Arg residue has been implicated as playing an important role in the interactions between galectins and the glucose moiety of lactose(10) . Furthermore, site-directed mutagenesis studies (9) indicate that this conserved Arg is indispensable for sugar binding. The presence of Ile (instead of an Arg) at the N-terminal CRD of galectin-8 suggests that this domain might have a different sugar-binding specificity. In that respect galectin-8 resembles galectin-4 whose CRDs are distinct both in structure and sugar binding specificity(28) . Galectin-8 resembles as well a C-type lectin, that functions as the macrophage-mannose receptor (40) and contains eight domains with sequence homology to other C-type CRDs, while only one domain has mannose binding activity by itself when expressed in isolation(2) . The presence of two CRDs with potential different sugar-binding specificity might be required to achieve high affinity binding to multivalent glycoprotein ligands possessing different sugar moieties.

Like other galectins(5) , galectin-8 lacks a classical signal sequence or a transmembrane segment. Indeed, galectin-8 was isolated from the cytosolic fraction of rat liver. These findings do not exclude the possibility that galectin-8, like other galectins(5, 6, 7) , could be externalized by an atypical secretory mechanism. Immunohistochemical studies revealed that secreted galectins are concentrated in evaginations of the plasma membrane, which pinch off to form labile lectin-rich extracellular vesicles which may interact with cell surface proteins(17, 41) . This property of galectins is not unique, as other cytoplasmic proteins, such as thymosin, interleukin-1(42) , and fibroblast growth factor(43) , lack a signal sequence, yet are externalized and function extracellularly. Expression of galectin-8 seems to be developmentally regulated. Very low levels of expression were noted in whole embryos, while high levels of expression were noted in adult tissues. In that respect galectin-8 might resemble other galectins that were implicated as regulators of cell growth(18, 19) and embryogenesis(21) .

Galectin-8 is a novel member of the rapidly growing family of galectins. Although its overall organization resembles that of galectin-4, several important features distinguish the two mammalian proteins. First, unlike galectin-4 that is specifically expressed in intestine and stomach, galectin-8 is expressed in several tissues including lung, liver, kidney, brain, hind limb, and cardiac muscle. Second, unlike galectin-4(28, 29) , but similar to galectins-1 and -2, galectin-8 contains 4 Cys residues. Third, the link peptide of galectin-8 is unique; it bears no similarity to the link peptide of galectin-4 or to the proline- and glycine-rich sequences within the N-terminal half of galectin-3, that contain Tyr or Phe residues at similar intervals(28) . Fourth, the N-terminal CRD of galectin-8 contains a unique WG-E-I motif instead of the consensus WG-E-R/K motif present in both CRDs of galectin-4. Hence, galectin-8 may represent a new ubiquitous subfamily of galectins, consisting of a tandem repeat of CRDs, joined by a linking peptide. Further studies are required to unravel the function of this newcomer to the galectin family.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U09824[GenBank].

§
Incumbent of the Philip Harris and Gerald Ronson Career Development Chair in Diabetes Research. To whom correspondence should be addressed. Fax: 972-8-342-380; Lizick{at}weizmann.weizmann.ac.il.

(^1)
The abbreviations used are: CRD, carbohydrate recognition domain; IRS-1, insulin receptor substrate-1; GST, glutathione S-transferase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; r-galectin-8, recombinant galectin-8.

(^2)
Y. R. Hadari, K. Paz, R. Dekel, T. Mestrovic, D. Accili, and Y. Zick, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Ronit Sagi-Eisenberg and Daniel Schindler for helpful discussions and a critical review of this manuscript.


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