RESUMO
T cell immunoglobulin and mucin-containing molecule 3 (TIM-3) exhibits unique, cell type- and context-dependent characteristics and functions. Here, we report that TIM-3 on myeloid cells plays essential roles in modulating lung inflammation. We found that myeloid cell-specific TIM-3 knock-in (FSF-TIM3/LysM-Cre+) mice have lower body weight and shorter lifespan than WT mice. Intriguingly, the lungs of FSF-TIM3/LysM-Cre+ mice display excessive inflammation and features of disease-associated pathology. We further revealed that galectin-3 levels are notably elevated in TIM-3-overexpressing lung-derived myeloid cells. Furthermore, both TIM-3 blockade and GB1107, a galectin-3 inhibitor, ameliorated lung inflammation in FSF-TIM3/LysM-Cre+/- mice. Using an LPS-induced lung inflammation model with myeloid cell-specific TIM-3 knock-out mice, we demonstrated the association of TIM-3 with both lung inflammation and galectin-3. Collectively, our findings suggest that myeloid TIM-3 is an important regulator in the lungs and that modulation of TIM-3 and galectin-3 could offer therapeutic benefits for inflammation-associated lung diseases.
Assuntos
Galectina 3 , Receptor Celular 2 do Vírus da Hepatite A , Células Mieloides , Pneumonia , Animais , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/genética , Galectina 3/metabolismo , Galectina 3/genética , Células Mieloides/metabolismo , Camundongos , Pneumonia/metabolismo , Pneumonia/patologia , Pneumonia/genética , Camundongos Knockout , Camundongos Endogâmicos C57BL , Galectinas/metabolismo , Galectinas/genética , Pulmão/patologia , Pulmão/metabolismoRESUMO
Essentially all plasma membrane proteins are glycosylated, and their activity is regulated by tuning their cell surface dynamics. This is achieved by glycan-binding proteins of the galectin family that either retain glycoproteins within lattices or drive their endocytic uptake via the clathrin-independent glycolipid-lectin (GL-Lect) mechanism. Here, we have used immunofluorescence-based assays to analyze how lattice and GL-Lect mechanisms affect the internalization of the cell adhesion and migration glycoprotein α5ß1 integrin. In retinal pigment epithelial (RPE-1) cells, internalized α5ß1 integrin is found in small peripheral endosomes under unperturbed conditions. Pharmacological compounds were used to competitively inhibit one of the galectin family members, galectin-3 (Gal3), or to inhibit the expression of glycosphingolipids, both of which are the fabric of the GL-Lect mechanism. We found that under acute inhibition conditions, endocytic uptake of α5ß1 integrin was strongly reduced, in agreement with previous studies on the GL-Lect driven internalization of the protein. In contrast, upon prolonged inhibitor treatment, the uptake of α5ß1 integrin was increased, and the protein was now internalized by alternative pathways into large perinuclear endosomes. Our findings suggest that under these prolonged inhibitor treatment conditions, α5ß1 integrin containing galectin lattices are dissociated, leading to an altered endocytic compartmentalization.
Assuntos
Endocitose , Galectina 3 , Integrina alfa5beta1 , Humanos , Galectina 3/metabolismo , Integrina alfa5beta1/metabolismo , Linhagem Celular , Endossomos/metabolismo , Adesão Celular , Galectinas/metabolismo , Proteínas SanguíneasRESUMO
We here report the purification of a novel member of the galectin family, the ß-galactoside-binding lectin hRTL, from the marine sponge Chondrilla australiensis. The hRTL lectin is a tetrameric proto-type galectin with a subunit molecular weight of 15.5 kDa, consisting of 141 amino acids and sharing 92% primary sequence identity with the galectin CCL from the congeneric species C. caribensis. Transcriptome analysis allowed for the identification of additional sequences belonging to the same family, bringing the total number of hRTLs to six. Unlike most other galectins, hRTLs display a 23 amino acid-long signal peptide that, according to Erdman degradation, is post-translationally cleaved, leaving an N-terminal end devoid of acetylated modifications, unlike most other galectins. Moreover, two hRTLs display an internal insertion, which determines the presence of an unusual loop region that may have important functional implications. The characterization of the glycan-binding properties of hRTL revealed that it had high affinity towards TF-antigen, sialyl TF, and type-1 N-acetyl lactosamine with a Galß1-3 structure. When administered to DLD-1 cells, a colorectal carcinoma cell line expressing mucin-associated TF-antigen, hRTL could induce glycan-dependent cytotoxicity.
