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1.
Microb Biotechnol ; 17(6): e14480, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38858807

RESUMO

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.


Assuntos
Hexosiltransferases , Proteínas de Membrana , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Hexosiltransferases/química , Glicosilação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Campylobacter/genética , Campylobacter/enzimologia , Campylobacter/metabolismo , Polissacarídeos/metabolismo , Sistema Livre de Células , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Glicoconjugados/metabolismo
2.
Life Sci ; 348: 122689, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38710281

RESUMO

Glycans and their glycoconjugates are complex biomolecules that are crucial for various biological processes. Glycoconjugates are found in all domains of life. They are covalently linked to key biomolecules such as proteins and lipids to play a pivotal role in cell signaling, adhesion, and recognition. The diversity of glycan structures and the associated complexity of glycoconjugates is the reason for their role in intricate biosynthetic pathways. Glycoconjugates play an important role in various diseases where they are actively involved in the immune response as well as in the pathogenicity of infectious diseases. In addition, various autoimmune diseases have been linked to glycosylation defects of different biomolecules, making them an important molecule in the field of medicine. The glycoconjugates have been explored for the development of therapeutics and vaccines, representing a breakthrough in medical science. They also hold significance in research studies to understand the mechanisms behind various biological processes. Finally, glycoconjugates have found an emerging role in various industrial and environmental applications which have been discussed here.


Assuntos
Glicoconjugados , Glicoconjugados/metabolismo , Glicoconjugados/química , Humanos , Polissacarídeos/química , Polissacarídeos/metabolismo , Glicosilação , Animais , Vacinas
3.
Int J Mol Sci ; 25(10)2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38791523

RESUMO

Glucose transporters GLUT1 belong to the major facilitator superfamily and are essential to human glucose uptake. The overexpression of GLUT1 in tumor cells designates it as a pivotal target for glycoconjugate anticancer drugs. However, the interaction mechanism of glycoconjugate drugs with GLUT1 remains largely unknown. Here, we employed all-atom molecular dynamics simulations, coupled to steered and umbrella sampling techniques, to examine the thermodynamics governing the transport of glucose and two glycoconjugate drugs (i.e., 6-D-glucose-conjugated methane sulfonate and 6-D-glucose chlorambucil) by GLUT1. We characterized the specific interactions between GLUT1 and substrates at different transport stages, including substrate recognition, transport, and releasing, and identified the key residues involved in these procedures. Importantly, our results described, for the first time, the free energy profiles of GLUT1-transporting glycoconjugate drugs, and demonstrated that H160 and W388 served as important gates to regulate their transport via GLUT1. These findings provide novel atomic-scale insights for understanding the transport mechanism of GLUT1, facilitating the discovery and rational design of GLUT1-targeted anticancer drugs.


Assuntos
Transportador de Glucose Tipo 1 , Glicoconjugados , Simulação de Dinâmica Molecular , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/química , Glicoconjugados/metabolismo , Glicoconjugados/química , Humanos , Glucose/metabolismo , Transporte Biológico , Termodinâmica
4.
Zebrafish ; 21(2): 177-180, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38621207

RESUMO

Lectins are carbohydrate-binding proteins with specific affinity to glycoconjugates expressed in various tissues. Lectins are of substantial utility as research, histochemical, and diagnostic tools in mammalian systems. Reactivity of 12 commonly used plant-based lectins was studied in zebrafish liver. Four lectins, tomato lectin (TL), wheat germ agglutinin, concanavalin A, and Jacalin showed strong reactivity to hepatic parenchymal structures. Importantly, TL reacted to glycoconjugates within segments of the larval and adult intrahepatic biliary network, from canaliculi to bile ducts. We provide evidence that lectins can serve as important histochemical tools to investigate the structural and functional characteristics of the zebrafish liver.


