C-terminal Carbohydrate Recognition Domain Research Articles

Ginseng-derived type I rhamnogalacturonan polysaccharide binds to galectin-8 and antagonizes its function

BackgroundPanax ginseng Meyer polysaccharides exhibit various biological functions, like antagonizing galectin-3-mediated cell adhesion and migration. Galectin-8 (Gal-8), with its linker-joined N- and C-terminal carbohydrate recognition domains (CRDs), is also crucial to these biological processes, and thus plays a role in various pathological disorders. Yet the effect of ginseng-derived polysaccharides in modulating Gal-8 function has remained unclear. MethodsP. ginseng-derived pectin was chromatographically isolated and enzymatically digested to obtain a series of polysaccharides. Biolayer Interferometry (BLI) quantified their binding affinity to Gal-8, and their inhibitory effects on Gal-8 was assessed by hemagglutination, cell migration and T-cell apoptosis. ResultsOur ginseng-derived pectin polysaccharides consist mostly of rhamnogalacturonan-I (RG-I) and homogalacturonan (HG). BLI shows that Gal-8 binding rests primarily in RG-I and its β-1,4-galactan side chains, with sub-micromolar KD values. Both N- and C-terminal Gal-8 CRDs bind RG-I, with binding correlated with Gal-8-mediated function. ConclusionP. ginseng RG-I pectin β-1,4-galactan side chains are crucial to binding Gal-8 and antagonizing its function. This study enhances our understanding of galectin-sugar interactions, information that may be used in the development of pharmaceutical agents targeting Gal-8.

Pathogenic bacteria defense and complement activation function analysis of Collectin-10 from Hexagrammos otakii.

With the tremendous success of the artificial breeding of Hexagrammos otakii, the yield has been substantially improved. However, intensive farming often results in bacterial diseases; hence it is imperative to find new antimicrobial molecules. In the present study, we identified a homologous cDNA fragment of collectin-10 from H. otakii, designated as HoCL-10. The cDNA length is 899 bp, which contains an open reading frame (ORF) of 816 bp encoding a secreted protein with 272 amino acid residues. The peptide of HoCL-10 contains an N-terminal collagen domain, a neck region, and a C-terminal carbohydrate recognition domain. The qRT-PCR results revealed that HoCL-10 mRNA was highest expressed in the liver and skin and was significantly induced post-LPS stimulation. The sugar and bacteria binding assay suggested that the recombinant HoCL-10 (rHoCL-10) could recognize various pathogen-associated molecular patterns (PAMPs) and bacteria. For effect on cells, rHoCL-10 enhanced the phagocytosis and migration ability of the macrophage indicated using pro-phagocytosis assay and trans-well assay. To determine the role of HoCL-10 in the complement system, the interaction between HoCL-10 and mannose-binding lectin associated serine protease 1, 2 (MASP-1, 2) were analyzed and demonstrated using ELISA and Far-western. And in vivo, the concentration of membrane-attack complex (MAC) in fish plasma was significantly down-regulated post-injection with HoCL-10 antibody. Finally, the bacteria challenge experiment was performed, implying that HoCL-10 may assist the host in defending against microbial invasion. The findings suggest that HoCL-10 may play crucial roles in host defense against microorganisms, possibly through opsonizing pathogens and activating the complement system.

COLEC10 Induces Endoplasmic Reticulum Stress by Occupying GRP78 and Inhibits Hepatocellular Carcinoma

