Galactose specific lectins from prostate tissue with different pathologies: biochemical and cellular studies.

BACKGROUND: Galectins-galactose-specific lectins are involved in various types of cell activities, including apoptosis, cell cycle regulation, inflammation and cell transformation. Galectins are implicated in prostate malignat transformation. It is not known yet if prostate glands with different grade of pathologies are expressing different galectins and if these galectins express different effects on the cell viability. METHODS: Cytosolic galactose-spesific lectin fractions from prostate tissue with different diagnosis were purified by affinity chromatography and analyzed by electrophoresis in polyacrylamide gel electrophoresis with sodium dodecyl sulphate. The lectin effects in a source-dependent maner were studied on cell viability on peripheral lymphocytes by MTT reduction method and on apoptosis by flow cytometry method. RESULTS: Affinity purified galactose-specific lectins fractions from normal and pathological tissue samples are characterized with different protein composition and they express different effects on cell viability and apoptosis. CONCLUSION: The effects of cytosolic galactose-specific lectins depend on the source of lectin fraction (glandular tissue disease). We suppose that the released cytosolic galectins from prostatic high grade intraepithelial neoplasia and adenocarcinoma tissue could suppress the immune status of the host patients.

Galactose-Binding C-Type Lectin Promotes Cellular Aggregation of Coelomocytes in Sea Cucumber.

Echinoderms have a large coelomic cavity containing coelomocytes. When the coelomic fluid is removed from the cavity, the cells aggregate immediately. We found that a fraction or an extract of the intestine of the sea cucumber, Apostichopus japonicus, markedly accelerated cellular movement and aggregation on a glass slide, and this effect was clearly inhibited by galactose. We successfully purified the aggregation-promoting factor, a 16 kDa protein, from the intestine. TOF-MS analysis followed by de novo sequencing revealed that the protein is a C-type lectin. RNA-seq data and cDNA cloning demonstrated the factor to be a novel lectin, named AjGBCL, consisting of 158 aa residues in the mature form. Microscopic observation revealed that most of the aggregating cells moved toward aggregates and not to an intestinal fragment, suggesting that AjGBCL is not a chemoattractant but a cellular aggregation-inducing factor that may induce aggregates to release chemoattractant. We report, for the first time, an endogenous molecule that promotes coelomocyte aggregation in echinoderms.

Cytotoxicity of Frutalin on Distinct Cancer Cells Is Independent of Its Glycosylation.

Frutalin is a plant lectin with beneficial immunobiological action, although the access to its active form is still restricted. Moreover, there is a knowledge gap on isoform activity and glycosylation impact on its bioactivity, and recombinant production protocols were seen as ineffective. Here, a simpler and faster production and purification protocol was developed, attaining a yield of purified frutalin 3.3-fold higher than that obtained previously. Hemagglutination assays confirmed that this frutalin isoform could not agglutinate rabbit erythrocytes, while maintaining the native tetrameric structure, as indicated by DLS analysis, and strong interaction with methyl-alpha-galactose, in fluorescence spectroscopy studies. The cytotoxicity of the recombinant frutalin isoform was shown in a broad panel of human cancer cells: colon (HCT116), melanoma (A375), triple-negative breast cancer (MDA-MB-231), and ovarian (IGROV-1). Treatment with 8.5-11.8 µM TrxFTL reduced proliferation of all cancer cells to half in 48 h. This anti-proliferative effect encompasses the p53 pathway since it was significantly reduced in p53-null colon cancer cells (HCT116 p53-/-; GI50 of 25.0 ± 3.0 µM), when compared to the isogenic p53-positive cells (HCT116 p53+/+; GI50 of 8.7 ± 1.8 µM; p < 0.002). This recombinantly produced frutalin isoform has relevant cytotoxic effect and its biological activity is not dependent on glycosylation. The developed E. coli production and purification protocol generates high yield of non-glycosylated frutalin isoform with potent cytotoxic activity, enabling the development of novel anticancer p53-targeting therapies.

