RESUMEN

During the development process of therapeutic monoclonal antibodies (mAbs), it is crucial to control (critical) quality attributes such as N-glycosylation influencing pharmacokinetics (PK) and Fc effector functions. Previous reports have shown that mAbs containing high-mannose N-glycans are cleared faster from blood circulation, leading to reduced half-lives. The high-mannose N-glycan content of mAbs can be influenced during the cell culture process by factors such as cell lines, process conditions, and media. Furthermore, mAbs have either one high mannose N-glycan (asymmetrical high-mannose glyco-pair) or two high mannose N-glycans (symmetrical high-mannose glyco-pair). The hypothesis that the mannose receptor (MR, CD206) accelerates clearance by facilitating their internalization and subsequent lysosomal degradation is widespread. However, the interaction between MR and mAbs has not been explicitly demonstrated. This study aimed to investigate this interaction, providing the first systematic demonstration of MR binding to the Fc region of mAbs with high-mannose N-glycans. Two novel analytical methods, MR surface plasmon resonance and MR affinity chromatography, were developed and applied to investigate the MR-mAb interaction. The interaction is found to be dependent on high-mannose content, but is independent of the mAb format or sequence. However, different glyco-pairs exhibited varying binding affinities to the MR, with the symmetrical high-mannose glyco-pair showing the strongest binding properties. These findings strengthen the hypothesis for the MR-mediated mAb interaction and contribute to a deeper understanding of the MR-mAb interaction, which could affect the criticality of high-mannose containing mAbs development strategies of IgG-based molecules and improve their PK profiles.

Asunto(s)

Anticuerpos Monoclonales , Lectinas Tipo C , Receptor de Manosa , Lectinas de Unión a Manosa , Manosa , Polisacáridos , Receptores de Superficie Celular , Polisacáridos/metabolismo , Polisacáridos/química , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Lectinas Tipo C/metabolismo , Manosa/metabolismo , Manosa/química , Humanos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/inmunología , Animales , Glicosilación , Cricetulus , Células CHO , Resonancia por Plasmón de Superficie , Unión Proteica

RESUMEN

Cryptosporidium is a globally distributed zoonotic protozoan parasite that can cause severe diarrhea in humans and animals. L-type lectins are carbohydrate-binding proteins involved in multiple pathways in animals and plants, including protein transportation, secretion, innate immunity, and the unfolded protein response signaling pathway. However, the biological function of the L-type lectins remains unknown in Cryptosporidium parvum. Here, we preliminarily characterized an L-type lectin in C. parvum (CpLTL) that contains a lectin-leg-like domain. Immunofluorescence assay confirmed that CpLTL is located on the wall of oocysts, the surface of the mid-anterior region of the sporozoite and the cytoplasm of merozoites. The involvement of CpLTL in parasite invasion is partly supported by experiments showing that an anti-CpLTL antibody could partially block the invasion of C. parvum sporozoites into host cells. Moreover, the recombinant CpLTL showed binding ability with mannose and the surface of host cells, and competitively inhibited the invasion of C. parvum. Two host cell proteins were identified by proteomics which should be prioritized for future validation of CpLTL-binding. Our data indicated that CpLTL is potentially involved in the adhesion and invasion of C. parvum.

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Title: Une protéine mono-transmembranaire, lectine de type L spécifique du mannose, potentiellement impliquée dans l'adhésion et l'invasion de Cryptosporidium parvum. Abstract: Cryptosporidium est un parasite protozoaire zoonotique répandu dans le monde entier qui peut provoquer de graves diarrhées chez les humains et les animaux. Les lectines de type L sont des protéines liant les glucides impliquées dans de multiples voies chez les animaux et les plantes, notamment le transport des protéines, la sécrétion, l'immunité innée et la voie de signalisation de la réponse protéique dépliée. Cependant, la fonction biologique des lectines de type L reste inconnue chez Cryptosporidium parvum. Ici, nous avons caractérisé de manière préliminaire une lectine de type L chez C. parvum (CpLTL) qui contient un domaine de type jambe de lectine. Le test d'immunofluorescence a confirmé que CpLTL est localisée sur la paroi des oocystes, la surface de la région médio-antérieure du sporozoïte et le cytoplasme des mérozoïtes. L'implication de CpLTL dans l'invasion parasitaire est en partie étayée par des expériences montrant qu'un anticorps anti-CpLTL peut bloquer partiellement l'invasion des sporozoïtes de C. parvum dans les cellules hôtes. De plus, la CpLTL recombinante a montré une capacité de liaison avec le mannose et la surface des cellules hôtes et a inhibé de manière compétitive l'invasion de C. parvum. Deux protéines de cellules hôtes ont été identifiées par protéomique et devraient être prioritaires pour la validation future de la liaison avec CpLTL. Nos données indiquent que CpLTL est potentiellement impliquée dans l'adhésion et l'invasion de C. parvum.

