RESUMO
Extracellular proteolysis critically regulates cellular and tissue responses and is often dysregulated in human diseases. The crosstalk between proteolytic processing and other major post-translational modifications (PTMs) is emerging as an important regulatory mechanism to modulate protease activity and maintain cellular and tissue homeostasis. Here, we focus on matrix metalloproteinase (MMP)-mediated cleavages and N-acetylgalactosamine (GalNAc)-type of O-glycosylation, two major PTMs of proteins in the extracellular space. We investigated the influence of truncated O-glycan trees, also referred to as Tn antigen, following the inactivation of C1GALT1-specific chaperone 1 (COSMC) on the general and MMP9-specific proteolytic processing in MDA-MB-231 breast cancer cells. Quantitative assessment of the proteome and N-terminome using terminal amine isotopic labelling of substrates (TAILS) technology revealed enhanced proteolysis by MMP9 within the extracellular proteomes of MDA-MB-231 cells expressing Tn antigen. In addition, we detected substantial modifications in the proteome and discovered novel ectodomain shedding events regulated by the truncation of O-glycans. These results highlight the critical role of mature O-glycosylation in fine-tuning proteolytic processing and proteome homeostasis by modulating protein susceptibility to proteolytic degradation. These data suggest a complex interplay between proteolysis and O-GalNAc glycosylation, possibly affecting cancer phenotypes.
Assuntos
Proteólise , Humanos , Glicosilação , Linhagem Celular Tumoral , Metaloproteinase 9 da Matriz/metabolismo , Antígenos Glicosídicos Associados a Tumores/metabolismo , Galactosiltransferases/metabolismo , Galactosiltransferases/genética , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteoma/análise , Chaperonas MolecularesRESUMO
BACKGROUND: Xenotransplantation using pig organs is now a clinical reality. However, the process for xenograft recipient screening lacks clarity and scientific rigor: no established thresholds exist to determine which levels of preformed antipig natural antibodies (Nabs) will be safe for clinical xenograft transplantation, and hyperacute rejection (HAR) or acute humoral xenograft rejection (AHXR), which still impacts pig-to-primate kidney xenograft survivals, may impede broader application of pig-to-human clinical xenograft transplantation. METHODS: We retrospectively examined 28 cases of pig-to-baboon kidney xenotransplantation using GalTKO±human complement regulatory protein (hCRP)-transgenic (Tg) pig donors, as well as 6 cases of triple-KO multi-Tg (10GE) pig donors, and developed screening algorithms to predict risk of HAR/AHXR based on recipient antipig Nab levels. Preformed Nabs were evaluated using both complement-dependent cytotoxicity and antibody (IgM and IgG) binding flow-cytometry assays. RESULTS: High complement-dependent cytotoxicity was associated with HAR/AHXR as expected. However, we also found that high levels of IgG were independently associated with HAR/AHXR, and we developed 2 indices to interpret and predict the risk of IgG-mediated HAR/AHXR. CONCLUSIONS: Based on the data in this study, we have established a new 2-step screening, which will be used for future clinical kidney xenotransplantation trials.
Assuntos
Animais Geneticamente Modificados , Rejeição de Enxerto , Sobrevivência de Enxerto , Transplante de Rim , Transplante Heterólogo , Animais , Transplante Heterólogo/efeitos adversos , Transplante de Rim/efeitos adversos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Estudos Retrospectivos , Suínos , Fatores de Risco , Imunoglobulina G/sangue , Galactosiltransferases/genética , Galactosiltransferases/imunologia , Galactosiltransferases/deficiência , Xenoenxertos , Imunidade Humoral , Imunoglobulina M/sangue , Humanos , Masculino , Anticorpos Heterófilos/imunologia , Doença AgudaRESUMO
Pregnane X receptor (PXR) has been reported to regulate glycolipid metabolism. The dysfunction of intestinal barrier contributes to metabolic disorders. However, the role of intestinal PXR in metabolic diseases remains largely unknown. Here, we show that activation of PXR by tributyl citrate (TBC), an intestinal-selective PXR agonist, improves high fat diet (HFD)-induced obesity. The metabolic benefit of intestinal PXR activation is associated with upregulation of ß-1,3 galactosyltransferase 5 (B3galt5). Our results reveal that B3galt5 mainly expresses in the intestine and is a direct PXR transcriptional target. B3galt5 knockout exacerbates HFD-induced obesity, insulin resistance and inflammation. Mechanistically, B3galt5 is essential to maintain the integrity of intestinal mucus barrier. B3galt5 ablation impairs the O-glycosylation of mucin2, destabilizes the mucus layer, and increases intestinal permeability. Furthermore, B3galt5 deficiency abolishes the beneficial effect of intestinal PXR activation on metabolic disorders. Our results suggest the intestinal-selective PXR activation regulates B3galt5 expression and maintains metabolic homeostasis, making it a potential therapeutic strategy in obesity.
