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Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by ß-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.
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Glicoproteínas , Polisacáridos , Polisacáridos/análisis , Polisacáridos/química , Polisacáridos/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Humanos , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Espectrometría de Masas/métodosRESUMEN
Sunitinib resistance creates a major clinical challenge for the treatment of advanced clear cell renal cell carcinoma (ccRCC) and functional and metabolic changes linked to sunitinib resistance are not fully understood. We sought to characterize the molecular and metabolic changes induced by the development of sunitinib resistance in ccRCC by developing and characterizing two human ccRCC cell lines resistant to sunitinib. Consistent with the literature, sunitinib-resistant ccRCC cell lines presented an aberrant overexpression of Axl and PD-L1, as well as a metabolic rewiring characterized by enhanced OXPHOS and glutamine metabolism. Therapeutic challenges of sunitinib-resistant ccRCC cell lines in vitro using small molecule inhibitors targeting Axl, AMPK and p38, as well as using PD-L1 blocking therapeutic antibodies, showed limited CTL-mediated cytotoxicity in a co-culture model. However, the AMPK activator metformin appears to sensitize the effect of PD-L1 blocking therapeutic antibodies and to enhance CTLs' cytotoxic effects on ccRCC cells. These effects were not broadly observed with the Axl and the p38 inhibitors. Taken together, these data suggest that targeting certain pathways aberrantly activated by sunitinib resistance such as the AMPK/PDL1 axis might sensitize ccRCC to immunotherapies as a second-line therapeutic approach.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Sunitinib/farmacología , Sunitinib/uso terapéutico , Carcinoma de Células Renales/patología , Antígeno B7-H1 , Neoplasias Renales/patología , Proteínas Quinasas Activadas por AMP , Línea Celular Tumoral , Resistencia a AntineoplásicosRESUMEN
The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR-LLL-HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.
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Linfocitos T CD8-positivos , COVID-19 , Humanos , SARS-CoV-2/metabolismo , Epítopos de Linfocito T , Receptores de Antígenos de Linfocitos T/metabolismo , Nucleocápside/metabolismo , Glicoproteína de la Espiga del CoronavirusRESUMEN
Opioids are commonly prescribed for extended periods of time to patients with advanced clear cell renal cell carcinoma to assist with pain management. Because extended opioid exposure has been shown to affect the vasculature and to be immunosuppressive, we investigated how it may affect the metabolism and physiology of clear cell renal cell carcinoma. RNA sequencing of a limited number of archived patients' specimens with extended opioid exposure or non-opioid exposure was performed. Immune infiltration and changes in the microenvironment were evaluated using CIBERSORT. A significant decrease in M1 macrophages and T cells CD4 memory resting immune subsets was observed in opioid-exposed tumors, whereas the changes observed in other immune cells were not statistically significant. Further RNA sequencing data analysis showed that differential expression of KEGG signaling pathways was significant between non-opioid-exposed specimens and opioid-exposed specimens, with a shift from a gene signature consistent with aerobic glycolysis to a gene signature consistent with the TCA cycle, nicotinate metabolism, and the cAMP signaling pathway. Together, these data suggest that extended opioid exposure changes the cellular metabolism and immune homeostasis of ccRCC, which might impact the response to therapy of these patients, especially if the therapy is targeting the microenvironment or metabolism of ccRCC tumors.
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Newborns are unable to reach the adult-level humoral immune response partly due to the potent immunoregulatory role of IL-10. Increased IL-10 production by neonatal B cells has been attributed to the larger population of IL-10-producting CD43+ B-1 cells in neonates. Here, we show that neonatal mouse CD43- non-B-1 cells also produce substantial amounts of IL-10 following B cell antigen receptor (BCR) activation. In neonatal mouse CD43- non-B-1 cells, BCR engagement activated STAT5 under the control of phosphorylated forms of signaling molecules Syk, Btk, PKC, FAK, and Rac1. Neonatal STAT5 activation led to IL-6 production, which in turn was responsible for IL-10 production in an autocrine/paracrine fashion through the activation of STAT3. In addition to the increased IL-6 production in response to BCR stimulation, elevated expression of IL-6Rα expression in neonatal B cells rendered them highly susceptible to IL-6-mediated STAT3 phosphorylation and IL-10 production. Finally, IL-10 secreted from neonatal mouse CD43- non-B-1 cells was sufficient to inhibit TNF-α secretion by macrophages. Our results unveil a distinct mechanism of IL-6-dependent IL-10 production in BCR-stimulated neonatal CD19+CD43- B cells.
