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Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates

Nature Chemical Biology volume 18, pages 332–341 (2022)Cite this article

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Abstract

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.

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Fig. 1: The BABS concept and lysosomal α-galactosidases.
Fig. 2: Decongested α-glyco-BABS probes show improved turnover.
Fig. 3: Glyco-BABS probes enable live-cell imaging of α-GALA and α-NAGAL activity.
Fig. 4: IC50 measurements in live cells.
Fig. 5: Quantification of activity and levels of α-GALA in patient fibroblasts.
Fig. 6: Quantitation of enzymatic activity in patient fibroblasts with measurement of IC50 and the chaperoning effect.

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Data availability

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.

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Acknowledgements

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.

Author information

Author notes

  1. Samy Cecioni

    Present address: Department of Chemistry, Université de Montréal, Québec, Canada

  2. Yang Wang

    Present address: Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China

  3. Yang Wang

    Present address: Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology (QNLM), Qingdao, China

  4. These authors contributed equally: Samy Cecioni, Roger A. Ashmus.

Authors and Affiliations

  1. Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada

    Samy Cecioni, Roger A. Ashmus, Pierre-André Gilormini, Sha Zhu, Xi Chen, Xiaoyang Shan, Christina Gros, Matthew C. Deen, Yang Wang, Robert Britton & David J. Vocadlo

  2. Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada

    Xiaoyang Shan & David J. Vocadlo

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Contributions

S.C., R.A.A., P.-A.G. and D.J.V. designed the research. R.A.A., S.C., S.Z. and M.C.D. optimized and performed chemical syntheses of glyco-BABS probes. S.C. performed in vitro characterization of BABS substrates. S.C., R.A.A., X.C. and P.-A.G. performed cell culture. S.C. and C.G. optimized and performed cell-based assays and fluorescence microscopy. X.S., P.-A.G. and R.A.A. performed co-localization experiments. P.-A.G. performed treatments and analysis of perturbogen experiments in live cells and in lysates. Y.W. and R.B. synthesized DGJNAc. S.C., R.A.A., P.-A.G. and D.J.V. analyzed data and wrote the manuscript.

Corresponding author

Correspondence to David J. Vocadlo.

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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.

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Extended data

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.

Source data

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.

Source data

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).

Source data

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).

Source data

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.

Source data

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.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–13, Supplementary Notes (Chemical methods, Synthesis schemes, Synthesis procedures, Compound characterization).

Reporting Summary

Supplementary Video 1

Fluorescence video of Gal-BABS treated live fibroblasts.

Source data

Source Data Fig. 2

Statistical source data from Fig. 2.

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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.

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Statistical source data from Fig. 5.

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Statistical source data from Fig. 6.

Source Data Extended Data Fig. 1

Statistical source data from Extended Data Fig. 1.

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Statistical source data from Extended Data Fig. 2.

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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.

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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

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