Understanding the function and regulation of enzymes within their physiologically relevant milieu requires quality tools that report on their cellular activities. Here we describe a strategy for glycoside hydrolases that overcomes several limitations in the field, enabling quantitative monitoring of their activities within live cells. We detail the design and synthesis of bright and modularly assembled bis-acetal-based (BAB) fluorescence-quenched substrates, illustrating this strategy for sensitive quantitation of disease-relevant human α-galactosidase and α-N-acetylgalactosaminidase activities. We show that these substrates can be used within live patient cells to precisely measure the engagement of target enzymes by inhibitors and the efficiency of pharmacological chaperones, and highlight the importance of quantifying activity within cells using chemical perturbogens of cellular trafficking and lysosomal homeostasis. These BAB substrates should prove widely useful for interrogating the regulation of glycosidases within cells as well as in facilitating the development of therapeutics and diagnostics for this important class of enzymes.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
The data that support the findings of this study are not deposited owing to the large amount of image-based data from 384-well microplates. All data are available from the corresponding author upon reasonable request. Source data are provided with this paper.
Varki, A., Cummings, R. & Esko, J. Essentials of Glycobiology 3rd edn (Cold Spring Harbor Laboratory Press, 2017).
Simon, G. M., Niphakis, M. J. & Cravatt, B. F. Determining target engagement in living systems. Nat. Chem. Biol. 9, 200–205 (2013).
Witte, M. D. et al. Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat. Chem. Biol. 6, 907–913 (2010).
Willems, L. I. et al. Potent and selective activity-based probes for GH27 human retaining α-galactosidases. J. Am. Chem. Soc. 136, 11622–11625 (2014).
Gao, Z., Thompson, A. J., Paulson, J. C. & Withers, S. G. Proximity ligation-based fluorogenic imaging agents for neuraminidases. Angew. Chem. Int. Ed. 57, 13538–13541 (2018).
Garland, M., Yim, J. J. & Bogyo, M. A bright future for precision medicine: advances in fluorescent chemical probe design and their clinical application. Cell Chem. Biol. 23, 122–136 (2016).
Burke, H. M., Gunnlaugsson, T. & Scanlan, E. M. Recent advances in the development of synthetic chemical probes for glycosidase enzymes. Chem. Commun. 51, 10576–10588 (2015).
Komatsu, T. et al. Design and synthesis of an enzyme activity-based labeling molecule with fluorescence spectral change. J. Am. Chem. Soc. 128, 15946–15947 (2006).
Ho, N.-H., Weissleder, R. & Tung, C.-H. A self-immolative reporter for β-galactosidase sensing. ChemBioChem 8, 560–566 (2007).
Hyun, J. Y., Kim, S., Lee, H. S. & Shin, I. A glycoengineered enzyme with multiple mannose-6-phosphates is internalized into diseased cells to restore its activity in lysosomes. Cell Chem. Biol. 25, 1255–1267 (2018).
Kamiya, M. et al. β-galactosidase fluorescence probe with improved cellular accumulation based on a spirocyclized rhodol scaffold. J. Am. Chem. Soc. 133, 12960–12963 (2011).
Lavis, L. D. & Raines, R. T. Bright building blocks for chemical biology. ACS Chem. Biol. 9, 855–866 (2014).
Yadav, A. K. et al. Fluorescence-quenched substrates for live cell imaging of human glucocerebrosidase activity. J. Am. Chem. Soc. 137, 1181–1189 (2015).
Cecioni, S. & Vocadlo, D. J. Carbohydrate bis-acetal-based substrates as tunable fluorescence-quenched probes for monitoring exo-glycosidase activity. J. Am. Chem. Soc. 139, 8392–8395 (2017).
Lombard, V. et al. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014).
Aerts, J. M. et al. Elevated globotriaosylsphingosine is a hallmark of Fabry disease. Proc. Natl Acad. Sci. USA 105, 2812–2817 (2008).
Germain, D. P. Fabry disease. Orphanet J. Rare Dis. 5, 30 (2010).
Guce, A. I., Clark, N. E., Rogich, J. J. & Garman, S. C. The molecular basis of pharmacological chaperoning in human α-galactosidase. Chem. Biol. 18, 1521–1526 (2011).
Clark, N. E. & Garman, S. C. The 1.9-Å structure of human α-N-acetylgalactosaminidase: the molecular basis of Schindler and Kanzaki diseases. J. Mol. Biol. 393, 435–447 (2009).
Asfaw, B. et al. Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases. J. Lipid Res. 43, 1096–1104 (2002).