Imitating evolution's tinkering by protein engineering reveals extension of human galectin-7 activity.

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.

Isolation, characterization of galactose-specific lectin from Odoiporus longicollis and its antibacterial and anticancer activities.

Lectins are renowned hemagglutinins and multivalent proteins with a well known quality for sugar-binding specificity that participate significantly in invertebrate defense functions. Studies on biological activity of lectin from coleopteran insect are very scarce. In this study, lectin from the hemolymph in the grub of banana pest, Odoiporus longicollis was subjected to purification, biochemical and functional characterizations. The lectin was purified by PEG precipitation and ion-exchange chromatography using Q-Sepharose as a matrix. The purified lectin showed hemagglutination activity against rat erythrocytes, heat-labile, cation independent and insensitive to EDTA. Further, the carbohydrate affinity of this lectin was found with mannitol, adonitol, L-arabinose, L-rhamnose, D-galactose and sorbitol. The native form of purified lectin was calculated as 360 kDa by FPLC system. Denatured gel electrophoresis of the purified lectin consisted of five distinct polypeptides with molecular weights approximately 160, 60, 52, 40 and 38 kDa, respectively. The amino acid sequences obtained through peptide mass fingerprinting analysis exhibited homologies to the known conserved regions of galactose binding lectins. Further, the purified lectin exhibited bacterial inhibition with LPS from Serratia marcescens. In addition, isolated lectin also exerted bacterial agglutination, antibacterial and anti-proliferative activity against Mycobacterium smegmatis, Bacillus pumilus and Neuro 2a cell line, respectively.

[Cell Function Research of ß-Trefoil Lectins from Mytilidae].

Two novel ß-trefoil lectins, MytiLec-1 and SeviL were found from mussels in the coast of Yokohama and Nagasaki. MytiLec-1 was purified from gill and mantle of Mytilus galloprovincialis. It was consisted of 149 amino acid residues and there was no similarity with any other proteins when it was discovered. We advocate for this "Mytilectin" as a new protein family because of their novelty of its primary structure and homologues were also found in other mussels. Glycan array analysis revealed that MytiLec-1 specifically bound to the Gb3 and Gb4 glycan which contained the α-galactoside. MytiLec-1 caused the apoptosis against the Burkitt's lymphoma cells through the interaction of Gb3 express in their cell surface. On the other hand, SeviL obtained from gill and mantle of Mytilisepta virgata showed the specific binding against GM1b, asialo GM1 and SSEA-4 which are known as glycosphingolipid glycan including the ß-galactoside. In addition, SeviL was identified as R type lectin by confirmation of QXW motif within its primary structure. Messenger RNA of SeviL like R type lectins was also found among the musssels including Mytilus galloprovincialis. SeviL also showed the apoptosis against asialo GM1 expressing cells. To apply the anticancer lectin as a novel molecular target drug, primary structure of MytiLec-1 was analyzed to enhance the stabilization of confirmation by computational design technique. It was succeeded to produce a monomeric artificial ß-trefoil lectin, Mitsuba-1 without losing the Gb3 binding ability. Comparison of biological function between Mitsuba-1 and MytiLec-1 is also described in this study.

Proteomic analysis shows that the main constituent of subepidermal localised cutaneous amyloidosis is not galectin-7.

Lichen or macular localised cutaneous amyloidoses have long been described as keratinic amyloidoses and believed to be due to the deposition of cytokeratin peptides originating from epidermis in the dermal papillae. However, recently it was suggested that galectin-7 is the causative protein for this type of amyloidosis. This was based on the detection of galectin-7 in a biopsy from a patient diagnosed with Bowen's disease and localised cutaneous amyloidosis. In this study we report mass spectrometry-based proteomic analysis of the protein composition of localised cutaneous amyloid deposits from seven patients using laser microdissection and show that basal keratins are the main constituents of the amyloid deposits. Galectin-7 was not present in the dermal amyloid deposits and was only present in the overlying Congo red negative epidermis.

