GRASP55 regulates sorting and maturation of the lysosomal enzyme β-hexosaminidase A

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.