Clark, N. E. et al. Pharmacological chaperones for human α-N-acetylgalactosaminidase. Proc. Natl Acad. Sci. USA 109, 17400–17405 (2012).
Germain, D. P. et al. Ten-year outcome of enzyme replacement therapy with agalsidase beta in patients with Fabry disease. J. Med. Genet. 52, 353–358 (2015).
Fan, J.-Q., Ishii, S., Asano, N. & Suzuki, Y. Accelerated transport and maturation of lysosomal α-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor. Nat. Med. 5, 112–115 (1999).
Germain, D. P. et al. Treatment of Fabry’s disease with the pharmacologic chaperone migalastat. N. Engl. J. Med. 375, 545–555 (2016).
Hughes, D. A. et al. Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study. J. Med. Genet. 54, 288–296 (2017).
Convertino, M., Das, J. & Dokholyan, N. V. Pharmacological chaperones: design and development of new therapeutic strategies for the treatment of conformational diseases. ACS Chem. Biol. 11, 1471–1489 (2016).
Schiffmann, R., Fuller, M., Clarke, L. A. & Aerts, J. M. F. G. Is it Fabry disease? Genet. Med. 18, 1181–1185 (2016).
Gal, A., Hughes, D. A. & Winchester, B. Toward a consensus in the laboratory diagnostics of Fabry disease—recommendations of a European expert group. J. Inherit. Metab. Dis. 34, 509–514 (2011).
Diyabalanage, H. V. K., Van de Bittner, G. C., Ricq, E. L. & Hooker, J. M. A chemical strategy for the cell-based detection of HDAC activity. ACS Chem. Biol. 9, 1257–1262 (2014).
Bhatia, G. S., Lowe, R. F., Pritchard, R. G. & Stoodley, R. J. Stereoselective epoxidations of vinylogous esters/carbonates directed by the 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl auxiliary: a route to near stereopure tertiary alcohols bearing functional arms. Chem. Commun. 1997, 1981–1982 (1997).
Yuan, J., Lindner, K. & Frauenrath, H. 1-O-vinyl glycosides via Tebbe olefination, their use as chiral auxiliaries and monomers. J. Org. Chem. 71, 5457–5467 (2006).
Corey, E. J. & Suggs, J. W. Method for catalytic dehalogenations via trialkyltin hydrides. J. Org. Chem. 40, 2554–2555 (1975).
Zhang, X., Zheng, N. & Rosania, G. R. Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines. J. Comput. Aided Mol. Des. 22, 629–645 (2008).
Asano, N. et al. In vitro inhibition and intracellular enhancement of lysosomal α-galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives. Eur. J. Biochem. 267, 4179–4186 (2000).
Benjamin, E. R. et al. The pharmacological chaperone 1-deoxygalactonojirimycin increases α-galactosidase A levels in Fabry patient cell lines. J. Inherit. Metab. Dis. 32, 424–440 (2009).
Yam, G. H.-F., Zuber, C. & Roth, J. A synthetic chaperone corrects the trafficking defect and disease phenotype in a protein misfolding disorder. FASEB J. 19, 12–18 (2005).
Yam, G. H.-F. et al. Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants. Am. J. Physiol. Cell Physiol. 290, C1076–C1082 (2006).
Ishii, S. et al. Aggregation of the inactive form of human α-galactosidase in the endoplasmic reticulum. Biochem. Biophys. Res. Commun. 220, 812–815 (1996).
Lukas, J. et al. Functional characterisation of α-galactosidase A mutations as a basis for a new classification system in Fabry disease. PLoS Genet. 9, e1003632 (2013).
Lukas, J. et al. Enzyme enhancers for the treatment of Fabry and Pompe disease. Mol. Ther. 23, 456–464 (2015).
Lippincott-Schwartz, J. et al. Brefeldin A’s effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell 67, 601–616 (1991).
Mollenhauer, H. H., Morré, D. J. & Rowe, L. D. Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity. Biochim. Biophys. Acta 1031, 225–246 (1990).
Yamamoto, A. et al. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Funct. 23, 33–42 (1998).
Shabbeer, J., Yasuda, M., Luca, E. & Desnick, R. J. Fabry disease: 45 novel mutations in the α-galactosidase A gene causing the classical phenotype. Mol. Genet. Metab. 76, 23–30 (2002).
Hamanaka, R. et al. Rescue of mutant α-galactosidase A in the endoplasmic reticulum by 1-deoxygalactonojirimycin leads to trafficking to lysosomes. Biochim. Biophys. Acta 1782, 408–413 (2008).
