GNPTAB Knockout Cell Line (Hela)

Mutations in underlie mucolipidosis II and mucolipidosis III α/β, which are inherited lysosomal storage disorders caused by a defective UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine phosphotransferase. As a result, newly synthesized acid hydrolases fail to acquire Mannose-6-Phosphate (Man-6-P) sorting signals, or do so to a lesser extent, and exhibit an impaired trafficking to lysosomes. Interestingly, we found that knockout HeLa cells are resistant to several cytotoxic agents: doxorubicin, chloroquine, staurosporine and paclitaxel. While we detected an increased trapping of weak bases in the expanded lysosomal population of these cells, which could reduce the effect of doxorubicin and chloroquine; the decreased cell response to staurosporine and paclitaxel suggested the involvement of alternative resistance mechanisms. Indeed, further investigation revealed that the hyperactivation of the Insulin-like Growth Factor 1 Receptor (IGF1R) pathway is a central player in the apoptosis resistance exhibited by Man-6-P sorting deficient cells.

The Golgi apparatus plays a crucial role in the delivery of lysosomal enzymes. Golgi reassembly stacking proteins, GRASP55 and GRASP65, are vital for maintaining Golgi structure and function. GRASP55 depletion results in the missorting and secretion of the lysosomal enzyme cathepsin D, though the mechanisms remain unclear. In this study, we conducted secretomic analyses of GRASP55 knockout cells and found a significant increase in lysosome-associated proteins in the extracellular medium. Using the lysosomal beta-hexosaminidase subunit alpha (HEXA) as a model, we found that GRASP55 depletion disrupted normal trafficking and processing of HEXA, resulting in increased secretion of the immature (pro-form) HEXA into the extracellular milieu, along with decreased levels of the mature form and enzymatic activity within the cell. GRASP55 depletion significantly reduced the complex formation between HEXA and mannose 6-phosphate (M6P) receptors (MPR), despite no overall change in MPR expression. Finally, we found there was a notable reduction in the expression of GNPTAB, leading to a reduction in M6P modification of HEXA, hindering its efficient targeting to lysosomes. These findings reveal the role of GRASP55 in regulating lysosomal enzyme dynamics, emphasizing its role in the sorting and trafficking of lysosomal proteins.

The Golgi-localized, gamma-ear containing, ADP-ribosylation factor-binding proteins (GGAs 1, 2, and 3) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) at the Golgi and play a role, along with adaptor protein complex 1 (AP-1), in the sorting of newly synthesized lysosomal hydrolases to the endolysosomal system. However, the relative importance of the two types of coat proteins in this process is still unclear. Here, we report that inactivation of all three GGA genes in HeLa cells decreased the sorting efficiency of cathepsin D from 97% to 73% relative to wild-type, with marked redistribution of the cation-independent MPR from peripheral punctae to the trans-Golgi network. In comparison, GNPTAB HeLa cells with complete inactivation of the mannose 6-phosphate pathway sorted only 20% of the cathepsin D. We conclude that the residual sorting of cathepsin D in the GGA triple-knockout cells is mediated by AP-1.