Collectin subfamily member 10 (COLEC10), a C-type lectin mainly expressed in the liver, is involved in the development of hepatocellular carcinoma (HCC). However, its underlying molecular mechanism in HCC progression remains unknown. In this study, reduced COLEC10 expression in tumor tissues was validated using various HCC cohorts and was associated with poor patient prognosis. COLEC10 overexpression attenuated HCC cell growth and migration abilities in vitro and in vivo. We identified that COLEC10 was a novel interactor of 78-kDa glucose-regulated protein (GRP78), a master modulator of the unfolded protein response in the endoplasmic reticulum (ER). COLEC10 overexpression potentiated ER stress in HCC cells, as demonstrated by elevated expression levels of phosphorylated protein kinase RNA-like ER kinase, phosphorylated inositol-requiring protein 1α, activating transcription factor 4, DNA damage-inducible transcript 3, and X-box-binding protein 1s. The ER in COLEC10-overexpressing cells also showed a dilated and fragmented pattern. Mechanistically, COLEC10 overexpression increases GRP78 occupancy through direct binding by the C-terminal carbohydrate recognition domain in the ER, which released and activated the ER stress transducers protein kinase RNA-like ER kinase and phosphorylated inositol-requiring protein 1α, triggering the unfolded protein response activity. COLEC10-overexpressing HCC cells generated a relatively high reactive oxygen species level and switched to apoptotic cell death under sorafenib-treated conditions. Our study provides the first novel view that COLEC10 inhibits HCC progression by regulating GRP78-mediated ER stress signaling and may serve as a promising therapeutic and prognostic biomarker.

Novel fusion protein PK5-RL-Gal-3C inhibits hepatocellular carcinoma via anti-angiogenesis and cytotoxicity

BackgroundGalectin-3 (Gal-3), the only chimeric β-galactosides-binding lectin, consists of Gal-3N (N-terminal regulatory peptide) and Gal-3C (C-terminal carbohydrate-recognition domain). Interestingly, Gal-3C could specifically inhibit endogenous full-length Gal-3 to exhibit anti-tumor activity. Here, we aimed to further improve the anti-tumor activity of Gal-3C via developing novel fusion proteins.MethodsPK5 (the fifth kringle domain of plasminogen) was introduced to the N-terminus of Gal-3C via rigid linker (RL) to generate novel fusion protein PK5-RL-Gal-3C. Then, we investigated the anti-tumor activity of PK5-RL-Gal-3C in vivo and in vitro by using several experiments, and figured out their molecular mechanisms in anti-angiogenesis and cytotoxicity to hepatocellular carcinoma (HCC).ResultsOur results show that PK5-RL-Gal-3C can inhibit HCC both in vivo and in vitro without obvious toxicity, and also significantly prolong the survival time of tumor-bearing mice. Mechanically, we find that PK5-RL-Gal-3C inhibits angiogenesis and show cytotoxicity to HCC. In detail, HUVEC-related and matrigel plug assays indicate that PK5-RL-Gal-3C plays an important role in inhibiting angiogenesis by regulating HIF1α/VEGF and Ang-2 both in vivo and in vitro. Moreover, PK5-RL-Gal-3C induces cell cycle arrest at G1 phase and apoptosis with inhibition of Cyclin D1, Cyclin D3, CDK4, and Bcl-2, but activation of p27, p21, caspase-3, -8 and -9.ConclusionNovel fusion protein PK5-RL-Gal-3C is potent therapeutic agent by inhibiting tumor angiogenesis in HCC and potential antagonist of Gal-3, which provides new strategy for exploring novel antagonist of Gal-3 and promotes their application in clinical treatment.

Galectin-3 Expression Promotes Pulmonary Hypertension Through Multiple Mechanisms

Pulmonary Hypertension is a progressive vascular disease resulting from the tapering of pulmonary arteries causing high pulmonary arterial blood pressure and ultimately right ventricular failure. A defining characteristic of Pulmonary Hypertension is the excessive remodeling of pulmonary arteries that includes increased proliferation, vascular fibrosis and inflammation. There is no outward cure for Pulmonary Hypertension nor are there interventions that effectively impede or reverse pulmonary arterial remodeling, and pulmonary vascular research over the past several decades has sought to identify novel molecular mechanisms to target for therapeutic benefit. Galectin-3 is a carbohydrate binding lectin that is unique for its chimeric structure, comprised of an N-terminal oligomerization domain and a C-terminal carbohydrate-recognition domain. Galectin-3 is a regulator of modifications in cell behavior that contribute to aberrant pulmonary arterial remodeling including cell proliferation, inflammation, and fibrosis, but its role in Pulmonary Hypertension is poorly understood. In this review, we define Galectin-3 and summarize specific topics regarding the role of Galectin-3 expression in the development of Pulmonary Hypertension by providing evidence which supports the ability of Galectin-3 to influence reactive oxygen species production, NADPH enzyme expression, vascular inflammation and vascular fibrosis, all phenomena which contribute to pulmonary arterial remodeling and the development of Pulmonary Hypertension.

