Cell cycle regulation by galectin-12, a new member of the galectin superfamily

Galectins are a family of beta-galactoside-binding animal lectins with conserved carbohydrate recognition domains (CRDs). Here we report the identification and characterization of a new galectin, galectin-12, which contains two domains that are homologous to the galectin CRD. The N-terminal domain contains all of the sequence elements predicted to form the two beta-sheets found in other galectins, as well as conserved carbohydrate-interacting residues. The C-terminal domain shows considerable divergence from the consensus sequence, and many of these conserved residues are not present. Nevertheless, the protein has lactose binding activity, most likely due to the contribution of the N-terminal domain. The mRNA for galectin-12 contains features coding for proteins with growth-regulatory functions. These include start codons in a context that are suboptimal for translation initiation and AU-rich motifs in the 3'-untranslated region, which are known to confer instability to mRNA. Galectin-12 mRNA is sparingly expressed or undetectable in many tissues and cell lines tested, but it is up-regulated in cells synchronized at the G(1) phase or the G(1)/S boundary of the cell cycle. Ectopic expression of galectin-12 in cancer cells causes cell cycle arrest at the G(1) phase and cell growth suppression. We conclude that galectin-12 is a novel regulator of cellular homeostasis.     

Molecular characterization of a novel proto-type antimicrobial protein galectin-1 from striped murrel

In this study, we reported a molecular characterization of a novel proto-type galectin-1 from the striped murrel Channa striatus (named as CsGal-1). The full length CsGal-1 was identified from an established striped murrel cDNA library and further we confirmed the sequence by cloning. The complete cDNA sequence of CsGal-1 is 590 base pairs (bp) in length and its coding region encoded a poly peptide of 135 amino acids. The polypeptide contains a galactoside binding lectin domain at 4–135. The domain carries a sugar binding site at 45–74 along with its signatures (H⁴⁵-X-Asn⁴⁷-X-Arg⁴⁹ and Trp⁶⁹-X-X-Glu⁷²-X-Arg⁷⁴). CsGal-1 shares a highly conserved carbohydrate recognition domain (CRD) with galectin-1 from other proto-type galectin of teleosts. The mRNA expressions of CsGal-1 in healthy and various immune stimulants including Aphanomyces invadans, Aeromonas hydrophila, Escherchia coli lipopolysaccharide and poly I:C injected tissues of C. striatus were examined using qRT-PCR. CsGal-1 mRNA is highly expressed in kidney and is up-regulated with different immune stimulants at various time points. To understand its biological activity, the coding region of CsGal-1 gene was expressed in an E. coli BL21 (DE3) cloning system and its recombinant protein was purified. The recombinant CsGal-1 protein was agglutinated with mouse erythrocytes at a concentration of 4 μg/mL in a calcium independent manner. CsGal-1 activity was inhibited by d-galactose at 25 mM⁻¹ and d-glucose and d-fructose at 100 mM⁻¹. The results of microbial binding assay showed that the recombinant CsGal-1 protein agglutinated only with the Gram-negative bacteria. Interestingly, we observed no agglutination against Gram-positive bacteria. Overall, the study showed that CsGal-1 is an important immune gene involved in the recognition and elimination of pathogens in C. striatus.     

An alternative non-tyrosine protein kinase product of the c-src gene in chicken skeletal muscle.

While the c-src locus is expressed as a 4.0-kilobase (kb) mRNA coding for pp60c-src in various chicken tissues, including embryonic muscle, it is expressed as a novel 3.0-kb mRNA in adult skeletal muscle. We have analyzed the primary structure of this alternatively transcribed and spliced c-src mRNA. The sequence revealed three open reading frames, with the previously defined c-src exons 1 through 5 or 6 comprising the third, on the 3' untranslated region of this 3-kb mRNA. The exons coding for the tyrosine kinase domain of pp60c-src were excluded. On the 5' side, 2 kb of sequence upstream from the previously defined exon 1 of the c-src gene was included in this mRNA. The start site for the 3-kb mRNA probably lies downstream of that for the 4-kb mRNA. The first reading frame of the 3.0-kb mRNA, called sur (for src upstream region), encoded a 24-kilodalton (kDa) protein product rich in cysteine and proline residues. In vitro analysis indicated that the 24-kDa sur protein was membrane associated. Antibodies to sur protein detected in vivo a 24-kDa muscle-specific protein which was developmentally regulated and corresponded to the switch from the 4-kb to the 3-kb c-src mRNA. A striking kinetic pattern of appearance of sur protein and disappearance of pp60c-src suggests that the expression of these two proteins is inversely related.     

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.     