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Interleucina-10 , Interleucina-6 , Animales , Ratones , Animales Recién Nacidos , Antígenos CD19/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Fosforilación , Receptores de Antígenos de Linfocitos B/metabolismo , Factor de Transcripción STAT5/metabolismo , Leucosialina/inmunologíaRESUMEN
Few live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are in pre-clinical or clinical development. We seek to attenuate SARS-CoV-2 (isolate WA1/2020) by removing the polybasic insert within the spike protein and the open reading frames (ORFs) 6-8, and by introducing mutations that abolish non-structural protein 1 (Nsp1)-mediated toxicity. The derived virus (WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A) replicates to 100- to 1000-fold-lower titers than the ancestral virus and induces little lung pathology in both K18-human ACE2 (hACE2) transgenic mice and Syrian hamsters. Immunofluorescence and transcriptomic analyses of infected hamsters confirm that three-pronged genetic modifications attenuate the proinflammatory pathways more than the removal of the polybasic cleavage site alone. Finally, intranasal administration of just 100 PFU of the WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A elicits robust antibody responses in Syrian hamsters and protects against SARS-CoV-2-induced weight loss and pneumonia. As a proof-of-concept study, we demonstrate that live but sufficiently attenuated SARS-CoV-2 vaccines may be attainable by rational design.
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COVID-19 , SARS-CoV-2 , Cricetinae , Ratones , Animales , Humanos , SARS-CoV-2/genética , Mesocricetus , Formación de Anticuerpos , Administración Intranasal , Vacunas contra la COVID-19 , COVID-19/prevención & control , Pulmón/patología , Ratones Transgénicos , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Glycoproteomic analysis of three Chinese hamster ovary (CHO) suspension host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in biopharmaceutical settings for recombinant protein production is reported. Intracellular and secreted glycoproteins were examined. We utilized an immobilization and chemoenzymatic strategy in our analysis. Glycoproteins or glycopeptides were first immobilized through reductive amination, and the sialyl moieties were amidated for protection. The desired N- or O-glycans and glycopeptides were released from the immobilization resin by enzymatic or chemical digestion. Glycopeptides were studied by Orbitrap Liquid chromatography-mass spectrometry (LC/MS), and the released glycans were analyzed by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Differences were detected in the relative abundances of N- and O-glycopeptide types, their resident and released glycans, and their glycoprotein complexity. Ontogeny analysis revealed key differences in features, such as general metabolic and biosynthetic pathways, including glycosylation systems, as well as distributions in cellular compartments. Host cell lines and subfraction differences were observed in both N- and O-glycan and glycoprotein pools. Differences were observed in sialyl and fucosyl glycan distributions. Key differences were also observed among glycoproteins that are problematic contaminants in recombinant antibody production. The differences revealed in this study should inform the choice of cell lines best suited for a particular bioproduction application.
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Productos Biológicos , Glicopéptidos , Animales , Células CHO , Cricetinae , Cricetulus , Glicopéptidos/análisis , Glicoproteínas/metabolismo , Polisacáridos/química , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens, and TCR repertoire size is believed to decline with age. However, the precise size of human TCR repertoires, in both total and subsets of T cells, as well as their changes with age, are not fully characterized. We conducted a longitudinal analysis of the human blood TCRα and TCRß repertoire of CD4+ and CD8+ T cell subsets using a unique molecular identifier-based (UMI-based) RNA-seq method. Thorough analysis of 1.9 × 108 T cells yielded the lower estimate of TCR repertoire richness in an adult at 3.8 × 108. Alterations of the TCR repertoire with age were observed in all 4 subsets of T cells. The greatest reduction was observed in naive CD8+ T cells, while the greatest clonal expansion was in memory CD8+ T cells, and the highest increased retention of TCR sequences was in memory CD8+ T cells. Our results demonstrated that age-related TCR repertoire attrition is subset specific and more profound for CD8+ than CD4+ T cells, suggesting that aging has a more profound effect on cytotoxic as opposed to helper T cell functions. This may explain the increased susceptibility of older adults to novel infections.