Assuntos
Antígenos Glicosídicos Associados a Tumores , Neoplasias Colorretais , Galectinas , Animais , Galectinas/farmacologia , Galectinas/metabolismo , Galectinas/isolamento & purificação , Galectinas/genética , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Linhagem Celular Tumoral , Antígenos Glicosídicos Associados a Tumores/metabolismo , Poríferos , Antineoplásicos/farmacologia , Antineoplásicos/química , Sequência de Aminoácidos , Amino AçúcaresRESUMO
Galectins are a class of lectins that are extensively expressed in all organisms. Galectins are involved in a range of functions, including early development, tissue regeneration, cancer and inflammation. It has been shown that galectin-8 is expressed in the villous and extravillous trophoblast (EVT) cells of the human placenta; however, its physiological role in pregnancy establishment has not been elucidated. Taking these factors into account, we investigated the functional role of galectin-8 in HTR-8/SVneo cells-a human EVT cell line-and human primary cytotrophoblast cells isolated from a first-trimester placenta. We analyzed the effects of recombinant human galectin-8 (rh galectin-8) on the adhesion, migration and invasion of HTR-8/SVneo cells. We used qPCR, cell-based ELISA (cELISA) and gelatin zymography to study the effects of galectin-8 on mediators of these processes, such as integrin subunits alpha-1 and beta-1 and matrix metalloproteinases (MMPs)-2 and -9, on the mRNA and protein levels. Further, we studied the effects of galectin-8 on primary cytotrophoblast cells' invasion. Galectin-8 stimulated the adhesion, migration and invasion of HTR-8/SVneo cells, as well as the invasion of primary cytotrophoblasts. In addition, the MMP-2 and -9 levels were increased, while the expression of integrins alpha-1 and beta-1 was not affected. Galectin-8 has the ability to positively affect EVTs' invasion, so it can be considered a significant factor in the trophoblast cell invasion process.
Assuntos
Adesão Celular , Movimento Celular , Galectinas , Metaloproteinase 2 da Matriz , Trofoblastos , Humanos , Trofoblastos/metabolismo , Trofoblastos/citologia , Galectinas/metabolismo , Movimento Celular/efeitos dos fármacos , Gravidez , Feminino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Linhagem Celular , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Placenta/metabolismo , Placenta/citologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/metabolismoRESUMO
Galectin-9 (Gal-9) expression in patients with acute-on-chronic liver failure and its correlation with prognosis remain unclear. This study investigated the relationship between liver failure prognosis and Gal-9 expression analysis in patients with acute-on-chronic liver failure. Patients with acute-on-chronic liver failure attributable to hepatitis B and those with chronic hepatitis B were included in this single-center prospective cohort study. The Gal-9 levels in the acute-on-chronic liver failure group were significantly higher than those in the chronic hepatitis B group, and there was an upregulation of Gal-9 and T-cell immunoglobulin domain and mucin domain-3 expressions in peripheral blood T cells. Gal-9 was localized in the regenerative areas of liver tissues in patients with acute-on-chronic liver failure, co-localizing with Kupffer cells. Kaplan-Meier survival curves showed that patients with Gal-9 levels < 9.6 ng/ml had a worse prognosis, with the area under the receiver operating characteristic curve (AUC-ROC) being similar to that of the Model for End-Stage Liver Disease score. The combined ROC curve of the two had better predictive performance, with an AUC of 0.945. High Gal-9 levels in liver regenerative areas can serve as a prognostic marker, indicating a better prognosis for patients with hepatitis B virus-acute-on-chronic liver failure.
