scholarly journals Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8)

2021 ◽  
Author(s):  
Roushu Zhang ◽  
Qiang Yang ◽  
Bhargavi M Boruah ◽  
Guanghui Zong ◽  
Chao Li ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Turnover Rate ◽  
Human Neutrophils ◽  
Complex Type ◽  
The Core ◽  
Km Value ◽  
High Mannose ◽  
Overall Efficiency ◽  
Core Fucosylation ◽  
Lysosomal Proteins

The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal β1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates.  Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the Km value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.

scholarly journals Substrate specificity of human liver neutral α-mannosidase

1992 ◽  
Vol 286 (1) ◽  
pp. 47-53 ◽  
Author(s):  
S al Daher ◽  
R De Gasperi ◽  
P Daniel ◽  
S Hirani ◽  
C Warren ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Human Liver ◽  
Core Structure ◽  
Catabolic Pathways ◽  
Mannose Residue ◽  
The Core ◽  
High Mannose

The digestion of radiolabelled natural oligosaccharide substrates by human liver neutral alpha-mannosidase has been studied by h.p.l.c. and h.p.t.l.c. The high-mannose oligosaccharides Man9GlcNAc and Man8GlcNAc are hydrolysed by the enzyme by two distinct non-random routes to a common product of composition Man6GlcNAc, which is then slowly converted into a unique Man5GlcNAc oligosaccharide, Man alpha(1----2)Man alpha(1----2)Man alpha(1----3)[Man alpha (1----6)] Man beta(1----4)GlcNAc. These pathways are different from the processing and lysosomal catabolic pathways for these structures. In particular, the alpha(1----2)-linked mannose residues attached to the core alpha(1----3)-linked mannose residue are resistant to hydrolysis. The key processing intermediate, Man alpha(1----3)[Man alpha(1----6)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc, is not produced in the digestion of high-mannose glycans by the neutral alpha-mannosidase, but it is hydrolysed by the enzyme by a non-random route to Man beta(1----4)GlcNAc via the core structure Man alpha(1----3)[Man alpha(1----6)]Man beta(1----4)GlcNAc. In contrast with its ready hydrolysis by lysosomal alpha-mannosidase, the core alpha(1----3)-mannosidic linkage is quite resistant to hydrolysis by neutral alpha-mannosidase. The precise specificity of neutral alpha-mannosidase towards high-mannose oligosaccharides suggests that it has a role in the modification of such structures in the cytosol.

scholarly journals Enzymatic Synthesis of a Fluorogenic Reporter Substrate and a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation

2019 ◽  
Author(s):  
Maxim Soroko ◽  
David Kwan
Keyword(s):  
Enzyme Activity ◽  
Enzymatic Synthesis ◽  
Enzyme Assay ◽  
Enzyme Reaction ◽  
Complex Type ◽  
The Core ◽  
Fluorogenic Probe ◽  
Tissue Lysate ◽  
High Throughput Assay ◽  
Core Fucosylation

We report a straight-forward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core-fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of <i>N</i>-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequential coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and very specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

scholarly journals High-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues are unique to porcine islets

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241249
Author(s):  
Yoshihide Nanno ◽  
Asif Shajahan ◽  
Roberto N. Sonon ◽  
Parastoo Azadi ◽  
Bernhard J. Hering ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Neuraminic Acid ◽  
Ionization Mass ◽  
Human Pancreas ◽  
Complex Type ◽  
Antibody Mediated Rejection ◽  
Porcine Islets ◽  
Porcine Islet ◽  
High Mannose ◽  
Core Fucosylation

Objectives Islet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection. Methods N- and O-glycans were isolated from human and adult porcine islets and analyzed using matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS). Results A total of 57 porcine and 34 human N-glycans and 21 porcine and 14 human O-glycans were detected from cultured islets. Twenty-eight of which were detected only from porcine islets, which include novel xenoantigens such as high-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues. Porcine islets have terminal N-glycolylneuraminic acid (NeuGc) residue in bi-antennary N-glycans and sialyl-Tn O-glycans. No galactose-α-1,3-galactose (α-Gal) or Sda epitope were detected on any of the islets. Conclusions These results provide important insights into the potential antigenic differences of N- and O-glycan profiles between human and porcine islets. Glycan differences may identify novel gene targets for genetic engineering to generate superior porcine islet donors.

scholarly journals Enzymatic Synthesis of a Fluorogenic Reporter Substrate and a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation

2019 ◽  
Author(s):  
Maxim Soroko ◽  
David Kwan
Keyword(s):  
Enzyme Activity ◽  
Enzymatic Synthesis ◽  
Enzyme Assay ◽  
Enzyme Reaction ◽  
Complex Type ◽  
The Core ◽  
Fluorogenic Probe ◽  
Tissue Lysate ◽  
High Throughput Assay ◽  
Core Fucosylation

We report a straight-forward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core-fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of <i>N</i>-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequential coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and very specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

Identification of high-mannose and multiantennary complex-type N-linked glycans containing α-galactose epitopes from Nurse shark IgM heavy chain

2009 ◽  
Vol 26 (8) ◽  
pp. 1055-1064 ◽  
Author(s):  
David J. Harvey ◽  
Max Crispin ◽  
Beryl E. Moffatt ◽  
Sylvia L. Smith ◽  
Robert B. Sim ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Complex Type ◽  
Nurse Shark ◽  
High Mannose

scholarly journals Analysis of site and structure specific core fucosylation in liver disease progression using exoglycosidase-assisted data-independent LC-MS/MS