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Linfocitos T CD8-positivos , Subgrupos de Linfocitos T , Adulto , Anciano , Linfocitos T CD4-Positivos , Humanos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
The immune checkpoint programmed death-ligand 1 (PD-L1) is expressed on the cell surface of tumor cells and is key for maintaining an immunosuppressive microenvironment through its interaction with the programmed death 1 (PD-1). Clear cell renal cell carcinoma (ccRCC) is a highly immunogenic cancer characterized by an aberrant aerobic glycolytic metabolism and is known to overexpress PD-L1. Multiple immunotherapies have been approved for the treatment of ccRCC, including cytokines and immune checkpoint inhibitors. Recently the intrinsic role of PD-L1 and interferon gamma (IFNγ) signaling have been studied in several types of tumor cells, yet it remains unclear how they affect the metabolism and signaling pathways of ccRCC. Using metabolomics, metabolic assays and RNAseq, we showed that IFNγ enhanced aerobic glycolysis and tryptophan metabolism in ccRCC cells in vitro and induced the transcriptional expression of signaling pathways related to inflammation, cell proliferation and cellular energetics. These metabolic and transcriptional effects were partially reversed following transient PD-L1 silencing. Aerobic glycolysis, as well as signaling pathways related to inflammation, were not induced by IFNγ when PD-L1 was silenced, however, tryptophan metabolism and activation of Jak2 and STAT1 were maintained. Our data demonstrate that PD-L1 expression is required to mediate some of IFNγ's effect in ccRCC cells and highlight the importance of PD-L1 signaling in regulating the metabolism of ccRCC cells in response to inflammatory signals.
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Despite the high safety profile demonstrated in clinical trials, the immunogenicity of adeno-associated virus (AAV)-mediated gene therapy remains a major hurdle. Specifically, T-cell-mediated immune responses to AAV vectors are related to loss of efficacy and potential liver toxicities. As post-translational modifications in T cell epitopes have the potential to affect immune reactions, the cellular immune responses to peptides derived from spontaneously deamidated AAV were investigated. Here, we report that highly deamidated sites in AAV9 contain CD4 T cell epitopes with a Th1 cytokine pattern in multiple human donors with diverse human leukocyte antigen (HLA) backgrounds. Furthermore, some peripheral blood mononuclear cell (PBMC) samples demonstrated differential T cell activation to deamidated or non-deamidated epitopes. Also, in vitro and in silico HLA binding assays showed differential binding to the deamidated or non-deamidated peptides in some HLA alleles. This study provides critical attributes to vector-immune-mediated responses, as AAV deamidation can impact the immunogenicity, safety, and efficacy of AAV-mediated gene therapy in some patients.
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Autophagy drives drug resistance and drug-induced cancer cell cytotoxicity. Targeting the autophagy process could greatly improve chemotherapy outcomes. The discovery of specific inhibitors or activators has been hindered by challenges with reliably measuring autophagy levels in a clinical setting. We investigated drug-induced autophagy in breast cancer cell lines with differing ER/PR/Her2 receptor status by exposing them to known but divergent autophagy inducers each with a unique molecular target, tamoxifen, trastuzumab, bortezomib or rapamycin. Differential gene expression analysis from total RNA extracted during the earliest sign of autophagy flux showed both cell- and drug-specific changes. We analyzed the list of differentially expressed genes to find a common, cell- and drug-agnostic autophagy signature. Twelve mRNAs were significantly modulated by all the drugs and 11 were orthogonally verified with Q-RT-PCR (Klhl24, Hbp1, Crebrf, Ypel2, Fbxo32, Gdf15, Cdc25a, Ddit4, Psat1, Cd22, Ypel3). The drug agnostic mRNA signature was similarly induced by a mitochondrially targeted agent, MitoQ. In-silico analysis on the KM-plotter cancer database showed that the levels of these mRNAs are detectable in human samples and associated with breast cancer prognosis outcomes of Relapse-Free Survival in all patients (RSF), Overall Survival in all patients (OS), and Relapse-Free Survival in ER+ Patients (RSF ER+). High levels of Klhl24, Hbp1, Crebrf, Ypel2, CD22 and Ypel3 were correlated with better outcomes, whereas lower levels of Gdf15, Cdc25a, Ddit4 and Psat1 were associated with better prognosis in breast cancer patients. This gene signature uncovers candidate autophagy biomarkers that could be tested during preclinical and clinical studies to monitor the autophagy process.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Antineoplásicos/uso terapéutico , Autofagia/efectos de los fármacos , Bortezomib/farmacología , Bortezomib/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Células MCF-7 , Compuestos Organofosforados/farmacología , Compuestos Organofosforados/uso terapéutico , Receptor ErbB-2/genética , Receptores de Estrógenos/genética , Receptores de Progesterona/genética , Análisis de Secuencia de ARN , Sirolimus/farmacología , Sirolimus/uso terapéutico , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Ubiquinona/análogos & derivados , Ubiquinona/farmacología , Ubiquinona/uso terapéuticoRESUMEN
The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without spike (S), membrane (M), and envelope (E) genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. IMPORTANCE SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. Additionally, our stable replicon cells represent a new model system to study SARS-CoV-2 replication.