Assuntos
Insuficiência Hepática Crônica Agudizada , Biomarcadores , Galectinas , Humanos , Galectinas/metabolismo , Galectinas/sangue , Insuficiência Hepática Crônica Agudizada/sangue , Insuficiência Hepática Crônica Agudizada/metabolismo , Insuficiência Hepática Crônica Agudizada/diagnóstico , Feminino , Masculino , Biomarcadores/sangue , Pessoa de Meia-Idade , Prognóstico , Adulto , Curva ROC , Estudos Prospectivos , Hepatite B Crônica/complicações , Fígado/metabolismo , Fígado/patologia , Estimativa de Kaplan-MeierRESUMO
Gestational diabetes mellitus (GDM) is a common condition during pregnancy. The prevalence of GDM is continuously increasing worldwide. Due to accessible diagnostic methods and a clear understanding of risk factors, GDM can be effectively diagnosed and managed. Galectins may influence immunomodulatory and inflammatory processes. This study examines the expression of galectin-7 in the placentas of women with gestational diabetes (GDM), compares it to its expression in healthy pregnancies, and evaluates the associated clinical outcomes. The placentas of 40 healthy women and 40 GDM placentas were included in the cohort. The expression level of galecin-7 was measured in the syncytiotrophoblast (SCT) and in the decidua of the placenta by immunohistochemistry and double immunofluorescence staining. The evaluation was performed by an immunoreactivity score (IRS). The study results show an increased expression of galectin-7 in the SCT and the decidua of GDM placentas as compared to the placentas of the control group. Elevated levels of galectin-7 were observed in both the nucleus and the cytoplasm. This study investigated the hypothesis that galectins are involved in pathophysiological processes of gestational diabetes. Statistical analysis of gene expression patterns confirmed that galectin-7 is indeed upregulated in GDM placentas. Further studies are needed to show the correlation of galectin-7 and the development and maintenance of gestational diabetes mellitus.
Assuntos
Diabetes Gestacional , Galectinas , Placenta , Humanos , Diabetes Gestacional/metabolismo , Diabetes Gestacional/genética , Feminino , Gravidez , Galectinas/metabolismo , Galectinas/genética , Placenta/metabolismo , Adulto , Trofoblastos/metabolismo , Decídua/metabolismo , Decídua/patologia , Estudos de Casos e ControlesRESUMO
Transforming growth factor (TGF)-ß signaling is critical for epithelial-mesenchymal transition (EMT) and colorectal cancer (CRC) metastasis. Disruption of Smad-depednent TGF-ß signaling has been shown in CRC cells. However, TGF-ß receptor remains expressed on CRC cells. Here, we investigated whether the cooperation between tumor-associated N-glycosylation and a glycan-binding protein modulated the TGF-ß-driven signaling and metastasis of CRC. We showed that galectin-8, a galactose-binding lectin, hampered TGF-ß-induced EMT by interacting with the type II TGF-ß receptor and competing with TGF-ß binding. Depletion of galectin-8 promoted the migration of CRC cells by increasing TGF-ß-receptor-mediated RAS and Src signaling, which was attenuated after recombinant galectin-8 treatment. Treatment with recombinant galectin-8 also induces JNK-dependent apoptosis in CRC cells. The anti-migratory effect of galectin-8 depended on ß4-galactosyltransferase-I (B4GALT1), an enzyme involved in N-glycan synthesis. Increased B4GALT1 expression was observed in clinical CRC samples. Depletion of B4GALT1 reduced the metastatic potential of CRC cells. Furthermore, inducible expression of galectin-8 attenuated tumor development and metastasis of CRC cells in an intra-splenic injection model. Our results thus demonstrate that galectin-8 alters non-canonical TGF-ß response in CRC cells and suppresses CRC progression.