Purification of Recombinant Galectins Expressed in Bacteria.

Galectins are soluble lectins that participate in many physiological and pathological functions. Since they can act extracellularly, the use of the recombinant protein is a recurrent strategy for studying their biological functions. Here, we provide a general protocol for the production of Galectins and their isolated or chimeric domains. We take advantage of their lectin activity and the 6xHis-tag addition for purification, thus obtaining a highly pure and active Galectin to use in both in vitro and in vivo assays. For complete details on the use and execution of this protocol, please refer to Cattaneo et al. (2011), Tribulatti et al. (2012), and Prato et al. (2020).

Galectin-3 is modulated in pancreatic cancer cells under hypoxia and nutrient deprivation.

Pancreatic ductal adenocarcinoma is one of the most aggressive tumors with a microenvironment marked by hypoxia and starvation. Galectin-3 has been evaluated in solid tumors and seems to present both pro/anti-tumor effects. So, this study aims to characterize the expression of Galectin-3 from pancreatic tumor cells and analyze its influence for cell survive and motility in mimetic microenvironment. For this, cell cycle and cell death were accessed through flow cytometry. Characterization of inside and outside Galectin-3 was performed through Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), immunofluorescence, Western blot, and ELISA. Consequences of Galectin-3 extracellular inhibition were investigated using cell death and scratch assays. PANC-1 showed increased Galectin-3 mRNA expression when cultivated in hypoxia for 24 and 48 h. After 24 h in simultaneously hypoxic/deprived incubation, PANC-1 shows increased Galectin-3 protein and secreted levels. For Mia PaCa-2, cultivation in deprivation was determinant for the increasing in Galectin-3 mRNA expression. When cultivated in simultaneously hypoxic/deprived condition, Mia PaCa-2 also presented increasing for the Galectin-3 secreted levels. Treatment of PANC-1 cells with lactose increased the death rate when cells were incubated simultaneously hypoxic/deprived condition. Therefore, it is possible to conclude that the microenvironmental conditions modulate the Galectin-3 expression on the transcriptional and translational levels for pancreatic cancer cells.

Metazoan Soluble ß-Galactoside-Binding Lectins, Galectins: Methods for Purification, Characterization of Their Carbohydrate-Binding Specificity, and Probing Their Ligands.

Galectins are a family of soluble ß-galactoside-binding proteins that share conserved carbohydrate recognition domain. Galectins are found in most multicellular organisms and exert various biological functions by binding to the surface glycoconjugates as lectins. In this chapter, we describe the general methods of purification of galectins, quality control of purified galectins, some example methods of evaluating their carbohydrate-binding activity, and use of galectin to search or detect galectin ligands as well as a series of precautions for the usage of galectins.

ß-Galactoside binding lectin from caddisfly larvae, Stenopsyche kodaikanalensis with selective modes of antibacterial activity: Purification and characterization.

Insects sustain the invading bacterial pathogens by inducing the production of lectin which participates in surveillance of non-self molecules. The antibacterial property of lectin is an inevitable aspect of innate immune system especially for the insects feeding the detritus organic matter. ß-galactoside binding lectin possessing antibacterial property was detected and purified from the hemolymph of larvae of caddisfly, Stenopsyche kodaikanalensis using affinity chromatography. The purified lectin exhibited highest hemagglutination titer value against buffalo erythrocytes and has affinity to lactose and fetuin which contains ß-galactoside linkages. It was found to be calcium independent, EDTA insensitive and heat labile. These reveal the characteristics features of S-Lac lectin. The molecular weight of lectin was 360 kDa with five distinct subunits such as 95, 90, 66, 62 and 47 kDa. The sequences acquired through MALDI-TOF-MS analysis shared homologies to the putative conserved region of leguminous lectin. Antibacterial studies were carried out with native soil bacterial isolates. It revealed that the lectin possessed the specific modes of action against bacteria that it can agglutinate the Bacillus subtilis and lyse the Bacillus flexus.