Asunto(s)

Cryptosporidium parvum , Manosa , Proteínas Protozoarias , Esporozoítos , Cryptosporidium parvum/fisiología , Cryptosporidium parvum/metabolismo , Cryptosporidium parvum/genética , Esporozoítos/fisiología , Esporozoítos/metabolismo , Animales , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Humanos , Manosa/metabolismo , Oocistos/fisiología , Criptosporidiosis/parasitología , Merozoítos/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Adhesión Celular , Proteómica

RESUMEN

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gut. There is growing evidence in Crohn's disease (CD) of the existence of a preclinical period characterized by immunological changes preceding symptom onset that starts years before diagnosis. Gaining insight into this preclinical phase will allow disease prediction and prevention. Analysis of preclinical serum samples, up to 6 years before IBD diagnosis (from the PREDICTS cohort), revealed the identification of a unique glycosylation signature on circulating antibodies (IgGs) characterized by lower galactosylation levels of the IgG fragment crystallizable (Fc) domain that remained stable until disease diagnosis. This specific IgG2 Fc glycan trait correlated with increased levels of antimicrobial antibodies, specifically anti-Saccharomyces cerevisiae (ASCA), pinpointing a glycome-ASCA hub detected in serum that predates by years the development of CD. Mechanistically, we demonstrated that this agalactosylated glycoform of ASCA IgG, detected in the preclinical phase, elicits a proinflammatory immune pathway through the activation and reprogramming of innate immune cells, such as dendritic cells and natural killer cells, via an FcγR-dependent mechanism, triggering NF-κB and CARD9 signaling and leading to inflammasome activation. This proinflammatory role of ASCA was demonstrated to be dependent on mannose glycan recognition and galactosylation levels in the IgG Fc domain. The pathogenic properties of (anti-mannose) ASCA IgG were validated in vivo. Adoptive transfer of antibodies to mannan (ASCA) to recipient wild-type mice resulted in increased susceptibility to intestinal inflammation that was recovered in recipient FcγR-deficient mice. Here we identify a glycosylation signature in circulating IgGs that precedes CD onset and pinpoint a specific glycome-ASCA pathway as a central player in the initiation of inflammation many years before CD diagnosis. This pathogenic glyco-hub may constitute a promising new serum biomarker for CD prediction and a potential target for disease prevention.

Asunto(s)

Enfermedad de Crohn , Inmunoglobulina G , Manosa , Polisacáridos , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Animales , Humanos , Glicosilación , Manosa/metabolismo , Manosa/inmunología , Ratones , Polisacáridos/inmunología , Polisacáridos/metabolismo , Femenino , Saccharomyces cerevisiae/inmunología , Masculino , Adulto , Anticuerpos Antifúngicos/sangre , Anticuerpos Antifúngicos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Biomarcadores/sangre , Persona de Mediana Edad , Fragmentos Fc de Inmunoglobulinas/inmunología , Glicoproteínas

RESUMEN

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

Asunto(s)

Manosa , Humanos , Glicosilación , Manosa/metabolismo , Especificidad por Sustrato , Glicoproteínas/metabolismo , Proteómica/métodos , Línea Celular , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Procesamiento Proteico-Postraduccional , Ingeniería Celular/métodos