Assuntos
Lectinas , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Histocitoquímica , Fígado/metabolismo , Glicoconjugados/metabolismo , Mamíferos/metabolismo
5.
Int J Biol Macromol ; 268(Pt 1): 131511, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38615867

RESUMO

This comprehensive review delves into the intricate landscape of glycans and glycoconjugates, unraveling their multifaceted roles across diverse biological dimensions. From influencing fundamental cellular processes such as signaling, recognition, and adhesion to exerting profound effects at the molecular and genetic levels, these complex carbohydrate structures emerge as linchpins in cellular functions and interactions. The structural diversity of glycoconjugates, which can be specifically classified into glycoproteins, glycolipids, and proteoglycans, underscores their importance in shaping the architecture of cells. Beyond their structural roles, these molecules also play key functions in facilitating cellular communication and modulating recognition mechanisms. Further, glycans and glycoconjugates prove invaluable as biomarkers in disease diagnostics, particularly in cancer, where aberrant glycosylation patterns offer critical diagnostic cues. Furthermore, the review explores their promising therapeutic applications, ranging from the development of glycan-based nanomaterials for precise drug delivery to innovative interventions in cancer treatment. This review endeavors to comprehensively explore the intricate functions of glycans and glycoconjugates, with the primary goal of offering valuable insights into their extensive implications in both health and disease. Encompassing a broad spectrum of biological processes, the focus of the review aims to provide a comprehensive understanding of the significant roles played by glycans and glycoconjugates.


Assuntos
Glicoconjugados , Polissacarídeos , Humanos , Polissacarídeos/química , Polissacarídeos/metabolismo , Glicoconjugados/química , Glicoconjugados/metabolismo , Animais , Neoplasias/metabolismo , Glicosilação , Glicoproteínas/química , Glicoproteínas/metabolismo
6.
Cells Tissues Organs ; 213(2): 147-160, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-36599327

RESUMO

The vomeronasal organ (VNO) is a tubular pheromone-sensing organ in which the lumen is covered with sensory and non-sensory epithelia. This study used immunohistochemistry and lectin histochemistry techniques to evaluate developmental changes, specifically of the glycoconjugate profile, in the horse VNO epithelium. Immunostaining analysis revealed PGP9.5 expression in some vomeronasal non-sensory epithelium (VNSE) cells and in the vomeronasal receptor cells of the vomeronasal sensory epithelium (VSE) in fetuses, young foals, and adult horses. Olfactory marker protein expression was exclusively localized in receptor cells of the VSE in fetuses, young foals, and adult horses and absent in VNSE. To identify the glycoconjugate type, lectin histochemistry was performed using 21 lectins. Semi-quantitative analysis revealed that the intensities of glycoconjugates labeled with WGA, DSL, LEL, and RCA120 were significantly higher in adult horse VSE than those in foal VSE, whereas the intensities of glycoconjugates labeled with LCA and PSA were significantly lower in adult horse VSE. The intensities of glycoconjugates labeled with s-WGA, WGA, BSL-II, DSL, LEL, STL, ConA, LCA, PSA, DBA, SBA, SJA, RCA120, jacalin, and ECL were significantly higher in adult horse VNSE than those in foal VNSE, whereas the intensity of glycoconjugates labeled with UEA-I was lower in adult horse VNSE. Histochemical analysis of each lectin revealed that various glycoconjugates in the VSE were present in the receptor, supporting, and basal cells of foals and adult horses. A similar pattern of lectin histochemistry was also observed in the VNSE of foals and adult horses. In conclusion, these results suggest that there is an increase in the level of N-acetylglucosamine (labeled by WGA, DSL, LEL) and galactose (labeled by RCA120) in horse VSE during postnatal development, implying that they may influence the function of VNO in adult horses.


Assuntos
Órgão Vomeronasal , Masculino , Humanos , Cavalos , Animais , Órgão Vomeronasal/metabolismo , Antígeno Prostático Específico/metabolismo , Epitélio/metabolismo , Lectinas/metabolismo , Glicoconjugados/análise , Glicoconjugados/metabolismo
7.
Front Biosci (Landmark Ed) ; 28(11): 305, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-38062836

RESUMO

Glycosylation is one of the most common post-translational modifications of proteins across all kingdoms of life. Diverse monosaccharides and polysaccharides can be attached to a range of amino acid residues generating N-glycosylation, O-glycosylation, C-glycosylation, S-glycosylation, as well as P-glycosylation. The functions of the eukaryotic glycosylation system during protein folding in the endoplasmic reticulum (ER) and Golgi are well-studied. Increasing evidence in the recent decade has demonstrated the presence of oligosaccharyltransferases (OSTs) in bacteria and archaea. In particular, the oligosaccharyltransferase (PglB) of Campylobacter jejuni and oligosaccharyltransferase (PglL) enzyme of Neisseria meningitidis are the most characterized OSTs that catalyze bacterial N-linked glycosylation and O-linked glycosylation, respectively. Glycoprotein administered as glycoconjugate vaccines have been shown to be effective prophylactic to protect against numerous pathogenic bacteria. The chemical synthesis of glycoproteins is complex and expensive, which limits its application to the development of glycoconjugate vaccines. However, studies have demonstrated that the biosynthesis of glycoproteins is realizable by transferring PglB, a plasmid encoding a substrate protein, or PglL, a plasmid encoding genes for glycan synthesis to Escherichia coli. This strategy can be applied to the development of glycoconjugate vaccines using engineered host E. coli. This review summarizes the structure and mechanism of action of the bacterial OSTs, PglB and PglL, and discusses their potential application to glycoconjugate vaccine design.


