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Glycobiology Mar 2022Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan...
Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce nonnative glycan chains that may not fulfill the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exoglycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis, a GH2 β-galactofuranosidase from Amesia atrobrunnea and the jack bean α-mannosidase. The extent of enzymatic O-deglycosylation was mapped against a full glycosyl linkage analysis of the O-glycosylated linker of cellobiohydrolase I from Trichoderma reesei (TrCel7A). Furthermore, the influence of deglycosylation on TrCel7A functionality was evaluated by kinetic characterization of native and O-deglycosylated forms of TrCel7A. This study expands structural knowledge on fungal O-glycosylation and presents a ready-to-use enzymatic approach for controlled O-glycan engineering in glycoproteins expressed in filamentous fungi.
Topics: Cellulose 1,4-beta-Cellobiosidase; Fungal Proteins; Glycosylation; Mannose; Mannosidases; alpha-Mannosidase
PubMed: 34939126
DOI: 10.1093/glycob/cwab127 -
Extremophiles : Life Under Extreme... May 2016Protein misfolding, aggregation and deposition in the brain, in the form of amyloid, are implicated in the etiology of several neurodegenerative disorders, such as... (Review)
Review
Protein misfolding, aggregation and deposition in the brain, in the form of amyloid, are implicated in the etiology of several neurodegenerative disorders, such as Alzheimer's, Parkinson's and prion diseases. Drugs available on the market reduce the symptoms, but they are not a cure. Therefore, it is urgent to identify promising targets and develop effective drugs. Preservation of protein native conformation and/or inhibition of protein aggregation seem pertinent targets for drug development. Several studies have shown that organic solutes, produced by extremophilic microorganisms in response to osmotic and/or heat stress, prevent denaturation and aggregation of model proteins. Among these stress solutes, mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, diglycerol phosphate and ectoine are effective in preventing amyloid formation by Alzheimer's Aβ peptide and/or α-synuclein in vitro. Moreover, mannosylglycerate is a potent inhibitor of Aβ and α-synuclein aggregation in living cells, and mannosylglyceramide and ectoine inhibit aggregation and reduce prion peptide-induced toxicity in human cells. This review focuses on the efficacy of stress solutes from hyper/thermophiles and ectoines to prevent amyloid formation in vitro and in vivo and their potential application in drug development against protein misfolding diseases. Current and envisaged applications of these extremolytes in neurodegenerative diseases and healthcare will also be addressed.
Topics: Amino Acids, Diamino; Amyloid; Animals; Archaea; Bacteria; Glyceric Acids; Glycerophosphates; Humans; Mannose; Proteostasis Deficiencies; Stress, Physiological
PubMed: 27071404
DOI: 10.1007/s00792-016-0828-8 -
Cancer Science Dec 2021Diverse metabolic changes are induced by various driver oncogenes during the onset and progression of leukemia. By upregulating glycolysis, cancer cells acquire a...
Diverse metabolic changes are induced by various driver oncogenes during the onset and progression of leukemia. By upregulating glycolysis, cancer cells acquire a proliferative advantage over normal hematopoietic cells; in addition, these changes in energy metabolism contribute to anticancer drug resistance. Because leukemia cells proliferate by consuming glucose as an energy source, an alternative nutrient source is essential when glucose levels in bone marrow are insufficient. We profiled sugar metabolism in leukemia cells and found that mannose is an energy source for glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. Leukemia cells express high levels of phosphomannose isomerase (PMI), which mobilizes mannose to glycolysis; consequently, even mannose in the blood can be used as an energy source for glycolysis. Conversely, suppression of PMI expression or a mannose load exceeding the processing capacity of PMI inhibited transcription of genes related to mitochondrial metabolism and the TCA cycle, therefore suppressing the growth of leukemia cells. High PMI expression was also a poor prognostic factor for acute myeloid leukemia. Our findings reveal a new mechanism for glucose starvation resistance in leukemia. Furthermore, the combination of PMI suppression and mannose loading has potential as a novel treatment for driver oncogene-independent leukemia.