Assuntos
Dieta Hiperlipídica , Galactosiltransferases , Resistência à Insulina , Mucosa Intestinal , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade , Receptor de Pregnano X , Animais , Obesidade/metabolismo , Obesidade/genética , Receptor de Pregnano X/metabolismo , Receptor de Pregnano X/genética , Galactosiltransferases/metabolismo , Galactosiltransferases/genética , Camundongos , Dieta Hiperlipídica/efeitos adversos , Mucosa Intestinal/metabolismo , Masculino , Intestinos , HumanosRESUMO
Glycosyltransferases are nature's key biocatalysts for the formation of glycosidic bonds. Discovery and characterization of new synthetically useful glycosyltransferases are critical for the development of efficient enzymatic and chemoenzymatic strategies for producing complex carbohydrates and glycoconjugates. Herein we report the identification of Pasteurella multocida PmNatB as a bifunctional single-catalytic-domain glycosyltransferase with both ß1-3-galactosyltransferase and ß1-3-N-acetylgalactosaminyltransferase activities. It is a novel glycosyltransferase for constructing structurally diverse GalNAcß3Galα/ßOR and Galß3GalNAcα/ßOR disaccharides in one-pot multienzyme systems with in situ generation of UDP-sugars.
Assuntos
Dissacarídeos , N-Acetilgalactosaminiltransferases , Pasteurella multocida , Pasteurella multocida/enzimologia , Dissacarídeos/química , Dissacarídeos/síntese química , Dissacarídeos/biossíntese , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/química , Galactosiltransferases/metabolismoRESUMO
Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and ß-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-ß-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-ß-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides.
Assuntos
Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , UTP-Hexose-1-Fosfato Uridililtransferase/genética , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/química , Expressão Gênica , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/química , Clonagem Molecular , Galactosefosfatos/metabolismo , Galactosefosfatos/genética , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Galactosiltransferases/químicaRESUMO
Introduction: Decreasing rates of blood donation and close margins between blood supply and demand pose challenges in healthcare. Genetically engineered pig red blood cells (pRBCs) have been explored as alternatives to human RBCs for transfusion, and triple-gene knockout (TKO) modification improves the compatibility of pRBCs with human blood in vitro. In this study, we assessed the efficacy and risks of transfusing wild-type (WT)- and TKO-pRBCs into nonhuman primates (NHPs). Methods: Blood from O-type WT and TKO pigs was processed to produce pRBCs for transfusion, which were transfused or not into NHPs (n=4 per group: WT, TKO, and control) after 25% total blood volume withdrawal: their biological responses were compared. Hematological, biochemical, and immunological parameters were measured before, immediately after, and at intervals following transfusion. Two months later, a second transfusion was performed in three NHPs of the transfusion group. Results: Transfusion of both WT- and TKO-pRBCs significantly improved RBC counts, hematocrit, and hemoglobin levels up to the first day post-transfusion, compared to the controls. The transfusion groups showed instant complement activation and rapid elicitation of anti-pig antibodies, as well as elevated liver enzyme and bilirubin levels post-transfusion. Despite the higher agglutination titers with WT-pRBCs in the pre-transfusion crossmatch, the differences between the WT and TKO groups were not remarkable except for less impairment of liver function in the TKO group. After the second transfusion, more pronounced adverse responses without any hematological gain were observed. Conclusions: WT- and TKO-pRBC transfusions effectively increased hematologic parameters on the first day, with rapid clearance from circulation thereafter. However, pRBC transfusion triggers strong antibody responses, limiting the benefits of the pRBC transfusion and increasing the risk of adverse reactions.
