RESUMO
Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.
Assuntos
Bactérias , Flagelina , Flagelina/genética , Flagelina/metabolismo , Estereoisomerismo , Bactérias/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Archaea/metabolismo , Flagelos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.
Assuntos
Culex , Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Ácidos Siálicos , Ligação Viral , Animais , Camundongos , Linhagem Celular , Culex/virologia , Culex/metabolismo , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Encefalite Japonesa/virologia , Encefalite Japonesa/metabolismo , Mosquitos Vetores/virologia , Neuraminidase/metabolismo , Neuraminidase/genética , Ácidos Siálicos/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Internalização do VírusRESUMO
Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid-binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell-specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas-deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.
Assuntos
Apoptose , Linfócitos B , Polissacarídeos , Ácidos Siálicos , Animais , Receptor do Fator Ativador de Células B/metabolismo , Linfócitos B/fisiologia , Sobrevivência Celular , Deleção de Genes , Camundongos , N-Acilneuraminato Citidililtransferase/genética , Polissacarídeos/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Ácidos Siálicos/metabolismoRESUMO
BACKGROUND: Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells. OBJECTIVE: We aimed to characterize Siglec-9 expression and function in human mast cells in vitro. METHODS: We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A (GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI). RESULTS: Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release. CONCLUSIONS: Siglec-9 and its ligands play an important role in limiting human mast cell activation in vitro.
Assuntos
Ácido Hialurônico , Mastócitos , Humanos , Ligantes , Ácido Hialurônico/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Imunoglobulina E/metabolismoRESUMO
Sialosides containing C8-modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid-interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para-nitrophenol-tagged α2-3- and α2-6-linked sialyl galactosides containing C8-acetamido, C8-azido, or C8-amino derivatized N-acetylneuraminic acid (Neu5Ac). High-throughput substrate specificity studies showed that the C8-modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8-azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8-acetamido-modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8-amino-modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8-amino or C8-acetamido-modified sialic acid can be used with other sialosides for diagnostic profiling of disease-causing sialidase-producing pathogens.
Assuntos
Neuraminidase , Ácidos Siálicos , Neuraminidase/metabolismo , Especificidade por Substrato , Humanos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Bactérias/enzimologia , Orthomyxoviridae/enzimologia , Vírus da Influenza A/enzimologiaRESUMO
The development of new methods for chemical glycosylation commonly includes comparison of various glycosyl donors. An attempted comparison of chemical properties of two sialic acid-based thioglycoside glycosyl donors, differing only in the substituent at O-9 (trifluoroacetyl vs chloroacetyl), at different concentrations (0.05 and 0.15 mol·L-1) led to mutually excluding conclusions concerning their relative reactivity and selectivity, which prevented us from revealing a possible influence of remote protective groups at O-9 on glycosylation outcome. According to the results of the supramer analysis of the reaction solutions, this issue might be related to the formation of supramers of glycosyl donors differing in structure hence chemical properties. These results seem to imply that comparison of chemical properties of different glycosyl donors may not be as simple and straightforward as it is usually considered.
RESUMO
The myelomonocytic receptor CD33 (Siglec-3) inhibits innate immune reactivity by extracellular V-set domain recognition of sialic acid (Sia)-containing "self-associated molecular patterns" (SAMPs). We earlier showed that V-set domain-deficient CD33-variant allele, protective against late-onset Alzheimer's Disease (LOAD), is derived and specific to the hominin lineage. We now report multiple hominin-specific CD33 V-set domain mutations. Due to hominin-specific, fixed loss-of-function mutation in the CMAH gene, humans lack N-glycolylneuraminic acid (Neu5Gc), the preferred Sia-ligand of ancestral CD33. Mutational analysis and molecular dynamics (MD)-simulations indicate that fixed change in amino acid 21 of hominin V-set domain and conformational changes related to His45 corrected for Neu5Gc-loss by switching to N-acetylneuraminic acid (Neu5Ac)-recognition. We show that human-specific pathogens Neisseria gonorrhoeae and Group B Streptococcus selectively bind human CD33 (huCD33) as part of immune-evasive molecular mimicry of host SAMPs and that this binding is significantly impacted by amino acid 21 modification. In addition to LOAD-protective CD33 alleles, humans harbor derived, population-universal, cognition-protective variants at several other loci. Interestingly, 11 of 13 SNPs in these human genes (including CD33) are not shared by genomes of archaic hominins: Neanderthals and Denisovans. We present a plausible evolutionary scenario to compile, correlate, and comprehend existing knowledge about huCD33-evolution and suggest that grandmothering emerged in humans.