Topics: Animals; Cell Line, Tumor; Citric Acid Cycle; Female; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; K562 Cells; Leukemia; Mannose; Mannose-6-Phosphate Isomerase; Mice; Pentose Phosphate Pathway; Prognosis; THP-1 Cells; Up-Regulation; Xenograft Model Antitumor Assays
PubMed: 34533861
DOI: 10.1111/cas.15138 -
Bioorganic & Medicinal Chemistry Aug 2022Naturally occurring N-glycans display much diversity in modifications, linkages, and peripheral presentation of the oligosaccharide chain. Despite continued advancements...
Naturally occurring N-glycans display much diversity in modifications, linkages, and peripheral presentation of the oligosaccharide chain. Despite continued advancements in oligosaccharide synthesis, synthetic access to these natural glycans remains challenging. Biologically relevant complex N-glycan mimetics with various natural and unnatural modifications are an alternate way for investigating glycan-protein interactions. Further supporting this pattern, we report here a new class of sialylated bi- and triantennary pseudo mannose N-glycans reproducing orientation of the underlying glycan chain and branching patterns and replacing the two inner mannopyranosyl units with 1,2,3-triazole rings. Such mimetics are straightforwardly generated by implementing multiple intermolecular Cu(I)-catalyzed azide-alkyne cycloaddition between chemoenzymatically synthesized azido sialosides and rationally designed C-3 and C-6 di-O- or C-2, C-3, and C-6 tri-O-alkynylated mannoside. Human recombinant Siglec-7-Fc fusion protein recognizes almost all sialylated pseudo mannose N-glycans in the microarray. However, a differential Sia-binding pattern was also observed. Given the library size, comparison of pairwise mannose N-glycan combinations showed that biantennary linear α(2,3)α(2,8)- and α(2,6)α(2,8)- or branched α(2,3)α(2,6)-, and triantennary branched α(2,3)α(2,6)-sialyl pseudo N-glycans possess similar binding capabilities and affinity to recombinant Siglec-7-Fc. While the full range of topological mannose arms remain elusive, the bi- and triantennary mimics are simpler structures for interrogating Siglec interactions.
Topics: Humans; Mannose; Oligosaccharides; Polysaccharides; Sialic Acid Binding Immunoglobulin-like Lectins
PubMed: 35640379
DOI: 10.1016/j.bmc.2022.116839 -
Langmuir : the ACS Journal of Surfaces... Nov 2020The surfaces of cells and pathogens are covered with short polymers of sugars known as glycans. Complex -glycans have a core of three mannose sugars with distal repeats...
The surfaces of cells and pathogens are covered with short polymers of sugars known as glycans. Complex -glycans have a core of three mannose sugars with distal repeats of -acetylglucosamine and galactose sugars terminating with sialic acid (SA). Long-range tough and short-range brittle self-adhesions were observed between SA and mannose residues, respectively, in ill-defined artificial monolayers. We investigated if and how these adhesions translate when the residues are presented in -glycan architecture with SA at the surface and mannose at the core and with other glycan sugars. Two pseudotyped viruses with complex -glycan shields were brought together in force spectroscopy (FS). At higher ramp rates, slime-like adhesions were observed between the shields, whereas Velcro-like adhesions were observed at lower rates. The higher approach rates compress the virus as a whole, and the self-adhesion between the surface SA is sampled. At the lower ramp rates, however, the complex glycan shield is penetrated and adhesion from the mannose core is accessed. The slime-like and Velcro-like adhesions were lost when SA and mannose were cleaved, respectively. While virus self-adhesion in forced contact was modulated by glycan penetrability, the self-aggregation of the freely diffusing virus was only determined by the surface sugar. Mannose-terminal viruses self-aggregated in solution, and SA-terminal ones required Ca ions to self-aggregate. Viruses with galactose or -acetylglucosamine surfaces did not self-aggregate, irrespective of whether or not a mannose core was present below the -acetylglucosamine surface. Well-defined rules appear to govern the self-adhesion and -aggregation of N-glycosylated surfaces, regardless of whether the sugars are presented in an ill-defined monolayer, or -glycan, or even polymer architecture.