Assuntos
Transfusão de Eritrócitos , Eritrócitos , Técnicas de Inativação de Genes , Animais , Transfusão de Eritrócitos/efeitos adversos , Transfusão de Eritrócitos/métodos , Suínos , Eritrócitos/imunologia , Eritrócitos/metabolismo , Animais Geneticamente Modificados , Hemoglobinas/metabolismo , Galactosiltransferases/genética , Galactosiltransferases/deficiência , Hematócrito , Feminino , Masculino , PrimatasRESUMO
INTRODUCTION: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs. METHODS: Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). RESULTS: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. CONCLUSION: Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.
Assuntos
Animais Geneticamente Modificados , Transplante de Coração , Inflamação , Papio , Transplante Heterólogo , Animais , Transplante Heterólogo/métodos , Transplante de Coração/métodos , Suínos , Inflamação/imunologia , Coagulação Sanguínea/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Humanos , Xenoenxertos/imunologia , Galactosiltransferases/genética , Imunossupressores/farmacologia , Citocinas/metabolismoRESUMO
BACKGROUND: Polyclonal rabbit antithymocyte globulins (ATGs) are commonly used in organ transplantation as induction. Anti- N -glycolylneuraminic acid carbohydrate antibodies which develop in response to rabbit carbohydrate antigens might lead to unwanted systemic inflammation. LIS1, the first new generation of antilymphocyte globulins (ALGs) derived from double knockout swine, lacking carbohydrate xenoantigens was already tested in nonhuman primates and rodent models. METHODS: This open-label, single-site, dose escalation, first-in-human, phase 1 study evaluated the safety, T cell depletion, pharmacokinetics, and pharmacodynamics of LIS1. In an ascending dose cohort (nâ =â 5), a primary kidney transplant recipient at low immunologic risk (panel reactive antibody [PRA]â <â 20%), received LIS1 for 5 d at either 0.6, 1, 3, 6, or 8 mg/kg. After each patient completed treatment, the data safety monitoring board approved respective dose escalation. In the therapeutic dose cohort (nâ =â 5) in patients with PRA <50% without donor specific antibodies, 2 patients received 8 mg/kg and 3 patients 10 mg/kg. RESULTS: CD3 + T cell depletion <100/mm 3 at day 2 was observed in all patients who received 6, 8, and 10 mg/kg of LIS1. The terminal half-life of LIS1 was 33.7 d with linearity in its disposition. Lymphocyte repopulation was fast and pretransplant lymphocyte subpopulation counts recovered within 2-4 wk. LIS1 was well tolerated, neither cytokine release syndrome nor severe thrombocytopenia or leukopenia were noticed. Antibodies to LIS1 were not detected. CONCLUSIONS: In this first-in-human trial, genome-edited swine-derived polyclonal LIS1 ALG was well tolerated, did not elicit antidrug antibodies, and caused time-limited T cell depletion in low- and medium-risk kidney transplant recipients.
Assuntos
Soro Antilinfocitário , Transplante de Rim , Transplante de Rim/efeitos adversos , Humanos , Animais , Soro Antilinfocitário/imunologia , Masculino , Pessoa de Meia-Idade , Suínos , Feminino , Adulto , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Depleção Linfocítica/métodos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Resultado do Tratamento , GalactosiltransferasesRESUMO
Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.
