New insights into the lysosomal glycoproteome

Lysosomes, essential catabolic cellular organelles packed with diverse lysosomal hydrolases, play critical roles in biomolecular degradation, autophagic processes and extracellular cell-cell communication. While the protein complement of the lysosome is well-studied, the lysosomal glycoproteome remain poorly understood despite the recognised importance of protein glycosylation including mannose-6-phosphorylation (M6P) in lysosome biology. To this end, we performed an in-depth glycoprofiling of both soluble and membrane protein fractions from isolated liver lysosomes (tritosomes) from wild-type (WT, n = 3-5) and acid-phosphatase 2/5 double knockout (ACP2/5-dKO, n = 3) mice using quantitative glycomics and glycoproteomics. ACP2/5-dKO mice were included in the study to also map the unprocessed lysosomal glycoproteome ahead of the final M6P removal from trafficking lysosomal glycoproteins. From WT lysosomes, we identified 1000+ unique N-glycopeptides covering 200+ N-glycosylation sites from 120 source glycoproteins comprising the expected lysosomal hydrolases. Glycomics identified 60 different N-glycan isomers spanning a surprisingly high abundance of paucimannose (Man1-3GlcNAc2Fuc0-1, >50% of the N-glycome) in addition to oligomannosidic and complex-type N-glycans. As expected, no M6P-tagged glycoproteins were found in the WT lysosomes. The WT lysosome displayed different N-glycome profiles across the soluble and membrane protein fractions indicating differential N-glycan processing of those protein subsets. Surprisingly, the glyco(proteo)mics data of ACP2/5-dKO lysosomes revealed very low levels of M6P (<10% of the N-glycoproteome) on only a few soluble lysosomal proteins (prosaposin, cathepsin D/L, dipeptidyl-peptidase-1). Similar to the WT lysosomes, the ACP2/5-dKO lysosomes instead displayed a paucimannose-rich glycophenotype (~50% of the N-glycome) and only relatively little oligomannosylation and complex-type N-glycosylation indicating that only a small proportion of the lysosomal hydrolases traffic to mouse liver lysosomes via the well-established M6P-tagging pathway. This study generates, for the first time, a comprehensive and quantitative N-glycoproteome map of the mouse liver lysosome representing an important resource to further unravel the glycobiology of lysosomes and their trafficking pathways.