Assuntos
Escherichia coli , Vacinas , Escherichia coli/genética , Proteínas de Bactérias/metabolismo , Glicoconjugados/metabolismo , Glicoproteínas/metabolismo , Bactérias/metabolismo
8.
J Virol ; 97(11): e0090623, 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-37843369

RESUMO

IMPORTANCE: It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.


Assuntos
Vírus da Influenza A , Influenza Humana , Pulmão , Receptores de Superfície Celular , Animais , Humanos , Proteínas de Transporte/metabolismo , Glicoconjugados/metabolismo , Vírus da Influenza A/metabolismo , Pulmão/virologia , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Açúcares/metabolismo , Influenza Aviária/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo
9.
Mol Biochem Parasitol ; 256: 111591, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37652240

RESUMO

Trypanosoma theileri maintains a long-term extracellular infection with a low parasitaemia in bovids. The surface of this parasite is predicted to be decorated with several surface molecules including membrane surface proteases (MSPs), trans-sialidases and T. theileri putative surface proteins (TTPSPs). However, there are no experimental data to verify this hypothesis. Here, we have purified and partially characterized the surface glycoconjugates of T. theileri using biochemical and mass spectrometry-based approaches. The glycoconjugates fall into two classes: glycoproteins and glycolipids. Proteomic analysis of the glycoprotein fraction demonstrated the presence of MSPs and abundant mucin-like TTPSPs, with most predicted to be GPI-anchored. Mass spectrometric characterization of the glycolipid fraction showed that these are mannose- and galactose-containing glycoinositolphospholipids (GIPLs) that are larger and more diverse than those of its phylogenetic relative T. cruzi, containing up to 10 hexose residues and carrying either alkylacyl-phosphatidylinositol or inositol-phospho-ceramide (IPC) lipid components.


Assuntos
Proteômica , Trypanosoma cruzi , Sequência de Carboidratos , Filogenia , Trypanosoma cruzi/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Glicoconjugados/química , Glicoconjugados/metabolismo , Glicolipídeos
10.
Chembiochem ; 24(20): e202300522, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37489880

RESUMO

Multicolor labeling for monitoring the intracellular localization of the same target type in the native environment using chemical fluorescent dyes is a challenging task. This approach requires both bioorthogonal and biocompatible ligations with an excellent fluorescence signal-to-noise ratio. Here, we present a metabolic glycan labeling technique that uses homemade fluorogenic dyes to investigate glycosylation patterns in live cells. These dyes allowed us to demonstrate rapid and efficient simultaneous multilabeling of glycoconjugates with minimum fluorescence noise. Our results demonstrate that this approach is capable of not only probing sialylation and GlcNAcylation in cells but also specifically labeling the cell-surface and intracellular sialylated glycoconjugates in live cells. In particular, we performed site-specific dual-channel fluorescence imaging of extra and intracellular sialylated glycans in HeLa and PC9 cancer cells as well as identified fluorescently labeled sialylated glycoproteins and glycans by a direct enrichment approach combined with an MS-based proteomic analysis in the same experiment. In conclusion, this study provides multilabeling tools in cellular systems for simultaneous site-specific glycan imaging and glycoproteomic analysis to study potential cancer- and disease-associated glycoconjugates.


Assuntos
Glicoproteínas , Proteômica , Humanos , Corantes Fluorescentes/metabolismo , Glicoconjugados/metabolismo , Polissacarídeos/metabolismo
11.
Biotechnol Adv ; 68: 108209, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37467868

