Endolysosomal N-glycan processing is critical to attain the most active form of the enzyme acid alpha-glucosidase

Abstract

Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, the deficiency of which leads to Pompe disease. Pompe disease can be treated with systemic recombinant human GAA (rhGAA) enzyme replacement therapy (ERT), but the current standard of care exhibits poor uptake in skeletal muscles, limiting its clinical efficacy. Furthermore, it is unclear how the specific cellular processing steps of GAA after delivery to lysosomes impact its efficacy. GAA undergoes both proteolytic cleavage and glycan trimming within the endolysosomal pathway, yielding an enzyme that is more efficient in hydrolyzing its natural substrate, glycogen. Here, we developed a tool kit of modified rhGAAs that allowed us to dissect the individual contributions of glycan trimming and proteolysis on maturation-associated increases in glycogen hydrolysis using in vitro and in cellulo enzyme processing, glycopeptide analysis by MS, and high-pH anion-exchange chromatography with pulsed amperometric detection for enzyme kinetics. Chemical modifications of terminal sialic acids on N-glycans blocked sialidase activity in vitro and in cellulo , thereby preventing downstream glycan trimming without affecting proteolysis. This sialidase-resistant rhGAA displayed only partial activation after endolysosomal processing, as evidenced by reduced catalytic efficiency. We also generated enzymatically deglycosylated rhGAA that was shown to be partially activated despite not undergoing proteolytic processing. Taken together, these data suggest that an optimal rhGAA ERT would require both N-glycan and proteolytic processing to attain the most efficient enzyme for glycogen hydrolysis and treatment of Pompe disease. Future studies should examine the amenability of next-generation ERTs to both types of cellular processing.

Long-term safety and efficacy of cipaglucosidase alfa plus miglustat in individuals living with Pompe disease: an open-label phase I/II study (ATB200-02)

“…A plateau is typically reached 2–3 years after treatment initiation, followed by progressive decline [ 9 – 12 , 18 , 19 ], highlighting a critical unmet need for new therapies with a more durable treatment response. The suboptimal outcomes observed with alglucosidase alfa led to research to better understand the possible mechanistic challenges of delivering a rhGAA to skeletal muscle [ 20 , 21 ]. First, rhGAA, which is most active at lysosomal pH, is less stable at the near-neutral pH of the bloodstream, resulting in rapid inactivation after infusion [ 20 ].…”

104-week efficacy and safety of cipaglucosidase alfa plus miglustat in adults with late-onset Pompe disease: a phase III open-label extension study (ATB200-07)

“…Cipaglucosidase alfa is a novel rhGAA with a high amount of CHO-cell derived bis-M6P glycans and enhanced glycosylation to ensure processing of the enzyme into a mature form with maximum catalytic activity. Combination with the enzyme stabilizer miglustat enhances the half-life of the enzyme in the blood to improve biodistribution and helps maintain catalytic activity prior to uptake into the muscle, where cipaglucosidase alfa dissociates from miglustat [ 19 – 21 ]. The results of PROPEL and the OLE support the clinical effectiveness of cipa + mig.…”

“…The variable and suboptimal long-term efficacy of alglucosidase alfa in LOPD prompted further research to better understand the challenges in delivering rhGAA to the lysosome of skeletal muscle. To date, three key challenges have been described in the literature: (1) despite large amounts of rhGAA infused into the blood (≥ 20 mg/kg), only a small percentage reaches the skeletal muscle due in part to clearance in the liver, suggesting that high-affinity binding to the cation-independent mannose 6-phosphate receptor (CI-MPR) is required for uptake of the remaining ERT into target muscle cells [ 19 , 20 ]; (2) rhGAA is delivered to the target tissue as a precursor that requires both proteolytic and N-glycan trimming to be converted into the version of GAA with the highest enzyme activity toward glycogen (7–10 × the activity of the precursor protein) [ 21 ]; (3) rhGAA is relatively unstable at the near-neutral pH of the blood and is rapidly inactivated following infusion [ 19 ].…”

Failure of Autophagy in Pompe Disease

“…Amicus Therapeutics has developed a next-generation rhGAA (Pombiliti™; cipaglucosidase alfa-atga + Opfolda™ (miglustat)) as a two-component therapy to improve the delivery of the enzyme to skeletal muscle lysosomes [113,119]. Unlike previous attempt to conjugate synthetic M6P-bearing oligosaccharides onto rhGAA, Pombiliti™ is a recombinant human GAA that is naturally expressed with high levels of bis-phosphorylated oligosaccharides.…”