RESUMEN

Glycans are carbohydrates present in every organism that bind to specific molecules such as lectins, a diverse group of proteins. Glycans are vital to cell proliferation and protein trafficking. In addition, embryogenesis is a critical phase in the development of marine organisms. This study investigated the effects of chilling and cryoprotective agents (CPAs) on glycans in the embryos of Stenopus hispidus. The glycan profiles of embryos of S. hispidus at the heartbeat stage were analyzed using lectin arrays. The results of analyses revealed that mannose was the most abundant glycan in the S. hispidus embryos; mannose is crucial to cell proliferation, providing the energy required for embryonic growth. Additionally, the results reveled that chilling altered the content of several glycans, including fucose and Gla-GlcNAc. Chilling may promote monosaccharide accumulation, facilitating osmotic regulation of cells and signal molecules to aid S. hispidus embryos in adapting to cold conditions. Changes were also observed in the lectins NPA, orysata, PALa, ASA, discoidin II, discoidin I, UDA, PA-IIL, and PHA-P after the samples were treated with different CPAs. DMSO may minimize cell damage during exposure to chilling by preserving cell structures, membrane properties, and functions. The present study is the first to investigate the profiles and functions of glycans in shrimp embryos subjected to low-temperature injuries. This study enhances the understanding of cell reproduction during embryogenesis and provides valuable information for the study of glycans in embryos.

Asunto(s)

Frío , Crioprotectores , Embrión no Mamífero , Lectinas , Polisacáridos , Animales , Polisacáridos/metabolismo , Crioprotectores/farmacología , Crioprotectores/metabolismo , Embrión no Mamífero/metabolismo , Lectinas/metabolismo , Criopreservación/métodos , Dimetilsulfóxido/farmacología , Manosa/metabolismo , Desarrollo Embrionario/efectos de los fármacos

RESUMEN

Enzymatic production of D-mannose attracts increasing attention because of the health effects and commercial values of D-mannose. Several kinds of epimerases or isomerases have been used for enzymatic production of D-mannose from D-glucose or D-fructose. D-Mannose epimerase (MEase), belonging to N-acyl-D-glucosamine 2-epimerase superfamily enzymes, catalyzes the C-2 epimerization between D-glucose and D-mannose. In this study, a novel MEase was identified from Cytophagaceae bacterium SJW1-29. Sequence and structure alignments indicate that it is highly conserved with the reported R. slithyformis MEase with the known crystal structure. It was a metal-independent enzyme, with an optimal pH of 8.0 and an optimal temperature of 40 °C. The specific activities on D-glucose and D-mannose were 2.90 and 2.96 U/mg, respectively. The Km, kcat, and kcat/Km on D-glucose were measured to be 194.9 mM, 2.72 s-1, and 0.014 mM-1 s-1, respectively. The purified enzyme produced 23.15 g/L of D-mannose from 100 g/L of D-glucose at pH 8.0 and 40 °C for 8 h, with a conversion rate of 23.15 %.

Asunto(s)

Carbohidrato Epimerasas , Glucosa , Manosa , Manosa/metabolismo , Glucosa/metabolismo , Especificidad por Sustrato , Cinética , Carbohidrato Epimerasas/metabolismo , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Concentración de Iones de Hidrógeno , Secuencia de Aminoácidos , Clonación Molecular , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Temperatura , Modelos Moleculares , Alineación de Secuencia

RESUMEN

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-ß-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

Asunto(s)

Proteínas Bacterianas , Bacteroides thetaiotaomicron , Microbioma Gastrointestinal , Polisacáridos , Polisacáridos/metabolismo , Humanos , Bacteroides thetaiotaomicron/metabolismo , Bacteroides thetaiotaomicron/enzimología , Bacteroides thetaiotaomicron/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Especificidad por Sustrato , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/genética , Manosa/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/genética , Familia de Multigenes

RESUMEN

Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n = 20) and WW (n = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden.