2020 ◽  
Author(s):  
Miloslav Sanda ◽  
Jaeil Ahn ◽  
Petr Kozlik ◽  
Radoslav Goldman
Keyword(s):  
Liver Disease ◽  
Disease Progression ◽  
Serum Proteins ◽  
Receptor Activation ◽  
Biological Macromolecules ◽  
The Core ◽  
Liver Disease Progression ◽  
Living Organisms ◽  
Protein Attachment ◽  
Core Fucosylation

ABSTRACTCarbohydrates form one of the major groups of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response, and receptor activation are regulated by glycosylation. Fucosylation of proteins regulates such processes and is associated with various diseases including autoimmunity and cancer. Mass spectrometry efficiently identifies structures of fucosylated glycans or sites of core fucosylated N-glycopeptides but quantification of the glycopeptides remains less explored. We performed experiments that facilitate quantitative analysis of the core fucosylation of proteins with partial structural resolution of the glycans and we present results of the mass spectrometric SWATH-type DIA analysis of relative abundances of the core fucosylated glycoforms of 45 glycopeptides derived from 18 serum proteins in liver disease of different etiologies. Our results show that a combination of soft fragmentation with exoglycosidases is efficient at the assignment and quantification of the core fucosylated N-glycoforms at specific sites of protein attachment. In addition, our results show that disease-associated changes in core fucosylation are peptide-dependent and further differ by branching of the core fucosylated glycans. Further studies are needed to verify whether tri- and tetra-antennary core fucosylated glycopeptides could be used as markers of liver disease progression.

Response of aggregating chick corneal cells to modifiers of N-linked oligosaccharides, endoglycosidase H and deoxymannojirimycin

1988 ◽  
Vol 89 (3) ◽  
pp. 405-413
Author(s):  
J. Overton
Keyword(s):  
Fine Structure ◽  
Present Report ◽  
Single Cells ◽  
Cellular Responses ◽  
Complex Type ◽  
Normal Complement ◽  
Sds Page ◽  
Moderate Rate ◽  
High Mannose ◽  
Endoglycosidase H

Chick corneal epithelium takes on its mature conformation between 11 and 16 days of incubation. Earlier work has shown that desmosome frequency increases during this period, reaching its highest rate at 15 1/2 days. In the present report aggregation rates of cells from embryos of 11 days and those of 15 1/2 days are compared. Younger cells, which form fewer desmosomes, aggregate at a more moderate rate than older cells. In addition, younger cells bind less concanavalin A (ConA) than older cells. To determine if increase in ConA binding could be related to these cellular responses, aggregating cells were exposed to endoglycosidase H (EndoH) and to deoxymannojirimycin. This treatment should permit comparison of the response of cells that have a normal complement of N-linked oligosaccharides with those that have reduced high-mannose or complex type sugars. The effectiveness of EndoH under the conditions used was confirmed by failure of treated glycoprotein after separation by SDS-PAGE and electroblotting to bind ConA. Aggregation rates of both older and younger cells were unaffected, as measured by disapperance of single cells, though older cells formed somewhat smaller aggregates at the highest dosage used. Desmosome formation was markedly reduced in the presence of the enzyme, even in the absence of other changes in the fine structure. At the highest dose of the enzyme the fine structure of older but not younger cells showed indications of blockage of transport. Deoxymannojirimycin appears to cause a build-up of high-mannose groups, since treated cells showed increased incorporation of [3H]mannose.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Man-Specific, GalNAc/T/Tn-Specific and Neu5Ac-Specific Seaweed Lectins as Glycan Probes for the SARS-CoV-2 (COVID-19) Coronavirus

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 543
Author(s):  
Annick Barre ◽  
Els J.M. Van Damme ◽  
Mathias Simplicien ◽  
Hervé Benoist ◽  
Pierre Rougé
Keyword(s):  
Influenza Virus ◽  
Antiviral Agents ◽  
Host Cells ◽  
Carbohydrate Binding ◽  
Complex Type ◽  
Enveloped Viruses ◽  
High Mannose ◽  
Hiv 1 ◽  
Biomedical Interest

Seaweed lectins, especially high-mannose-specific lectins from red algae, have been identified as potential antiviral agents that are capable of blocking the replication of various enveloped viruses like influenza virus, herpes virus, and HIV-1 in vitro. Their antiviral activity depends on the recognition of glycoprotein receptors on the surface of sensitive host cells—in particular, hemagglutinin for influenza virus or gp120 for HIV-1, which in turn triggers fusion events, allowing the entry of the viral genome into the cells and its subsequent replication. The diversity of glycans present on the S-glycoproteins forming the spikes covering the SARS-CoV-2 envelope, essentially complex type N-glycans and high-mannose type N-glycans, suggests that high-mannose-specific seaweed lectins are particularly well adapted as glycan probes for coronaviruses. This review presents a detailed study of the carbohydrate-binding specificity of high-mannose-specific seaweed lectins, demonstrating their potential to be used as specific glycan probes for coronaviruses, as well as the biomedical interest for both the detection and immobilization of SARS-CoV-2 to avoid shedding of the virus into the environment. The use of these seaweed lectins as replication blockers for SARS-CoV-2 is also discussed.

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