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Antivirales , Tratamiento Farmacológico de COVID-19 , Evaluación Preclínica de Medicamentos , SARS-CoV-2 , Animales , Antivirales/farmacología , Línea Celular , Células Clonales , Cricetinae , Evaluación Preclínica de Medicamentos/métodos , ARN Viral , Replicón , SARS-CoV-2/efectos de los fármacos , Replicación ViralRESUMEN
Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting humans and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein, which may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either the bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins.ImportanceThe four-residue insert (PRRA) at the boundary between the S1and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2. We compared the usage of 10 ACE2 orthologs and found that the presence of PRRA not only affects the cellular tropism of SARS-CoV-2 but also modulates the usage of ACE2 orthologs by the closely related bat RaTG13 S protein. The binding of pseudovirions carrying RaTG13 S with a PRRA insert to mouse ACE2 was nearly 2-fold higher than that of pseudovirions carrying RaTG13 S.
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BACKGROUND: Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIIIFT ) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIIIFT can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated. OBJECTIVE: To isolate and characterize the FVIIIFT fraction in rFVIII products. METHODS: Protein fractions unable (FVIIIFT ) and able (FVIIIEL ) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. RESULTS AND CONCLUSIONS: A robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIIIFT was found at various contents (0.4%-21.5%) in all products. Compared with FVIIIEL , FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIIIFT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low-density lipoprotein receptor-related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIIIFT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.
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Hemofilia A , Hemostáticos , Animales , Pruebas de Coagulación Sanguínea , Factor VIII , Hemofilia A/tratamiento farmacológico , Ratones , Factor de von WillebrandRESUMEN
Very little is known about disease transmission via the gut microbiome. We hypothesized that certain inflammatory features could be transmitted via the gut microbiome and tested this hypothesis using an animal model of inflammatory diseases. Twelve-week-old healthy C57 Bl/6 and Germ-Free (GF) female and male mice were fecal matter transplanted (FMT) under anaerobic conditions with TNFΔARE-/+ donors exhibiting spontaneous Rheumatoid Arthritis (RA) and Inflammatory Bowel Disease (IBD) or with conventional healthy mice control donors. The gut microbiome analysis was performed using 16S rRNA sequencing amplification and bioinformatics analysis with the HIVE bioinformatics platform. Histology, immunohistochemistry, ELISA Multiplex analysis, and flow cytometry were conducted to confirm the inflammatory transmission status. We observed RA and IBD features transmitted in the GF mice cohort, with gut tissue disruption, cartilage alteration, elevated inflammatory mediators in the tissues, activation of CD4/CD8+ T cells, and colonization and transmission of the gut microbiome similar to the donors' profile. We did not observe a change or transmission when conventional healthy mice were FMT with TNFΔARE-/+ donors, suggesting that a healthy microbiome might withstand an unhealthy transplant. These findings show the potential involvement of the gut microbiome in inflammatory diseases. We identified a cluster of bacteria playing a role in this mechanism.
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O-Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in the release of O-glycans. Moreover, chemical releasing methods, such as ß-elimination/Michael addition, are not specific to O-glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions. Recently, the O-protease OpeRATOR, derived from intestinal bacteria and expressed in Escherichia coli, has become commercially available. The digestion of O-glycoprotein yields O-glycopeptides cleaved at the N-terminal end of serine and threonine, with O-glycan remaining intact. The enzyme has a broad substrate specificity and includes mammalian cores 1-8. However, OpeRATOR is not fully active toward sialylated glycoproteins, and it has been suggested that this acidic residue be removed prior to digestion, thus sacrificing structural information. In this study, we investigated the performance of OpeRATOR under a range of conditions, including buffer selection, varying pH, sialic acid modification, and digestion temperature, in order to optimize the enzymatic activity, with a special emphasis on sialylated glycosites. Conditions derived in this work facilitate the OpeRATOR digestion of fully sialylated O-glycopeptides that are mass tagged to identify the sialyl linkage, thus facilitating the analysis of these charged O-glycopeptides, which are often important in biological processes.