Asunto(s)

Manosa , Polisacáridos , Humanos , Femenino , Polisacáridos/metabolismo , Polisacáridos/química , Manosa/metabolismo , Manosa/química , Persona de Mediana Edad , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Glicómica , Mama/metabolismo , Mama/química , Mama/patología , Fucosa/metabolismo , Fucosa/química , Adulto , Microambiente Tumoral

RESUMEN

Due to the shallow and hydrophilic binding sites of carbohydrate-binding proteins, the design of glycomimetics is often complicated by high desolvation costs as well as competition with solvent. Therefore, a careful optimization of interaction vectors and ligand properties is required in the design and optimization of glycomimetics. Here, we employ thermodynamics-guided design to optimize mannose-based glycomimetics targeting the human C-type lectin receptor dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN), a pathogenic host factor in viral infections. By exploring ligand rigidification and hydrogen bond engineering, a monovalent glycomimetic with an unprecedented affinity for DC-SIGN in the low µM range was discovered. A matched molecular pair analysis based on microcalorimetric data revealed a stereospecific hydrogen bond interaction with Glu358/Ser360 as the origin of this cooperative and enthalpically dominated interaction. This detailed insight into the binding mechanism paves the way for an improvement of monovalent glycomimetics targeting DC-SIGN.

Asunto(s)

Moléculas de Adhesión Celular , Enlace de Hidrógeno , Lectinas Tipo C , Receptores de Superficie Celular , Termodinámica , Lectinas Tipo C/metabolismo , Lectinas Tipo C/química , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/química , Humanos , Diseño de Fármacos , Manosa/química , Manosa/metabolismo , Ligandos , Modelos Moleculares , Sitios de Unión

RESUMEN

The parasitic helminth Schistosoma mansoni is a potent inducer of type 2 immune responses by stimulating dendritic cells (DCs) to prime T helper 2 (Th2) responses. We previously found that S. mansoni soluble egg antigens (SEA) promote the synthesis of Prostaglandin E2 (PGE2) by DCs through ERK-dependent signaling via Dectin-1 and Dectin-2 that subsequently induces OX40L expression, licensing them for Th2 priming, yet the ligands present in SEA involved in driving this response and whether specific targeting of PGE2 synthesis by DCs could affect Th2 polarization are unknown. We here show that the ability of SEA to bind Dectin-2 and drive ERK phosphorylation, PGE2 synthesis, OX40L expression, and Th2 polarization is impaired upon cleavage of high-mannose glycans by Endoglycosidase H treatment. This identifies high-mannose glycans present on glycoproteins in SEA as important drivers of this signaling axis. Moreover, we find that OX40L expression and Th2 induction are abrogated when microsomal prostaglandin E synthase-1 (mPGES) is selectively inhibited, but not when a general COX-1/2 inhibitor is used. This shows that the de novo synthesis of PGE2 is vital for the Th2 priming function of SEA-stimulated DCs as well as points to the potential existence of other COX-dependent lipid mediators that antagonize PGE2-driven Th2 polarization. Lastly, specific PGE2 inhibition following immunization with S. mansoni eggs dampened the egg-specific Th cell response. In summary, our findings provide new insights in the molecular mechanisms underpinning Th2 induction by S. mansoni and identify druggable targets for potential control of helminth driven-Th2 responses.

Asunto(s)

Dinoprostona , Lectinas Tipo C , Manosa , Polisacáridos , Schistosoma mansoni , Células Th2 , Animales , Ratones , Antígenos Helmínticos/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Lectinas Tipo C/metabolismo , Lectinas Tipo C/inmunología , Manosa/metabolismo , Manosa/inmunología , Ratones Endogámicos C57BL , Óvulo/inmunología , Óvulo/metabolismo , Ligando OX40/metabolismo , Polisacáridos/inmunología , Polisacáridos/metabolismo , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/metabolismo , Esquistosomiasis mansoni/parasitología , Células Th2/inmunología , Células Th2/metabolismo

RESUMEN

Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man6 and higher glycans and a decrease in Man5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation.

Asunto(s)

Trastornos Congénitos de Glicosilación , Fibroblastos , Liposomas , Manosafosfatos , Fosfotransferasas (Fosfomutasas) , Humanos , Glicosilación/efectos de los fármacos , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/tratamiento farmacológico , Trastornos Congénitos de Glicosilación/metabolismo , Trastornos Congénitos de Glicosilación/patología , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Manosafosfatos/metabolismo , Fosfotransferasas (Fosfomutasas)/genética , Fosfotransferasas (Fosfomutasas)/metabolismo , Fosfotransferasas (Fosfomutasas)/deficiencia , Proteómica , Manosa/metabolismo