Assuntos
Avós , Hominidae , Alelos , Aminoácidos , Animais , Cognição , Hominidae/genética , HumanosRESUMO
Subtype H7 avian influenza A viruses (IAVs) are enzootic in wild aquatic birds and have caused sporadic spillovers into domestic poultry and humans. Here, we determined the distribution of fucosylated α2,3 sialoglycan (i.e., sialyl Lewis X [SLeX]) in chickens and five common dabbling duck species and the association between SLeX and cell/tissue/host tropisms of H7 IAVs. Receptor binding analyses showed that H7 IAVs bind to both α2,3-linked (SA2,3Gal) and α2,6-linked sialic acids (SA2,6Gal), but with a higher preference for SLeX; H7 IAVs replicated more efficiently in SLeX-overexpressed than SLeX-deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLeX was detected in both respiratory and gastrointestinal tissues of chickens and mallard ducks and in only the respiratory tissues of gadwall, green-wing teal, and northern shoveler but not in wood ducks. Viral-tissue binding assays showed that H7 IAVs bind to chicken colon crypt cells that express SLeX but fewer bind to mallard colon crypt cells, which do not express SLeX; H7 IAVs bind efficiently to epithelial cells of all tissues expressing SA2,3Gal. High viral replication was identified in both chickens and mallards infected with an H7 virus, regardless of SLeX expression, and viruses were detected in all cells to the same degree as viruses detected in the viral-tissue binding assays. In summary, this study suggests that SLeX facilitates infection of H7 viruses, but other types of SA2,3Gal glycan receptors shape the tissue/host tropisms of H7 IAVs. IMPORTANCE In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLeX expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLeX expression is not the only determinant of viral replication at either the tissue or host level. This study suggested that access to heterologous SA2,3Gal glycan receptors, including fucosylated α2,3-linked sialoglycans, shape tissue and host tropism of H7 IAVs in aquatic wild birds.
Assuntos
Vírus da Influenza A , Influenza Aviária , Antígeno Sialil Lewis X , Tropismo Viral , Animais , Animais Selvagens/virologia , Galinhas/virologia , Cães , Patos/virologia , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Polissacarídeos , Ácidos Siálicos , Antígeno Sialil Lewis X/metabolismoRESUMO
Sialic acid sugar-carrying glycans, sialoglycans, are aberrantly expressed on many tumor cells and have emerged as potent regulatory molecules involved in creating a tumor-supportive microenvironment. Sialoglycans can be recognized by sialic acid-binding immunoglobulin-like lectins (Siglecs), a family of immunomodulatory receptors. Most mammalian Siglecs transmit inhibitory signals comparable with the immune checkpoint inhibitor programmed death protein 1 (PD-1), but some are activating. Recent studies have shown that tumor cells can exploit sialoglycan-Siglec interactions to modulate immune cell function, contributing to an immunosuppressive tumor microenvironment (TME). Interference with sialoglycan synthesis or sialoglycan-Siglec interactions might improve antitumor immunity. Many questions regarding specificity, signaling, and regulatory function of sialoglycan-Siglec interactions remain. We posit that sialoglycans and Siglecs present as potential glyco-immune 'checkpoints' for cancer immunotherapy.
