In vivo and in vitro analyses of LYSET functions for skeletal and cellular homeostasis

• Applicant: Professor Dr. Thomas Braulke
• Subject Area: Orthopaedics, Traumatology, Reconstructive Surgery
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564153147

Lysosomes play a major role in the degradation and recycling of different classes of intra- and extracellular macromolecules by about 70 soluble enzymes that are transported from the Golgi apparatus to lysosomes in a mannose 6-phosphate (M6P)-dependent and receptor-mediated manner. The formation M6P tags on oligosaccharides of the newly synthesized lysosomal enzymes is catalyzed by a hexameric N-acetylglucosaminyl-1-phosphotransferase (GNPT) complex in the cis-Golgi. Using genetic screens we have recently discovered a novel membrane protein, lysosomal enzyme trafficking factor (LYSET), that retains GNPT in the Golgi. The deficiency of LYSET leads to the lysosomal degradation of GNPT and subsequently to the inability to modify lysosomal enzymes with M6P tags resulting in their hypersecretion and associated lysosomal dysfunctions. Patients of two unrelated families have been reported with mutations in the LYSET gene who had progressive skeletal alterations but differ in their clinical features and severity of the disease. We have generated a new Lyset knock-in (ki) mouse (c.215dupA; p.Y72Ter) corresponding to a homozygous mutation detected in one of the described patients and want to investigate the physiological role of Lyset in bone development and remodeling in vivo and the pathogenesis of skeletal abnormalities. In addition to deep skeletal phenotyping, morphological and functional studies of cultured osteoblasts and osteoblasts of control and Lyset ki mice will be performed in combination with lysosomal proteomes and secretomes. Another objective of this project focuses on the role of M6P-independent receptors in LYSET-deficient cells to allow the targeting of select lysosomal enzymes lacking M6P tags to lysosomes in order to maintain residual degradative functions. Preliminary proteomic data suggest that sortilin appears to be a promising candidate of an M6P-independent cargo receptor. We plan to increase the endogenous expression level of sortilin by CRISPR-activation or stable, inducible overexpression in LYSET-deficient cells followed by proteomic analysis of isolated lysosomes and secretomes to determine the total number, extent and substrate specificity of targeted lysosomal enzymes, as well as their impact on the accumulation of non-degraded material. This project will yield insights into i) the cell type-specific function of LYSET for bone development and pathomechanisms of skeletal alterations in vivo and ii) the impact of alternate M6P-independent receptors, exemplified by sortilin, as a compensatory transport protein that might be translationally applicable to other M6P pathway-related diseases.

• DFG Programme: Research Grants
• Co-Investigators: Professorin Dr. Sabrina Jabs; Professor Thorsten Schinke, Ph.D.