Avian surfactant protein (SP)-A2 first arose in an early tetrapod before the divergence of amphibians and gradually lost the collagen domain

The air-liquid interface of the mammalian lung is lined with pulmonary surfactants, a mixture of specific proteins and lipids that serve a dual purpose-enabling air-breathing and protection against pathogens. In mammals, surfactant proteins A (SP-A) and D (SP -D) are involved in innate defence of the lung. Birds seem to lack the SP-D gene, but possess SP-A2, an additional SP-A-like gene. Here we investigated the evolution of the SP-A and SP-D genes using computational gene prediction, homology, simulation modelling and phylogeny with published avian and other vertebrate genomes. PCR was used to confirm the identity and expression of SP-A analogues in various tissue homogenates of zebra finch and turkey. In silico analysis confirmed the absence of SP-D-like genes in all 47 published avian genomes. Zebra finch and turkey SP-A1 and SP-A2 sequences, confirmed by PCR of lung homogenates, were compared with sequenced and in silico predicted vertebrate homologs to construct a phylogenetic tree. The collagen domain of avian SP-A1, especially that of zebra finch, was dramatically shorter than that of mammalian SP-A. Amphibian and reptilian genomes also contain avian-like SP-A2 protein sequences with a collagen domain. NCBI Gnomon-predicted avian and alligator SP-A2 proteins all lacked the collagen domain completely. Both avian SP-A1 and SP-A2 sequences form separate clades, which are most closely related to their closest relatives, the alligators. The C-terminal carbohydrate recognition domain (CRD) of zebra finch SP-A1 was structurally almost identical to that of rat SP-A. In fact, the CRD of SP-A is highly conserved among all the vertebrates. Birds retained a truncated version of mammalian type SP-A1 as well as a non-collagenous C-type lectin, designated SP-A2, while losing the large collagenous SP-D lectin, reflecting their evolutionary trajectory towards a unidirectional respiratory system. In the context of zoonotic infections, how these evolutionary changes affect avian pulmonary surface protection is not clear.

Molecular characterization and expression profiling of tandem-repeat galectin-8 from red-spotted grouper (Epinephelus akaara): Potential antibacterial, antiviral, and wound healing activities

Galectin-8 is a typical β-galactoside binding lectin, which primarily functions as a pattern recognition receptor and/or danger receptor that is engaged in pathogen recognition by the host innate immune system. Although several fish galectins have been identified, the role of galectin-8 in teleost immunity is still not fully understood. In this study, molecular, transcriptional, and immune-related functions of galectin-8 (EaGal8) from red-spotted grouper (Epinephelus akaara) were analyzed. The open reading frame of EaGal8 comprised 960 bp encoding 319 amino acids of a ∼35 kDa protein, composed of the N- and C-terminal carbohydrate recognition domains joined by a short hinge peptide. Phylogenetic analysis revealed that EaGal8 was closely related to the Epinephelus lanceolatus galectin-8-like protein. Although EaGal8 showed ubiquitous tissue expression, the highest expression level was observed in the blood. Immunostimulants, including lipopolysaccharide, poly(I:C), and nervous necrosis virus, significantly upregulated the EaGal8 transcription level in a time-dependent manner (p < 0.05). Furthermore, recombinant EaGal8 (rEaGal8) showed a binding affinity toward seven different carbohydrates in a concentration-dependent manner. In addition, rEaGal8 caused strong agglutination of fish red blood cells and several gram-positive and gram-negative bacteria, including Streptococcus iniae, Streptococcus parauberis, Lactococcus garvieae, Escherichia coli, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa. For the first time in teleosts, we report the wound healing ability of galectin-8 in this study. At low concentrations, rEaGal8 showed potential wound healing responses in FHM cells, in vitro. Thus, this study reinforces the role of EaGal8 in innate immune responses against bacterial and viral infections and wound healing in red-spotted grouper.

