Conserved Carbohydrate Recognition Domain Research Articles

Molecular cloning and functional characterization of eight galectin genes from Takifugu obscurus

Galectins are a family of animal lectins involved in the immune response against pathogens. However, the roles of fish galectins during pathogen infection require comprehensive studies. In the present research, eight different galectin genes from Takifugu obscurus (named ToGalec1-8) were identified and characterized. ToGalec1-8 encoded proteins of 240, 182, 373, 145, 452, 135, 359 and 346 amino acids, respectively. All predicted ToGalec1-8 proteins possessed one or more conserved carbohydrate recognition domains (CRDs). Phylogenetic analysis revealed that ToGalec1-8 were evolutionarily closely related to their counterparts in other selected vertebrates, hinting their genetic relationship. Tissue distribution analysis showed that most ToGalec genes were distributed ubiquitously in all detected tissues, with relatively high expression in immune tissues. After stimulation by Vibrio harveyi and Staphylococcus aureus, the mRNA transcripts of ToGalec1-8 in liver and kidney were significantly upregulated. In addition, RNA interference experiments indicated that knockdown of ToGalec1 and ToGalec7 inhibited the clearance of bacteria in vivo. Taken together, these obtained results suggested that ToGalec1-8 play an important role in innate immunity and defense against bacterial infection in T. obscurus.

Largemouth bass galectin, MsGal-9: Mediating various functions as a pattern recognition receptor and a potential damage-associated molecular pattern

Galectins are lectins that bind to β-galactose and are widely expressed in immune system tissues, playing pivotal roles in innate immunity through their conserved carbohydrate-recognition domains (CRDs). In this present investigation, a tandem-repeat galectin was discovered in the largemouth bass, Micropterus salmoides (designated as MsGal-9). The open reading frame of MsGal-9 encodes two CRDs, each containing two consensus motifs that are essential for ligand binding. MsGal-9 is expressed in various tissues of the largemouth bass, with particularly high expression levels in the liver and spleen. The full-length form of MsGal-9, as well as the N-terminal (MsGal-9-N) and C-terminal (MsGal-9-C) CRDs, were individually recombined. Their ability for nonself recognition was studied. The three recombinant proteins were able to bind to glucan (GLU), peptidoglycan (PGN), and lipopolysaccharide (LPS), with MsGal-9 displaying the highest binding activity. Furthermore, rMsGal-9-N exhibited higher binding activity towards GLU in comparison to rMsGal-9-C. Further investigations revealed that the full-length rMsGal-9 could significantly bind to Gram-positive bacteria, Gram-negative bacteria, and fungi, while rMsGal-9-C specifically bound to Escherichia coli. However, rMsGal-9-N did not exhibit significant binding activity towards any microbes. These findings indicate that MsGal-9 requires both CRDs to cooperate in order to fulfill its nonself recognition function. All three recombinant proteins demonstrated agglutination activity towards various microbes, with MsGal-9 and MsGal-9-N displaying a similar broad binding spectrum, while MsGal-9-C agglutinated three types of bacteria. Moreover, both MsGal-9 and MsGal-9-N were capable of coagulating largemouth bass red blood cells, whereas MsGal-9-C lacked this ability. However, MsGal-9-C played a significant role in enhancing the encapsulation of leukocytes in comparison to MsGal-9-N. All three proteins acted as potential damage-associated molecular patterns (DAMPs), inducing apoptosis in leukocytes.

Molecular characterization and functional study of a galectin-9 from a teleost fish, Boleophthalmus pectinirostris

