Abstract
Acid hydrolases utilize a carbohydrate-dependent mechanism for lysosomal targeting. These hydrolases acquire a mannose 6-phosphate tag by the action of the GlcNAc-1-phosphotransferase enzyme, allowing them to bind receptors and traffic to endosomes. Loss of GlcNAc-1-phosphotransferase results in hydrolase hypersecretion and profound lysosomal storage. Little, however, is known about how these cellular phenotypes affect the trafficking, activity, and localization of surface glycoproteins. To address this question, we profiled the abundance of surface glycoproteins in WT and CRISPR-mediated GNPTAB-/- HeLa cells and identified changes in numerous glycoproteins, including the uptake receptor LRP1 and multiple receptor tyrosine kinases. Decreased cell surface LRP1 in GNPTAB-/- cells corresponded with a reduction in its steady-state level and less amyloid-β-40 (Aβ40) peptide uptake. GNPTAB-/- cells displayed elevated activation of several kinases including Met receptor. We found increased Met phosphorylation within both the kinase and the docking domains and observed that lower concentrations of pervanadate were needed to cause an increase in phospho-Met in GNPTAB-/- cells. Together, these data suggested a decrease in the activity of the receptor and non-receptor protein-tyrosine phosphatases that down-regulate Met phosphorylation. GNPTAB-/- cells exhibited elevated levels of reactive oxygen species, known to inactivate cell surface and cytosolic phosphatases by oxidation of active site cysteine residues. Consistent with this mode of action, peroxide treatment of parental HeLa cells elevated phospho-Met levels whereas antioxidant treatment of GNPTAB-/- cells reduced phospho-Met levels. Collectively, these findings identify new mechanisms whereby impaired lysosomal targeting can impact the activity and recycling of receptors.
Highlights
Acid hydrolases utilize a carbohydrate-dependent mechanism for lysosomal targeting
Differences were noted in the profile of the major labeled glycoproteins, with the most striking changes noted between parental HeLa and GNPTABϪ/Ϫ cells (Fig. 1A)
In light of the differences noted in the profile of labeled glycoproteins, we focused the proteomic analysis on the parental and GNPTABϪ/Ϫ HeLa cells
Summary
Acid hydrolases utilize a carbohydrate-dependent mechanism for lysosomal targeting. These hydrolases acquire a mannose 6-phosphate tag by the action of the GlcNAc-1-phosphotransferase enzyme, allowing them to bind receptors and traffic to endosomes. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Higher levels of secreted glycosidases in GNPTABϪ/Ϫ cells because of loss of M6P targeting may result in increased processing and altered cell surface residence of glycoproteins by affecting their interaction with galectins or other factors [21, 22] Defining these mechanisms is likely to provide important new clues to the molecular pathogenesis of lysosomal diseases. Our findings reveal changes in the abundance of multiple cell surface glycoproteins, including reduced levels of the uptake receptor LRP1 and receptor tyrosine kinase Met. Characterization of the functional consequences and molecular basis for these changes revealed new mechanisms of action whereby loss of lysosomal targeting and lysosomal storage alter receptor activity and receptor-mediated uptake
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