Cloning, Expression, and Purification of Galectins for In Vitro Studies.

Galectins are best known for their ability to bind glycoconjugates containing β-galactose, but classification of these small proteins within the galectin family is also defined by amino acid homology within structural domains and exon/intron junctions within genes. As galectins are expressed by organisms as diverse as some fungi, C. elegans, fish, birds and mammals, and biological activities attributed to galectins are equally diverse, it becomes essential to identify, clone, and characterize galectins from many sources. Glutathione S-transferase (GST) fused to the amino-terminus of galectin cDNAs has proven to be especially useful for the preparation of recombinant galectins in bacteria for use on glycan arrays, in experiments with cultured or isolated cells, and in pull-down assays with immunopurified glycoproteins. Many galectins are stabilized by reducing reagents, such that binding and elution of GST-galectins from glutathione-conjugated Sepharose with excess glutathione is both efficient and innocuous. The ability to bind and elute GST-galectins from lactose-conjugated Sepharose with excess lactose provides a relatively easy means to insure that galectins are competent for glycoconjugate binding prior to experimentation. This chapter focuses primarily on the varied approaches to use GST-galectin binding to glutathione- and lactose-conjugated Sepharose to purify recombinant galectins and then develop effective experimental protocols to characterize the specificity, interactions and function of galectins cloned from any source. We provide one example where a pull-down assay with all the GST-tagged canine galectins reveals that the C-terminal carbohydrate recognition domain of galectin-9 (Gal-9C) specifically recognizes the glycan-dependent apical targeting signal from the glycoprotein MUC1.

Blood Levels of Galectin-9, an Immuno-Regulating Molecule, Reflect the Severity for the Acute and Chronic Infectious Diseases.

Galectin-9 (Gal-9) is a β-galactoside-binding lectin capable of promoting or suppressing the progression of infectious diseases. This protein is susceptible to cleavage of its linker-peptides by several proteases, and the resulting cleaved forms, N-terminal carbohydrate recognition domain (CRD) and C-terminal CRD, bind to various glycans. It has been suggested that full-length (FL)-Gal-9 and the truncated (Tr)-Gal-9s could exert different functions from one another via their different glycan-binding activities. We propose that FL-Gal-9 regulates the pathogenesis of infectious diseases, including human immunodeficiency virus (HIV) infection, HIV co-infected with opportunistic infection (HIV/OI), dengue, malaria, leptospirosis, and tuberculosis (TB). We also suggest that the blood levels of FL-Gal-9 reflect the severity of dengue, malaria, and HIV/OI, and those of Tr-Gal-9 markedly reflect the severity of HIV/OI. Recently, matrix metallopeptidase-9 (MMP-9) was suggested to be an indicator of respiratory failure from coronavirus disease 2019 (COVID-19) as well as useful for differentiating pulmonary from extrapulmonary TB. The protease cleavage of FL-Gal-9 may lead to uncontrolled hyper-immune activation, including a cytokine storm. In summary, Gal-9 has potential to reflect the disease severity for the acute and chronic infectious diseases.

Galectin-3 Promotes ROS, Inflammation, and Vascular Fibrosis in Pulmonary Arterial Hypertension.