Galectin-9, a tandem-repeat galectin, plays an important role in the regulation of innate immune response against various microbial infections. Here, galectin-9 from mudskipper (Boleophthalmus pectinirostris) was identified and named as BpGal-9. Putative BpGal-9 contains two conserved carbohydrate recognition domains (CRDs), one CRD within N-terminal (N-CRD) and the other one within C-terminal (C-CRD). Multi-alignment analysis indicated that BpGal-9 shared the highest amino acid sequence identity of 64.3 % with that of Southern platyfish (Xiphophorus maculatus). Phylogenetic analysis showed that BpGal-9 grouped tightly with other teleosts galectin-9 and was most closely related to that of Southern platyfish. BpGal-9 transcripts were more abundant in the intestine, and its expression upregulated significantly in the intestine, kidney, spleen, gills, and skin after Edwardsiella tarda infection. Meanwhile, BpGal-9 expression significantly increased in hemocytes and serum of mudskipper infected by E. tarda. The recombinant BpGal-9 (rBpGal-9) and rBpGal-9C-CRD could agglutinate all tested bacteria, whereas rBpGal-9N-CRD could only agglutinate three kinds of bacteria. When targeting the same bacteria, rBpGal-9 showed stronger agglutinating activities than rBpGal-9C-CRD or rBpGal-9N-CRD. In addition, the induction effect of three recombinant proteins on the mRNA expression of anti-inflammatory cytokines (BpIL-10 and BpTGF-β) was better than that on the pro-inflammatory cytokines (BpIL-1β and BpTNF-α). Our result suggested that the N-CRD and C-CRD of galectin-9 contribute differently to its multiple functions in innate immunity in teleosts.

Isomerization of proline-46 in the N-terminal tail of galectin-3 enhances T cell apoptosis via the ROS-ERK pathway

Galectin-3 (Gal-3) is unique in the galectin family, due to the presence of a long N-terminal tail (NT) arising from its conserved carbohydrate recognition domain (CRD). Although functional significance of the NT has remained elusive, our previous studies demonstrated the importance of NT prolines to Gal-3 function. Here, we show that during the time Gal-3 stands in solution for three or more days, Gal-3 NT undergoes a slow, intra-molecular, time-dependent conformational/dynamical change associated with proline cis-trans isomerization. From initial dissolution of Gal-3 in buffer to three days in solution, Gal-3-mediated T cell apoptosis is enhanced from 23 % to 37 %. Western blotting and flow cytometry show that the enhancement occurs via the ROS-ERK pathway, and not by the PKC-ERK pathway. To assess which proline(s) is (are) responsible for this effect, we individually mutated all 14 NT prolines within the first 68 residues to alanines, and assessed their effect on ROS production. Our study shows that isomerization of P46 alone is responsible for the upregulation of ROS and T cell apoptosis. NMR studies show that this unique effect is mediated by a change in dynamic interactions between the NT and CRD F-face, which in turn leads to this change in Gal-3 function.

A galectin-9 involved in the microbial recognition and haemocyte autophagy in the Pacific oyster Crassostrea gigas

Galectin-9 is a tandem-repeat type member of galectin family participating in various immune responses, such as cell agglutination, phagocytosis, and autophagy. In the present study, a tandem repeat galectin-9 (defined as CgGal-9) was identified from Pacific oyster Crassostrea gigas, which consisted of two conserved carbohydrate recognition domains (CRDs) joined by a linker peptide. CgGal-9 was closely clustered with CaGal-9 from C. angulata, and they were assigned into the branch of invertebrate galectin-9s in the phylogenetic tree. The mRNA transcripts of CgGal-9 were detected in all the tested tissues, with the highest expression level in haemocytes. The mRNA expressions of CgGal-9 in haemocytes increased significantly after lipopolysaccharide (LPS) and Vibrio splendidus stimulation. The recombinant CgGal-9 was able to bind all the examined pathogen-associated molecular patterns (LPS, peptidoglycan, and mannose) and microbes (V. splendidus, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, and Pichia pastoris), and agglutinated most of them in the presence of Ca2+. In CgGal-9-RNAi oysters, the mRNA expressions of autophagy related genes (CgBeclin1, CgATG5, CgP62 and CgLC3) in haemocytes decreased significantly while that of CgmTOR increased significantly at 3 h after V. splendidus stimulation. The autophagy level and mRNA expressions of autophagy related genes decreased in haemocytes after CgGal-9 was blocked by the corresponding antibody. These results revealed that CgGal-9 was able to bind different microbes and might be involved in haemocyte autophagy in the immune response of oyster.