Assuntos
Sistemas CRISPR-Cas , Centrifugação , Eletroporação , Edição de Genes , Animais , Edição de Genes/métodos , Suínos , Eletroporação/métodos , Sistemas CRISPR-Cas/genética , Embrião de Mamíferos/metabolismo , Zona Pelúcida/metabolismo , Zigoto/metabolismo , Blastocisto/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , Mutação/genética , GalactosiltransferasesRESUMO
Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (ß-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.
Assuntos
Animais Geneticamente Modificados , Galactosiltransferases , Técnicas de Inativação de Genes , Rejeição de Enxerto , Sobrevivência de Enxerto , Transgenes , Transplante Heterólogo , Animais , Sobrevivência de Enxerto/imunologia , Humanos , Suínos , Galactosiltransferases/genética , Galactosiltransferases/deficiência , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Macaca mulatta , Transplante de RimRESUMO
Larsen of La Réunion Island syndrome (LRS) is an autosomal recessive condition associated with multiple large joint dislocations, clubfeet, severe dwarfism, and distinctive facial features. LRS is caused by a recurrent homozygous variant in B4GALT7 gene with a founder effect in La Réunion population. Proteoglycans (PG) that are a major component of the extracellular matrix, are composed of a core protein connected to a glycosaminoglycans side chain via a tetrasaccharide linker region. B4GALT7 encodes galactosyltransferase I, one of the enzymes involved in the biosynthesis of the linker region. Conditions caused by pathogenic biallelic variants in genes implicated in the synthesis of the tetrasaccharide linker of PG are known as linkeropathies. Prenatal features are rarely described in this group of chondrodysplasias. We present a series of 12 unpublished patients having LRS and describe the perinatal phenotype. All the patients had a prenatal growth restriction with brevity of limbs. The other features revealed by ultrasounds were increased nuchal translucency at 10-12 weeks of gestation (50 %), feet abnormalities (clubfeet or metatarsus varus) (25 %), dislocation affecting at least one large joint (elbow, knee, wrist) (25 %). Bilateral bowing of femora was noted for two fetuses. Fibular hypertrophy was noted for one fetus. Prenatal helical computed tomography (CT) performed in three pregnancies showed additional data such as bowing of the forearm bones, proximal radio-ulnar synostosis, or dislocation of large joints. Prenatal sonographic and helical CT findings led to the prenatal diagnosis of LRS in four patients. We confirm that the neonatal clinical picture of LRS has an important overlap with that reported in patients with B4GALT7 deficiency outside La Réunion Island and other linkeropathies. The core of the phenotypic spectrum combines low birth height, micromelia, hypermobility, dislocation of at least one large joint, facial features with prominent eyes, microstomia, depressed nasal bridge, and midface hypoplasia. Other clinical features include clubfeet (33%), bifid thumb in one patient, and cardiac abnormalities in two patients. Radiological findings include radio-ulnar synostosis (75%), metaphyseal flaring, precocious carpal ossification, and a Swedish key appearance of the proximal femora. Finally, we also report radiological features rarely described in B4GALT7-linkeropathies, including bowing of the femora and fibular hypertrophy. Our results confirm the phenotypic continuum of LRS within linkeropathies with some additional findings, including a high frequency of clubfeet usually described in B3GALT6-linkeropathies, the presence of congenital heart diseases usually described in B3GAT3-linkeropathies, and a high frequency of metaphyseal flaring usually reported in B3GALT6 or XITLT1-linkeropathies. This is the first study that describes the perinatal phenotype in a cohort of patients with LRS. This study can help improve the prenatal diagnosis of the linkeropathies and add this group of conditions to the differential diagnosis of chondrodysplasias with multiple dislocations. In view of the founder effect for LRS in La Réunion Island, this disease should be suspected in fetuses with growth restriction and micromelia. Thus in case of LOH which include B4GALT7 identified in SNP-array, we recommend performing a targeted Sanger sequencing for the recurrent mutation c.808C > T; p. (Arg270Cys).