RESUMO

Glycoconjugates are the ubiquitous components of mammalian cells, mainly synthesized by covalent bonds of carbohydrates to other biomolecules such as proteins and lipids, with a wide range of potential applications in novel vaccines, therapeutic peptides and antibodies (Ab). Considering the emerging developments in glycoscience, renewable production of glycoconjugates is of importance and lignocellulosic biomass (LCB) is a potential source of carbohydrates to produce synthetic glycoconjugates in a sustainable pathway. In this review, recent advances in glycobiology aiming on glycoconjugates production is presented together with the recent and cutting-edge advances in the therapeutic properties and application of glycoconjugates, including therapeutic glycoproteins, glycosaminoglycans (GAGs), and nutraceuticals, emphasizing the integral role of glycosylation in their function and efficacy. Special emphasis is given towards the potential exploration of carbon neutral feedstocks, in which LCB has an emerging role. Techniques for extraction and recovery of mono- and oligosaccharides from LCB are critically discussed and influence of the heterogeneous nature of the feedstocks and different methods for recovery of these sugars in the development of the customized glycoconjugates is explored. Although reports on the use of LCB for the production of glycoconjugates are scarce, this review sets clear that the potential of LCB as a source for the production of valuable glycoconjugates cannot be underestimated and encourages that future research should focus on refining the existing methodologies and exploring new approaches to fully realize the potential of LCB in glycoconjugate production.


Assuntos
Glicoconjugados , Glicoproteínas , Animais , Biomassa , Glicoconjugados/química , Glicoconjugados/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Carboidratos/química , Mamíferos
12.
Biotechnol Adv ; 67: 108180, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37236328

RESUMO

The recent advancement in the human glycome and progress in the development of an inclusive network of glycosylation pathways allow the incorporation of suitable machinery for protein modification in non-natural hosts and explore novel opportunities for constructing next-generation tailored glycans and glycoconjugates. Fortunately, the emerging field of bacterial metabolic engineering has enabled the production of tailored biopolymers by harnessing living microbial factories (prokaryotes) as whole-cell biocatalysts. Microbial catalysts offer sophisticated means to develop a variety of valuable polysaccharides in bulk quantities for practical clinical applications. Glycans production through this technique is highly efficient and cost-effective, as it does not involve expensive initial materials. Metabolic glycoengineering primarily focuses on utilizing small metabolite molecules to alter biosynthetic pathways, optimization of cellular processes for glycan and glycoconjugate production, characteristic to a specific organism to produce interest tailored glycans in microbes, using preferably cheap and simple substrate. However, metabolic engineering faces one of the unique challenges, such as the need for an enzyme to catalyze desired substrate conversion when natural native substrates are already present. So, in metabolic engineering, such challenges are evaluated, and different strategies have been developed to overcome them. The generation of glycans and glycoconjugates via metabolic intermediate pathways can still be supported by glycol modeling achieved through metabolic engineering. It is evident that modern glycans engineering requires adoption of improved strain engineering strategies for creating competent glycoprotein expression platforms in bacterial hosts, in the future. These strategies include logically designing and introducing orthogonal glycosylation pathways, identifying metabolic engineering targets at the genome level, and strategically improving pathway performance (for example, through genetic modification of pathway enzymes). Here, we highlight current strategies, applications, and recent progress in metabolic engineering for producing high-value tailored glycans and their applications in biotherapeutics and diagnostics.


Assuntos
Produtos Biológicos , Humanos , Produtos Biológicos/metabolismo , Polissacarídeos/química , Glicosilação , Glicoconjugados/genética , Glicoconjugados/metabolismo , Engenharia Metabólica/métodos , Bactérias/genética
13.
Glycoconj J ; 40(3): 295-303, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37052731

RESUMO

Sialylation is an important terminal modification of glycoconjugates that mediate diverse functions in physiology and disease. In this review we focus on how altered cell surface sialylation status is sensed by cytosolic galectins when the integrity of intracellular vesicles or organelles is compromised to expose luminal glycans to the cytosolic milieu, and how this impacts galectin-mediated cellular responses. In addition, we discuss the roles of mammalian sialidases on the cell surface, in the organelle lumen and cytosol, and raise the possibility that intracellular glycan processing may be critical in controlling various galectin-mediated responses when cells encounter stress.