Identification of two galectin-4 proteins (PcGal4-L and PcGal4-L-CRD) and their function in AMP expression in Procambarus clarkii

Galectins, a family of lectins that bind to β-galactoside, possess conserved carbohydrate recognition domains (CRDs) and play a crucial role in recognizing and eliminating pathogens in invertebrates. Two galectin-4 genes (PcGal4) isoforms, named PcGal4-L and PcGal4-L-CRD, were cloned from the cDNA library of Procambarus clarkia in our study. PcGal4-L contains an open reading frame (ORF, 1089 bp), which encodes a protein consisting of 362 amino acids including a single CRD and six low complexity regions. The full-length cDNA of PcGal4-L-CRD contains a 483 bp ORF that encodes a protein of 160 amino acids, with a single CRD and a low-complexity region. The difference between the two PcGal4 isoforms is that PcGal4-L has 202 additional amino acids after the CRD compared to the PcGal4-L-CRD. These two isoforms are grouped together with other galectins from crustaceans through phylogenetic analysis. Further study revealed that total PcGal4 (including PcGal4-L and PcGal4-L-CRD) was primarily expressed in the muscle, gills and intestine. The mRNA levels of total PcGal4 in gills and hemocytes were significantly induced after challenge with Aeromonas hydrophila. Both recombinant PcGal4-L and its spliced isoform, PcGal4-L-CRD, could directly bind to lipopolysaccharides, peptidoglycan and five tested microorganisms, inducing a wide spectrum of microbial agglutination. The spliced isoform PcGal4-L-CRD showed a stronger binding ability than PcGal4-L. In addition, when the PcGal4 was knockdown, transcriptions of seven antimicrobial peptides (AMPs) genes (ALF5, ALF6, ALF8, CRU1, CRU2, CRU3 and CRU4) in gills and seven AMPs genes (ALF5, ALF6, ALF8, ALF9, CRU1, CRU3 and CRU4) in hemocytes were significantly decreased. Meanwhile, the survival rate of P. clarkii decreased in the PcGal4-dsRNA group. In summary, these results indicate that PcGal4 can mediate the innate immunity in P. clarkii by bacterial recognition and agglutination, as well as regulating AMP expression, thus recognition and understanding of the functions of galectin in crustaceans in immune resistance.

Molecular characterization, immune responses, and functional aspects of atypical prototype galectin from redlip mullet (Liza haematocheila) as a pattern recognition receptor in host immune defense system

Galectins are a family of lectins that are widely distributed β-galactoside-binding proteins identified in diverse organisms. Galectin family have appeared as pattern recognition receptors (PRRs) responsible for initiating and controlling the innate immunity. The present study aimed to study the binding ability and potential role in PRRs of galectin-related protein B-like (LhGal B-like) from redlip mullet (Liza haematocheila) involved in the host immune responses. We constructed a cDNA library of redlip mullet and identified the LhGal B-like sequence. By sequence analysis and multiple sequence alignment, we revealed that LhGal B-like contains a conserved carbohydrate recognition domain (CRD) and consists of 135 amino acids with a predicted molecular weight of 16.07 kDa. In addition, pairwise comparison results showed that LhGal B-like shares higher sequence identity (82.2–95.2%) and similarity (89–95.9%) with fish species than those (34.1–37.8% and 57.2–58.1%, respectively) with other species. The phylogenetic tree showed that LhGal B-like clustered into the fish group and was evolutionally related to Mastacembelus armatus. The tissue distribution results revealed that LhGal B-like was expressed ubiquitously in all the tested tissues, where it was highly expressed in the brain, followed by gills and muscle. The immune modulated expression of LhGal B-like was observed by injecting lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). According to the results, in the gills, the mRNA expression of LhGal B-like was significantly upregulated upon LPS treatment after 48 h and upon poly I:C treatment after 48 and 72 h. In addition, the result showed significant upregulations upon LPS and poly I:C treatment after 24 h. However, significant downregulation was also shown in the earlier phase after injection of poly I:C and L. garvieae in gills. Further, the binding affinity of recombinant LhGal B-like (rLhGal B-like) was evaluated using carbohydrate, pathogen-associated molecular patterns (PAMP) and bacterial binding assays. The rLhGal B-like could bind all the examined carbohydrates but had a higher affinity to α-lactose. PAMPs and bacterial binding experiments verified a wide range of PAMP molecules and bacterial strains that rLhGal B-like could bind to. Moreover, we examined the agglutination activity of rLhGal B-like, and the result showed that it could aggregate all the gram-positive and gram-negative bacteria. Taken together, our findings reveal the functional aspects of LhGal B-like as a PRR and the potential involvement of LhGal B-like in the innate immunity of redlip mullet.

