Inhibition of O-GlcNAcylation Reduces Cell Viability and Autophagy and Increases Sensitivity to Chemotherapeutic Temozolomide in Glioblastoma.

Glioblastoma (GB) is the most aggressive primary malignant brain tumor and is associated with short survival. O-GlcNAcylation is an intracellular glycosylation that regulates protein function, enzymatic activity, protein stability, and subcellular localization. Aberrant O-GlcNAcylation is related to the tumorigenesis of different tumors, and mounting evidence supports O-GlcNAc transferase (OGT) as a potential therapeutic target. Here, we used two human GB cell lines alongside primary human astrocytes as a non-tumoral control to investigate the role of O-GlcNAcylation in cell proliferation, cell cycle, autophagy, and cell death. We observed that hyper O-GlcNAcylation promoted increased cellular proliferation, independent of alterations in the cell cycle, through the activation of autophagy. On the other hand, hypo O-GlcNAcylation inhibited autophagy, promoted cell death by apoptosis, and reduced cell proliferation. In addition, the decrease in O-GlcNAcylation sensitized GB cells to the chemotherapeutic temozolomide (TMZ) without affecting human astrocytes. Combined, these results indicated a role for O-GlcNAcylation in governing cell proliferation, autophagy, cell death, and TMZ response, thereby indicating possible therapeutic implications for treating GB. These findings pave the way for further research and the development of novel treatment approaches which may contribute to improved outcomes and increased survival rates for patients facing this challenging disease.

Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19.

BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

GM2/GM3 controls the organizational status of CD82/Met microdomains: further studies in GM2/GM3 complexation.

At cell surface gangliosides might associate with signal transducers proteins, grown factor receptors, integrins, small G-proteins and tetraspanins establishing microdomains, which play important role in cell adhesion, cell activation, motility, and growth. Previously, we reported that GM2 and GM3 form a heterodimer that interacts with the tetraspanin CD82, controlling epithelial cell mobility by inhibiting integrin-hepatocyte growth factor-induced cMet tyrosine kinase signaling. By using molecular dynamics simulations to study the molecular basis of GM2/GM3 interaction we demonstrate, here, that intracellular levels of Ca2+ mediate GM2/GM3 complexation via electrostatic interaction with their carboxyl groups, while hydrogen bonds between the ceramide groups likely aid stabilizing the complex. The presence of GM2/GM3 complex alters localization of CD82 on cell surface and therefore downstream signalization. These data contribute for the knowledge of how glycosylation may control signal transduction and phenotypic changes.

Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19.

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

CD43 sialoglycoprotein modulates cardiac inflammation and murine susceptibility to Trypanosoma cruzi infection.

CD43 (leukosialin) is a large sialoglycoprotein abundantly expressed on the surface of most cells from the hematopoietic lineage. CD43 is directly involved in the contact between cells participating in a series of events such as signaling, adherence and host parasite interactions. In this study we examined the role of CD43 in the immune response against Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, a potential life-threatening illness endemic in 21 Latin American countries according to the WHO. The acute stage of infection is marked by intense parasitemia and cardiac tissue parasitism, resulting in the recruitment of inflammatory cells and acute damage to the heart tissue. We show here that CD43-/- mice were more resistant to infection due to increased cytotoxicity of antigen specific CD8+ T cells and reduced inflammatory infiltration in the cardiac tissue, both contributing to lower cardiomyocyte damage. In addition, we demonstrate that the induction of acute myocarditis involves the engagement of CD43 cytoplasmic tripeptide sequence KRR to ezrin-radixin-moiesin cytoskeletal proteins. Together, our results show the participation of CD43 in different events involved in the pathogenesis of T. cruzi infection, contributing to a better overall understanding of the mechanisms underlying the pathogenesis of acute chagasic cardiomyopathy.

Emerging role of glycosylation in the polarization of tumor-associated macrophages.

Tumors are formed by several cell types interacting in a complex environment of soluble and matrix molecules. The crosstalk between the cells and extracellular components control tumor fate. Macrophages are highly plastic and diverse immune cells that are known to be key regulators of this complex network, which is mostly because they can adjust their metabolism and reprogram their phenotype and effector function. Here, we review the studies that disclose the central role of metabolism and tumor microenvironment in shaping the phenotype and function of macrophages, highlighting the importance of the hexosamine biosynthetic pathway. We further discuss growing evidence of nutrient-sensitive protein modifications such as O-GlcNAcylation and extracellular glycosylation in the function and polarization of tumor-associated macrophages.

