Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis.

Galectin-1 (GAL-1), a member of a family of conserved beta-galactoside-binding proteins, has been shown to induce in vitro apoptosis of activated T cells and immature thymocytes. We assessed the therapeutic effects and mechanisms of action of delivery of GAL-1 in a collagen-induced arthritis model. A single injection of syngeneic DBA/1 fibroblasts engineered to secrete GAL-1 at the day of disease onset was able to abrogate clinical and histopathological manifestations of arthritis. This effect was reproduced by daily administration of recombinant GAL-1. GAL-1 treatment resulted in reduction in anticollagen immunoglobulin (Ig)G levels. The cytokine profile in draining lymph node cells and the anticollagen IgG isotypes in mice sera at the end of the treatment clearly showed inhibition of the proinflammatory response and skewing towards a type 2-polarized immune reaction. Lymph node cells from mice engaged in the gene therapy protocol increased their susceptibility to antigen-induced apoptosis. Moreover, GAL-1-expressing fibroblasts and recombinant GAL-1 revealed a specific dose-dependent inhibitory effect in vitro in antigen-dependent interleukin 2 production to an A(q)-restricted, collagen type 2-specific T cell hybridoma clone. Thus, a correlation between the apoptotic properties of GAL-1 in vitro and its immunomodulatory properties in vivo supports its therapeutic potential in the treatment of T helper cell type 1-mediated autoimmune disorders.

Cloning and nucleotide sequence of a full-length cDNA for human 14 kDa beta-galactoside-binding lectin.

A full-length cDNA for a 14K-type human lung beta-galactoside-binding lectin was cloned. The cDNA includes a 405 bp open reading frame coding 135 amino acids including the initiator methionine, and having a single internal EcoRI site and a polyadenylation signal. The deduced amino-acid sequence agreed completely with the sequence of a human placenta lectin determined by direct amino-acid sequence analysis (Hirabayashi, J. and Kasai, K. (1988) J. Biochem. 104, 1-4). It showed extensive sequence similarity with other vertebrate 14K-type lectins and a 35K-type lectin (carbohydrate-binding protein 35) of mouse 3T3 cell. Search of a Genbank sequence data base revealed significant sequence similarity between the beta-galactoside-binding lectins and the carboxyl-terminal half of an IgE-binding protein, the cDNA of which has been cloned from rat basophilic leukemia cells. Thus, 14K-type lectin, 35K-type lectin and IgE-binding protein appeared to form a superfamily of proteins. Almost all invariant residues are located in the central region of the 14K-type lectins, so this region may constitute an essential part of the lectins, such as the sugar-binding domain.

Immunological cross-reactivity between beta-galactoside-binding lectins of vertebrates. Possible relationship of antigenicity to oligomeric structure.

Structural relationships among five beta-galactoside-binding lectins isolated from human, mouse and chick were studied using immunochemical methods. The lectins examined were human placenta lectin with a 14-kDa subunit (human 14K lectin), two types of mouse lectin (mouse 15K and mouse 16K lectin), and two types of chick lectin (chick 14K and chick 16K lectin). Five polyclonal antibodies raised against these lectins were used. Antibody to human 14K lectin cross-reacted with mouse 15K and chick 14K lectins. Antibodies to both mouse 15K and chick 14K lectins cross-reacted with human 14K and chick 16K lectins. Antibody to chick 16K lectin cross-reacted with mouse 15K lectin. An immunological relationship was not found between human 14K and chick 16K lectins, or between mouse 15K and chick 14K lectins. Mouse 16K lectin did not show any immunological relationship with any of the other lectins. A monoclonal antibody raised against chick 14K lectin cross-reacted with chick 16K lectin. These results cannot be explained simply in terms of phylogenic distance but suggest that vertebrate beta-galactoside-binding lectins can be classified into two structural groups on the basis of their antigenicities. One group, which is characterized as a monomer type, includes human 14K and chick 14K lectins. The other group, which is characterized as a dimer type, includes mouse 15K and chick 16K lectins.