PSI-11 Effect of Garlic Barrier on Fecal egg Count and Protein Secretion in Goats

Abstract The present study was carried out to determine the effect of garlic on egg counts and secretion of Galectins (Gals) in goat blood. Garlic (Allium sativum) has been used as medicinal herb since time immemorial in almost every known civilization. It has antibiotic, anti-helminthic and anti-inflammatory properties. Galectins (Gals) are a family of β-galactosidase binding proteins that have a conserved carbohydrate recognition domain. They are involved in cell adhesion, migration, activation, proliferation, apoptosis and modulate pathological processes such as inflammation. Twelve (N=12) adult female clinically healthy Boer x Spanish goats were divided into two treatment groups (N=6 each) used. Treatment group 1 was drenched with 10 ml of Garlic barrier as recommended by the manufacturer (Garlic Barrier, Glendale, CA) and Treatment group 2 was drenched with water over a four-week period. Fecal and blood samples were collected weekly. Eggs per gram was evaluated using Modified McMaster method and the total plasma protein concentration was assessed using the BCA assay (Pierce). All variables were assessed using a t-test (SAS analysis software version 9.0 at p≤0.05). Garlic treatment affected the fecal egg count and plasma protein concentration when compared to untreated controls. The fecal egg count was reduced in animals treated with garlic. The effect varied over time. Garlic EPG in weeks 1 (p<0.004) and 3 (p<0.027) of treatment. A significant increase in total protein concentration (p<0.004) was observed. Garlic treatment has an effect on GIN levels and the inflammatory response. The time dependent effects may indicate parasite, infection stage specific targets and need further study.

Exploring the Molecular Interactions of Symmetrical and Unsymmetrical Selenoglycosides with Human Galectin-1 and Galectin-3.

Galectins (Gals) are small cytosolic proteins that bind β-galactoside residues via their evolutionarily conserved carbohydrate recognition domain. Their dysregulation has been shown to be associated with many diseases. Consequently, targeting galectins for clinical applications has become increasingly relevant to develop tailored inhibitors selectively for one galectin. Accordingly, binding studies providing the molecular details of the interaction between galectin and inhibitor may be useful for the rational design of potent and selective antagonists. Gal-1 and Gal-3 are among the best-studied galectins, mainly for their roles in cancer progression; therefore, the molecular details of their interaction with inhibitors are demanded. This work gains more value by focusing on the interaction between Gal-1 and Gal-3 with the selenylated analogue of the Gal inhibitor thiodigalactose, characterized by a selenoglycoside bond (SeDG), and with unsymmetrical diglycosyl selenides (unsym(Se). Gal-1 and Gal-3 were produced heterologously and biophysically characterized. Interaction studies were performed by ITC, NMR spectroscopy, and MD simulation, and thermodynamic values were discussed and integrated with spectroscopic and computational results. The 3D complexes involving SeDG when interacting with Gal-1 and Gal-3 were depicted. Overall, the collected results will help identify hot spots for the design of new, better performing, and more specific Gal inhibitors.