Assuntos
Galactosiltransferases , Osteocondrodisplasias , Fenótipo , Humanos , Feminino , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Masculino , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Recém-Nascido , GravidezRESUMO
BACKGROUND: Neuroimmune plays an important role in major depressive disorders (MDD). N-linked protein glycosylation (NLG) might contribute to depression by regulating the neuroinflammatory response. As microglia is the main executor of neuroimmune function in the central neural system (CNS), targeting the process of N-linked protein glycosylation of microglia in the mice used for studying depression might potentially offer new avenues for the strategy for MDD. METHODS: The chronic unpredictable mild stress (CUMS) mouse model was established for the whole brain microglia isolating. Then, RNA samples of microglia were extracted for transcriptome sequencing and mRNA analysis. Immunofluorescence (IF) was used to identify the expression level of NLG-related enzyme, B4galt1, in microglia. RESULTS: The data showed that NLG was positively related to depression. Moreover, the NLG-related gene, B4galt1 increased expression in the microglia of CUMS mice. Then, the inhibition of NLG reversed the depressive behavior in CUMS mice. The expression level of B4galt1 in CUMS mice was upregulating following the NLG-inhibitor treatment. Similar results haven't been observed in neurons. Information obtained from these experiments showed increasing expression of B4galt1 in microglia following depressive-like behaviors. CONCLUSIONS: These findings indicate that NLG in microglia is associated with MDD, and suggest that therapeutically targeting NLG might be an effective strategy for depression. LIMITATIONS: How to modulate the B4galt1 or NLG pathways in microglia efficiently and economically request new technologies.
Assuntos
Transtorno Depressivo Maior , Modelos Animais de Doenças , Microglia , Animais , Camundongos , Microglia/metabolismo , Glicosilação , Transtorno Depressivo Maior/metabolismo , Masculino , Estresse Psicológico/metabolismo , Estresse Psicológico/imunologia , Depressão/metabolismo , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Encéfalo/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Gene-gene interactions likely contribute to the etiology of multifactorial diseases such as cerebral venous thrombosis (CVT) and could be one of the main sources of known missing heritability. We explored Factor XI (F11) and ABO gene interactions among patients with CVT. METHODS: Patients with CVT of European ancestry from the large Bio-Repository to Establish the Aetiology of Sinovenous Thrombosis (BEAST) international collaboration were recruited. Codominant modelling was used to determine interactions between genome-wide identified F11 and ABO genes with CVT status. RESULTS: We studied 882 patients with CVT and 1,205 ethnically matched control participants (age: 42 ± 15 vs 43 ± 12 years, p = 0.08: sex: 71% male vs 68% female, p = 0.09, respectively). Individuals heterozygous (AT) for the risk allele (T) at both loci (rs56810541/F11 and rs8176645/ABO) had a 3.9 (95% CI 2.74-5.71, p = 2.75e-13) increase in risk of CVT. Individuals homozygous (TT) for the risk allele at both loci had a 13.9 (95% CI 7.64-26.17, p = 2.0e-15) increase in risk of CVT. The presence of a non-O blood group (A, B, AB) combined with TT/rs56810541/F11 increased CVT risk by OR = 6.8 (95% CI 4.54-10.33, p = 2.00e15), compared with blood group-O combined with AA. DISCUSSION: Interactions between factor XI and ABO genes increase risk of CVT by 4- to 14-fold.
Assuntos
Sistema ABO de Grupos Sanguíneos , Fator XI , Humanos , Sistema ABO de Grupos Sanguíneos/genética , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Fator XI/genética , Trombose Venosa/genética , Trombose Intracraniana/genética , Epistasia Genética/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , GalactosiltransferasesRESUMO
The interleukin (IL)-22 cytokine can be protective or inflammatory in the intestine. It is unclear if IL-22 receptor (IL-22Ra1)-mediated protection involves a specific type of intestinal epithelial cell (IEC). By using a range of IEC type-specific Il22Ra1 conditional knockout mice and a dextran sulfate sodium (DSS) colitis model, we demonstrate that IL-22Ra1 signaling in MATH1+ cells (goblet and progenitor cells) is essential for maintaining the mucosal barrier and intestinal tissue regeneration. The IL-22Ra1 signaling in IECs promotes mucin core-2 O-glycan extension and induces beta-1,3-galactosyltransferase 5 (B3GALT5) expression in the colon. Adenovirus-mediated expression of B3galt5 is sufficient to rescue Il22Ra1IEC mice from DSS colitis. Additionally, we observe a reduction in the expression of B3GALT5 and the Tn antigen, which indicates defective mucin O-glycan, in the colon tissue of patients with ulcerative colitis. Lastly, IL-22Ra1 signaling in MATH1+ progenitor cells promotes organoid regeneration after DSS injury. Our findings suggest that IL-22-dependent protective responses involve O-glycan modification, proliferation, and differentiation in MATH1+ progenitor cells.