Production and purification of a recombinant human 14 kDa beta-galactoside-binding lectin.

The cDNA for a 14 kDa human beta-galactoside-binding lectin was inserted into a plasmid carrying a taq promoter, and the lectin protein was expressed in E. coli cells. The recombinant lectin was extracted from the cells and purified to apparent homogeneity by a single-step chromatography on an asialofetuin-agarose column. Subunit molecular mass (14 kDa), hemagglutinating activity and antigenicity were indistinguishable from those of the human placental lectin. Though the N-terminal of the placental lectin is blocked with an acetyl group, the recombinant lectin was found to have a free amino group. However, the N-terminal amino acid sequences were identical. The recombinant lectin was considered to have the same three-dimensional structure as the placental lectin.

Expression of endogenous galectin-1 and galectin-3 in intrahepatic cholangiocarcinoma.

Galectins, a family of beta-galactoside-binding animal lectins, might be involved in tumor progression. In this study, the expression patterns of galectin-1 and -3 were examined immunohistochemically in intrahepatic cholangiocarcinoma (ICC), with emphasis on its development and progression as well as its histopathologic features, by use of samples of normal intrahepatic bile duct (n = 20), biliary epithelial dysplasia (n = 15), ICC (n = 40), and a cholangiocarcinoma cell line, CCKS1. In normal intrahepatic bile ducts, galectin-3 was constitutively but weakly expressed, whereas galectin-1 was not expressed. In hepatolithiasis, biliary epithelial dysplasia was strongly positive for galectin-3 but negative for galectin-1. Galectin-3 was frequently and strongly expressed in the cytoplasm of well-differentiated ICCs, and its expression was significantly decreased and less intense or even absent in poorly differentiated ICCs. Galectin-1 was expressed in carcinoma cells in ICC, and its incidence and extent were correlated with histologic dedifferentiation of ICC. Proliferative cell nuclear antigen (PCNA) labeling index (LI) was higher in ICC cases positive for galectin-1 than in those that were negative. Galectin-1 was strongly expressed in cancerous stroma of ICC, and this stromal expression was related to histologic dedifferentiation of ICC. In the carcinoma cell line CCKS1, galectin-1 and -3 were expressed in the cytoplasm of carcinoma cells, and galectin-1 was additionally detected in the culture medium. These results suggest that galectin-1 was newly expressed on carcinoma cells of ICC, and its overexpression seems to be associated with neoplastic progression and proliferative activities, and the expression of galectin-1 in cancerous stroma may also be related to the progression of ICC. Galectin-3 expression in epithelial cells is up-regulated in the preneoplastic and early neoplastic stages of ICC, although galectin-3 tends to disappear at later stages of ICC. HUM PATHOL 32:302-310.

Complete amino acid sequence of a beta-galactoside-binding lectin from human placenta.

The complete amino acid sequence of a beta-galactoside-binding lectin from human placenta was determined at protein level. The lectin consists of 134 amino acids and its N-terminal alanine is blocked with acetate. The lectin shows about 50% similarity with chick 14K lectin, which was the first vertebrate beta-galactoside-binding lectin completely sequenced. Only 14 residues proved to be different from those of rat lung lectin, the sole mammalian lectin of which the complete sequence has been reported.

Galectins: a family of animal lectins that decipher glycocodes.

Galectins, animal lectins exhibiting specificity for galactosides, are now known to be widely distributed from lower invertebrates, such as sponges and nematodes, to higher vertebrates. The origin of the family can be traced back to the Precambrian era. They are classified into proto-, chimera-, and tandem-repeat types on the basis of protein architecture. The molecular functions of these types should be different because they can cross-link pairs of biomolecules of different combinations. Their biological significance, however, is not yet fully understood because they are involved in too many phenomena, such as differentiation, morphogenesis, metastasis, etc., and too many problems remain unsolved, such as those regarding their controversial cellular localization, mechanism of externalization, etc. Nevertheless, such difficulties seem to indicate their importance as household equipment and their common roles throughout the animal kingdom. They are likely to be responsible for recognizing the N-acetyllactosamine (LacNAc) structure, which is included in various glycoconjugates and considered to be an important glycocode, and then carry out appropriate tasks under given circumstances. Recently, crystallographic studies revealed that galectins and legume lectins such as concanavalin A have a common topology in spite of the absence of sequence homology. This suggests a possible relationship between animal and plant lectins, and the existence of a lectin super family. Studies on the galectin family are becoming increasingly important for glycobiology.