Galectin-1 activates carbonic anhydrase IX and modulates glioma metabolism

Galectins are a family of β-galactose-specific binding proteins residing within the cytosol or nucleus, with a highly conserved carbohydrate recognition domain across many species. Accumulating evidence shows that Galectin 1 (Gal-1) plays an essential role in cancer, and its expression correlates with tumor aggressiveness and progression. Our preliminary data showed Gal-1 promotes glioma stem cell (GSC) growth via increased Warburg effect. mRNA expression and clinical data were obtained from The Cancer Genome Atlas database. The immunoblot analysis conducted using our cohort of human glioblastoma patient specimens (hGBM), confirmed Gal-1 upregulation in GBM. GC/MS analysis to evaluate the effects of Gal-1 depletion showed elevated levels of α-ketoglutaric acid, and citric acid with a concomitant reduction in lactic acid levels. Using Biolog microplate-1 mitochondrial functional assay, we confirmed that the depletion of Gal-1 increases the expression levels of the enzymes from the TCA cycle, suggesting a reversal of the Warburg phenotype. Manipulation of Gal-1 using RNA interference showed reduced ATP, lactate levels, cell viability, colony-forming abilities, and increased expression levels of genes implicated in the induction of apoptosis. Gal-1 exerts its metabolic role via regulating the expression of carbonic anhydrase IX (CA-IX), a surrogate marker for hypoxia. CA-IX functions downstream to Gal-1, and co-immunoprecipitation experiments along with proximity ligation assays confirm that Gal-1 physically associates with CA-IX to regulate its expression. Further, silencing of Gal-1 in mice models showed reduced tumor burden and increased survival compared to the mice implanted with GSC controls. Further investigation of Gal-1 in GSC progression and metabolic reprogramming is warranted.

Bruceine A protects against diabetic kidney disease via inhibiting galectin-1

Bruceine A is a natural quassinoid compound extracted from the fruit of the Traditional Chinese Medicine Brucea javanica (L.) Merr. that has various types of various biological activities. However, whether the compound has a protective effect on diabetic kidney disease remains unknown. Galectin-1 is actively involved in a variety of chronic inflammation-relevant human diseases including diabetic kidney disease. Here, we identified Bruceine A as a kidney protective molecule against a model of diabetic kidney disease in db/db mice with potent anti-inflammatory activity both invitro and invivo. Mechanistically, by selectively binding to the conserved carbohydrate-recognition domain of galectin-1 and disrupting the interaction between galectin-1 and the receptor for activated protein C kinase 1, Bruceine A was found to inhibit galectin-1-mediated inflammatory signal transduction under high glucose stress in rat mesangial HBZY-1 cells. Thus, our findings reveal Bruceine A as an unidentified galectin-1 inhibitor affording significant protection against diabetic kidney disease and may provide novel pharmacological therapeutics for the disease.

Molecular characterization and antibacterial ability of galectin-3 and galectin-9 in Onychostoma macrolepis

Galectins belong to the β-galactoside binding protein family, which have conserved carbohydrate-recognition domains (CRDs) and participate in innate and acquired immunity in animals. In this study, two galectin genes were cloned from Onychostoma macrolepis, OmGal-3 (galectin-3) and OmGal-9 (galectin-9). The open reading frames (ORFs) of OmGal-3 and OmGal-9 contain 732 and 978 base pairs, encoding 243 and 325 amino acids, respectively. OmGal-3 contains a C-terminal CRD, but OmGal-9 contains an N-terminal CRD and a C-terminal CRD. Two galectins were expressed at varying levels in all tissues examined, with the liver showing the highest expression. The relative gene expression levels of OmGal-3 and OmGal-9 following Aeromonas hydrophila infection were significantly up-regulated in the liver and spleen, and OmGal-9 had a greater increase than OmGal-3. The recombinant OmGal-3 and OmGal-9 proteins (rOmGal-3 and rOmGal-9) were authenticated and verified by SDS-PAGE and western blotting. ROmGal-3 and rOmGal-9 agglutinated all tested bacteria, including 3 g-positive bacteria (Aeromonas hydrophila, Escherichia coli, and Vibrio parahaemolyticus) and 3 g-negative bacteria (Streptococcus agalactiae, Staphylococcus aureus, and Bacillus cereus) in vivo without Ca2+. ROmGal-3 showed strong binding both to gram-positive and gram-negative bacteria and OmGal-9 had a stronger binding activity against gram-positive bacteria. Furthermore, rOmGal-3 and rOmGal-9 exhibited dose-dependent binding capability to two classic pathogens associated molecular pattern (LPS and PGN) and two sugars (d-lactose and d-galactose), and rOmGal-3 has better binding activity at lower concentrations in LPS and PGN than rOmGal-3. The integrated analyses indicate that the two galectins probably play an important role in innate immune defense by binding to bacterial cells via the CRD domain against pathogen infection.