Pulmonary Arterial Hypertension (PAH) is a progressive vascular disease arising from the narrowing of pulmonary arteries (PA) resulting in high pulmonary arterial blood pressure and ultimately right ventricular (RV) failure. A defining characteristic of PAH is the excessive remodeling of PA that includes increased proliferation, inflammation, and fibrosis. There is no cure for PAH nor interventions that effectively impede or reverse PA remodeling, and research over the past several decades has sought to identify novel molecular mechanisms of therapeutic benefit. Galectin-3 (Gal-3; Mac-2) is a carbohydrate-binding lectin that is remarkable for its chimeric structure, comprised of an N-terminal oligomerization domain and a C-terminal carbohydrate-recognition domain. Gal-3 is a regulator of changes in cell behavior that contribute to aberrant PA remodeling including cell proliferation, inflammation, and fibrosis, but its role in PAH is poorly understood. Herein, we summarize the recent literature on the role of Gal-3 in the development of PAH and provide experimental evidence supporting the ability of Gal-3 to influence reactive oxygen species (ROS) production, NOX enzyme expression, inflammation, and fibrosis, which contributes to PA remodeling. Finally, we address the clinical significance of Gal-3 as a target in the development of therapeutic agents as a treatment for PAH.

The therapeutic potential of galectin-3 inhibition in fibrotic disease.

Galectin-3 is a beta-galactoside-binding mammalian lectin and part of the 15 member galectin family that are evolutionarily highly conserved. It is the only chimeric protein with a C-terminal carbohydrate recognition domain (CRD) linked to a proline, glycine, and tyrosine rich additional N-terminal domain. Galectin-3 binds several cell surface glycoproteins via its CRD domain as well as undergoing oligomerization, via binding at the N-terminal or the CRD, resulting in the formation of a galectin-3 lattice on the cell surface. The galectin-3 lattice has been regarded as being a crucial mechanism whereby extracellular galectin-3 modulates cellular signalling by prolonging retention time or retarding lateral movement of cell surface receptors in the plasma membrane. As such galectin-3 can regulate various cellular functions such as diffusion, compartmentalization and endocytosis of plasma membrane glycoproteins and glycolipids and the functionality of membrane receptors. In multiple models of organ fibrosis, it has been demonstrated that galectin-3 is potently pro-fibrotic and modulates the activity of fibroblasts and macrophages in chronically inflamed organs. Increased galectin-3 expression also activates myofibroblasts resulting in scar formation and may therefore impact common fibrotic pathways leading to fibrosis in multiple organs. Over the last decade there has been a marked increase in the scientific literature investigating galectin-3 in a range of fibrotic diseases as well as the clinical development of new galectin-3 inhibitors. In this review we will examine the role of galectin-3 in fibrosis, the therapeutic strategies for inhibiting galectin-3 in fibrotic disease and the clinical landscape to date.

Expression and antibacterial analysis of galectin-8 and -9 genes in mandarin fish, Siniperca chuatsi

Galectin-8 and galectin-9 belong to tandem repeat-type galectins, and in the present study, these two genes were cloned in mandarin fish Siniperca chuatsi. The open reading frame (ORF) of the mandarin fish galectin-8 and galectin-9 contains 942, and 1008 bp, encoding 313 and 335 amino acids, respectively. As a conserved feature, an N-terminal carbohydrate recognition domain (CRD), and a C-terminal CRD were observed in each of the two galectins in mandarin fish. In healthy fish, galectin-8 and -9 were constitutively expressed in all organs/tissues examined, and their expression can be induced following the stimulation of LPS and poly(I:C). It is obvious that galectin-8 had a higher increase at mRNA level following the stimulation of poly(I:C). It is further demonstrated that mandarin fish galectin-8 inhibited the growth of Flavobacterium columnare and Streptococcus agalactiae, and in addition to the two species of bacteria, galectin-9 inhibited also the growth of Edwardsiella piscicida, which provides the basis for further understanding their antibacterial role in immune response of mandarin fish.

Galectin-8 Enhances T cell Response by Promotion of Antigen Internalization and Processing.

