Tagless LysoIP for immunoaffinity enrichment of native lysosomes from clinical samples
Daniel Saarela, … , Monther Abu-Remaileh, Esther M. Sammler
Daniel Saarela, … , Monther Abu-Remaileh, Esther M. Sammler
Published December 26, 2024
Citation Information: J Clin Invest. 2025;135(4):e183592. https://doi.org/10.1172/JCI183592.
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Research Article Cell biology Neuroscience

Tagless LysoIP for immunoaffinity enrichment of native lysosomes from clinical samples

Abstract

Lysosomes are implicated in a wide spectrum of human diseases, including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration, and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in patients remains challenging. Here, we report the development of the ‘tagless LysoIP’ method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell–derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients’ lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms.

Authors

Daniel Saarela, Pawel Lis, Sara Gomes, Raja S. Nirujogi, Wentao Dong, Eshaan Rawat, Sophie Glendinning, Karolina Zeneviciute, Enrico Bagnoli, Rotimi Fasimoye, Cindy Lin, Kwamina Nyame, Fanni A. Boros, Friederike Zunke, Frederic Lamoliatte, Sadik Elshani, Matthew Jaconelli, Judith J.M. Jans, Margriet A. Huisman, Christian Posern, Lena M. Westermann, Angela Schulz, Peter M. van Hasselt, Dario R. Alessi, Monther Abu-Remaileh, Esther M. Sammler

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Figure 5

Tagless LysoIP enriches intact and functional lysosomes from healthy donor PBMCs.

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Tagless LysoIP enriches intact and functional lysosomes from healthy don...
(A) Workflow of flow cytometry analysis of magnetic beads bound to lysosomes enriched via tagless LysoIPs or MockIPs from PBMC homogenates pretreated with and without the V-ATPase inhibitor bafilomycin A1 (200 nM), prior to staining with Red Lysotracker (50 nM). (B) Representative scatter plot from 1 of the 3 donors, (Y-axis) and bead size (Forward scatter, FSC, X-axis), (C) Quantification of the percentage of beads positive for Lysotracker fluorescence. Data presented as mean ± SD (n = 3 donors) and analysed by ordinary 2-way ANOVA with Dunnet’s multiple comparison test. (D) GCase (glucocerebrosidase, GBA1) protein enrichment quantified by DIA mass-spectrometry in LysoIPs from 6 donors. Data presented as mean ± SD (n = 6). 2-tailed unpaired t test. (E) GCase activity measured by 4-methylumbelliferone (4-MU) assay in lysosomes enriched from PBMCs from 6 donors. Data presented as mean ± SD and 2-way ANOVA with Dunnet’s test for multiple comparison.