Structure-based identification of galectin-1 selective modulators in dietary food polyphenols: a pharmacoinformatics approach

In this study, a set of dietary polyphenols was comprehensively studied for the selective identification of the potential inhibitors/modulators for galectin-1. Galectin-1 is a potent prognostic indicator of tumor progression and a highly regarded therapeutic target for various pathological conditions. This indicator is composed of a highly conserved carbohydrate recognition domain (CRD) that accounts for the binding affinity of β-galactosides. Although some small molecules have been identified as galectin-1 inhibitors/modulators, there are limited studies on the identification of novel compounds against this attractive therapeutic target. The extensive computational techniques include potential drug binding site recognition on galectin-1, binding affinity predictions of ~ 500 polyphenols, molecular docking, and dynamic simulations of galectin-1 with selective dietary polyphenol modulators, followed by the estimation of binding free energy for the identification of dietary polyphenol-based galectin-1 modulators. Initially, a deep neural network-based algorithm was utilized for the prediction of the druggable binding site and binding affinity. Thereafter, the intermolecular interactions of the polyphenol compounds with galectin-1 were critically explored through the extra-precision docking technique. Further, the stability of the interaction was evaluated through the conventional atomistic 100 ns dynamic simulation study. The docking analyses indicated the high interaction affinity of different amino acids at the CRD region of galectin-1 with the proposed five polyphenols. Strong and consistent interaction stability was suggested from the simulation trajectories of the selected dietary polyphenol under the dynamic conditions. Also, the conserved residue (His44, Asn46, Arg48, Val59, Asn61, Trp68, Glu71, and Arg73) associations suggest high affinity and selectivity of polyphenols toward galectin-1 protein.Graphic

Characterization and functional analysis of tandem threonine containing C-type lectin (Thr-Lec) from the ridgetail white prawn Exopalaemon carinicauda

As an important pattern-recognition receptor (PRR), C-type lectins (CTLs) play significant roles in recognizing microbes and battle against pathogenic microorganism in innate immunity. In this study, two tandem threonine containing CTLs (designated as EcThr-LecA and EcThr-LecB) were identified from Exopalaemon carinicauda. The full-length cDNA of EcThr-LecA and EcThr-LecB consisted of 1521 and 1518 bp with 1251 and 1242 bp open reading frame encoding a protein with 412 and 413 amino acids, respectively. The genome structure of EcThr-LecA included 10 exons and 9 introns, and the sequences of intron6 and intron7 were variable. The nucleotide sequence of intron2 in EcThr-LecB was specific and different with that of EcThr-LecA. EcThr-LecA and EcThr-LecB proteins were predicted to have a signal peptide, two conserved carbohydrate recognition domain (CRD), and tandem threonine region. The expression levels of EcThr-LecA and EcThr-LecB in the intestine were significantly up-regulated after Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge. RNA interference (RNAi) was used to explore the effects of EcThr-LecB silencing on the mRNA expression of anti-lipopolysaccharide factor (ALF), crustin (CRU), and lysozyme (LYSO). Knock down of EcThr-LecB could evidently down-regulate the expression of eight different antibacterial peptides (AMPs), including EcALF2, EcCRU1, EcCRU3, EcCRU4, EcLYSO1, EcLYSO2, EcLYSO3, and EcLYSO4, whereas make no effect on the transcription of EcALF1, EcALF3, EcCRU2, and EcLYSO5. The recombinant two CRD domains and tandem threonine region (RLecB) of EcThr-LecB could bind diverse bacteria, lipopolysaccharide, and peptidoglycans in vitro. In addition, RLecB could accelerate the clearance of V. parahaemolyticus in vivo. The present data indicated that new-found tandem threonine containing CTLs in E. carinicauda may act as PRR to participate in the innate immune defense against pathogens by the recognition of non-self, regulation of AMPs, and clearance of invaders.