Neuropharmacological characterization of frutalin in mice: Evidence of an antidepressant-like effect mediated by the NMDA receptor/NO/cGMP pathway.

In this study we evaluated the effect of frutalin (FTL) on mouse behavior. Mice (n=6/group) were treated (i.p.) with FTL (0.25; 0.5 or 1mg/kg) or vehicle and submitted to several tests (hole-board/HBT, elevated plus maze/PMT, open field/OFT, tail suspension/TST, or forced swimming/FST). Yohimbine, ketamine, l-NAME, aminoguanidine, 7-NI, methylene blue, l-arginine or dl-serine was administered 30min before FTL (0.5mg/kg). To evaluate the subchronic effect, animals were injected with FTL or vehicle for 7days and submitted to the FST. Molecular docking was simulated using FTL against NOS and the NMDA receptor. No changes were observed in the HBT or the OFT. FTL (0.25mg/kg) increased the number of entries into enclosed arms in the PMT. FTL reduced immobility in the TST (0.25 and 0.5mg/kg) and the FST (0.25mg/kg; 0.5mg/kg). The effect of FTL was dependent on carbohydrate interaction and protein structure integrity and was reduced by ketamine, l-NAME, aminoguanidine, 7-NI and methylene blue, but not by l-arginine, yohimbine or dl-serine. The antidepressant-like effect remained after subchronic treatment. The molecular docking study revealed a strong interaction between FTL and NOS and NMDA. FTL was found to have an antidepressant-like effect mediated by the NMDA receptor/NO/cGMP pathway.

Galectin-10: a new structural type of prototype galectin dimer and effects on saccharide ligand binding.

Galectin-10 (Gal-10) which forms Charcot-Leyden crystals in vivo, is crucial to regulating lymph cell function. Here, we solved the crystal structures of Gal-10 and eight variants at resolutions of 1.55-2.00 Å. Structural analysis and size exclusion chromatography demonstrated that Gal-10 dimerizes with a novel global shape that is different from that of other prototype galectins (e.g., Gal-1, -2 and -7). In the Gal-10 dimer, Glu33 from one subunit modifies the carbohydrate-binding site of another, essentially inhibiting disaccharide binding. Nevertheless, glycerol (and possibly other small hydroxylated molecules) can interact with residues at the ligand binding site, with His53 being the most crucial for binding. Alanine substitution of the conserved Trp residue (Trp72) that is crucial to saccharide binding in other galectins, actually leads to enhanced erythrocyte agglutination, suggesting that Trp72 negatively regulates Gal-10 ligand binding. Overall, our crystallographic and biochemical results provide insight into Gal-10 ligand binding specificity.

SL15: A seminal plasma-derived lectin from the sperm of llama (Lama glama).

The oviductal sperm reservoir of South American camelids is formed when sperm bind to N-acetylgalactosamine (GalNAc) on the surface of oviductal epithelium. The aim of this study was to characterize the GalNAc-binding proteins on llama sperm, and to establish their origin. Sperm-adsorbed proteins were extracted with 0.5 M KCl in Hepes-balanced salts. Sperm-adsorbed and seminal plasma proteins were then subjected to ligand blotting for their GalNAc affinity, and the labeled bands were identified by mass spectrometry. Three proteins were identified in seminal plasma versus only one in the sperm-adsorbed population; SL15, a seminal lectin, was common to both. SL15 is a homologue of Zymogen granule protein 16, homolog B-like, which belongs to the Jacalin-related lectin family. This lectin is likely presented to sperm via seminal plasma since epididymal sperm are not capable of binding GalNAc, whereas ejaculated sperm does, and its transcript was enriched predominantly in the prostate and bulbourethral glands. This is the first report of a seminal lectin in South American camelids that originates in the male reproductive tract, and is probably involved in sperm reservoir formation.

Frutalin reduces acute and neuropathic nociceptive behaviours in rodent models of orofacial pain.

