Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the a1,6-linked mannosyl residue (M7A, M6 and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6 and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major a1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other a1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.

Repurposing of Miglustat to inhibit the coronavirus Severe Acquired Respiratory Syndrome SARS-CoV-2

Repurposing clinically available drugs to treat the new coronavirus disease COVID-19 is an urgent need in these early stages of the SARS-CoV-2 pandemic, when very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated for SARS-CoV-2 at concentrations achievable in the plasma by current clinical regimens without cytotoxicity. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious virus. The mechanism resides in the inhibitory activity towards α-glucosidases that are involved in early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. The wealth of available data on the clinical use of Miglustat for the treatment of lysosomal storage disorders and the antiviral properties against SARS-CoV-2 make it an ideal candidate for drug repurposing.

Anti-melanogenic activity of salacinol by inhibition of tyrosinase oligosaccharide processing

Hyperpigmentation that manifests through melasma and solar lentigo (age spots), although mostly harmless for health, bothers many people. Controlling the rate-limiting activity of tyrosinase is most effective for suppressing excessive melanin formation and accordingly recent research has focused on the maturation of tyrosinase. Salacia, a medicinal plant, has been used to treat diabetes in India and Sri Lanka. Salacia extract reportedly contains components that inhibit the activity of α-glucosidase. Salacinol, the active ingredient in Salacia extract, has unique thiosugar sulphonium sulphate inner salt structure. Here, we observed that the salacinol component of Salacia extract possesses anti-melanogenic activity in comparison to various existing whitening agents. Although the anti-melanogenic mechanism of salacinol is presumably medicated by inhibition of tyrosinase activity, which is often found in existing whitening agents, salacinol did not inhibit tyrosinase activity in vitro. Analysis of the intracellular state of tyrosinase showed a decrease in the mature tyrosinase form due to inhibition of N-linked oligosaccharide processing. Salacinol inhibited the processing glucosidase I/II, which are involved in the initial stage of N-linked glycosylation. Owing to high activity, low cytotoxicity and high hydrophilicity, salacinol is a promising candidate compound in whitening agents aimed for external application on skin.

Genomic Potential for Polysaccharide Deconstruction in Bacteria

ABSTRACTGlycoside hydrolases are important enzymes that support bacterial growth by enabling the degradation of polysaccharides (e.g., starch, cellulose, xylan, and chitin) in the environment. Presently, little is known about the overall phylogenetic distribution of the genomic potential to degrade these polysaccharides in bacteria. However, knowing the phylogenetic breadth of these traits may help us predict the overall polysaccharide processing in environmental microbial communities. In order to address this, we identified and analyzed the distribution of 392,166 enzyme genes derived from 53 glycoside hydrolase families in 8,133 sequenced bacterial genomes. Enzymes for oligosaccharides and starch/glycogen were observed in most taxonomic groups, whereas glycoside hydrolases for structural polymers (i.e., cellulose, xylan, and chitin) were observed in clusters of relatives at taxonomic levels ranging from species to genus as determined by consenTRAIT. The potential for starch and glycogen processing, as well as oligosaccharide processing, was observed in 85% of the strains, whereas 65% possessed enzymes to degrade some structural polysaccharides (i.e., cellulose, xylan, or chitin). Potential degraders targeting one, two, and three structural polysaccharides accounted for 22.6, 32.9, and 9.3% of genomes analyzed, respectively. Finally, potential degraders targeting multiple structural polysaccharides displayed increased potential for oligosaccharide deconstruction. This study provides a framework for linking the potential for polymer deconstruction with phylogeny in complex microbial assemblages.

Endoplasmic Reticulum α-Glycosidases of Candida albicans Are Required for N Glycosylation, Cell Wall Integrity, and Normal Host-Fungus Interaction

ABSTRACT The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc3Man9GlcNAc2 N-glycan is processed by α-glucosidases I and II and α1,2-mannosidase to generate Man8GlcNAc2. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1 encode for α-glucosidase I, α-glucosidase II catalytic subunit, and α1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated β-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.

Man2C1, an α-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin–proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic α-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an α-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic α-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-β-N-acetylglucosaminidase and α-mannosidase may represent the common ‘non-lysosomal’ catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.