CLN3 is required for the clearance of glycerophosphodiesters from lysosomes

Laqtom, Nouf N.; Dong, Wentao; Medoh, Uche N.; Cangelosi, Andrew L.; Dharamdasani, Vimisha; Chan, Sze Ham; Kunchok, Tenzin; Lewis, Caroline A.; Heinze, Ivonne; Tang, Rachel; Grimm, Christian; Dang, An; Porter, Forbes D.; Ori, Alessandro; Sabatini, David M.; Abu-Remaileh, Monther

doi:10.1038/s41586-022-05221-y

Cited by 73 publications

(102 citation statements)

References 53 publications

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“…( E ) Schematic of mucin proteolysis assay with purified mouse liver lysosomes. ( F ) Immunoblots of organelle markers comparing whole tissue lysate and Lyso-Tag IPs of livers from mice with and without the Lyso-Tag ( 33 ). ( G ) Lysosomes were purified from C57BL/6 mouse liver as described in Materials and Methods .…”

Section: Resultsmentioning

confidence: 99%

“…Given the observed acidic pH optimum for MUC16 degradation, we hypothesized that lysosomal cathepsin D was chiefly responsible for the observed activity in cell lysate. Using the Lyso-Tag method ( 33 , 34 ), we purified lysosomes from C57BL/6 mouse liver and lysed them in pure water for activity assays or 1% Triton for immunoblotting ( Materials and Methods and Fig. 2 E ).…”

Section: Resultsmentioning

confidence: 99%

“…Lyso-Tag–expressing mice were generated via cross of Rosa26; lox-stop-lox-Tmem192-3xHA with CMV-Cre transgenic mice of the C57BL/6J background (The Jackson Laboratory, strain no. 035401) ( 33 , 34 ). Tissues were collected from Lyso-Tag mice under Stanford APLAC protocol No.…”

Section: Methodsmentioning

confidence: 99%

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Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals

Pedram

Laqtom

Shon

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N -glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O -glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O -glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals

Pedram

Laqtom

Shon

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…Structurally, CLN3 encodes a transmembrane lysosome protein whose C- and N-ends localise to the cytoplasm 106 . While CLN3 function remains not fully elucidated, recent studies have demonstrated that it is required for glycerophospholipid catabolism 107,108 . Glycerophospholipids are key structural components of cell membranes, but also play a variety of regulatory roles, including in innate immunity 109 .…”

Section: Discussionmentioning

confidence: 99%

High-resolution promoter interaction analysis in Type 3 Innate Lymphoid Cells implicates Batten Disease geneCLN3in Crohn’s Disease aetiology

Malysheva

Ray-Jones

Cazares

et al. 2022

Preprint

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Innate lymphoid cells (ILCs) are rare tissue-resident “helper” lymphocytes that do not express diversified antigen receptors. Type 3 ILCs (ILC3s) are an important class of these cells enriched in the respiratory and intestinal mucosa, where they regulate inflammation and mucosal homeostasis. To gain insight into the cis-regulatory circuitries underlying ILC3 function, we used high-resolution Capture Hi-C to profile promoter-anchored chromosomal contacts in human primary ILC3s. Combining significant interaction detection with the Activity-By-Contact approach adapted to Capture Hi-C, we reveal a multitude of contacts between promoters and distal regulatory elements and obtain evidence for distinct regulatory wiring of alternative promoters. We find that promoter-interacting regions in ILC3s are enriched for genetic variants associated with multiple immune diseases. Focusing on Crohn’s disease (CD), in which ILC3s are established mediators, we used a Bayesian approach that incorporates multivariate fine-mapping to link CD-associated genetic variants with putative target genes. We identify known and previously unimplicated genes in conferring genetic risk of CD through activity in ILC3s. This includes the CLN3gene that is mutated in the neurodegenerative disorder Batten disease. UsingCln3mutant mice, we show thatCLN3is a putative negative regulator of IL-17 production in an inflammatory subset of ILC3s. This finding suggests a functional role forCLN3in ILC3 biology, with mechanistic implications for both Crohn’s and Batten diseases.

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“…While traditional techniques for purifying the Golgi from mammalian cells, such as densitybased centrifugation, helped shape our current understanding of its cellular roles, such methods are too slow to preserve what is likely a labile Golgi metabolome and transient protein interactions that regulate its functions. To overcome this, we used insights from our work and other's to purify cellular organelles (10)(11)(12)(13)(14)(15)(16)(17)(18) to develop a novel method for the rapid capture of intact Golgi from human cells using organelle-specific immunoprecipitation (Golgi-IP).…”

Section: Introductionmentioning

confidence: 99%

Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content

Fasimoye

Dong

Nirujogi

et al. 2022

Preprint

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The Golgi is a membrane-bound organelle that is essential for protein and lipid biosynthesis. It represents a central trafficking hub that sorts proteins and lipids to various destinations or for secretion from the cell. The Golgi has emerged as a docking platform for cellular signalling pathways including LRRK2 kinase whose deregulation leads to Parkinson disease. Golgi dysfunction is associated with a broad spectrum of diseases including cancer, neurodegeneration, and cardiovascular diseases. To allow the study of the Golgi at high resolution, we report a rapid immunoprecipitation technique (Golgi-IP) to isolate intact Golgi mini-stacks for subsequent analysis of their content. By fusing the Golgi resident protein TMEM115 to three tandem HA epitopes (GolgiTAG), we purified the Golgi using Golgi-IP with minimal contamination from other compartments. We then established an analysis pipeline using liquid chromatography coupled with mass spectrometry to characterize the human Golgi proteome, metabolome and lipidome. Subcellular proteomics confirmed known Golgi proteins and identified novel ones. Metabolite profiling established the first known human Golgi metabolome and revealed the selective enrichment of uridine-diphosphate (UDP) sugars and their derivatives, which is consistent with their roles in protein and lipid glycosylation. Furthermore, targeted metabolomics validated SLC35A2 as the subcellular transporter for UDP-hexose. Finally, lipidomics analysis showed that phospholipids including phosphatidylcholine, phosphatidylinositol and phosphatidylserine are the most abundant Golgi lipids and that glycosphingolipids are enriched in this compartment. Altogether, our work establishes a comprehensive molecular map of the human Golgi and provides a powerful method to study the Golgi with high precision in health and disease states.

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CLN3 is required for the clearance of glycerophosphodiesters from lysosomes

Cited by 73 publications

References 53 publications

Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals

Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals

High-resolution promoter interaction analysis in Type 3 Innate Lymphoid Cells implicates Batten Disease geneCLN3in Crohn’s Disease aetiology

Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content

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