Assuntos
Colite , Sulfato de Dextrana , Interleucina 22 , Interleucinas , Receptores de Interleucina , Animais , Interleucinas/metabolismo , Camundongos , Glicosilação , Colite/metabolismo , Colite/patologia , Colite/induzido quimicamente , Receptores de Interleucina/metabolismo , Mucinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Humanos , Transdução de Sinais , Camundongos Endogâmicos C57BL , Inflamação/patologia , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos Knockout , Galactosiltransferases/metabolismo , Galactosiltransferases/genética , Células-Tronco/metabolismoRESUMO
PURPOSE: Core 1ß1,3-galactosyltransferase 1 (C1GALT1) exhibits elevated expression in multiple cancers. The present study aimed to elucidate the clinical significance of C1GALT1 aberrant expression and its impact on radiosensitivity in lung adenocarcinoma (LUAD). METHODS: The C1GALT1 expression and its clinical relevance were investigated through public databases and LUAD tissue microarray analyses. A549 and H1299 cells with either C1GALT1 knockdown or overexpression were further assessed through colony formation, gamma-H2A histone family member X immunofluorescence, 5-ethynyl-2'-deoxyuridine incorporation, and flow cytometry assays. Bioinformatics analysis was used to explore single cell sequencing data, revealing the influence of C1GALT1 on cancer-associated cellular states. Vimentin, N-cadherin, and E-cadherin protein levels were measured through western blotting. RESULTS: The expression of C1GALT1 was significantly higher in LUAD tissues than in adjacent non-tumor tissues both at mRNA and protein level. High expression of C1GALT1 was correlated with lymph node metastasis, advanced T stage, and poor survival, and was an independent risk factor for overall survival. Radiation notably upregulated C1GALT1 expression in A549 and H1299 cells, while radiosensitivity was increased following C1GALT1 knockdown and decreased following overexpression. Experiment results showed that overexpression of C1GALT1 conferred radioresistance, promoting DNA repair, cell proliferation, and G2/M phase arrest, while inhibiting apoptosis and decreasing E-cadherin expression, alongside upregulating vimentin and N-cadherin in A549 and H1299 cells. Conversely, C1GALT1 knockdown had opposing effects. CONCLUSION: Elevated C1GALT1 expression in LUAD is associated with an unfavorable prognosis and contributes to increased radioresistance potentially by affecting DNA repair, cell proliferation, cell cycle regulation, and epithelial-mesenchymal transition (EMT).