Molecular characterization and functional study of galectin-related proteins from the Japanese flounder (Paralichthys olivaceus)

Galectins are a family of lectins that bind β-galactoside sugars through their conserved carbohydrate-recognition domains (CRDs) and play integral roles in the innate immune systems of vertebrates and invertebrates. Accumulating amounts of evidence have shown that galectin-related protein (GRP) plays a crucial role in fish immunity. In this study, the GRP cDNA with a 546 bp open reading frame from Paralichthys olivaceus was identified and characterized and was designated as PoGRP. PoGRP was 92.82% similar to the GRP sequences of Seriola dumerili and Anabas testudineus. PoGRP was found to be widely expressed in all tested P. olivaceus tissues, with the highest expression levels being observed in the brain, spleen and liver. Additionally, after this fish species was challenged with Edwardsiella tarda, PoGRP expression was significantly upregulated. The recombinant PoGRP protein (rPoGRP) exhibited binding to various pathogen-associated molecular patterns (PAMPs) and to gram-negative and gram-positive bacteria. In addition, rPoGRP exhibited the ability to agglutinate different bacteria in the presence of Ca2+. In summary, our findings indicated that PoGRP plays a vital role in the immune system to protect against bacterial infection. This study lays the foundation for further functional characterization of GRP in aquatic animal immunity.

Generation and Use of Recombinant Galectins.

Galectins are soluble carbohydrate binding proteins that can bind β-galactose-containing glycoconjugates by means of a conserved carbohydrate recognition domain (CRD). In mammalian systems, galectins have been shown to mediate very important roles in innate and adaptive immunity as well as facilitating host-pathogen relationships. Many of these studies have relied on purified recombinant galectins to uncover key features of galectin biology. A major limitation to this approach is that certain recombinant galectins purified using standard protocols are easily susceptible to loss of glycan-binding activity. As a result, biochemical studies that employ recombinant galectins can be misleading if the overall activity of a galectin remains unknown in a given assay condition. This article examines fundamental considerations when purifying galectins by lactosyl-sepharose and nickel-NTA affinity chromatography using human galectin-4N and -7 as examples, respectively. As other approaches are also commonly applied to galectin purification, we also discuss alternative strategies to galectin purification, using human galectin-1 and -9 as examples. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Purification of galectins using lactosyl-sepharose affinity chromatography Basic Protocol 2: Purification of human galectin-7 using a nickel-NTA affinity chromatography column Alternate Protocol 1: Iodoacetamide alkylation of free sulfhydryls on galectin-1 Alternate Protocol 2: Purification of human galectin-9 using lactosyl-sepharose column chromatography.

A Fish Galectin-8 Possesses Direct Bactericidal Activity.

Galectins are a family of animal lectins with high affinity for β-galactosides. Galectins are able to bind to bacteria, and a few mammalian galectins are known to kill the bound bacteria. In fish, no galectins with direct bactericidal effect have been reported. In the present study, we identified and characterized a tandem repeat galectin-8 from tongue sole Cynoglossus semilaevis (designated CsGal-8). CsGal-8 possesses conserved carbohydrate recognition domains (CRDs), as well as the conserved HXNPR and WGXEE motifs that are critical for carbohydrate binding. CsGal-8 was constitutively expressed in nine tissues of tongue sole and up-regulated in kidney, spleen, and blood by bacterial challenge. When expressed in HeLa cells, CsGal-8 protein was detected both in the cytoplasm and in the micro-vesicles secreted from the cells. Recombinant CsGal-8 (rCsGal-8) bound to lactose and other carbohydrates in a dose dependent manner. rCsGal-8 bound to a wide range of gram-positive and gram-negative bacteria and was co-localized with the bound bacteria in animal cells. Lactose, fructose, galactose, and trehalose effectively blocked the interactions between rCsGal-8 and different bacteria. Furthermore, rCsGal-8 exerted potent bactericidal activity against some gram-negative bacterial pathogens by directly damaging the membrane and structure of the pathogens. Taken together, these results indicate that CsGal-8 likely plays an important role in the immune defense against some bacterial pathogens by direct bacterial interaction and killing.