Orofacial pain is a highly prevalent clinical condition, yet difficult to control effectively with available drugs. Much attention is currently focused on the anti-inflammatory and antinociceptive properties of lectins. The purpose of this study was to evaluate the antinociceptive effect of frutalin (FTL) using rodent models of inflammatory and neuropathic orofacial pain. Acute pain was induced by formalin, glutamate or capsaicin (orofacial model) and hypertonic saline (corneal model). In one experiment, animals were pretreated with l-NAME and naloxone to investigate the mechanism of antinociception. The involvement of the lectin domain in the antinociceptive effect of FTL was verified by allowing the lectin to bind to its specific ligand. In another experiment, animals pretreated with FTL or saline were submitted to the temporomandibular joint formalin test. In yet another, animals were submitted to infraorbital nerve transection to induce chronic pain, followed by induction of thermal hypersensitivity using acetone. Motor activity was evaluated with the rotarod test. A molecular docking was performed using the TRPV1 channel. Pretreatment with FTL significantly reduced nociceptive behaviour associated with acute and neuropathic pain, especially at 0.5 mg/kg. Antinociception was effectively inhibited by l-NAME and d-galactose. In line with in vivo experiments, docking studies indicated that FTL may interact with TRPV1. Our results confirm the potential pharmacological relevance of FTL as an inhibitor of orofacial nociception in acute and chronic pain mediated by TRPA1, TRPV1 and TRPM8 receptor.

Characterization of a dual-CRD galectin in the silkworm Bombyx mori.

Galectins (S-type lectins) are an ancient family of lectins with the ß-galactoside binding activity. In mammals, galectins play essential roles in many biological processes, such as development, immune homeostasis and tumor progression. However, few studies have been devoted to their functions in insects. Here, we characterized the only dual-CRD galectin in the silkworm Bombyx mori (BmGalectin-4). BmGalectin-4 cDNA possesses an open reading frame of 1089 bp, which encodes a putative galectin of 363 amino acids containing tandem carbohydrate recognition domains (CRDs). BmGalectin-4 was expressed in various tissues but the protein was most abundant in fertilized eggs. Its transcript level in fertilized eggs was upregulated upon bacterial challenge. Recombinant BmGalectin-4 purified from Escherichia coli bound to bacterial cell wall components and bacterial cells. In addition, the recombinant protein induced bacterial agglutination, but did not have antibacterial activity against selected microorganisms. Taken together, our results suggest that BmGalectin-4 may function as a pattern recognition receptor primarily in silkworm fertilized eggs.

Galectins are human milk glycan receptors.

The biological recognition of human milk glycans (HMGs) is poorly understood. Because HMGs are rich in galactose we explored whether they might interact with human galectins, which bind galactose-containing glycans and are highly expressed in epithelial cells and other cell types. We screened a number of human galectins for their binding to HMGs on a shotgun glycan microarray consisting of 247 HMGs derived from human milk, as well as to a defined HMG microarray. Recombinant human galectins (hGal)-1, -3, -4, -7, -8 and -9 bound selectively to glycans, with each galectin recognizing a relatively unique binding motif; by contrast hGal-2 did not recognize HMGs, but did bind to the human blood group A Type 2 determinants on other microarrays. Unlike other galectins, hGal-7 preferentially bound to glycans expressing a terminal Type 1 (Galß1-3GlcNAc) sequence, a motif that had eluded detection on non-HMG glycan microarrays. Interactions with HMGs were confirmed in a solution setting by isothermal titration microcalorimetry and hapten inhibition experiments. These results demonstrate that galectins selectively bind to HMGs and suggest the possibility that galectin-HMG interactions may play a role in infant immunity.

A galectin related protein from Oplegnathus fasciatus: Genomic, molecular, transcriptional features and biological responses against microbial pathogens.