RESUMEN

INTRODUCTION: Fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG) is a rare autosomal recessive inborn error of metabolism characterized by a decreased flux through the salvage pathway of GDP-fucose biosynthesis due to a block in the recycling of L-fucose that exits the lysosome. FCSK-CDG has been described in 5 individuals to date in the medical literature, with a phenotype comprising global developmental delays/intellectual disability, hypotonia, abnormal myelination, posterior ocular disease, growth and feeding failure, immune deficiency, and chronic diarrhea, without clear therapeutic recommendations. PATIENT AND METHODS: In a so far unreported FCSK-CDG patient, we studied proteomics and glycoproteomics in vitro in patient-derived fibroblasts and also performed in vivo glycomics, before and after treatment with either D-Mannose or L-Fucose. RESULTS: We observed a marked increase in fucosylation after D-mannose supplementation in fibroblasts compared to treatment with L-Fucose. The patient was then treated with D-mannose at 850 mg/kg/d, with resolution of the chronic diarrhea, resolution of oral aversion, improved weight gain, and observed developmental gains. Serum N-glycan profiles showed an improvement in the abundance of fucosylated glycans after treatment. No treatment-attributed adverse effects were observed. CONCLUSION: D-mannose is a promising new treatment for FCSK-CDG.

Asunto(s)

Trastornos Congénitos de Glicosilación , Fibroblastos , Manosa , Humanos , Trastornos Congénitos de Glicosilación/tratamiento farmacológico , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/patología , Trastornos Congénitos de Glicosilación/metabolismo , Manosa/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Masculino , Fucosa/metabolismo , Glicosilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Femenino , Proteómica

RESUMEN

Metabolic reprogramming is a hallmark of cancer. In addition to metabolic alterations in the tumor cells, multiple other metabolically active cell types in the tumor microenvironment (TME) contribute to the emergence of a tumor-specific metabolic milieu. Here, we defined the metabolic landscape of the TME during the progression of head and neck squamous cell carcinoma (HNSCC) by performing single-cell RNA sequencing on 26 human patient specimens, including normal tissue, precancerous lesions, early stage cancer, advanced-stage cancer, lymph node metastases, and recurrent tumors. The analysis revealed substantial heterogeneity at the transcriptional, developmental, metabolic, and functional levels in different cell types. SPP1+ macrophages were identified as a protumor and prometastatic macrophage subtype with high fructose and mannose metabolism, which was further substantiated by integrative analysis and validation experiments. An inhibitor of fructose metabolism reduced the proportion of SPP1+ macrophages, reshaped the immunosuppressive TME, and suppressed tumor growth. In conclusion, this work delineated the metabolic landscape of HNSCC at a single-cell resolution and identified fructose metabolism as a key metabolic feature of a protumor macrophage subpopulation. Significance: Fructose and mannose metabolism is a metabolic feature of a protumor and prometastasis macrophage subtype and can be targeted to reprogram macrophages and the microenvironment of head and neck squamous cell carcinoma.

Asunto(s)

Progresión de la Enfermedad , Neoplasias de Cabeza y Cuello , Análisis de la Célula Individual , Carcinoma de Células Escamosas de Cabeza y Cuello , Microambiente Tumoral , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/metabolismo , Animales , Ratones , Manosa/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/inmunología , Fructosa/metabolismo , Plasticidad de la Célula , Masculino

RESUMEN

Mucins are major components of the mucus. Besides the highly O-glycosylated tandem repeat domains, mucins contain Cys domains (CysDs). CysDs contain conserved disulfide-forming cysteine residues as well as a WxxW motif. Since this is the consensus sequence for tryptophan C-mannosylation, mucin CysDs have been suggested to be targets for C-mannosyltransferases, but this has never been directly shown. Here, we recombinantly expressed human mucin CysDs in Chinese hamster ovary (CHO) cells and analyzed the C-mannosylation status. Mass spectrometric analysis revealed that the putative C-mannose site is not or only barely C-mannosylated. However, mutation of the adjacent cysteine residues enabled C-mannosylation to occur. In contrast to mucin CysDs, the homologous CysD of human cartilage intermediate layer protein 1 (CILP1) lacks these cysteine residues preceding the WxxW motif. We show that CILP1 CysD is C-mannosylated, but introducing a cysteine at the -2 position causes this modification to be lost. We thus conclude that the presence of cysteine residues prevents the modification of the WxxW motif in CysDs.