Assuntos
Polissacarídeos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Microambiente Tumoral , Animais , Humanos , Imunidade , Imunoterapia , Neoplasias/terapia , Polissacarídeos/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Microambiente Tumoral/imunologiaRESUMO
A subclass of the sialic acid family consists of intramolecular lactones that may function as key indicators of physiological and pathological states. However, the existence of these compounds in free form is highly improbable, since they are unlikely to exist in an aqueous solution due to their lability. Current analytical method used to detect them in biological fluids has not recognized their reactivity in solution and is prone to misidentification. However, recent advances in synthetic methods for 1,7-lactones have allowed the preparation of these sialic acid derivatives as authentic reference standards. We report here the development of a new HPLC-MS method for the simultaneous detection of the 1,7-lactone of N-acetylneuraminic acid, its γ-lactone derivative, and N-acetylneuraminic acid that overcomes the limitations of the previous analytical procedure for their identification.
Assuntos
Ácido N-Acetilneuramínico , Ácidos Siálicos , Ácidos Siálicos/análise , Lactonas , Cromatografia Líquida de Alta PressãoRESUMO
Sialic acids are nine-carbon sugars that frequently cap glycans at the cell surface in cells of vertebrates as well as cells of certain types of invertebrates and bacteria. The nine-carbon backbone of sialic acids can undergo extensive enzymatic modification in nature and O-acetylation at the C-4/7/8/9 position in particular is widely observed. In recent years, the detection and analysis of O-acetylated sialic acids have advanced, and sialic acid-specific O-acetyltransferases (SOATs) and O-acetylesterases (SIAEs) that add and remove O-acetyl groups, respectively, have been identified and characterized in mammalian cells, invertebrates, bacteria, and viruses. These advances now allow us to draw a more complete picture of the biosynthetic pathway of the diverse O-acetylated sialic acids to drive the generation of genetically and biochemically engineered model cell lines and organisms with altered expression of O-acetylated sialic acids for dissection of their roles in glycoprotein stability, development, and immune recognition, as well as discovery of novel functions. Furthermore, a growing number of studies associate sialic acid O-acetylation with cancer, autoimmunity, and infection, providing rationale for the development of selective probes and inhibitors of SOATs and SIAEs. Here, we discuss the current insights into the biosynthesis and biological functions of O-acetylated sialic acids and review the evidence linking this modification to disease. Furthermore, we discuss emerging strategies for the design, synthesis, and potential application of unnatural O-acetylated sialic acids and inhibitors of SOATs and SIAEs that may enable therapeutic targeting of this versatile sialic acid modification.
Assuntos
Acetiltransferases/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Acetilação , Animais , Vias Biossintéticas , Doença , Glicoproteínas/metabolismo , Humanos , Ácido N-Acetilneuramínico/química , Polissacarídeos/químicaRESUMO
Cellular interactions between endothelial cells and macrophages regulate macrophage localization and phenotype, but the mechanisms underlying these interactions are poorly understood. Here we explored the role of sialoglycans on lymphatic endothelial cells (LEC) in interactions with macrophage-expressed Siglec-1 (CD169). Lectin-binding assays and mass spectrometric analyses revealed that LEC from human skin express more sialylated glycans than the corresponding blood endothelial cells. Higher amounts of sialylated and/or sulfated glycans on LEC than BEC were consistently observed in murine skin, lung and lymph nodes. The floor LEC of the subcapsular sinus (SCS) in murine lymph nodes (LN) displayed sialylated glycans at particularly high densities. The sialoglycans of LN LEC were strongly bound by Siglec-1. Such binding plays an important role in the localization of Siglec-1+ LN-SCS macrophages, as their numbers are strongly reduced in mice expressing a Siglec-1 mutant that is defective in sialoglycan binding. The residual Siglec-1+ macrophages are less proliferative and have a more anti-inflammatory phenotype. We propose that the densely clustered, sialylated glycans on the SCS floor LEC are a key component of the macrophage niche, providing anchorage for the Siglec-1+ LN-SCS macrophages.