The two lectin domains of the tandem-repeat 32-kDa galectin of the nematode Caenorhabditis elegans have different binding properties. Studies with recombinant protein.

Some properties of recombinant proteins derived from the 32-kDa galectin isolated from the nematode Caenorhabditis elegans, which lectin is composed of two tandemly repeated homologous domains [Hirabayashi et al. (1992) J. Biol. Chem. 267, 15485], were studied in order to elucidate the function of this unique polypeptide architecture. We expressed the whole molecule (N32), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) in Escherichia coli using the expression vector pET21a. All of the recombinant proteins were bound by asialofetuin-Sepharose. CD spectra of the recombinant proteins indicated all of them to be rich in beta-structure and properly refolded. Gel filtration on an HPLC column suggested that all of them existed as monomers. Neither Nh nor Ch seemed to form dimers, in contrast to vertebrate proto-type galectins. Only N32 showed hemagglutination activity towards trypsinized rabbit erythrocytes. Comparison of the affinity of N32, Nh, and Ch for asialofetuin-Sepharose by frontal affinity chromatography [Kasai et al. (1986) J. Chromatogr. 376, 33] showed that Ch has 7-fold weaker affinity than N32, and Nh proved to have still weaker affinity. Since the Asn residue in the CRD (carbohydrate recognition domain), which is conserved in all other galectins, is substituted by Ser in the case of Nh, these data suggest that the two CRDs in this tandem-repeat galectin have different sugar binding properties and that the 32-kDa galectin may serve as a heterobifunctional crosslinker.

Monoclonal antibodies against a beta-galactoside-binding lectin of chick embryo.

Monoclonal antibodies against an endogenous beta-galactoside-binding lectin (monomer molecular weight 14,000, 14K lectin) of chick embryo were prepared and characterized. The inhibitory activities against hemagglutination, antigenic determinants and binding specificities were examined. Monoclonal antibody S1A4-5 strongly inhibited the hemagglutination activity of this lectin. This antibody did not bind to any cyanogen bromide (BrCN) fragment of the lectin. Another monoclonal antibody, S1A4-3, bound to one of the BrCN fragments (residues 34-66). However, this antibody inhibited hemagglutination only weakly. The bindings to isolectins of beta-galactoside-binding lectin, namely 14K lectin (monomer molecular weight 16,000) and a third species which is assumed to be a hybrid molecule consisting of 14K and 16K lectin subunits, were examined. The antibody SIA4-5 bound to 14K lectin but not to 16K lectin. In the case of the third species, intermediate binding was observed.

Further characterization and structural studies on human placenta lectin.

The properties of a previously purified beta-galactoside-binding lectin of human placenta were studied in detail. Isoelectric focusing gave multiple bands around pH 4.9, although the lectin preparation was homogenous in SDS-polyacrylamide gel electrophoresis. High-performance gel chromatography suggested that the lectin exists mainly as the monomer and that a small fraction forms a dimer. From all the criteria examined, human placenta lectin resembles one of the chick lectins obtained from embryonic skin or adult intestine (subunit molecular weight: 14,000). The lectin was inactivated by thiol-modifying reagents, p-chloromercuribenzoic acid and N-ethylmaleimide. Reduced and carboxymethylated lectin contained five carboxymethylated cysteines per subunit, and five free thiol groups were titrated by using 5,5'-dithio-bis(2-nitrobenzoic acid). Preliminary sequence analysis showed the presence of a region highly homologous to the corresponding region of the chick lectin (13 identical residues out of 18 from number 70 to 87 of the chick lectin), suggesting a close evolutionary relation between these lectins and the importance of this conserved region in the function of the lectins.