Assuntos
Movimento Celular , Neoplasias Colorretais , Transição Epitelial-Mesenquimal , Galactosiltransferases , Galectinas , Metástase Neoplásica , Humanos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Galectinas/metabolismo , Galectinas/genética , Galactosiltransferases/metabolismo , Galactosiltransferases/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Animais , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Linhagem Celular Tumoral , Transdução de Sinais , Camundongos , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Camundongos Nus , Ligação Proteica , Apoptose/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Premature rupture of the membranes (PROM) is a key cause of preterm birth and represents a major cause of neonatal mortality and morbidity. Natural products N-acetyl-d-galactosamine (GalNAc), which are basic building blocks of important polysaccharides in biological cells or tissues, such as chitin, glycoproteins, and glycolipids, may improve possible effects of wound healing. METHODS: An in vitro inflammation and oxidative stress model was constructed using tumor necrosis-α (TNF-α) and lipopolysaccharide (LPS) action on WISH cells. Human amniotic epithelial cells (hAECs) were primarily cultured by digestion to construct a wound model. The effects of GalNAc on anti-inflammatory and anti-oxidative stress, migration and proliferation, epithelial-mesenchymal transition (EMT), glycosaminoglycan (GAG)/hyaluronic acid (HA) production, and protein kinase B (Akt) pathway in hAECs and WISH cells were analyzed using the DCFH-DA fluorescent probe, ELISA, CCK-8, scratch, transwell migration, and western blot to determine the mechanism by which GalNAc promotes amniotic wound healing. RESULTS: GalNAc decreased IL-6 expression in TNF-α-stimulated WISH cells and ROS expression in LPS-stimulated WISH cells (P < 0.05). GalNAc promoted the expression of Gal-1 and Gal-3 with anti-inflammatory and anti-oxidative stress effects. GalNAc promoted the migration of hAECs (50% vs. 80%) and WISH cells through the Akt signaling pathway, EMT reached the point of promoting fetal membrane healing, and GalNAc did not affect the activity of hAECs and WISH cells (P > 0.05). GalNAc upregulated the expression of sGAG in WISH cells (P < 0.05) but did not affect HA levels (P > 0.05). CONCLUSIONS: GalNAc might be a potential target for the prevention and treatment of PROM through the galectin pathway, including (i) inflammation; (ii) epithelial-mesenchymal transition; (iii) proliferation and migration; and (iv) regression, remodeling, and healing.
Assuntos
Acetilgalactosamina , Movimento Celular , Transição Epitelial-Mesenquimal , Ruptura Prematura de Membranas Fetais , Galectinas , Transdução de Sinais , Cicatrização , Humanos , Ruptura Prematura de Membranas Fetais/metabolismo , Acetilgalactosamina/metabolismo , Acetilgalactosamina/análogos & derivados , Galectinas/metabolismo , Gravidez , Células Epiteliais/metabolismo , Linhagem Celular , Estresse Oxidativo , Feminino , Âmnio/metabolismo , Âmnio/citologia , Proliferação de Células , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.
Assuntos
Galectinas , Imunidade Inata , Infecções por Klebsiella , Klebsiella pneumoniae , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/metabolismo , Humanos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Galectinas/metabolismo , Galectinas/imunologia , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/microbiologia , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Lectinas/metabolismo , Lectinas/imunologia , Ligação ProteicaRESUMO
During neuronal pruning, phagocytes engulf shed cellular debris to avoid inflammation and maintain tissue homeostasis. How phagocytic receptors recognize degenerating neurites had been unclear. Here, we identify two glucosyltransferases Alg8 and Alg10 of the N-glycosylation pathway required for dendrite fragmentation and clearance through genetic screen. The scavenger receptor Draper (Drpr) is N-glycosylated with complex- or hybrid-type N-glycans that interact specifically with galectins. We also identify the galectins Crouching tiger (Ctg) and Hidden dragon (Hdg) that interact with N-glycosylated Drpr and function in dendrite pruning via the Drpr pathway. Ctg and Hdg are required in hemocytes for expression and function, and are induced during dendrite injury to localize to injured dendrites through specific interaction with exposed phosphatidylserine (PS) on the surface membrane of injured dendrites. Thus, the galectins Ctg and Hdg bridge the interaction between PS and N-glycosylated Drpr, leading to the activation of phagocytosis.
Assuntos
Dendritos , Galectinas , Hemócitos , Fagocitose , Fosfatidilserinas , Animais , Fosfatidilserinas/metabolismo , Glicosilação , Galectinas/metabolismo , Hemócitos/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genéticaRESUMO
With the ongoing advancement of immune checkpoint research, targeting tumors through immune checkpoint blockade has emerged as a crucial approach in cancer therapy. T cell immunoglobulin and mucin-domain containing protein 3 (TIM-3) functions as a negative immune checkpoint. It has been demonstrated that the interaction of TIM-3 with its ligand galectin-9 (Gal-9) can promote immune escape in a variety of cancers. In hematologic, digestive, and respiratory tumors, it affects different signaling pathways by blocking TIM-3/Gal-9 interaction, thereby regulating the growth of T cells, B cells, dendritic cells, NK cells, and monocytes/macrophages, and inhibiting regulatory T cells to exert anti-tumor effects. TIM-3 antibodies have potential therapeutic value as immune checkpoint inhibitors in molecularly-targeted anti-tumor therapy. This article reviews the mechanisms of anti-tumor effects of TIM-3/Gal-9 in the tumor microenvironment of various cancers.