Assuntos
Células Endoteliais/metabolismo , Linfonodos/metabolismo , Macrófagos/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Pele/metabolismo , Animais , Células CHO , Cricetulus , Células Endoteliais/citologia , Humanos , Linfonodos/citologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Pele/citologiaRESUMO
Sialic-acid binding lectin from bullfrog (Rana catesbeiana) eggs, cSBL, is a cytotoxic ribonuclease (RNase) belonging to the RNase A superfamily. cSBL is cytotoxic to tumor cells, such as malignant pleural mesothelioma by inducing apoptotic cell death caused by the degradation of RNA in tumor cells. In addition, we have reported some data that cSBL could be involved in the endoplasmic reticulum stress pathway, and it was also assumed to cause apoptotic cell death. The most significant property of cSBL is its specificity toward malignant cells. Furthermore, since the antitumor activity of cSBL was confirmed by in vivo experiments using mouse xenograft models, it is expected to be a candidate for clinical chemotherapy. Here, we summarize the history of cSBL, alternatively called "leczyme," with its present and future.
Assuntos
Antineoplásicos , Apoptose , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Lectinas/metabolismo , Camundongos , Rana catesbeiana/metabolismo , Ribonucleases/metabolismo , Ribonucleases/farmacologia , Ribonucleases/uso terapêuticoRESUMO
Finding convenient ways for the stereoselective α-sialylation is important due to the high practical significance of α-sialic acid-containing glycans and neoglycoconjugates. It was proposed that sialylation stereoselectivity is determined by the structure of the sialyl cation (also known in biochemistry as "sialosyl cation"), a supposed intermediate in this reaction. Here we design a new approach for studying the conformational space of highly flexible sialyl cation and find 1625 unique conformers including those stabilized by covalent remote participation (also known as long-range participation) of 4-O-acetyl (4-OAc), 5-N-trifluoroacetyl (5-NTFA), as well as 7,8,9-OAc from both α and ß sides. The most energetically stable sialyl cation conformers are featured by 4-OAc participation, closely followed by 5-NTFA- and 7-OAc-stabilized conformers; unstabilized sialyl cation conformers are â¼10â kcal mol-1 less stable than the 4-OAc-stabilized ones. Analysis of all the obtained conformers by means of substituents positions, side chain conformations and ring puckering led us to a new "eight-conformer hypothesis" which describes interconversions among the most important sialyl cation conformers and predicts that stronger remote participation of acyl groups favors ß-anomers. Thus, selective synthesis of the desired α-sialosides requires minimization of acyl groups participation.
Assuntos
Conformação Molecular , CátionsRESUMO
In late 2015, an epizootic of Highly Pathogenic Avian Influenza (H5Nx) was registered in Southwestern France, including more than 70 outbreaks in commercial poultry flocks. Phylogenetic analyses suggested local emergence of H5 viruses which differed from A/goose/Guangdong/1/1996 clade 2.3.4.4b lineage and shared a unique polybasic cleavage site in their hemagglutinin protein. The present work provides an overview of the pathobiological picture associated with this epizootic in naturally infected chickens, guinea fowls and ducks. Upon necropsy examination, selected tissues were sampled for histopathology, immunohistochemistry and quantitative Real Time Polymerase Chain Reaction. In Galliformes, HPAIVs infection manifested as severe acute systemic vasculitis and parenchymal necrosis and was associated with endothelial expression of viral antigen. In ducks, lesions were mild and infrequent, with sparse antigenic detection in respiratory and digestive mucosae and leukocytes. Tissue quantifications of viral antigen and RNA were higher in chickens and guinea fowls compared to duck. Subsequently, recombinant HA (rHA) was generated from a H5 HPAIV isolated from an infected duck to investigate its glycan-binding affinity for avian mucosae. Glycan-binding analysis revealed strong affinity of rHA for 3'Sialyl-LacNAc and low affinity for Sialyl-LewisX, consistent with a duck-adapted virus similar to A/Duck/Mongolia/54/2001 (H5N2). K222R and S227R mutations on rHA sequence shifted affinity towards Sialyl-LewisX and led to an increased affinity for chicken mucosa, confirming the involvement of these two mutations in the glycan-binding specificity of the HA. Interestingly, the rHA glycan binding pattern of guinea fowl appeared intermediate between duck and chicken. The present study presents a unique pathobiological description of the H5 HPAIVs outbreaks that occurred in 2015-2016 in Southwestern France.