SummaryGalectin-8 (Gal-8) is a mammalian lectin endowed with immunostimulatory ability. In the present work, we demonstrate that Gal-8-glycan interactions on the surface of antigen-presenting cells (APCs) promote antigen binding and internalization, independently from antigen nature. Both Gal-8 and antigen were together internalized and localized in early endosomes. Interestingly, antigen processing by APCs was also accelerated in the presence of Gal-8 as a separate mechanism, distinct from the increased antigen internalization. Moreover, APCs pulsed together with antigen and Gal-8 were able to activate cognate CD4+ T cells more efficiently than those pulsed with antigen alone. This enhanced antigen presentation was still evident in the absence of costimulatory signals and APCs-derived soluble mediators. Therefore, our results provide evidence for as yet unrecognized mechanism by which Gal-8 stimulates the elicitation of the immune response in a lectin-dependent manner, by inducing antigen uptake and processing upon lattice formation at APCs surface.

Characterization and functional study of Galectin3 from Japanese flounder (Paralichthys olivaceus)

Galectins belong to the β-galactoside binding protein family and participate in both innate and acquired immunity. In this study, we described the molecular characteristics of Galectin3 gene from Japanese flounder (Paralichthys olivaceus), designed as PoGalectin3. Its open reading frame was 1128 bp, encoding a protein composed of 375 amino acids. PoGalectin3 belongs to chimeric galactose agglutinin, which contains a C-terminal carbohydrate recognition domain (CRD) (L250-P372), and its N-terminal is rich in proline (P) and glycine (G). Multiple sequence alignment and phylogenetic tree showed that PoGalectin3 was conservative in different aquatic animals. Tissue distribution confirmed that PoGalectin3 showed significantly highest expression in brain, moderate expression in liver, intestine and muscle. PoGalectin3 was significantly increased post infection with Edwardsiella tarda from intestine tissue of P. olivaceus. In order to investigate the binding ability of PoGalectin3 to pathogen-associated molecular patterns, the recombinant PoGalectin3 protein (rPoGalectin3) was successfully expressed and purified, and an Enzyme linked immunosorbent assay (ELISA) experiment was performed. ELISA refers to the qualitative and quantitative detection method of immune response by combining soluble antigen or antibody with solid-phase carrier. It was confirmed that rPoGalectin3 exhibited high affinity to lipopolysaccharide and peptidoglycan. The rPoGalectin3 also exhibited a concentration dependent binding capacity with Gram-positive bacteria (Bacillus pumilus, Bacillus subtilis, Bacillus cereus) and Gram-negative bacteria (Aeromonas salmonicida, E. tarda, Vibrio vulnificus). In addition, the results of microbial agglutination experiment showed that rPoGalectin3 could agglutinate Gram-positive bacteria (B. pumilus, B. subtilis) and Gram-negative bacteria (A. salmonicida, E. tarda) in the presence of Ca2+. In conclusion, this research laid an important foundation for the specific function analysis of PoGalectin3, which provide theoretical basis for the prevention and control of aquatic diseases.

Modulation of plasma protein expression in bullfrog (Rana catesbeiana) tadpoles during seasonal acclimatization and thermal acclimation

Biological activities in ectothermic vertebrates depend to a great extent on ambient temperature. Adapting their biological systems to annual or short-term alterations in temperature may play an important role in thermal resistance or overwintering survival. Using SDS-PAGE and western blot, we examined plasma proteins in bullfrog (Rana catesbeiana) tadpoles that were seasonally acclimatized (winter vs. summer) or thermally acclimated (4 °C vs. 21 °C) and identified two season-responsive proteins. The first, transthyretin (TTR), is a plasma thyroid hormone distributor protein that was abundant in summer, and the second is a protein containing C-type lectin-like domain (CTLD) that was abundant in winter and cold acclimation of 4 weeks. Sequence analysis revealed that the C-terminal carbohydrate recognition domain of this CTLD protein (termed collectin X) was highly similar to those of the collectin family members, which participate in complement activation of the innate immune system; however, it lacked most of collagen-like domain. Among the hepatic genes involved in the thyroid system, ttr and dio3 were up-regulated, whereas thra and thrb were down-regulated, in summer acclimatization or warm acclimation. In contrast, the collectin X gene (colectx), as well as colect10 and colect11 in the collectin family involved in the innate immune system, were down-regulated during warm acclimation, although fcn2 in the ficolin family was up-regulated during summer acclimatization and warm acclimation. These findings indicate that seasonal acclimatization and thermal acclimation differentially affect some components of the thyroid and innate immune systems at protein and transcript levels.