Assuntos
Galectinas , Polissacarídeos , Animais , Galectinas/metabolismo , Citosol/metabolismo , Polissacarídeos/metabolismo , Glicoconjugados/metabolismo , Organelas , Mamíferos/metabolismo
14.
Carbohydr Polym ; 311: 120736, 2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-37028871

RESUMO

Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate against Group A Streptococcus infections. Native GAC consists of a polyrhamnose (polyRha) backbone with N-acetylglucosamine (GlcNAc) at every second rhamnose residue. Both native GAC and the polyRha backbone have been proposed as vaccine components. Here, chemical synthesis and glycoengineering were used to generate a panel of different length GAC and polyrhamnose fragments. Biochemical analyses were performed confirming that the epitope motif of GAC is composed of GlcNAc in the context of the polyrhamnose backbone. Conjugates from GAC isolated and purified from a bacterial strain and polyRha genetically expressed in E. coli and with similar molecular size to GAC were compared in different animal models. The GAC conjugate elicited higher anti-GAC IgG levels with stronger binding capacity to Group A Streptococcus strains than the polyRha one, both in mice and in rabbits. This work contributes to the development of a vaccine against Group A Streptococcus suggesting GAC as preferable saccharide antigen to include in the vaccine.


Assuntos
Acetilglucosamina , Vacinas , Camundongos , Animais , Coelhos , Acetilglucosamina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Carboidratos , Streptococcus pyogenes/metabolismo , Glicoconjugados/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-37018937

RESUMO

Raw materials or bioactive ingredients trigger mechanisms to assimilate nutrients and activate metabolic pathways that promote growth, immune function, or energy storage. Our understanding of these processes at a molecular level remains limited in aquaculture, especially in shrimp. Here, hepatopancreas proteomics and haemolymph metabolomics were used to investigate the post-prandial response of black tiger shrimps (Penaeus monodon) fed a conventional fishmeal diet (FM); a diet supplemented with the microbial biomass Novacq™ (NV); krill meal (KM); or, fasted (FS). Using FM as a control, a 2-fold change in abundance threshold was implemented to determine the significance of proteins and metabolites. NV fed shrimp showed preference for energy derived from carbohydrates indicated by a strong signature of glycoconjugate metabolism and activation of the amino- and nucleotide sugar metabolic pathway. KM activated the glyoxylate and dicarboxylate pathway that denoted shrimp preference for lipidic energy. KM also influenced energy generation by the TCA cycle inferred from higher abundance of the metabolites succinic semialdehyde, citric acid, isocitrate, alpha ketoglutarate and ATP and downregulation of the enzyme isocitrate dehydrogenase that catalyses oxidative decarboxylation of isocitrate. FS shrimp displayed down-regulation of oxidative phosphorylation and resorted to internal lipid reserves for energy homeostasis displaying a strong signature of autophagy. Pyrimidine metabolism was the preferred energy strategy in this group. Our study also provided evidence that during fasting or consumption of specific ingredients, shrimp share common pathways to meet their energy requirements, however, the intensity at which these pathways were impacted was diet dependent.


Assuntos
Penaeidae , Animais , Isocitratos/metabolismo , Hepatopâncreas/metabolismo , Dieta , Metabolismo Energético , Quitina/metabolismo , Glicoconjugados/metabolismo , Autofagia , Imunidade
16.
Appl Environ Microbiol ; 89(4): e0191922, 2023 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-36951572

RESUMO

The bacterium Leptothrix cholodnii generates cell chains encased in sheaths that are composed of woven nanofibrils. The nanofibrils are mainly composed of glycoconjugate repeats, and several glycosyltransferases (GTs) are required for its biosynthesis. However, only one GT (LthA) has been identified to date. In this study, we screened spontaneous variants of L. cholodnii SP6 to find those that form smooth colonies, which is one of the characteristics of sheathless variants. Genomic DNA sequencing of an isolated variant revealed an insertion in the locus Lcho_0972, which encodes a putative GT family 8 protein. We thus designated this protein LthB and characterized it using deletion mutants and antibodies. LthB localized adjacent to the cell envelope. ΔlthB cell chains were nanofibril free and thus sheathless, indicating that LthB is involved in nanofibril biosynthesis. Unlike the ΔlthA mutant and the wild-type strain, which often generate planktonic cells, most ΔlthB organisms presented as long cell chains under static conditions, resulting in deficient pellicle formation, which requires motile planktonic cells. These results imply that sheaths are not required for elongation of cell chains. Finally, calcium depletion, which induces cell chain breakage due to sheath loss, abrogated the expression of LthA, but not LthB, suggesting that these GTs cooperatively participate in glycoconjugate biosynthesis under different signaling controls. IMPORTANCE In recent years, the regulation of cell chain elongation of filamentous bacteria via extracellular signals has attracted attention as a potential strategy to prevent clogging of water distribution systems and filamentous bulking of activated sludge in industrial settings. However, a fundamental understanding of the ecology of filamentous bacteria remains elusive. Since sheath formation is associated with cell chain elongation in most of these bacteria, the molecular mechanisms underlying nanofibril sheath formation, including the intracellular signaling cascade in response to extracellular stimuli, must be elucidated. Here, we isolated a sheathless variant of L. cholodnii SP6 and thus identified a novel glycosyltransferase, LthB. Although mutants with deletions of lthA, encoding another GT, and lthB were both defective for nanofibril formation, they exhibited different phenotypes of cell chain elongation and pellicle formation. Moreover, LthA expression, but not LthB expression, was influenced by extracellular calcium, which is known to affect nanofibril formation, indicating the functional diversities of LthA and LthB. Such molecular insights are critical for a better understanding of ecology of filamentous bacteria, which, in turn, can be used to improve strategies to control filamentous bacteria in industrial facilities.