Assuntos
Galectinas , Receptor Celular 2 do Vírus da Hepatite A , Neoplasias , Humanos , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Galectinas/metabolismo , Galectinas/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Microambiente Tumoral/imunologia , Transdução de SinaisRESUMO
Galectins are ß-galactoside-binding animal lectins involved in various biological functions, such as host defense. Galectin-2 and -3 are members of the galectin family that are expressed in the stomach, including the gastric mucosa and surface mucous cells. Galectin-3 exhibits aggregation and bactericidal activity against Helicobacter pylori in a ß-galactoside-dependent manner. We previously reported that galectin-2 has the same activity under neutral pH conditions. In this study, the H. pylori aggregation activity of galectin-2 was examined under weakly acidic conditions, in which H. pylori survived. Galectin-2 agglutinated H. pylori even at pH 6.0, but not at pH 5.0, correlating with its structural stability, as determined using circular dichroism. Additionally, galectin-2 binding to the lipopolysaccharide (LPS) of H. pylori cultured under weakly acidic conditions was investigated using affinity chromatography and Western blotting. Galectin-2 could bind to H. pylori LPS containing H type I, a Lewis antigen, in a ß-galactoside-dependent manner. In contrast, galectin-3 was structurally more stable than galectin-2 under acidic conditions and bound to H. pylori LPS containing H type I and Lewis X. In conclusion, galectin-2 and -3 might function cooperatively in the defense against H. pylori in the stomach under different pH conditions.
Assuntos
Galectina 2 , Helicobacter pylori , Lipopolissacarídeos , Helicobacter pylori/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/química , Concentração de Íons de Hidrogênio , Galectina 2/metabolismo , Galectina 2/química , Humanos , Galectina 3/metabolismo , Galectina 3/química , Ligação Proteica , Aglutinação , Galectinas/metabolismo , Galectinas/químicaRESUMO
Galectin-3 (Gal-3) is a carbohydrate binding protein that has been implicated in the development and progression of fibrotic diseases. Proof-of-principal animal models have demonstrated that inhibition of Gal-3 is a potentially viable pathway for the treatment of fibrosisâwith small molecule Gal-3 inhibitors advanced into clinical trials. We hereby report the discovery of novel galactose-based monosaccharide Gal-3 inhibitors comprising 2-methyl-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (compound 20) and 4-phenyl-4H-1,2,4-triazole (compound 15). Notably, hindered rotation caused by steric interaction between the 3-thione and ortho-trifluoromethyl group of compounds 20, 21 induced formation of thermodynamically stable atropisomers. Distinct X-ray cocrystal structures of 20 and 21 were obtained, which clearly demonstrated that the configuration of 21 proscribes a key halogen bonding σ-hole interaction of 3-chloro with carbonyl oxygen of Gly182, thereby leading to significant loss in potency. Ultimately, 20 and 15 were evaluated in mouse pharmacokinetic studies, and both compounds exhibited oral exposures suitable for further in vivo assessment.
Assuntos
Galactose , Galectina 3 , Triazóis , Triazóis/química , Triazóis/farmacologia , Triazóis/síntese química , Triazóis/farmacocinética , Galactose/química , Galactose/metabolismo , Animais , Humanos , Galectina 3/antagonistas & inibidores , Galectina 3/metabolismo , Camundongos , Relação Estrutura-Atividade , Cristalografia por Raios X , Tionas/química , Tionas/farmacologia , Tionas/síntese química , Tionas/farmacocinética , Proteínas Sanguíneas/metabolismo , Galectinas/antagonistas & inibidores , Galectinas/metabolismo , Modelos MolecularesRESUMO
Galectins (Gals) are a type of S-type lectin that are widespread and evolutionarily conserved among metazoans, and can act as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). In this study, 10 Gals (ToGals) were identified in the Golden pompano (Trachinotus ovatus), and their conserved domains, motifs, and collinearity relationships were analyzed. The expression of ToGals was regulated following infection to Cryptocaryon irritans and Streptococcus agalactiae, indicating that ToGals participate in immune responses against microbial pathogens. Further analysis was conducted on one important member, Galectin-3, subcellular localization showing that ToGal-3like protein is expressed both in the nucleus and cytoplasm. Recombinant protein obtained through prokaryotic expression showed that rToGal-3like can agglutinate red blood cells of rabbit, carp and golden pompano and also agglutinate and kill Staphylococcus aureus, Bacillus subtilis, Vibrio vulnificus, S. agalactiae, Pseudomonas aeruginosa, and Aeromonas hydrophila. This study lays the foundation for further research on the immune roles of Gals in teleosts.