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Endopeptidasas/química , Glicopéptidos/análisis , Glicoproteínas/análisis , Polisacáridos/análisis , Ácidos Siálicos/química , Animales , Secuencia de Carbohidratos , Bovinos , Escherichia coli/enzimología , Etildimetilaminopropil Carbodiimida/química , Fetuínas/análisis , Fetuínas/química , Glicoproteínas/química , Lactoferrina/análisis , Lactoferrina/química , Mucinas/análisis , Mucinas/química , Polisacáridos/química , Triazoles/químicaRESUMEN
In mammalian cells, N-glycans may include multiple N-acetyllactosamine (poly-LacNAc) units that can play roles in various cellular functions and properties of therapeutic recombinant proteins. Previous studies indicated that ß-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß-1,4-galactotransferase 1 (B4GALT1) are two of the primary glycosyltransferases involved in generating LacNAc units. In the current study, knocking out sialyltransferase genes slightly enhanced the LacNAc content (≥4 repeats per glycan) on recombinant EPO protein. Next, the role of single and dual-overexpression of B3GNT2 and B4GALT1 was explored in recombinant EPO-expressing Chinese hamster ovary (CHO) cells. While overexpression of B4GALT1 slightly enhanced the levels of large glycans on recombinant EPO, overexpression of B3GNT2 in EPO-expressing CHO cells significantly decreased the recombinant EPO LacNAc content, resulting in N-glycans terminating primarily with GlcNAc structures, a limited number of Gals, and nearly undetectable sialylation, which was also observed in sialyltransferases knock-out-B3GNT2 overexpression cell lines. Considering the nature of the binding domain motifs present on B3GNT2, which evolved from ß1,3-galactosyltransferases, its overexpression may have competed and inhibited endogenous ß1,4-galactosyltransferases for exposed GlcNAc residues on the N-glycans, resulting in premature termination of many N-glycans at GlcNAc. Furthermore, B3GNT2 overexpression enhanced intracellular UDP-GlcNAc and CMP-Neu5Ac content while slightly lowering UDP-Gal content. The presence of a sink for UDP-GlcNAc in the form of B3GNT2 with no disposition may have also elevated the intracellular levels of this nucleotide as well as its downstream product, CMP-Neu5Ac. Furthermore, we were unable to overexpress B4GALT1 at either the transcriptional or translational levels following initial B3GNT2 expression. Expression of B3GNT2 following initial expression of B4GALT1 was also problematic in that transcriptional and translational analysis indicated the accumulation of truncated B3GNT2 missing a section of the B3GNT2 trans-Golgi lumen domain while transmembrane and cytoplasmic domains were present. Given that glycosylation is a very complex intra-network process, the addition of one or more recombinant glycosyltransferases may have an unexpected influence on the expression and activities of glycosyltransferases, which can disrupt the nucleotide sugar levels and lead to unexpected modifications of the resulting N-glycan patterns.
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Metabolismo de los Hidratos de Carbono , Glicosiltransferasas , Ingeniería Metabólica , Polisacáridos , Animales , Células CHO , Cricetulus , Glicosilación , Glicosiltransferasas/biosíntesis , Glicosiltransferasas/genética , Polisacáridos/biosíntesis , Polisacáridos/genéticaRESUMEN
To evaluate the expression of immune checkpoint genes, their concordance with expression of IFNγ, and to identify potential novel ICP related genes (ICPRG) in colorectal cancer (CRC), the biological connectivity of six well documented ("classical") ICPs (CTLA4, PD1, PDL1, Tim3, IDO1, and LAG3) with IFNγ and its co-expressed genes was examined by NGS in 79 CRC/healthy colon tissue pairs. Identification of novel IFNγ- induced molecules with potential ICP activity was also sought. In our study, the six classical ICPs were statistically upregulated and correlated with IFNγ, CD8A, CD8B, CD4, and 180 additional immunologically related genes in IFNγ positive (FPKM > 1) tumors. By ICP co-expression analysis, we also identified three IFNγ-induced genes [(IFNγ-inducible lysosomal thiol reductase (IFI30), guanylate binding protein1 (GBP1), and guanylate binding protein 4 (GBP4)] as potential novel ICPRGs. These three genes were upregulated in tumor compared to normal tissues in IFNγ positive tumors, co-expressed with CD8A and had relatively high abundance (average FPKM = 362, 51, and 25, respectively), compared to the abundance of the 5 well-defined ICPs (Tim3, LAG3, PDL1, CTLA4, PD1; average FPKM = 10, 9, 6, 6, and 2, respectively), although IDO1 is expressed at comparably high levels (FPKM = 39). We extended our evaluation by querying the TCGA database which revealed the commonality of IFNγ dependent expression of the three potential ICPRGs in 638 CRCs, 103 skin cutaneous melanomas (SKCM), 1105 breast cancers (BC), 184 esophageal cancers (ESC), 416 stomach cancers (STC), and 501 lung squamous carcinomas (LUSC). In terms of prognosis, based on Pathology Atlas data, correlation of GBP1 and GBP4, but not IFI30, with 5-year survival rate was favorable in CRC, BC, SKCM, and STC. Thus, further studies defining the role of IFI30, GBP1, and GBP4 in CRC are warranted.