Assuntos
Adenocarcinoma de Pulmão , Galactosiltransferases , Neoplasias Pulmonares , Tolerância a Radiação , Humanos , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/radioterapia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/mortalidade , Prognóstico , Tolerância a Radiação/genéticaRESUMO
AIMS/INTRODUCTION: To investigate the long-term efficacy of various encapsulated xenogeneic islet transplantation, and to explore the impact of different donor porcine genetic traits on islet transplantation outcomes. MATERIALS AND METHODS: Donor porcine islets were obtained from wild-type, α1,3-galactosyltransferase knockout (GTKO) and GTKO with overexpression of membrane cofactor protein genotype. Naked, alginate, alginate-chitosan (AC), alginate-perfluorodecalin (A-PFD) and AC-perfluorodecalin (AC-PFD) encapsulated porcine islets were transplanted into diabetic mice. RESULTS: In vitro assessments showed no differences in the viability and function of islets across encapsulation types and donor porcine islet genotypes. Xenogeneic encapsulated islet transplantation with AC-PFD capsules showed the most favorable long-term outcomes, maintaining normal blood glucose levels for 180 days. A-PFD capsules showed comparable results to AC-PFD capsules, followed by AC capsules and alginate capsules. Conversely, blood glucose levels in naked islet transplantation increased to >300 mg/dL within a week after transplantation. Naked islet transplantation outcomes showed no improvement based on donor islet genotype. However, alginate or AC capsules showed delayed increases in blood glucose levels for GTKO and GTKO with overexpression of membrane cofactor protein porcine islets compared with wild-type porcine islets. CONCLUSION: The AC-PFD capsule, designed to ameliorate both hypoxia and inflammation, showed the highest long-term efficacy in xenogeneic islet transplantation. Genetic modifications of porcine islets with GTKO or GTKO with overexpression of membrane cofactor protein did not influence naked islet transplantation outcomes, but did delay graft failure when encapsulated.
Assuntos
Diabetes Mellitus Experimental , Transplante das Ilhotas Pancreáticas , Transplante Heterólogo , Transplante das Ilhotas Pancreáticas/métodos , Animais , Suínos , Camundongos , Transplante Heterólogo/métodos , Diabetes Mellitus Experimental/terapia , Alginatos , Galactosiltransferases/genética , Sobrevivência de Enxerto , Ilhotas Pancreáticas , Glicemia/análise , Masculino , Genótipo , Doadores de TecidosRESUMO
Drought is the most severe form of stress experienced by plants worldwide. Cucumber is a vegetable crop that requires a large amount of water throughout the growth period. In our previous study, we identified that overexpression of CsHSFA1d could improve cold tolerance and the content of endogenous jasmonic acid in cucumber seedlings. To explore the functional diversities of CsHSFA1d, we treat the transgenic plants under drought conditions. In this study, we found that the heat shock transcription factor HSFA1d (CsHSFA1d) could improve drought stress tolerance in cucumber. CsHSFA1d overexpression increased the expression levels of galactinol synthase (CsGolS3) and raffinose synthase (CsRS) genes, encoding the key enzymes for raffinose family oligosaccharide (RFO) biosynthesis. Furthermore, the lines overexpressing CsHSFA1d showed higher enzymatic activity of GolS and raffinose synthase to increase the content of RFO. Moreover, the CsHSFA1d-overexpression lines showed lower reactive oxygen species (ROS) accumulation and higher ROS-scavenging enzyme activity after drought treatment. The expressions of antioxidant genes CsPOD2, CsAPX1 and CsSOD1 were also upregulated in CsHSFA1d-overexpression lines. The expression levels of stress-responsive genes such as CsRD29A, CsLEA3 and CsP5CS1 were increased in CsHSFA1d-overexpression lines after drought treatment. We conclude that CsHSFA1d directly targets and regulates the expression of CsGolS3 and CsRS to promote the enzymatic activity and accumulation of RFO to increase the tolerance to drought stress. CsHSFA1d also improves ROS-scavenging enzyme activity and gene expression indirectly to reduce drought-induced ROS overaccumulation. This study therefore offers a new gene target to improve drought stress tolerance in cucumber and revealed the underlying mechanism by which CsHSFA1d functions in the drought stress by increasing the content of RFOs and scavenging the excessive accumulation of ROS.