Complete amino acid sequence of 14 kDa beta-galactoside-binding lectin of chick embryo.

The complete amino acid sequence of a soluble beta-galactoside-binding lectin (subunit MW 14,500) of chick embryo was determined. The protein consists of 134 amino acids beginning with serine and ending with glutamic acid, and its N-terminal was blocked with acetate. The agreement of the present result with that obtained from nucleotide sequence analysis (Y. Ohyama et al. (1986) Biochem. Biophys. Res. Commun. 134, 51-56) indicates the lack of a cleavable leader sequence. Internal homologies were observed in several regions along the polypeptide chain. The highest homology (55% identity) was found between residues 42-58 and residues 112-128. This suggests that chick 14 kDa lectin may have evolved via several gene duplications.

Purification and characterization of beta-galactoside-binding proteins from Caenorhabditis elegans.

Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble beta-galactoside-binding lectins which had previously been only found in vertebrates.

Ligand-binding properties of annexin from Caenorhabditis elegans (annexin XVI, Nex-1).

Annexins are structurally related proteins that bind phospholipids in a calcium-dependent manner. Recently, we showed that annexins IV, V, and VI also bind glycosaminoglycans in a calcium-dependent manner. Annexins are widely distributed from lower to higher eukaryotes, and the nematode Caenorhabditis elegans has been found to contain Nex-1, an annexin homologue. Here, we characterize the ligand-binding properties of Nex-1 using recombinant Nex-1. Nex-1 binds to liposomes containing phosphatidylserine. The apparent K(d) was calculated by Biacore to be 4.4 nM. Compared to mammalian annexins, the Nex-1 phospholipid-binding specificities were similar whereas the K(d) values were one order of magnitude larger. The Nex-1 glycosaminoglycan-binding specificities were investigated by affinity chromatography and solid-phase assays. Nex-1 binds to heparin, heparan sulfate, and chondroitin sulfate but not to chondroitin and chemically N- or O-desulfated heparin. Besides phospholipids, heparan sulfate and/or chondroitin (sulfate), probably on perlecan, could be endogenous ligands of Nex-1.

Release of cytotoxin by macrophages on treatment with human placenta lectin.

Human lectin purified from placenta induced release of cytotoxin from a murine macrophage cell line and human peritoneal monocytes. This activity was not due to contamination of the lectin preparation with lipopolysaccharide.

Applied slalom chromatography improved DNA separation by the use of columns developed for reversed-phase chromatography.

Improved resolution in slalom chromatography, a novel size-fractionation method discovered recently for relatively large DNA molecules (> 5 kpb), was obtained by using columns generally employed for reversed-phase chromatography: i.e., two types of Capcell-Pak (methyl or phenyl-derivatized 5-microns microbeads), and five types of Hypersil-3 packings (trimethylsilyl, dimethyloctyl, cyanopropyl, octadecyl or phenyl-derivatized 3-microns microbeads). The resolution of 5-15-kbp DNA was significantly improved by employing these columns, though the separation characteristics differed. When Capcell-Pak columns were used with a normal low-salt eluting solvent (10 mM sodium phosphate, pH 6.8, 1 mM EDTA), chromatograms were obtained for lambda/HindIII fragments (a mixture of 0.1, 0.5, 2.0, 2.3, 4.4, 6.6, 9.4 and 23.1-kbp fragments) similar to those obtained previously with Asahipak GS-310 5-microns size-exclusion packings. However, when up to 0.2 M NaCl was added to the solvent, the DNA was increasingly retarded, particularly the 4.4, 6.6 and 9.4-kbp fragments, resulting in improved resolution in the low to middle molecular-mass range. The effect of salt was more significant with Capcell-Pak Phe than C1, although various features characteristic of slalom chromatography were preserved with both columns; i.e., dependency on DNA size, flow-rate, and temperature. This suggests that a mixed mode of separation, that is, slalom mode and hydrophobic-interaction mode, was operating. Although all of the Hypersil-3 packings showed significant adsorption of lambda/HindIII fragments under low-salt conditions, the fragments could be eluted with satisfactory yield and resolution by adding acetonitrile (> 5%) to the solvent. Notably, these Hypersil-3 packings allowed resolution of a 4.4-kbp lambda/HindIII fragment from the flow-through fraction for the first time, possibly due to their small particle size. Thus, various packing materials developed for high-performance liquid chromatography proved to be applicable for slalom chromatography, though the eluting conditions still need to be refined. The results support the concept that slalom chromatography is based on a hydrodynamic phenomenon.