Muntau, A. C. et al. Innovative strategies to treat protein misfolding in inborn errors of metabolism: pharmacological chaperones and proteostasis regulators. J. Inherit. Metab. Dis. 37, 505–523 (2014).
Kovarik, M. L. & Allbritton, N. L. Measuring enzyme activity in single cells. Trends Biotechnol. 29, 222–230 (2011).
Whitfield, P. D. et al. Monitoring enzyme replacement therapy in Fabry disease—role of urine globotriaosylceramide. J. Inherit. Metab. Dis. 28, 21–33 (2005).
Liu, H.-C. et al. Globotriaosylsphingosine (lyso-Gb3) might not be a reliable marker for monitoring the long-term therapeutic outcomes of enzyme replacement therapy for late-onset Fabry patients with the Chinese hotspot mutation (IVS4+919G>A). Orphanet J. Rare Dis. 9, 111 (2014).
Khan, A. et al. Lentivirus-mediated gene therapy for Fabry disease. Nat. Commun. 12, 1178 (2021).
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (Discovery-RGPIN298406) and the Networks of Centres of Excellence GlycoNet (Team Grant RG-1). D.J.V. acknowledges the Canada Research Chairs (CRC) Program for support as a Tier I CRC in Chemical Biology. R.A.A. thanks the Michael Smith Foundation for Health Research for a Fellowship. S.C. thanks the CIHR for a postdoctoral fellowship, and S.C. is now supported by NSERC (grants nos. RGPIN2019-05451 and DGECR2019-00076) and by the Fonds de Recherche du Québec – Nature et Technologies (2021-NC-281486). P.-A.G. thanks the CIHR, the Michael Smith Foundation for Health Research and the Pacific Parkinson’s Research Institute for postdoctoral fellowships. We thank the Center for High-Throughput Chemical Biology (Simon Fraser University) for access to core facilities.
A patent application (US application number 16/622,870) with inventors including R.A.A., S.C. and D.J.V. has been filed and covers the probes and applications described herein. The remaining authors declare no competing interests.
Peer review information
Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Fluorescence Quenching of the Glyco-BABS probes.
Comparison of the fluorescence of the fluorophores alone versus the same concentration of the Glyco-BABS alone was used to determine the quenching efficiency for each substrate probe. We used the ratio of slopes to calculate quenching efficiencies of: 99.3 % for α-Gal-OH-EDANS, 99.5 % for α-Gal-H-EDANS, 99.7 % for α-GalNAc-H-EDANS, 98.7 % for α-Gal-H-TMR and 98.8 % for α-GalNAc-H-TMR. Data are presented as mean values ± SD with n = 3 technical replicates.
Extended Data Fig. 2 In vitro kinetic analyses for α-Gal-H-TMR and α-GalNAc-H-TMR BABS probes with α-GALA and α-NAGAL.
Top: Titration of α-GALA and α-NAGAL with α-Gal-H-TMR (left, 20 μM) and α-GalNAc-H-TMR (right, 20 μM). Similar to EDANS-based BABS probes, this showed that both α-GALA and α-NAGAL can process α-Gal-H-TMR in vitro and only α-NAGAL can process α-GalNAc-H-TMR. Bottom: Kinetics of Glyco-BABS-TMR with α-GALA (Left) and with α-NAGAL (Right). Not corrected for IFE. Data are presented as mean values + /- SD with n = 3 technical replicates. Second-order rate constants were determined from linear regression through origin.
Extended Data Fig. 3 Representative images for co-localization of Glyco-BABS probes in live SK-N-SH cells.
Live cells (2 independent experiments) were incubated with vehicle (DMSO) or inhibitor (migalastat or DGJNAc) and treated with Hoechst 33342, Lysotracker Green and the α-Gal-H-TMR (top) or α-GalNAc-H-TMR (bottom). Scale bars represent 10 μm.
Extended Data Fig. 4 Dose and Time-dependent optimization of Glyco-BABS probes in live cells.
Live SK-N-SH cells plated in 384-well plates were treated with a range of α-Gal-H-TMR and α-GalNAc-H-TMR concentrations (10, 5, 2.5, 1.25 μM) at various times (t – 4hrs, t – 3hrs, t – 2hrs, t – 1 hr, t – 30 mins) before washing cells and imaging of fluorescence. Data are presented as mean values + /- SD with n = 3 measurements (3 independent wells).