Cellular glycosylation senses metabolic changes and modulates cell plasticity during epithelial to mesenchymal transition.

Epithelial to mesenchymal transition (EMT) is a developmental program reactivated by tumor cells that leads to the switch from epithelial to mesenchymal phenotype. During EMT, cells are transcriptionally regulated to decrease E-cadherin expression while expressing mesenchymal markers such as vimentin, fibronectin, and N-cadherin. Growing body of evidences suggest that cells engaged in EMT undergo a metabolic reprograming process, redirecting glucose flux toward hexosamine biosynthesis pathway (HBP), which fuels aberrant glycosylation patterns that are extensively observed in cancer cells. HBP depends on nutrient availability to produce its end product UDP-GlcNAc, and for this reason is considered a metabolic sensor pathway. UDP-GlcNAc is the substrate used for the synthesis of major types of glycosylation, including O-GlcNAc and cell surface glycans. In general, the rate limiting enzyme of HBP, GFAT, is overexpressed in many cancer types that present EMT features as well as aberrant glycosylation. Moreover, altered levels of O-GlcNAcylation can modulate cell morphology and favor EMT. In this review, we summarize some of the current knowledge that correlates glucose metabolism, aberrant glycosylation and hyper O-GlcNAcylation supported by HBP that leads to EMT activation. Developmental Dynamics 247:481-491, 2018. © 2017 Wiley Periodicals, Inc.

Evidences for the involvement of cell surface glycans in stem cell pluripotency and differentiation.

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal ß-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed ß-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.

Increase of O-glycosylated oncofetal fibronectin in high glucose-induced epithelial-mesenchymal transition of cultured human epithelial cells.

Growing evidences indicate that aberrant glycosylation can modulate tumor cell invasion and metastasis. The process termed "epithelial-mesenchymal transition" (EMT) provides a basic experimental model to shed light on this complex process. The EMT involves a striking decline in epithelial markers, accompanied by enhanced expression of mesenchymal markers, culminating in cell morphology change and increased cell motility. Few recent studies have established the participation glycosylation during EMT. Studies now come into knowledge brought to light the involvement of a site-specific O-glycosylation in the IIICS domain of human oncofetal fibronectin (onfFN) during the EMT process. Herein we show that high glucose induces EMT in A549 cells as demonstrated by TGF-ß secretion, cell morphology changes, increased cellular motility and the emergence of mesenchymal markers. The hyperglycemic conditions increased onfFN protein levels, promoted an up regulation of mRNA levels for ppGalNAc-T6 and FN IIICS domain, which contain the hexapeptide (VTHPGY) required for onfFN biosynthesis. Glucose effect involves hexosamine (HBP) biosynthetic pathway as overexpression of glutamine: fructose-6-phosphate amidotransferase increases mesenchymal markers, onfFN levels and mRNA levels for FN IIICS domain. In summary, our results demonstrate, for the first time that the metabolism of glucose through HBP promotes O-glycosylation of the oncofetal form of FN during EMT modulating tumorogenesis.

Trypanosoma cruzi subverts host cell sialylation and may compromise antigen-specific CD8+ T cell responses.

Upon activation, cytotoxic CD8(+) T lymphocytes are desialylated exposing beta-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8(+) T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8(+) T cell surface, thereby dampening antigen-specific CD8(+) T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8(+) T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8(+) T cell surface. The cytotoxic activity of antigen-experienced CD8(+) T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase-mediated resialylation in vitro and in vivo. Inhibition of the parasite's native trans-sialidase activity during infection strongly decreased CD8(+) T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8(+) T cell interactions with peptide-major histocompatibility complex class I complexes. CD8(+) T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism.

Novel 1,3,4-thiadiazolium-2-phenylamine chlorides derived from natural piperine as trypanocidal agents: chemical and biological studies.

We herein describe the synthesis and characterization of nine new 1,3,4-thiadiazolium-2-phenylamine chlorides derived from natural piperine. We evaluate their toxic effects against the different evolutive forms of Trypanosoma cruzi, and the host cell (murine macrophages). The results obtained show that mesoionic hydrochloride MI possesses the best activity profile. Compound MI may be a prototype for use in the development of a new chemotherapeutic agent with high efficiency, which may be employed in the treatment of Chagas' disease.