A Rapid Protocol for Preparing 8-Aminopyrene-1,3,6-Trisulfonate-Labeled Glycans for Capillary Electrophoresis-Based Enzyme Assays.

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene-1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, a technique amenable to equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel by simple diffusion and subsequent solid-phase extraction (SPE)-based desalting, affords a single glycan species free of excess labeling reagents and buffer components. The described protocol also offers a simple, rapid method for the simultaneous removal of excess APTS and unlabeled glycan material from reaction mixtures. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.

The primary familial brain calcification-associated protein MYORG is an α-galactosidase with restricted substrate specificity.

Primary familial brain calcification (PFBC) is characterised by abnormal deposits of calcium phosphate within various regions of the brain that are associated with severe cognitive impairments, psychiatric conditions, and movement disorders. Recent studies in diverse populations have shown a link between mutations in myogenesis-regulating glycosidase (MYORG) and the development of this disease. MYORG is a member of glycoside hydrolase (GH) family 31 (GH31) and, like the other mammalian GH31 enzyme α-glucosidase II, this enzyme is found in the lumen of the endoplasmic reticulum (ER). Though presumed to act as an α-glucosidase due to its localization and sequence relatedness to α-glucosidase II, MYORG has never been shown to exhibit catalytic activity. Here, we show that MYORG is an α-galactosidase and present the high-resolution crystal structure of MYORG in complex with substrate and inhibitor. Using these structures, we map detrimental mutations that are associated with MYORG-associated brain calcification and define how these mutations may drive disease progression through loss of enzymatic activity. Finally, we also detail the thermal stabilisation of MYORG afforded by a clinically approved small molecule ligand, opening the possibility of using pharmacological chaperones to enhance the activity of mutant forms of MYORG.

Large-Scale Reassessment of In-Vineyard Smoke-Taint Grapevine Protection Strategies and the Development of Predictive Off-Vine Models.

Smoke taint in wine is thought to be caused by smoke-derived volatile phenols (VPs) that are absorbed into grape tissues, trapped as conjugates that are imperceptible by smell, and subsequently released into wines as their free odor-active forms via metabolism by yeasts during fermentation. Blocking VP uptake into grapes would, therefore, be an effective way for vineyards to protect ripening grape crops exposed to smoke. Here, we re-evaluated a biofilm that had previously shown promise in pilot studies in reducing levels of smoke-derived VPs. A suite of nine free and acid-labile VPs were quantitated in Pinot Noir grapes that had been exposed to smoke after being coated with the biofilm one, seven or 14 days earlier. In contrast with earlier studies, our results demonstrated that in all cases, the biofilm treatments led to increased concentrations of both free and total VPs in smoke-exposed grapes, with earlier applications elevating concentrations of some VPs more than the later time points. Tracking VP concentrations through the grape ripening process demonstrated that some (phenol, p/m-cresol, and guaiacol) were not entirely sequestered in grapes as acid-labile conjugates, suggesting the presence of VP storage forms beyond simple glycosides. Free VPs in grapes, though a minor portion of the total, most clearly correlated with concentrations present in the resulting wines. Finally, red table grapes, available year round, were observed to replicate the effects of the biofilm treatments and were capable of transforming most VPs into acid-labile conjugates in under 24 h, indicating that they might be an effective model for rapidly assessing smoke-taint prophylactic products in the laboratory.