Assuntos
Galectinas , Filogenia , Animais , Galectinas/genética , Galectinas/imunologia , Galectinas/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Família Multigênica , Streptococcus agalactiae/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Peixes/imunologia , Peixes/genética , Perciformes/imunologia , Perciformes/genética , Perfilação da Expressão GênicaRESUMO
Galectin-8 is a small soluble lectin with two carbohydrate recognition domains (CRDs). N- and C-terminal CRDs of Gal-8 differ in their specificity for glycan ligands. Here, we wanted to find out whether oligomerization of individual CRDs of galectin-8 affects its biological activity. Using green fluorescent protein polygons (GFPp) as an oligomerization scaffold, we generated intrinsically fluorescent CRDs with altered valency. We show that oligomers of C-CRD are characterized by significant cell surface affinity. Furthermore, the multivalency of the resulting variants has an impact on cellular activities such as cell signaling, heparin binding and proliferation. Our data indicates that tunable valence is a useful tool for modifying the biological activity of CRDs of galectins.
Assuntos
Galectinas , Galectinas/metabolismo , Galectinas/química , Humanos , Ligantes , Ligação Proteica , Engenharia de Proteínas/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/química , Multimerização Proteica , Proliferação de Células , Heparina/química , Heparina/metabolismoRESUMO
IgA nephropathy (IgAN) is the most common type of glomerulonephritis that frequently progresses to kidney failure. However, the molecular pathogenesis underlying IgAN remains largely unknown. Here, we investigated the role of galectin-3 (Gal-3), a galactoside-binding protein in IgAN pathogenesis, and showed that Gal-3 expression by the kidney was significantly enhanced in patients with IgAN. In both TEPC-15 hybridoma-derived IgA-induced, passive, and spontaneous "grouped" ddY IgAN models, Gal-3 expression was clearly increased with disease severity in the glomeruli, peri-glomerular regions, and some kidney tubules. Gal-3 knockout (KO) in the passive IgAN model had significantly improved proteinuria, kidney function and reduced severity of kidney pathology, including neutrophil infiltration and decreased differentiation of Th17 cells from kidney-draining lymph nodes, despite increased percentages of regulatory T cells. Gal-3 KO also inhibited the NLRP3 inflammasome, yet it enhanced autophagy and improved kidney inflammation and fibrosis. Moreover, administration of 6-de-O-sulfated, N-acetylated low-molecular-weight heparin, a competitive Gal-3 binding inhibitor, restored kidney function and improved kidney lesions in passive IgAN mice. Thus, our results suggest that Gal-3 is critically involved in IgAN pathogenesis by activating the NLRP3 inflammasome and promoting Th17 cell differentiation. Hence, targeting Gal-3 action may represent a new therapeutic strategy for treatment of this kidney disease.