Asunto(s)

Cricetulus , Cisteína , Manosa , Cisteína/metabolismo , Cisteína/genética , Cisteína/química , Humanos , Animales , Células CHO , Manosa/metabolismo , Manosa/química , Glicosilación , Mucinas/metabolismo , Mucinas/química , Mucinas/genética , Dominios Proteicos , Secuencia de Aminoácidos , Secuencias de Aminoácidos , Secuencia Conservada , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química

RESUMEN

Biochemical analysis of human normal bronchial cells (BEpiC) and human cancer lung cells (A549) has been performed by using Raman spectroscopy and Raman imaging. Our approach provides a biochemical compositional mapping of the main cell components: nucleus, mitochondria, lipid droplets, endoplasmic reticulum, cytoplasm and cell membrane. We proved that Raman spectroscopy and Raman imaging can distinguish successfully BEpiC and A549 cells. In this study, we have focused on the role of mannose in cancer development. It has been shown that changes in the concentration of mannose can regulate some metabolic processes in cells. Presented results suggest lipids and proteins can be considered as Raman biomarkers during lung cancer progression. Analysis obtained for bands 1444 cm-1, and 2854 cm-1 characteristic for lipids and derivatives proved that the addition of mannose reduced levels of these compounds. Results obtained for protein compounds based on bands 858 cm-1, 1004 cm-1 and 1584 cm-1 proved that the addition of mannose increases the values of protein in BEpiC cells and blocks protein glycolisation in A549 cells. Noticing Raman spectral changes in BEpiC and A549 cells supplemented with mannose can help to understand the mechanism of sugar metabolism during cancer development and could play in the future an important role in clinical treatment.

Asunto(s)

Metabolismo de los Lípidos , Manosa , Espectrometría Raman , Humanos , Espectrometría Raman/métodos , Manosa/metabolismo , Manosa/química , Células A549 , Proteínas/metabolismo , Proteínas/análisis , Bronquios/metabolismo , Bronquios/citología

RESUMEN

Non-alcoholic fatty liver disease (NAFLD), a chronic liver condition and metabolic disorder, has emerged as a significant health issue worldwide. D-mannose, a natural monosaccharide widely existing in plants and animals, has demonstrated metabolic regulatory properties. However, the effect and mechanism by which D-mannose may counteract NAFLD have not been studied. In this study, network pharmacology followed by molecular docking analysis was utilized to identify potential targets of mannose against NAFLD, and the leptin receptor-deficient, genetically obese db/db mice was employed as an animal model of NAFLD to validate the regulation of D-mannose on core targets. As a result, 67 targets of mannose are predicted associated with NAFLD, which are surprisingly centered on the mechanistic target of rapamycin (mTOR). Further analyses suggest that mTOR signaling is functionally enriched in potential targets of mannose treating NAFLD, and that mannose putatively binds to mTOR as a core mechanism. Expectedly, repeated oral gavage of supraphysiological D-mannose ameliorates liver steatosis of db/db mice, which is based on suppression of hepatic mTOR signaling. Moreover, daily D-mannose administration reduced hepatic expression of lipogenic regulatory genes in counteracting NAFLD. Together, these findings reveal D-mannose as an effective and potential NAFLD therapeutic through mTOR suppression, which holds translational promise.

Asunto(s)

Manosa , Farmacología en Red , Enfermedad del Hígado Graso no Alcohólico , Serina-Treonina Quinasas TOR , Animales , Ratones , Hígado/metabolismo , Hígado/efectos de los fármacos , Manosa/farmacología , Manosa/metabolismo , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Patterning and growth are fundamental features of embryonic development that must be tightly coordinated. To understand how metabolism impacts early mesoderm development, we used mouse embryonic stem-cell-derived gastruloids, that co-expressed glucose transporters with the mesodermal marker T/Bra. We found that the glucose mimic, 2-deoxy-D-glucose (2-DG), blocked T/Bra expression and abolished axial elongation in gastruloids. However, glucose removal did not phenocopy 2-DG treatment despite a decline in glycolytic intermediates. As 2-DG can also act as a competitive inhibitor of mannose in protein glycosylation, we added mannose together with 2-DG and found that it could rescue the mesoderm specification both in vivo and in vitro. We further showed that blocking production and intracellular recycling of mannose abrogated mesoderm specification. Proteomics analysis demonstrated that mannose reversed glycosylation of the Wnt pathway regulator, secreted frizzled receptor Frzb. Our study showed how mannose controls mesoderm specification in mouse gastruloids.