Topics: Mannose; N-Acetylneuraminic Acid; Polysaccharides; Sugars; Viruses
PubMed: 33186493
DOI: 10.1021/acs.langmuir.0c01953 -
Molecules (Basel, Switzerland) Aug 2021-Mannosylation is a post-translational modification of proteins in the endoplasmic reticulum. Monomeric α-mannose is attached to specific Trp residues at the first Trp... (Review)
Review
-Mannosylation is a post-translational modification of proteins in the endoplasmic reticulum. Monomeric α-mannose is attached to specific Trp residues at the first Trp in the Trp-x-x-Trp/Cys (W-x-x-W/C) motif of substrate proteins, by the action of -mannosyltransferases, -related gene products. The acceptor substrate proteins are included in the thrombospondin type I repeat (TSR) superfamily, cytokine receptor type I family, and others. Previous studies demonstrated that -mannosylation plays critical roles in the folding, sorting, and/or secretion of substrate proteins. A -mannosylation-defective gene mutation was identified in humans as the disease-associated variant affecting a -mannosylation motif of W-x-x-W of ADAMTSL1, which suggests the involvement of defects in protein -mannosylation in human diseases such as developmental glaucoma, myopia, and/or retinal defects. On the other hand, monomeric -mannosyl Trp (-Man-Trp), a deduced degradation product of -mannosylated proteins, occurs in cells and extracellular fluids. Several studies showed that the level of -Man-Trp is upregulated in blood of patients with renal dysfunction, suggesting that the metabolism of -Man-Trp may be involved in human kidney diseases. Together, protein -mannosylation is considered to play important roles in the biosynthesis and functions of substrate proteins, and the altered regulation of protein -manosylation may be involved in the pathophysiology of human diseases. In this review, we consider the biochemical and biomedical knowledge of protein -mannosylation and -Man-Trp, and introduce recent studies concerning their significance in biology and medicine.
Topics: Humans; Mannose; Protein C; Protein Processing, Post-Translational; Tryptophan
PubMed: 34500691
DOI: 10.3390/molecules26175258 -
Journal of Separation Science Jun 2022We developed a simple high-performance liquid chromatography assay to monitor high-mannose glycans in monoclonal antibodies by monitoring terminal alpha-mannose as a...
We developed a simple high-performance liquid chromatography assay to monitor high-mannose glycans in monoclonal antibodies by monitoring terminal alpha-mannose as a surrogate marker. Analysis of glycan data of therapeutic monoclonal antibodies by 2-aminobenzamide assay showed a linear relationship between high mannose and terminal mannose of Fc glycans. Concanavalin A has a strong affinity to alpha-mannose in glycans of typical therapeutic monoclonal antibodies. To show that terminal mannose binds specifically to Concanavalin A column, exoglycosidase-treated monoclonal antibodies were serially blended with untreated monoclonal antibodies. Linear responses of terminal-mannose binding to the column and comparable data trending with high mannose levels by 2-aminobenzamide assay confirmed that terminal-mannose levels measured by the Concanavalin A column can be used as a surrogate for the prediction of high-mannose levels in monoclonal antibodies. The assay offers a simple, fast, and specific capability for the prediction of high-mannose content in samples compared with traditional glycan profiling by 2-aminobenzamide or mass spectrometry-based methods. When the Concanavalin A column was coupled with protein A column for purification of antibodies from cell culture samples in a fully automated two-dimensional analysis, high-mannose data could be relayed to the manufacturing team in less than 30 min, allowing near-real-time monitoring of high-mannose levels in the cell culture process.
Topics: Antibodies, Monoclonal; Cell Culture Techniques; Chromatography, Affinity; Concanavalin A; Lectins; Mannose; Polysaccharides
PubMed: 35043561
DOI: 10.1002/jssc.202100903 -
Metabolism: Clinical and Experimental Jan 2020Mannose is a glucose-associated serum metabolite mainly released by the liver. Recent studies have shown several unexpected pleiotropic effects of mannose including...