Purification, characterization, structural analysis and protein chemistry of a buffalo heart galectin-1

A soluble β-galactoside-binding lectin was purified by gel filtration chromatography from Bubalus bubalis heart. Its metal-independent nature, molecular weight of 14.5 kDa, preferential affinity for β-d-lactose, and 87–92% identity with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. Stokes radii determination using gel filtration under reducing and non-reducing conditions revealed its homo-dimeric nature, further confirming its Gal-1 nomenclature. The purified lectin was found to be the most stable mammalian heart galectin purified till date, suggesting its preferential use in various recognition studies. Treatment of the purified lectin with oxidizing agent, thiol blocking reagents, denaturants, and detergents resulted in significant changes in UV–VIS, fluorescence, CD and FTIR spectra, which strongly emphasized the important aspect of regular secondary structure of galectins for the maintenance of their active conformation. Reduction of the activity of the purified lectin after oxidation by H₂O₂, with remarkable fluorescence quenching, may suggest potential role for galectin-1 in free radical-induced, oxidative stress-mediated cardiovascular disorders. The predictions of bioinformatics studies were found to be in accordance with the results obtained in wet lab.     

Structural basis for distinct binding properties of the human galectins to Thomsen-Friedenreich antigen

The Thomsen-Friedenreich (TF or T) antigen, Galβ1-3GalNAcα1-O-Ser/Thr, is the core 1 structure of O-linked mucin type glycans appearing in tumor-associated glycosylation. The TF antigen occurs in about 90% of human cancer cells and is a potential ligand for the human endogenous galectins. It has been reported that human galectin-1 (Gal-1) and galectin-3 (Gal-3) can perform their cancer-related functions via specifically recognizing TF antigen. However, the detailed binding properties have not been clarified and structurally characterized. In this work, first we identified the distinct TF-binding abilities of Gal-1 and Gal-3. The affinity to TF antigen for Gal-3 is two orders of magnitude higher than that for Gal-1. The structures of Gal-3 carbohydrate recognition domain (CRD) complexed with TF antigen and derivatives, TFN and GM1, were then determined. These structures show a unique Glu-water-Arg-water motif-based mode as previously observed in the mushroom galectin AAL. The observation demonstrates that this recognition mode is commonly adopted by TF-binding galectins, either as endogenous or exogenous ones. The detailed structural comparisons between Gal-1 and Gal-3 CRD and mutagenesis experiments reveal that a pentad residue motif ((51)AHGDA(55)) at the loop (g1-L4) connecting β-strands 4 and 5 of Gal-1 produces a serious steric hindrance for TF binding. This motif is the main structural basis for Gal-1 with the low affinity to TF antigen. These findings provide the intrinsic structural elements for regulating the TF-binding activity of Gal-1 in some special conditions and also show certain target and approach for mediating some tumor-related bioactivities of human galectins.     

Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro

Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin-stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.     

Molecular cloning of a mouse scavenger receptor with C-type lectin (SRCL)(1), a novel member of the scavenger receptor family.

The cDNA clone encoding a mouse scavenger receptor with C-type lectin (SRCL), a novel member of the scavenger receptor family, has been isolated from a mouse embryonic cDNA library. The predicted cDNA sequence contains a 2226 bp open reading frame encoding a coiled-coil, collagen-like, C-type lectin/carbohydrate recognition domain with an overall sequence identity of 92% to human SRCL. In contrast to human, mouse SRCL mRNA was expressed ubiquitously in various adult tissues including the liver and spleen, in which human SRCL mRNA was under detection limits. Mouse SRCL mRNA was expressed in the macrophage cell line J774A.1 cells at a high level and in the embryo as early as E9.     

Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens

Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward a glycan microarray containing hundreds of structurally diverse glycans, and we compared these results to binding determinants on cells. All three galectins exhibited differences in glycan binding characteristics. On both the microarray and on cells, Gal-2 and Gal-3 exhibited higher binding than Gal-1 to fucose-containing A and B blood group antigens. Gal-2 exhibited significantly reduced binding to all sialylated glycans, whereas Gal-1 bound alpha2-3- but not alpha2-6-sialylated glycans, and Gal-3 bound to some glycans terminating in either alpha2-3- or alpha2-6-sialic acid. The effects of sialylation on Gal-1, Gal-2, and Gal-3 binding to cells also reflected differences in cellular sensitivity to Gal-1-, Gal-2-, and Gal-3-induced phosphatidylserine exposure. Each galectin exhibited higher binding for glycans with poly-N-acetyllactosamine (poly(LacNAc)) sequences (Galbeta1-4GlcNAc)(n) when compared with N-acetyllactosamine (LacNAc) glycans (Galbeta1-4GlcNAc). However, only Gal-3 bound internal LacNAc within poly(LacNAc). These results demonstrate that each of these galectins mechanistically differ in their binding to glycans on the microarrays and that these differences are reflected in the determinants required for cell binding and signaling. The specific glycan recognition by each galectin underscores the basis for differences in their biological activities.     

Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM

A novel 36-kDa endochitinase named chit36 has been isolated and characterized from Trichoderma harzianum Rifai TM. Partial amino acid sequences from the purified protein were used to clone the fungal cDNA, based on polymerase chain reaction with degenerate primers. The complete open reading frame encodes a 344-amino acid protein which shows 84% similarity to a putative chitinase from Streptomyces coelicolor. Chit36 was overexpressed under the pki1 constitutive promoter from Trichoderma reesei via biolistic transformation of T. harzianum TM. Stable transformants showed expression and endochitinase activity of chit36 in glucose-rich medium. Culture filtrates containing secreted CHIT36 as the sole chitinolytic enzyme completely inhibited the germination of Botrytis cinerea conidia. Growth of Fusarium oxysporum f. sp. melonis and Sclerotium rolfsii were significantly inhibited on agar plates on which the Trichoderma transformants had previously been grown.     

Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.

The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466, 1991) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUF1. Using antisera specific for these polypeptides, we demonstrate that the 37- and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation.     

Dimeric Galectin-8 induces phosphatidylserine exposure in leukocytes through polylactosamine recognition by the C-terminal domain

Human galectins have distinct and overlapping biological roles in immunological homeostasis. However, the underlying differences among galectins in glycan binding specificity regulating these functions are unclear. Galectin-8 (Gal-8), a tandem repeat galectin, has two distinct carbohydrate recognition domains (CRDs) that may cross-link cell surface counter receptors. Here we report that each Gal-8 CRD has differential glycan binding specificity and that cell signaling activity resides in the C-terminal CRD. Full-length Gal-8 and recombinant individual domains (Gal-8N and Gal-8C) bound to human HL60 cells, but only full-length Gal-8 signaled phosphatidylserine (PS) exposure in cells, which occurred independently of apoptosis. Although desialylation of cells did not alter Gal-8 binding, it enhanced cellular sensitivity to Gal-8-induced PS exposure. By contrast, HL60 cell desialylation increased binding by Gal-8C but reduced Gal-8N binding. Enzymatic reduction in surface poly-N-acetyllactosamine (polyLacNAc) glycans in HL60 cells reduced cell surface binding by Gal-8C but did not alter Gal-8N binding. Cross-linking and light scattering studies showed that Gal-8 is dimeric, and studies on individual subunits indicate that dimerization occurs through the Gal-8N domain. Mutations of individual domains within full-length Gal-8 showed that signaling activity toward HL60 cells resides in the C-terminal domain. In glycan microarray analyses, each CRD of Gal-8 showed different binding, with Gal-8N recognizing sulfated and sialylated glycans and Gal-8C recognizing blood group antigens and polyLacNAc glycans. These results demonstrate that Gal-8 dimerization promotes functional bivalency of each CRD, which allows Gal-8 to signal PS exposure in leukocytes entirely through C-terminal domain recognition of polyLacNAc glycans.     

A new isoform of human membrane-bound IgE.

The epsilon-chain of membrane-bound IgE on the surface of B lymphocytes is known to contain a membrane-anchoring peptide segment that is encoded by two membrane exons, me.1 and me.2. In analyzing pertinent segments in mRNA from human IgE-expressing B cells by using PCR methods and Northern blotting analyses, we have identified three species of mRNA of epsilon-chain with variations in the splicing of the membrane exons. The conventional species (m/s) contains the predicted me.1 and me.2; species m/1 harbors 156 extra nucleotides 5' of me.1 with unaltered reading frame; species s/t lacks me.1 and hence the segment encoding the hydrophobic transmembrane stretch and contains a shifted me.2 reading frame. Rabbit antibodies, which were prepared by immunization using a peptide of 36 amino acid residues representing an encoded segment unique to mRNA species m/l, could specifically bind to human IgE-expressing B cell lines and react with an epsilon-chain on Western immunoblots. These results indicate that there exists a previously unidentified isoform of human membrane-bound IgE.     