Characterization and expression of galectin-3 in grass carp (Ctenopharyngodon idella)

Galectins are members of evolutionary conserved lectin family and play important roles in the innate and adaptive immunity of both vertebrates and invertebrates. Galectin-3 is the only chimera galectin with one C-terminal carbohydrate recognition domain (CRD) connected to the N-terminal end. Here, a galectin-3 (named CiGal3) from grass carp was identified and characterized, which encodes polypeptides 362 amino acids with a predicted molecular mass of 36.45 kDa and theoretical isoelectric point of 4.91. The sugar binding motifs involved in carbohydrate binding activity (H–N-R, V–N and W--E-R) were detected in CRD. In comparison to other species, CiGal3 showed the highest similarity and identity to Cyprinus carpio (95.3% sequence similarity and 92.5% sequence identity). The subcellular localization of CiGal3 was distributed in the cytoplasm and nucleus of transfected cells. The CiGal3 transcripts were ubiquitously expressed in all checked tissues and highly expressed in immune tissues. In addition, the expression of CiGal3 in liver and spleen was induced post grass carp reovirus (GCRV), lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly I:C) challenge. These results suggest that CiGal3 plays a vital role in the immune system.

Atomic-resolution crystal structures of the immune protein conglutinin from cow reveal specific interactions of its binding site with N-acetylglucosamine

Bovine conglutinin is an immune protein that is involved in host resistance to microbes and parasites and interacts with complement component iC3b, agglutinates erythrocytes, and neutralizes influenza A virus. Here, we determined the high-resolution (0.97–1.46 Å) crystal structures with and without bound ligand of a recombinant fragment of conglutinin's C-terminal carbohydrate-recognition domain (CRD). The structures disclosed that the high-affinity ligand N-acetyl-d-glucosamine (GlcNAc) binds in the collectin CRD calcium site by interacting with the O3′ and O4′ hydroxyls alongside additional specific interactions of the N-acetyl group oxygen and nitrogen with Lys-343 and Asp-320, respectively. These residues, unique to conglutinin and differing both in sequence and in location from those in other collectins, result in specific, high-affinity binding for GlcNAc. The binding pocket flanking residue Val-339, unlike the equivalent Arg-343 in the homologous human surfactant protein D, is sufficiently small to allow conglutinin Lys-343 access to the bound ligand, whereas Asp-320 lies in an extended loop proximal to the ligand-binding site and bounded at both ends by conserved residues that coordinate to both calcium and ligand. This loop becomes ordered on ligand binding. The electron density revealed both α and β anomers of GlcNAc, consistent with the added α/βGlcNAc mixture. Crystals soaked with α1–2 mannobiose, a putative component of iC3b, reported to bind to conglutinin, failed to reveal bound ligand, suggesting a requirement for presentation of mannobiose as part of an extended physiological ligand. These results reveal a highly specific GlcNAc-binding pocket in conglutinin and a novel collectin mode of carbohydrate recognition.

The expression of galectin-3 in breast cancer and its association with chemoresistance: a systematic review of the literature.