Mannose is a glucose-associated serum metabolite mainly released by the liver. Recent studies have shown several unexpected pleiotropic effects of mannose including increased regulatory T cells (Tregs), prevention of auto-immune disease and ability to reduce growth of human cancer cells. We have previously shown in large cohorts that elevated serum mannose levels are associated with future development of type 2 diabetes (T2D) and cardiovascular disease. However, potential direct effects of mannose on insulin sensitivity in vivo or in vitro are unknown. We here show that administration of mannose (0.1 g/kg BW twice daily) for one week in man did not elicit negative effects on meal-modified glucose tolerance, markers of inflammation or insulin levels. Tregs number and insulin signaling in human liver cells were unchanged. These data suggest that mannose is a marker, and not a mediator, of insulin resistance. To verify this, we examined serum mannose levels during long-term euglycemic hyperinsulinemic clamps in non-diabetic and T2D individuals. Mannose was reduced by insulin infusion in proportion to whole-body insulin sensitivity. Thus, mannose is a biomarker of insulin resistance which may be useful for the early identification of diabetic individuals with insulin resistance and increased risk of its complications.
Topics: Aged; Biomarkers; Case-Control Studies; Female; Glucose Clamp Technique; Glucose Tolerance Test; Humans; Insulin; Insulin Resistance; Male; Mannose; Middle Aged; Signal Transduction
PubMed: 31682799
DOI: 10.1016/j.metabol.2019.153974 -
Journal of Chemical Information and... Oct 2022Despite their fundamental biological relevance, structure-property relationships in -glycans are fundamentally lacking, and their highly multidimensional compositional...
Despite their fundamental biological relevance, structure-property relationships in -glycans are fundamentally lacking, and their highly multidimensional compositional and conformational phase spaces remain largely unexplored. The torsional flexibility of the glycosidic linkages and the ring dynamics result in wide, rugged free-energy landscapes that are difficult to sample in molecular dynamics simulations. We show that a novel enhanced-sampling scheme combining replica exchange with solute and collective-variable tempering, enabling transitions over all relevant energy barriers, delivers converged distributions of solvated -glycan conformers. Several dimensionality-reduction algorithms are compared and employed to generate conformational free-energy maps in two dimensions. Together with an originally developed conformation-based nomenclature scheme that uniquely identifies glycan conformers, our modeling procedure is applied to reveal the effect of chemical substitutions on the conformational ensemble of selected high-mannose-type and complex glycans. Moreover, the structure-prediction capabilities of two commonly used glycan force fields are assessed via the theoretical prediction of experimentally available nuclear magnetic resonance J-coupling constants. The results especially confirm the key role of ω and ψ torsion angles in discriminating between different conformational states and suggest an intriguing correlation between the torsional and ring-puckering degrees of freedom that may be biologically relevant.
Topics: Mannose; Rationalization; Molecular Conformation; Molecular Dynamics Simulation; Polysaccharides
PubMed: 36179122
DOI: 10.1021/acs.jcim.2c01049 -
Nature Chemical Biology Apr 2021Tryptophan C-mannosylation is an unusual co-translational protein modification performed by metazoans and apicomplexan protists. The prevalence and biological functions...
Tryptophan C-mannosylation is an unusual co-translational protein modification performed by metazoans and apicomplexan protists. The prevalence and biological functions of this modification are poorly understood, with progress in the field hampered by a dearth of convenient tools for installing and detecting the modification. Here, we engineer a yeast system to produce a diverse array of proteins with and without tryptophan C-mannosylation and interrogate the modification's influence on protein stability and function. This system also enabled mutagenesis studies to identify residues of the glycosyltransferase and its protein substrates that are crucial for catalysis. The collection of modified proteins accrued during this work facilitated the generation and thorough characterization of monoclonal antibodies against tryptophan C-mannosylation. These antibodies empowered proteomic analyses of the brain C-glycome by enriching for peptides possessing tryptophan C-mannosylation. This study revealed many new modification sites on proteins throughout the secretory pathway with both conventional and non-canonical consensus sequences.
Topics: Amino Acid Sequence; Antibodies; Glycosylation; Glycosyltransferases; Mannose; Peptides; Protein Engineering; Protein Processing, Post-Translational; Protein Stability; Proteomics; Saccharomyces cerevisiae; Saccharomycetales; Tryptophan
PubMed: 33542533
DOI: 10.1038/s41589-020-00727-w