Assuntos
Cucumis sativus , Galactosiltransferases , Regulação da Expressão Gênica de Plantas , Oligossacarídeos , Proteínas de Plantas , Plantas Geneticamente Modificadas , Rafinose , Espécies Reativas de Oxigênio , Cucumis sativus/genética , Cucumis sativus/fisiologia , Cucumis sativus/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rafinose/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Oligossacarídeos/metabolismo , Galactosiltransferases/metabolismo , Galactosiltransferases/genética , Secas , Fatores de Transcrição de Choque Térmico/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Estresse Fisiológico/genéticaRESUMO
Novel therapeutic strategies are urgently required for osteosarcoma, given the early age at onset and persistently high mortality rate. Modern transcriptomics techniques can identify differentially expressed genes (DEGs) that may serve as biomarkers and therapeutic targets, so we screened for DEGs in osteosarcoma. We found that osteosarcoma cases could be divided into fair and poor survival groups based on gene expression profiles. Among the genes upregulated in the poor survival group, siRNA-mediated knockdown of the glycosylation-related gene C1GALT1 suppressed osteosarcoma cell proliferation in culture. Gene expression, phosphorylation, and glycome array analyses also demonstrated that C1GALT1 is required to maintain ERK signaling and cell cycle progression. Moreover, the C1GALT1 inhibitor itraconazole suppressed osteosarcoma cell proliferation in culture, while doxycycline-induced shRNA-mediated knockdown reduced xenograft osteosarcoma growth in mice. Elevated C1GALT1 expression is a potential early predictor of poor prognosis, while pharmacological inhibition may be a feasible treatment strategy for osteosarcoma.
Assuntos
Ciclo Celular , Proliferação de Células , Galactosiltransferases , Sistema de Sinalização das MAP Quinases , Osteossarcoma , Osteossarcoma/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Humanos , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Camundongos , Ciclo Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Masculino , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos NusRESUMO
Galactinol synthase (GolS), which catalyses the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR/Cas9 gene-editing technology in tomato (Solanum lycopersicum) to create the gols2 mutant showing uniformly green fruits without dark-green shoulders, and promoting fruit ripening. Analysis indicated that galactinol was undetectable in the ovaries and fruits of the mutant, and the accumulation of chlorophyll and chloroplast development was suppressed in the fruits. RNA-sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development were down-regulated, including PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, GOLDEN 2-LIKE 2, and CHLOROPHYLL A/B-BINDING PROTEINS. In addition, early color transformation and ethylene release was prompted in the gols2 lines by regulation of the expression of genes involved in carotenoid and ethylene metabolism (e.g. PHYTOENE SYNTHASE 1, CAROTENE CIS-TRANS ISOMERASE, and 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2/4) and fruit ripening (e.g. RIPENING INHIBITOR, NON-RIPENING, and APETALA2a). Our results provide evidence for the involvement of GolS2 in pigment and ethylene metabolism of tomato fruits.
Assuntos
Carotenoides , Clorofila , Etilenos , Frutas , Proteínas de Plantas , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/enzimologia , Carotenoides/metabolismo , Clorofila/metabolismo , Frutas/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Etilenos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Galactosiltransferases/metabolismo , Galactosiltransferases/genética , Regulação da Expressão Gênica de PlantasRESUMO
The glycoprotein-N-acetylgalactosamine ß1,3-galactosyltransferase, known as T-synthase (EC 2.4.1.122), plays a crucial role in the synthesis of the T-antigen, which is the core 1 O-glycan structure. This enzyme transfers galactose from UDP-Gal to GalNAc-Ser/Thr. The T-antigen has significant functions in animal development, immune response, and recognition processes. Molluscs are a successful group of animals that inhabit various environments, such as freshwater, marine, and terrestrial habitats. They serve important roles in ecosystems as filter feeders and decomposers but can also be pests in agriculture and intermediate hosts for human and cattle parasites. The identification and characterization of novel carbohydrate active enzymes, such as T-synthase, can aid in the understanding of molluscan glycosylation abilities and their adaptation and survival abilities. Here, the T-synthase enzymes from the snail Pomacea canaliculata and the oyster Crassostrea gigas are identified, cloned, expressed, and characterized, with a focus on structural elucidation. The synthesized enzymes display core 1 ß1,3-galactosyltransferase activity using pNP-α-GalNAc as substrate and exhibit similar biochemical parameters as previously characterised T-synthases from other species. While the enzyme from C. gigas shares the same structural parameters with the other enzymes characterised so far, the T-synthase from P. canaliculata lacks the consensus sequence CCSD, which was previously considered indispensable.