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1. The unusual cell wall of the Lyme disease spirochaete Borrelia burgdorferi is shaped by a tick sugar

   https://doi.org/10.1038/s41564-021-01003-w  

   DeHart, Tanner G.; Kushelman, Mara R.; Hildreth, Sherry B.; Helm, Richard F.; Jutras, Brandon L. (December 2021, Nature Microbiology)

   Abstract Peptidoglycan—a mesh sac of glycans that are linked by peptides—is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N- acetylglucosamine (Glc N Ac) β-(1–4)-linked to N -acetylmuramic acid (Mur N Ac). Borrelia burgdorferi , the tick-borne Lyme disease pathogen, produces glycan chains in which Mur N Ac is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography–mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain Glc N Ac–Glc N Ac. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility. 

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2. Kifunensine-sensitive ADP-ribosylation factor A1E G69R mutant reveals coordination of protein glycosylation and vesicle transport pathways

   https://doi.org/10.1093/jxb/eraf017  

   Nagashima, Yukihiro; Sharma, Vinita; Reekers, Lea-Franziska; von_Schaewen, Antje; Koiwa, Hisashi; Napier, ed., Richard (January 2025, Journal of Experimental Botany)

   Abstract Complex N-glycans are asparagine (N)-linked branched sugar chains attached to secretory proteins in eukaryotes. They are produced by modification of N-linked oligosaccharide structures in the endoplasmic reticulum and Golgi apparatus. Complex N-glycans formed in the Golgi apparatus are often assigned specific roles unique to the host organism, with their roles in plants remaining largely unknown. Using inhibitor (kifunensine, KIF) hypersensitivity as read out, we identified Arabidopsis mutants that require complex N-glycan modification. Among >100 KIF-sensitive mutants, one showing abnormal secretory organelles and a salt-sensitive phenotype contained a point mutation leading to amino acid replacement (G69R) in ARFA1E, a small Arf1-GTPase family protein presumably involved in vesicular transport. In vitro assays showed that the G69R exchange interferes with protein activation. In vivo, ARFA1EG69R caused dominant-negative effects, altering the morphology of the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). Post-Golgi transport (endocytosis/endocytic recycling) of the essential glycoprotein KORRIGAN1, one of the KIF sensitivity targets, is slowed down constitutively as well as under salt stress in the ARFA1EG69R mutant. Because regulated cycling of plasma membrane proteins is required for stress tolerance of the host plants, the ARFA1EG69R mutant established a link between KIF-targeted luminal glycoprotein functions/dynamics and cytosolic regulators of vesicle transport in endosome-/cell wall-associated tolerance mechanisms. 

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3. Synthetic glycosidases for the precise hydrolysis of oligosaccharides and polysaccharides

   https://doi.org/10.1039/D0SC05338D  

   Li, Xiaowei; Zhao, Yan (January 2021, Chemical Science)

   null (Ed.)

   Glycosidases are an important class of enzymes for performing the selective hydrolysis of glycans. Although glycans can be hydrolyzed in principle by acidic water, hydrolysis with high selectivity using nonenzymatic catalysts is an unachieved goal. Molecular imprinting in cross-linked micelles afforded water-soluble polymeric nanoparticles with a sugar-binding boroxole in the imprinted site. Post-modification installed an acidic group near the oxygen of the targeted glycosidic bond, with the acidity and distance of the acid varied systematically. The resulting synthetic glycosidase hydrolyzed oligosaccharides and polysaccharides in a highly controlled fashion simply in hot water. These catalysts not only broke down amylose with similar selectivities to those of natural enzymes, but they also could be designed to possess selectivity not available with biocatalysts. Substrate selectivity was mainly determined by the sugar residues bound within the active site, including their spatial orientations. Separation of the product was accomplished through in situ dialysis, and the catalysts left behind could be used multiple times with no signs of degradation. This work illustrates a general method to construct synthetic glycosidases from readily available building blocks via self-assembly, covalent capture, and post-modification. In addition, controlled, precise, one-step hydrolysis is an attractive way to prepare complex glycans from naturally available carbohydrate sources. 

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4. Chemical inhibitors of hexokinase‐2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures

   https://doi.org/10.1002/bit.28417  

   Naik, Harnish_Mukesh; Kumar, Swetha; Reddy, Jayanth_Venkatarama; Gonzalez, Jacqueline_E; McConnell, Brian_O; Dhara, Venkata_Gayatri; Wang, Tiexin; Yu, Marcella; Antoniewicz, Maciek_R; Betenbaugh, Michael_J (May 2023, Biotechnology and Bioengineering)

   Abstract Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by‐product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase‐2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6‐phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, andN‐glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2‐deoxy‐d‐glucose (2DG) and 5‐thio‐d‐glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%–45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO‐Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO‐Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes.N‐glycan analysis of EPO‐Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG‐supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi‐, tri‐, and tetra‐antennary structures and up to 50% lower EPO‐Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2‐deoxy‐hexose (2DH) on EPO‐FcN‐glycans and addition of 5TG resulted in the first‐ever observedN‐glycan incorporation of 5‐thio‐hexose (5TH). Six percent to 23% ofN‐glycans included 5TH moieties, most likely 5‐thio‐mannose and/or 5‐thio‐galactose and/or possibly 5‐thio‐N‐acetylglucosamine, and 14%–33% ofN‐glycans included 2DH moieties, most likely 2‐deoxy‐mannose and/or 2‐deoxy‐galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism,N‐glycosylation processing, and formation of alternative glycoforms. 

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5. Phase behavior of lyotropic alkyl thioglycolipid surfactants: Effects of sugar headgroup and alkyl tail length

   https://doi.org/10.1002/jsde.12746  

   Kegel, Laurel_L; Wang, Yu‐Cheng; Szabó, Lajos_Z; Polt, Robin; Pemberton, Jeanne_E (March 2024, Journal of Surfactants and Detergents)

   Abstract The lyotropic properties of alkyl thioglycosides with varying sugar headgroup (lactose, cellobiose, maltose, galactose, or glucose) and alkyl chain length (octyl, decyl, or dodecyl chains) are investigated by surface tensiometry, visual observation, and fluorescence spectroscopy. The results substantiate that the glycosidic S‐linkage confers considerably different solution aggregation behavior on these surfactants relative to their O‐linked counterparts, where the properties of the latter are known. The materials properties of the aggregated structures from the alkyl thioglycosides vary considerably. Micelles are formed by octyl thiocellobioside and all alkyl thiomaltosides. Turbid aggregate solutions are formed by the alkyl thioglucosides and octyl thiogalactoside, whereas the longer chain alkyl thiogalactosides are minimally soluble. Fluorescence spectroscopy of these systems confirms their aggregation in lamellar‐like structures. The alkyl thiocellobiosides and alkyl thiolactosides form hydrogels from these low‐molecular weight materials at concentrations almost an order of magnitude lower than gels using other low‐molecular weight materials. Here, hydrogels form at concentrations <0.3 wt% with some forming hydrogels at concentrations as low as 0.03 wt% from alkyl thiocellobiosides and thiolactosides, with hydrogel properties differing significantly with this slight change in the sugar headgroup. Alkyl thiocellobiosides form a nanofiber network and alkyl thiolactosides form globular hydrogels. Overall, these results clearly document materials properties that can readily be controlled and designed depending on molecular structure. 

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