Assuntos
Modelos Animais de Doenças , Galectina 3 , Glomerulonefrite por IGA , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Células Th17 , Glomerulonefrite por IGA/patologia , Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/metabolismo , Glomerulonefrite por IGA/genética , Animais , Galectina 3/metabolismo , Galectina 3/genética , Galectina 3/antagonistas & inibidores , Humanos , Células Th17/imunologia , Células Th17/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Masculino , Feminino , Inflamassomos/metabolismo , Inflamassomos/imunologia , Autofagia/efeitos dos fármacos , Fibrose , Linfócitos T Reguladores/imunologia , Diferenciação Celular , Galectinas/genética , Galectinas/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Camundongos Endogâmicos C57BL , Glomérulos Renais/patologia , Glomérulos Renais/imunologia , Imunoglobulina A/metabolismo , Imunoglobulina A/imunologiaRESUMO
Glycans cover the cell surface to form the glycocalyx, which governs a myriad of biological phenomena. However, understanding and regulating glycan functions is extremely challenging due to the large number of heterogeneous glycans that engage in intricate interaction networks with diverse biomolecules. Glycocalyx-editing techniques offer potent tools to probe their functions. In this study, we devised a HaloTag-based technique for glycan manipulation, which enables the introduction of chemically synthesized glycans onto a specific protein (protein of interest, POI) and concurrently incorporates fluorescent units to attach homogeneous, well-defined glycans to the fluorescence-labeled POIs. Leveraging this HaloTag-based glycan-display system, we investigated the influence of the interactions between Gal-3 and various N-glycans on protein dynamics. Our analyses revealed that glycosylation modulates the lateral diffusion of the membrane proteins in a structure-dependent manner through interaction with Gal-3, particularly in the context of the Gal-3-induced formation of the glycan network (galectin lattice). Furthermore, N-glycan attachment was also revealed to have a significant impact on the extracellular vesicle-loading of membrane proteins. Notably, our POI-specific glycan introduction does not disrupt intact glycan structures, thereby enabling a functional analysis of glycans in the presence of native glycan networks. This approach complements conventional glycan-editing methods and provides a means for uncovering the molecular underpinnings of glycan functions on the cell surface.
Assuntos
Vesículas Extracelulares , Galectinas , Proteínas de Membrana , Polissacarídeos , Polissacarídeos/química , Polissacarídeos/metabolismo , Glicosilação , Galectinas/metabolismo , Galectinas/química , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Humanos , Difusão , Membrana Celular/metabolismo , Membrana Celular/químicaRESUMO
Structural characterization of protein-ligand binding interfaces at atomic resolution is essential for improving the design of specific and potent inhibitors. Herein, we explored fast 19F- and 1H-detected magic angle spinning NMR spectroscopy to investigate the interaction between two fluorinated ligand diastereomers with the microcrystalline galectin-3 carbohydrate recognition domain. The detailed environment around the fluorine atoms was mapped by 2D 13C-19F and 1H-19F dipolar correlation experiments and permitted characterization of the binding interface. Our results demonstrate that 19F MAS NMR is a powerful tool for detailed characterization of protein-ligand interfaces and protein interactions at the atomic level.
Assuntos
Flúor , Galectina 3 , Galectinas , Galectina 3/química , Galectina 3/metabolismo , Ligantes , Flúor/química , Galectinas/química , Galectinas/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Humanos , Ligação Proteica , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Modelos Moleculares , Domínios Proteicos , Carboidratos/químicaRESUMO
Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as ß-galactoside binding lectins. However, certain members of this family have shown selective affinity toward specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7, and -12) toward a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I versus type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity toward poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance experiments. Particularly, we identified oligosaccharide blood group A antigen tetraose 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited interleukin-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses.
Assuntos
Antígenos de Grupos Sanguíneos , Galectina 4 , Galectinas , Leite Humano , Oligossacarídeos , Humanos , Leite Humano/metabolismo , Leite Humano/química , Oligossacarídeos/metabolismo , Oligossacarídeos/química , Antígenos de Grupos Sanguíneos/metabolismo , Antígenos de Grupos Sanguíneos/química , Galectinas/metabolismo , Galectinas/química , Ligantes , Galectina 4/metabolismo , Galectina 4/química , Ligação Proteica , Interleucina-6/metabolismoRESUMO
Endometrial cancer (EC) is a common gynecological cancer worldwide. Treatment has been improved in recent years; however, in advanced stages, therapeutic options are still limited. The expression of galectins is increased in several tumor types and that they are involved in important cell processes. Large studies on endometrial cancer are still pending; Specimens of 225 patients with EC were immunohistochemically stained with antibodies for Gal-8 and Gal-9. Expression was correlated with histopathological variables. The cytosolic expression of both galectins is associated with grading and survival. Cytosolic Galectin-8 expression is a positive prognostic factor for overall survival (OS) and progression-free survival (PFS), while nuclear Gal-8 expression correlates only to OS. The cytosolic presence of Galectin-9 is correlated with a better prognosis regarding OS. Our results suggest that expression of both galectins is associated with OS and PFS in EC. Further studies are needed to understand the underlying molecular mechanisms.