Galectin-3 is a Mr 31,000 protein that belongs to a family of carbohydrate-binding proteins. Galectin-3 has already been associated with protection against apoptosis through cell to cell or cell to matrix adhesion processes. It seems that galectin-3 plays an important role in tumor progression, cell growth, invasion and metastasis. Galectin-3 is the only member of the chimeric galectins that has an N-terminal glycine and proline domain and a C-terminal carbohydrate recognition domain that allows galectin-3 to accommodate larger structures such us polylactosaminoglycans and intervene to DNA damage repair process. In this systematic review, our primary goal is to identify the effect of galectin-3 expression in association with drug resistance and apoptosis inhibition in breast cancer. Scopus and PubMed databases were searched on 26 November 2018 using the following combination of keywords: (galectin-3 OR gal-3 OR LGALS3) AND (breast cancer) AND (chemoresistance OR (drug resistance) OR chemosensitivity). All the articles in English, regardless the time of publication, text availability and species included were initially accepted. In the majority of the included studies, the expression of galectin-3 had a protective role in cell survival via different pathways such as the response to DNA damage and repair or the inhibition of apoptosis after treatment with a chemotherapeutic agent. Galectin-3 expression in breast tumors might be an important factor in the selection of the most suitable treatment.

Identification and Characterization of a Novel Quadruple-Domain Galectin from the Hong Kong Oyster Crassostrea hongkongensis

Galectins, i.e. β-galactoside-binding animal lectins, play essential roles in the innate immunity systems of vertebrates and invertebrates. In the present study, a full-length cDNA coding for a galectin from the Hong Kong oyster Crassostrea hongkongensis (designated ChGal) was cloned and characterized. The complete cDNA of ChGal included an open reading frame (ORF) of 1668 bp, as well as 5′- and 3′-untranslated regions (UTRs) of 75 bp and 302 bp, respectively. The ORF coded for a putative protein of 555 amino acids with an estimated molecular mass of 63.4 kDa and a theoretical isoelectric point of 5.0. Sequence analysis revealed that ChGal contains four carbohydrate recognition domains (CRDs), each containing the conserved carbohydrate-binding motifs H-NPR and WG-ER. Phylogenetic analysis produced an unrooted tree with four clades: single CRDs, C-terminal CRDs with tandem-repeat galectin clade, N-terminal CRDs with tandem-repeat galectin clade, and quadruple-CRD clusters. ChGal clustered in the quadruple-domain galectins, which are divided into four subgroups on the basis of the position of each individual CRD. Recombinant ChGal exhibited strong agglutinating activity against Escherichia coli, Vibrio alginolyticus, and Bacillus thuringiensis. ChGal mRNA was constitutively expressed in all tissues tested, with the highest level detected in the digestive gland. The expression of ChGal significantly increased in haemocytes in response to V. alginolyticus challenge. These results suggest that ChGal play an important role in the innate immune responses of the Hong Kong oyster.

Targeting the C-terminus of galectin-9 induces mesothelioma apoptosis and M2 macrophage depletion

ABSTRACT Galectin-9 has emerged as a promising biological target for cancer immunotherapy due to its role as a regulator of macrophage and T-cell differentiation. In addition, its expression in tumor cells modulates tumor cell adhesion, metastasis, and apoptosis. Malignant mesothelioma (MM) is an aggressive neoplasm of the mesothelial cells lining the pleural and peritoneal cavities, and in this study, we found that both human MM tissues and mouse MM cells express high levels of galectin-9. Using a novel monoclonal antibody (mAb) (Clone P4D2) that binds the C-terminal carbohydrate recognition domain (CRD) of galectin-9, we demonstrate unique agonistic properties resulting in MM cell apoptosis. Furthermore, the P4D2 mAb reduced tumor-associated macrophages differentiation toward a protumor phenotype. Importantly, these effects exerted by the P4D2 mAb were observed in both human and mouse in vitro experiments and not observed with another antigalectin-9 specific mAb (clone P1D9) that engages the N-terminus CRD of galectin-9. In syngeneic murine models of MM, P4D2 mAb treatment inhibited tumor growth and improved survival, with tumors from P4D2-treated mice exhibited reduced infiltration of tumor-associated M2 macrophages. This was consistent with an increased production of inducible nitric oxide synthase, which is a major enzyme-regulating macrophage inflammatory response to cancer. These data suggest that using an antigalectin 9 mAb with agonistic properties similar to those exerted by galectin-9 may provide a novel multitargeted strategy for the treatment of mesothelioma and possibly other galectin-9 expressing tumors.