Galectins, a family of ß-galactoside-binding lectins, are pattern recognition receptors that recognize pathogen-associated molecular patterns and are subsequently involved in the opsonization, phagocytosis, complement activation, and killing of microbes. Here, we report a novel galectin related protein (GRP) identified from rock bream (Oplegnathus fasciatus), designated OfGal like B. The cDNA of OfGal like B is 517 bp with an open reading frame (ORF) of 438 bp, encoding 145 amino acids, with a single carbohydrate recognition domain (CRD). However, only two of the seven critical residues responsible for carbohydrate recognition were identified in the CRD. There was no signal peptide identified in the OfGal like B protein. The genomic structure of OfGal like B, determined using a bacterial artificial chromosome (BAC) genomic library, consists of four exons and three introns. Homology assessment, multiple sequence alignment, and phylogenetic analysis indicated that OfGal like B is an evolutionarily conserved lectin that is closely related to the proto-type galectins. OfGal like B mRNA was constitutively expressed in a wide range of tissues in healthy rock breams. When challenged with bacterial or viral stimulants, OfGal like B was up-regulated in the gills and spleen of rock breams, indicating that it likely plays an important role during bacterial and viral infections. Furthermore, recombinant OfGal like B (rOfGal like B) lacked carbohydrate-binding activity but was able to recognize and agglutinate bacteria, including Streptococcus iniae, Listeria monocytogenes, Vibrio tapetis, Escherichia coli, and Edwardsiella tarda, and a ciliate parasite, Miamiensis avidus. These results collectively suggest that OfGal like B is involved in pathogen recognition and plays a significant role(s) in the innate defense mechanism of rock bream.

Integrating Qualitative and Quantitative Tools for the Detection and Identification of Lectins in Major Human Diseases.

Lectins are the (glyco)proteins that recognize and bind to specific sugar moieties without altering their structure. Galectins are mammalian lectins characterized by the presence of conserved 134 amino acids carbohydrate recognition domain and specificity for ß-galactosides. The involvement of lectins in diverse biological spectrum, especially some deadly human diseases like cancer, neurological disorders and cardiovascular disorders has proclaimed them as one of the important components of glycobiology, thereby seeking the methods of their detection and identification heavily desirable. In the present manuscript, we have provided a comprehensive outline of various methods of detection and identification of lectins employed till date, with their needs and usage varying according to the level of infrastructure of laboratories and around the world. In addition, a vision for some quick, highly sensitive and advanced methods for lectin detection and identification for diagnostic and therapeutic of various diseases is also provided.

Molecular cloning, expression of a galectin gene in Pacific white shrimp Litopenaeus vannamei and the antibacterial activity of its recombinant protein.

Galectins play crucial roles in innate immune responses in invertebrate by recognizing and eliminating microinvaders. In this study, a cDNA encoding a galectin in the Pacific white shrimp (L. vannamei) was identified and characterized. A recombinant variant of this lectin, rLvgalectin, was expressed in the model organism P. pastoris and its expression was confirmed by Western blot. Biochemical assays indicated that the recombinant protein antibacterial rLvgalectin activity and was expressed in all of the organism's tested tissues Injection of the bacterium V. alginolyticus into L. vannamei induced hemocytes upregulation of Lvgalectin. The recombinant Lvgalectin protein (rLvgalectin) could bind various microorganism including Gram-positive bacteria, Gram-negative bacteria and yeast. And it revealed antimicrobial activity against the test Gram-positive bacteria, Gram-negative bacteria, but did not inhibit the growth of fungus Pichia pastoris. Moreover, rLvgalectin could significantly enhance the clearance activity of V. alginolyticus in vivo. In vivo challenge experiments showed that the recombinant rLvgalectin protein can significantly reduce the mortalities of V. alginolyticus injection. Furthermore, Compared to their wild-type counterparts, Lvgalectin-silenced shrimp exhibited increased mortality and hemocyte apoptosis, decreased bacterial clearance ability and total hemocyte counts, and stronger expression of Lvp53, LvproPO, LvPEN3, and LvCrustin following V. alginolyticus challenge. Taken together, these results suggest that galectin is important in the innate immune response of shrimp to pathogens infection.