Assuntos
Glicosiltransferases , Leptothrix , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Leptothrix/fisiologia , Cálcio/metabolismo , Análise de Sequência de DNA , Glicoconjugados/metabolismo
17.
Chembiochem ; 24(14): e202200783, 2023 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-36892311

RESUMO

The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic α-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 α-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α-dystroglycanopathy.


Assuntos
Distroglicanas , Galectinas , Galectinas/metabolismo , Glicoconjugados/metabolismo , Glicopeptídeos
19.
J Phycol ; 59(2): 309-322, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36471567

RESUMO

Interactions between marine diatoms and bacteria have been studied for decades. However, the visualization of physical interactions between these diatoms and their colonizers is still limited. To enhance our understanding of these specific interactions, a new Thalassiosira rotula isolate from the North Sea (strain 8673) was characterized by scanning electron microscopy and confocal laser scanning microscopy (CLSM) after staining with fluorescently labeled lectins targeting specific glycoconjugates. To investigate defined interactions of this strain with bacteria the new strain was made axenic and co-cultivated with a natural bacterial community and in two- or three-partner consortia with different bacteria of the Roseobacter group, Gammaproteobacteria and Bacteroidetes. The CLSM analysis of the consortia identified six out of 78 different lectins as very suitable to characterize glycoconjugates of T. rotula. The resulting images show that fucose-containing threads were the dominant glycoconjugates secreted by the T. rotula cells but chitin and to a lesser extent other glycoconjugates were also identified. Bacteria attached predominantly to the fucose glycoconjugates. The colonizing bacteria showed various attachment patterns such as adhering to the diatom threads in aggregates only or attaching to both the surfaces and the threads of the diatom. Interestingly the colonization patterns of single bacteria differed strikingly from those of bacterial co-cultures, indicating that interactions between two bacterial species impacted the colonization of the diatom. Our observations help to better understand physical interactions and specific colonization patterns of distinct bacterial mono- and co-cultures with an abundant diatom of costal seas.


Assuntos
Diatomáceas , Diatomáceas/metabolismo , Fucose/metabolismo , Bactérias/metabolismo , Ecossistema , Glicoconjugados/metabolismo , Lectinas/metabolismo
20.
Adv Neurobiol ; 29: 1-39, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255670

RESUMO

This chapter provides an overview of structures and functions of complex carbohydrates (commonly called glycans) that are covalently linked to proteins or lipids to form glycoconjugates known as glycoproteins, glycolipids, and proteoglycans. To understand the complexity of the glycan structures, the nature of their monosaccharide building blocks, how the monomeric units are covalently linked to each other, and how the resulting glycans are attached to proteins or lipids are discussed. Then, the classification, nomenclature, structural features, and functions of the glycan moieties of animal glycoconjugates are briefly described. All three classes of glycoconjugates are constituents of plasma membranes of all animal cells, including those of the nervous system. Glycoproteins and proteoglycans are also found abundantly as constituents of tissue matrices. Additionally, glycan-rich mucin glycoproteins are the major constituents of mucus secretions of epithelia of various organs. Furthermore, the chapter draws attention to the incredible structural complexity and diversity of the glycan moieties of cell surface and extracellular glycoconjugates. Finally, the involvement of glycans as informational molecules in a wide range of essential functions in almost all known biological processes, which are crucial for development, differentiation, and normal functioning of animals, is discussed.


Assuntos
Carboidratos , Glicoconjugados , Animais , Glicoconjugados/química , Glicoconjugados/metabolismo , Carboidratos/química , Glicoproteínas/química , Glicoproteínas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Glicolipídeos/química , Proteoglicanas/química , Monossacarídeos , Membrana Celular/metabolismo , Mucinas
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