Galectin-3 regulates RasGRP4-mediated activation of N-Ras and H-Ras

Galectin-3 (Gal-3) is a pleiotropic beta-galactoside-binding protein expressed at relatively high levels in human neoplasms. Its carbohydrate recognition domain (CRD) contains a hydrophobic pocket that can accommodate the farnesyl moiety of K-Ras. Binding of K-Ras to Gal-3 stabilizes K-Ras in its active (GTP-bound) state. Gal-3, which does not interact with N-Ras, was nevertheless shown to reduce N-Ras-GTP in BT-549 cells by an unknown mechanism that we explored here. First, comparative analysis of various cancer cell lines (glioblastomas, breast cancer cells and ovarian carcinomas) showed a positive correlation between low N-Ras-GTP/high K-Ras-GTP phenotype and Gal-3 expression levels. Next we found that epidermal growth factor-stimulated GTP loading of N-Ras, but not of K-Ras, is blocked in cells expressing high levels of Gal-3. Activation of Ras guanine nucleotide releasing proteins (RasGRPs) by phorbol 12-myristate 13-acetate (PMA) or downregulation of Gal-3 by Gal-3 shRNA increased the levels of N-Ras-GTP in Gal-3 expressing cells. We further show that the N-terminal domain of Gal-3 interacts with and inhibits RasGRP4-mediated GTP loading on N-Ras and H-Ras proteins. Growth of BT-549 cells stably expressing the Gal-3 N-terminal domain was strongly attenuated. Overall, these experiments demonstrate a new control mechanism of Ras activation in cancer cells whereby the Gal-3 N-terminal domain inhibits activation of N-Ras and H-Ras proteins.     

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.     

Cloning, mapping and genomic organization of a fish C-type lectin gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Utilizing a splenic cDNA library and rapid amplification of cDNA 5' ends (5'-RACE), a C-type lectin gene was cloned from a homozygous cloned rainbow trout. The 1176 bp cDNA contains a 714 bp open reading frame from which a 238-amino-acid (aa) (27 kDa) protein was deduced. It was confirmed that this protein belongs to the C-type animal lectins, and is a type II membrane receptor. The predicted protein from this sequence contains a 48 aa cytoplasmic domain, a 20 aa transmembrane domain (TM), a 46 aa stalk region and a 124 aa carbohydrate-recognition domain (CRD). The stalk region contains a leucine-zipper, and an N-glycosylation site was also found in the CRD. Sequence alignment and phylogenetic analysis of the CRD indicate that the protein has similarity with human dendritic cell immunoreceptor (DCIR), gp120 binding C-type lectin (gp120BCL) and mammalian hepatic lectins. The N-terminus (aa 4-183) has similarity with NKG2, a group of C-type lectin receptors important in human natural killer cell function. The genomic DNA (gDNA) containing this gene was amplified and sequenced. The 4569 bp gDNA contains five exons and four introns. The first three exons encode the cytoplasmic domain, the TM and stalk region, respectively. Unlike the other type II C-type lectin receptors in which the CRD was encoded by three exons, the CRD of this lectin was encoded by two exons. A transposon Tc1-like fragment was found in intron III. Intron IV is composed of a simple repeat. Tissue-specific expression of the gene was studied by RT-PCR, and it was mainly expressed in spleen and peripheral blood leukocyte (PBL). Using AluI to digest the fragment containing exon I, intron I and exon II, an RFLP was produced between the sequences of this gene in two cloned fish, OSU 142 and Arlee (AR). Seventy-one doubled haploids (DH) of OSU X AR were screened, and the gene was mapped to linkage group XIV on the published map (Young et al., Genetics 148 (1998) 839).     

cDNA cloning and mRNA expression of a tandem-repeat galectin (PoGal2) from the pearl oyster, Pinctada fucata

Galectins can recognize and specifically bind to β-galactoside residues, playing crucial roles in innate immune responses of vertebrates and invertebrates. We cloned the cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated as PoGal2). PoGal2 cDNA is 1347 bp long and consists of a 5'-untranslated region (UTR) of 3 bp, a 3'-UTR of 297 bp with one cytokine RNA instability motif (ATTTA), and an open reading frame of 1047 bp, encoding a polypeptide of 349 amino acids, with an estimated molecular mass of 38.1 kDa and a theoretical isoelectric point of 8.5. PoGal2 contains two carbohydrate recognition domains (CRDs); both have the conserved carbohydrate-binding motifs H-NPR and WG-EE. PoGal2 shares 50.6 and 50.9% identity with those of abalone (Haliotis discus) and the Manila clam (Venerupis philippinarum), respectively. Phylogenetic analysis revealed that the tandem-repeat galectins formed two clades for the different species. Molluscan tandem-repeat galectins were clustered into a single clade, and nematode tandem-repeat galectins were clustered into another single clade. In both clades, CRD-N and CRD-C were divided into different groups. PoGal2 mRNA was constitutively expressed in all tissues analyzed, and the expression level of PoGal2 mRNA was found to be significantly up-regulated in digestive glands, gills and hemocytes after Vibrio alginolyticus stimulation/infection. Expression profile analysis showed that the expression level of PoGal2 mRNA was significantly up-regulated at 8, 12 and 24 h after V. alginolyticus infection. These results suggest that PoGal2 is a constitutive and inducible acute-phase protein involved in the innate immune response of pearl oysters.