Assuntos
Anseriformes , Galliformes , Vírus da Influenza A Subtipo H5N2 , Influenza Aviária , Animais , Anseriformes/metabolismo , Galinhas/metabolismo , Patos/metabolismo , Galliformes/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H5N2/genética , FilogeniaRESUMO
Sugar additions to biomolecules, or glycans, are some of the most abundant biomolecule modifications in biology because they enable cells to adapt to changing nutrient and stress conditions. An unmet challenge for the field of glycobiology is the study of glycan biosynthetic pathways with chemical control, especially in live cell settings. The objective of this study was to create biocompatible glycan precursors with controlled release properties. Here, we report eleven "caged" sugar probes that release glycan biosynthetic precursor molecules upon light exposure. The specific sugar pathways we target with our probes regulate the addition of the N-acetyl sugars GlcNAc, GalNAc, and sialic acid onto biomolecules in cells, each of which has the potential to alter glycan processes involved in cell morphology, signaling, and behavior. We hypothesized that our glycan precursor probes would remain biologically inert until light-initiated decaging conditions were met, avoiding biological activities including metabolism and cytotoxicity. The photocaged analogs of GlcNAc, GalNAc, and ManNAc (sialic acid precursor) sugars, which we call "photo-sugars," were released within minutes of light exposure at their optimal wavelengths. During the course of the study, we characterized the cell compatibility of these sugars under their respective decaging conditions, and found highly cell compatible GlcNAc, GalNAc, and ManNAc photocaged precursors. Release of GlcNAc-1-phosphate precursors led to altered ATP levels in cells, demonstrating preliminary metabolic engineering. We envision these probes as useful additions to the chemical glycobiology field that will enable spatiotemporal control over glycosylation pathways in living mammalian cells.
Assuntos
Engenharia Metabólica , Polissacarídeos , Animais , Mamíferos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/biossíntese , Polissacarídeos/metabolismo , Açúcares/metabolismoRESUMO
Terminal sialic acids (Sia) on soluble glycoprotein of saliva play an important role in the clearance of influenza virus. The aim of this study is to investigate the alteration of sialylation on the salivary proteins of women during the lactation period and its effect on the saliva binding ability to virus. In total, 210 saliva samples from postpartum women with and without breastfeeding were collected, and the expression level of α2-3/6-linked Sia on the whole salivary proteins and specific glycoproteins of IgA and MUC5B from different groups were tested and verified using lectin microarray, blotting analysis and ELISA based method. The H1N1 vaccine and three strains of Avian influenza virus (AIV) were used for the saliva binding assay. Results showed that the variation in salivary expression level of α2-3-linked Sia was much more obvious than the α2-6-linked Sia, which was up-regulated significantly in the breastfeeding groups compared to the non-breastfeeding groups at the same postpartum stage. Furthermore, the binding abilities of salivary glycoproteins to AIV strains and H1N1 vaccine were increased in breastfeeding groups accordingly. This finding adds new evidence for the maternal benefit of breastfeeding and provides new thinking to protect postpartum women from AIV infection.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Aviária , Animais , Feminino , Glicoproteínas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A/metabolismo , Ácidos SiálicosRESUMO