Galectin-1 production is elevated in hypertrophic scar.

Upon healing, burn wounds often leave hypertrophic scars (HTSs) marked by excess collagen deposition, dermal and epidermal thickening, hypervascularity, and an increased density of fibroblasts. The Galectins, a family of lectins with a conserved carbohydrate recognition domain, function intracellularly and extracellularly to mediate a multitude of biological processes including inflammatory responses, angiogenesis, cell migration and differentiation, and cell-ECM adhesion. Galectin-1 (Gal-1) has been associated with several fibrotic diseases and can induce keratinocyte and fibroblast proliferation, migration, and differentiation into fibroproliferative myofibroblasts. In this study, Gal-1 expression was assessed in human and porcine HTS. In a microarray, galectins 1, 4, and 12 were upregulated in pig HTS compared to normal skin (fold change = +3.58, +6.11, and +3.03, FDR <0.01). Confirmatory qRT-PCR demonstrated significant upregulation of Galectin-1 (LGALS1) transcription in HTS in both human and porcine tissues (fold change = +7.78 and +7.90, P <.05). In pig HTS, this upregulation was maintained throughout scar development and remodeling. Immunofluorescent staining of Gal-1 in human and porcine HTS showed significantly increased fluorescence (202.5 ± 58.2 vs 35.2 ± 21.0, P <.05 and 276.1 ± 12.7 vs 69.7 ± 25.9, P <.01) compared to normal skin and co-localization with smooth muscle actin-expressing myofibroblasts. A strong positive correlation (R = .948) was observed between LGALS1 and Collagen type 1 alpha 1 mRNA expression. Gal-1 is overexpressed in HTS at the mRNA and protein levels and may have a role in the development of scar phenotypes due to fibroblast over-proliferation, collagen secretion, and dermal thickening. The role of galectins shows promise for future study and may lead to the development of a pharmacotherapy for treatment of HTS.

Characterization and functional analysis of a galectin-related protein B from Nile tilapia involved in the immune response to bacterial infection.

Galectin-related protein is a kind of lectin without canonical activity that regulates cell adhesion and cell growth. In this study, a novel galectin-related protein B (OnGRPB) was identified from Nile tilapia (Oreochromis niloticus). The open reading frame of OnGRPB was 438bp and encoded a peptide of 145 amino acids. The deduced protein sequence of OnGRPB possessed a conserved carbohydrate recognition domain (CRD) with partial sugar binding sites (N-R, V-N and W-E) and shared high identities with other fish GRPB proteins. The qRT-PCR analysis found that OnGRPB was widely distributed in various tissues and monocyte/macrophages (Mo/MΦ) of healthy tilapia. After Streptococcus agalactiae infection, OnGRPB transcripts were significantly up-regulated in liver, spleen, head kidney and Mo/MΦ. The recombinant OnGRPB protein (rOnGRPB) had the binding activity and agglutination ability to bacteria. Also, rOnGRPB could modulate antibacterial activity and inflammatory factor expression of Mo/MΦ. These data collectively indicate that OnGRPB plays roles in the immune response of Nile tilapia against bacterial infection.

Galectins and Ovarian Cancer.

Ovarian cancer is known for its aggressive pathological features, including the capacity to undergo epithelial to mesenchymal transition, promoting angiogenesis, metastatic potential, chemoresistance, inhibiting apoptosis, immunosuppression and promoting stem-like features. Galectins, a family of glycan-binding proteins defined by a conserved carbohydrate recognition domain, can modulate many of these processes, enabling them to contribute to the pathology of ovarian cancer. Our goal herein was to review specific galectin members identified in the context of ovarian cancer, with emphasis on their association with clinical and pathological features, implied functions, diagnostic or prognostic potential and strategies being developed to disrupt their negative actions.