Extended Data Fig. 5 Sequential repetitive imaging reveals excellent signal stability over time after treatment with BABS probes.
After treatments of cells with substrate probes and incubation for 2 hours (37 °C, 5% CO2), cells are washed and imaging media containing α-GALA / α-NAGAL inhibitors was dispensed in all wells according to the live cells methods described above. First images were acquired (t0) and the plate was kept in the high-content imager (environmentally controlled) for 3+ hours. Repeated imaging of the plate at different timepoints was used to monitor signal stability. Data are presented as mean values ± SD with n = 4 measurements (4 independent wells).
Extended Data Fig. 6 α-GALA mutants show perturbed localization and glycosylation patterns.
a, Representative immunocytochemistry fluorescence images (4 independent replicates) of patient fibroblasts after fixation and staining using anti-α-GALA (abcam ab1683341) and anti-GCase (RnD MAB7410) antibodies. Image were acquired on an ImageXpress Micro XLS (40X). Blue channel shows nuclei as stained with Hoechst 33342 (DAPI Channel), green shows α-GALA (FITC channel) and red shows a different lysosomal β-glucosidase GCase (TRITC channel). Scale bars represent 50 μm. b, Western blot analysis of α-GALA levels in response to proteasome inhibitor MG-132 in both WT and R301G fibroblasts. Data are presented as mean values + /- SD with n = 3 biological replicates. Statistical significance was tested using unpaired t-test (one-tailed; in WT P = 0.095, in R301G, P = 0.035). c, Analysis of glycosylation profile of endogenous α-GALA in WT vs. R301G fibroblasts as indicated by treatments of cell lysates with endoglycosidases EndoH or PNGase F (2 biological replicates). Endo H cleaves high mannose and early N-glycans but is inactive on complex glycans. As such Endo H treatment is used as a proxy to reveal glycoproteins at the ER stage of glycosylation processing. PNGase F is able to cleave all N-Glycans and can be used to evaluate the proportion of mature glycoprotein. αGalA from the R301G appears to be more sensitive to EndoH than its counterpart in Wild Type cells, suggesting that the fraction of non-mature non-fully glycosylated protein is more important in the R301G mutant than in the Wild Type.
Extended Data Fig. 7 Measurements of α-GALA activity in R301G fibroblasts and comparison of live cell lysosomal activity with standard lysate measurements.
R301G fibroblasts were treated with vehicle, Brefeldin A (100 nM), Monensin (20 μM) or Bafilomycin (50 nM) for 4 hours before lysate activity analysis. Center bars represent medians and expand to the first and third quartiles; whiskers extend to Min/Max data points. Statistical significance was tested using one-way ANOVA and multiple comparisons with vehicle using Dunnett’s post hoc test (for vehicle vs. monensin lysate assay, P = 0.189; for all other vehicle vs. treatment comparisons, P < 0.0001). n.s. not significant, *P < 0.05, ****P < 0.0001. Data are presented as mean values + /- SD with n = 4 measurements (4 independent wells) for the live cell assay and n = 8 technical replicates for the lysate assay.
Supplementary Figs. 1–13, Supplementary Notes (Chemical methods, Synthesis schemes, Synthesis procedures, Compound characterization).
Supplementary Video 1
Fluorescence video of Gal-BABS treated live fibroblasts.
Source Data Fig. 2
Statistical source data from Fig. 2.
Source Data Fig. 3
Statistical source data from Fig. 3.
Source Data Fig. 4
Statistical source data from Fig. 4.
Source Data Fig. 5
Unprocessed western blot from Fig. 5b.
Source Data Fig. 5
Statistical source data from Fig. 5.
Source Data Fig. 6
Statistical source data from Fig. 6.
Source Data Extended Data Fig. 1
Statistical source data from Extended Data Fig. 1.
Source Data Extended Data Fig. 2
Statistical source data from Extended Data Fig. 2.
Source Data Extended Data Fig. 4
Statistical source data from Extended Data Fig. 4.
Source Data Extended Data Fig. 5
Statistical source data from Extended Data Fig. 5.
Source Data Extended Data Fig. 6
Statistical source data from Extended Data Fig. 6.
Source Data Extended Data Fig. 6
Unprocessed western blots.
Source Data Extended Data Fig. 7
Statistical source data from Extended Data Fig. 7.
Rights and permissions
About this article
Cite this article
Cecioni, S., Ashmus, R.A., Gilormini, PA. et al. Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates. Nat Chem Biol 18, 332–341 (2022). https://doi.org/10.1038/s41589-021-00960-x