Asunto(s)

Manosa , Mesodermo , Animales , Mesodermo/metabolismo , Ratones , Manosa/metabolismo , Glicosilación , Desoxiglucosa/metabolismo , Desoxiglucosa/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Gástrula/metabolismo , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Receptores Frizzled/metabolismo , Receptores Frizzled/genética

RESUMEN

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella's type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-ß-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella's interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods.

Asunto(s)

Manosa , Salmonella typhimurium , Salmonella typhimurium/genética , Manosa/metabolismo , Spinacia oleracea , Proteínas Fimbrias/genética , Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Adhesinas Bacterianas/genética , Adhesión Bacteriana/genética

RESUMEN

d-mannose has been widely used in food, medicine, cosmetic, and food-additive industries. To date, chemical synthesis or enzymatic conversion approaches based on iso/epimerization reactions for d-mannose production suffered from low conversion rate due to the reaction equilibrium, necessitating intricate separation processes for obtaining pure products on an industrial scale. To circumvent this challenge, this study showcased a new approach for d-mannose synthesis from glucose through constructing a phosphorylation-dephosphorylation pathway in an engineered strain. Specifically, the gene encoding phosphofructokinase (PfkA) in glycolytic pathway was deleted in Escherichia coli to accumulate fructose-6-phosphate (F6P). Additionally, one endogenous phosphatase, YniC, with high specificity to mannose-6-phosphate, was identified. In ΔpfkA strain, a recombinant synthetic pathway based on mannose-6-phosphate isomerase and YniC was developed to direct F6P to mannose. The resulting strain successfully produced 25.2 g/L mannose from glucose with a high conversion rate of 63% after transformation for 48 h. This performance surpassed the 15% conversion rate observed with 2-epimerases. In conclusion, this study presents an efficient method for achieving high-yield mannose synthesis from cost-effective glucose.

Asunto(s)

Escherichia coli , Glucosa , Manosa , Manosa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fosforilación , Glucosa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Manosafosfatos/metabolismo , Ingeniería Metabólica , Fructosafosfatos/metabolismo , Manosa-6-Fosfato Isomerasa/metabolismo , Manosa-6-Fosfato Isomerasa/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Glucólisis

RESUMEN

Host survival depends on the elimination of virus and mitigation of tissue damage. Herein, we report the modulation of D-mannose flux rewires the virus-triggered immunometabolic response cascade and reduces tissue damage. Safe and inexpensive D-mannose can compete with glucose for the same transporter and hexokinase. Such competitions suppress glycolysis, reduce mitochondrial reactive-oxygen-species and succinate-mediated hypoxia-inducible factor-1α, and thus reduce virus-induced proinflammatory cytokine production. The combinatorial treatment by D-mannose and antiviral monotherapy exhibits in vivo synergy despite delayed antiviral treatment in mouse model of virus infections. Phosphomannose isomerase (PMI) knockout cells are viable, whereas addition of D-mannose to the PMI knockout cells blocks cell proliferation, indicating that PMI activity determines the beneficial effect of D-mannose. PMI inhibition suppress a panel of virus replication via affecting host and viral surface protein glycosylation. However, D-mannose does not suppress PMI activity or virus fitness. Taken together, PMI-centered therapeutic strategy clears virus infection while D-mannose treatment reprograms glycolysis for control of collateral damage.

Asunto(s)

Manosa-6-Fosfato Isomerasa , Manosa , Animales , Ratones , Manosa-6-Fosfato Isomerasa/metabolismo , Glicosilación , Manosa/metabolismo , Glucosa/metabolismo , Antivirales/farmacología