Effects of DNA topology, temperature and solvent viscosity on DNA retardation in slalom chromatography.

Slalom chromatography is a unique size-fractionation method applicable to large DNA molecules [>5 kilobase pairs (kbp)]. The method was first developed by using columns packed with microbeads (diameter, <20 microm) used for high-performance liquid chromatography and by applying a relatively fast flow-rate (>0.3 ml/min). Previous studies suggested that the separation is attributed to a hydrodynamic rather than to an equilibrium phenomenon (J. Hirabayashi and K. Kasai, Anal. Biochem. 178 (1989) 336; J. Hirabayashi, N. Itoh, K. Noguchi and K. Kasai, Biochemistry, 29 (1990) 9515). In the present report, the results of a systematic study on the effects of DNA topology, temperature, and solvent viscosity on DNA retardation are described. Firstly, the behaviour of circular (super-coiled) and linearized forms of charomid DNAs (20-42 kbp) was studied. Circular-form DNA molecules were found to be fractionated size-dependently similarly to linear forms in a flow-rate dependent manner. However, the extent of retardation of the circular form DNA was apparently less than that of the corresponding linear forms. Circular DNAs showed almost the same retardation (e.g., 42 kbp) as DNA fragments (e.g., 20 kbp) having approximately half of the size of the former. This observation indicates that DNA retardation is basically related to physical length, not to mass. Secondly, to study the effect of temperature with special reference to solvent viscosity, we carried out chromatographic analysis at various temperatures ranging from 6 to 65 degrees C in both the absence and presence of sucrose (10 or 20%, w/v). The results showed that it is the solvent viscosity that determines the extent of retardation. Taken together, all of physicochemical parameters that define hydrodynamic properties, i.e., particle size, flow-rate and solvent viscosity, proved to be critical in slalom chromatography as well as the potential physical length of the DNA, thus supporting the concept that slalom chromatography is based on a hydrodynamic principle.

Reinforcement of frontal affinity chromatography for effective analysis of lectin-oligosaccharide interactions.

Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 microm diameter), and packed into a miniature column (e.g., 10 x 4.0 mm, 0.126 ml). Twelve pyridylaminated oligosaccharides were applied to the column through a 2-ml sample loop, and their elution patterns were monitored by fluorescence. The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V0); and the dissociation constant, Kd, was calculated according to the literature [K. Kasai, Y. Oda, M. Nishikawa, S. Ishii, J. Chromatogr. 376 (1986) 33]. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The relative affinity of various galectins for asialofetuin could be easily compared in terms of the extent of retardation. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 microg/ml for protein) analyses of lectin-carbohydrate interactions. It should become a powerful tool for elucidation of biomolecular interactions, in particular for functional analysis of a large number of proteins that should be the essential issues of post-genome projects.

Application of reinforced frontal affinity chromatography and advanced processing procedure to the study of the binding property of a Caenorhabditis elegans galectin.

Frontal affinity chromatography is a very useful and simple method to analyze molecular interactions between an analyte and an immobilized ligand by calculating the extent of "retardation" of the elution front. We developed a very simple and efficient data-processing procedure that enables the measurement of very small differences in retardation with precision. This procedure was successfully applied to comparison of the binding properties of recombinant C. elegans galectins for their ligand.