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 allowed major discoveries in the field, however studying lysosomal dysfunction in human patients remains challenging. Here, we report the development of the “tagless LysoIP method” to enable 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 (iPSCs) derived neurons). Isolated lysosomes are intact and suitable for subsequent multimodal omics analyses. To validate our approach, we employed the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells (PBMCs) derived from fresh blood from patients with CLN3 disease, a neurodegenerative LSD. Metabolic profiling of isolated lysosomes showed 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 novel biomarkers and discover human-relevant disease mechanisms.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
One Sentence Summary:
We describe and validate the tagless LysoIP approach to enable the isolation and study of native lysosomes from patient peripheral blood mononuclear cells for analysis of biomarkers and human-relevant disease mechanisms.
We have made minor edits to the text, updated links to https files (Curtain) and otherwise updated the Volcano blots. No major changes have been made that would change the results or interpretation. Figure 1: the drawing shows a-BSA as mockIP. This was changed to 'BSA' Figure 2: gray dots on Volcano blot labeled as 'Background' plot. This was changed to 'other proteins'. Figure 3; A) same for a-BSA. Figure 4. In the legend add a dot after this sentence: (B) The chemical structure of the annotated GPDs in this study SFig2: older version of this figure had been uploaded, this has now been rectified and 'background' changed to other proteins as above in volcano blot SFig3: a-BSA changed to BSA SFig4: a-BSA changed to BSA. C was put in upper case. The legend was corrected to separate B and C. SFig5: Background in E. As before
