Tagless LysoIP for immunoaffinity enrichment of native lysosomes from clinical samples
Daniel Saarela, Paweł Lis, Sara Gomes, Raja Sekhar Nirujogi, Wentao Dong, Eshaan S. Rawat, S. G. Glendinning, Karolina Zeneviciute, Enrico Bagnoli, Rotimi Fasimoye, Cindy Lin, Kwamina Nyame, Fanni Annamária Boros, Friederike Zunke, Frédéric Lamoliatte, Sadik Elshani, Matthew Jaconelli, Judith Jans, Margriet Huisman, Christian Posern, Lena Marie Westermann, Angela Schulz, Peter M. van Hasselt, Dario R. Alessi, Monther Abu-Remaileh, Esther Sammler
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.