Desialylation of cell surface glycoproteins carried out by sialidases affects various immunological processes. However, the role of neuraminidase 1 (NEU1), one of the four mammalian sialidases, in inflammation and autoimmune disease is not completely unraveled to date. In this study, we analyzed the retinal expression of NEU1 in equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis. Mass spectrometry revealed significantly higher abundance of NEU1 in retinal Müller glial cells (RMG) of ERU-diseased horses compared to healthy controls. Immunohistochemistry uncovered NEU1 expression along the whole Müller cell body in healthy and uveitic states and confirmed higher abundance in inflamed retina. Müller glial cells are the principal macroglial cells of the retina and play a crucial role in uveitis pathogenesis. To determine whether higher expression levels of NEU1 in uveitic RMG correlate with the desialylation of retinal cells, we performed lectin-binding assays with sialic acid-specific lectins. Through these experiments, we could demonstrate a profound loss of both α2-3- and α2-6-linked terminal sialic acids in uveitis. Hence, we hypothesize that the higher abundance of NEU1 in uveitic RMG plays an important role in the pathogenesis of uveitis by desialylation of retinal cells. As RMG become activated in the course of uveitis and actively promote inflammation, we propose that NEU1 might represent a novel activation marker for inflammatory RMG. Our data provide novel insights in the expression and implication of NEU1 in inflammation and autoimmune disease.
Assuntos
Doenças Autoimunes , Uveíte , Animais , Doenças Autoimunes/veterinária , Cavalos , Imuno-Histoquímica , Mamíferos , Neurônios/metabolismo , Retina/química , Retina/metabolismo , Uveíte/metabolismo , Uveíte/veterináriaRESUMO
Understanding how influenza viruses traverse the mucus and recognize host cells is critical for evaluating their zoonotic potential, and for prevention and treatment of the disease. The surface of the influenza A virus is covered with the receptor-binding protein hemagglutinin and the receptor-cleaving enzyme neuraminidase, which jointly control the interactions between the virus and the host cell. These proteins are organized in closely spaced trimers and tetramers to facilitate multivalent interactions with sialic acid-terminated glycans. This review shows that the individually weak multivalent interactions of influenza viruses allow superselective binding, virus-induced recruitment of receptors, and the formation of dynamic complexes that facilitate molecular walking. Techniques to measure the avidity and receptor specificity of influenza viruses are reviewed, and the pivotal role of multivalent interactions with their emergent properties in crossing the mucus and entering host cells is discussed. A model is proposed for the initiation of cell entry through virus-induced receptor clustering. The multivalent interactions of influenza viruses are maintained in a dynamic regime by a functional balance between binding and cleaving.
Assuntos
Vírus da Influenza A , Influenza Humana , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , Ácido N-Acetilneuramínico , Neuraminidase , Receptores ViraisRESUMO
Metabolic glycoengineering (MGE) is an established method to incorporate chemical reporter groups into cellular glycans for subsequent bioorthogonal labeling. The method has found broad application for the visualization and isolation of glycans allowing their biological roles to be probed. Furthermore, targeting of drugs to cancer cells that present high concentrations of sialic acids on their surface is an attractive approach. We report the application of a labeling reaction using 1,2-diamino-4,5-methylenedioxybenzene for the quantification of sialic acid derivates after MGE with various azide- and alkene-modified ManNAc, GlcNAc, and GalNAc derivatives. We followed the time course of sialic acid production and were able to detect sialic acids modified with the chemical reporter group - not only after addition of ManNAc derivatives to the cell culture. A cyclopropane-modified ManNAc derivative, being a model for the corresponding cyclopropene analog, which undergoes fast inverse-electron-demand Diels-Alder reactions with 1,2,4,5-tetrazines, resulted in the highest incorporation efficiency. Furthermore, we investigated whether feeding the cells with natural and unnatural ManNAc derivative results in increased levels of sialic acids and found that this is strongly dependent on the investigated cell type and cell fraction. For HEK 293T cells, a strong increase in free sialic acids in the cell interior was found, whereas cell-surface sialic acid levels are only moderately increased.