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Endolysosomal processing of N-glycans on acid alpha-glucosidase is critical to attain the most active enzyme for hydrolyzing glycogen

ABSTRACT: Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, a deficiency in 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 has poor uptake in skeletal muscles, limiting clinical efficacy. Further, it is unclear how the specific cellular processing steps of GAA post-delivery to lysosomes impact efficacy. GAA undergoes both proteolytic cleavage and glycan trimming within the endolysosomal pathway, yielding a more active enzyme for hydrolyzing its natural substrate glycogen. The relative contributions for each of these processing steps for increasing rhGAA glycogen hydrolytic activity are unclear. 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 hydrolytic activity on glycogen. 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 following endolysosomal processing, as evidenced by lower catalytic efficiency on glycogen. 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 kinetics for glycogen hydrolysis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: M. Osman Sheikh

LAB HEAD: Dr. M. Osman Sheikh

PROVIDER: PXD025285 | Pride | 2021-05-05

REPOSITORIES: Pride

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			Action	DRS
	12-31-20_NeuAc_Oxi.xlsx	Xlsx
	AOAA___Neuramin__RA.xlsx	Xlsx
	GAAkif_10uM_EndoH_10days_Sites_6_and_7.xlsx	Xlsx
	GAAkif_EndoH-10days.raw	Raw
	Lum___Neuramin__RA.xlsx	Xlsx

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Project description:Infantile-onset Pompe disease is an inherited disorder that is normally diagnosed within the first months of life. It is caused by lack of or defect in an enzyme (a special protein that carries out normal chemical reactions within the body) called acid alpha-glucosidase (GAA). GAA normally breaks down glycogen (stored sugar) in lysosomes (the part of the cell that digests food and other chemicals). Pompe disease is one of many lysosomal storage diseases (LSDs). LSDs are diseases caused by the malfunction of the lysosome or one of their digestive enzymes. Patients with Pompe disease cannot break down lysosomal glycogen. This causes glycogen to build up and damage cells throughout the body, especially in the heart and muscles. Current treatment for Pompe disease involves enzyme replacement therapy (ERT). In this treatment, the drug alglucosidase alfa (Myozyme) is put into your blood. The drug provides a form of the GAA enzyme to replace the enzyme that is missing or not working properly in the patient's blood. This treatment has allowed babies to live longer and achieve developmental milestones. In this study, researchers will learn about the patient's ability to tolerate ERT. Cross-Reactive Immunological Material (CRIM) is a measurement of natural GAA production. A patient's CRIM status (either positive or negative) is an important factor that affects how he or she responds to ERT with Myozyme. Children who produce some natural GAA are classified as CRIM+, while children who do not produce any natural GAA are classified as CRIM-. Children who are CRIM+ generally tolerate ERT well. But, children who are CRIM-, and some children classified as CRIM+, have a poor response to ERT. Patients who have a poor response to ERT have complications because their body sees Myozyme as "foreign" and triggers an immune response to try to remove it from the body. Treatments are currently being developed to stop this immune response and prevent complications from ERT. We will enroll patients with Infantile Pompe disease in this longitudinal natural history (observational) study. The specific aims of this study are: <ol> <li>To determine and correlate Cross-Reactive Immunological Material (CRIM) status with the GAA gene mutations found on these patients</li> <li>To validate an approach for determining CRIM status from whole blood sample, with the gold standard determination of CRIM status by skin fibroblasts and mutation analysis</li> <li>To explore the clinical treatment response and natural history of CRIM-positive and CRIM-negative Pompe disease patients with and without immune modulation</li> <li>To investigate the role of immune response to treatment</li> </ol>

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Endolysosomal processing of N-glycans on acid alpha-glucosidase is critical to attain the most active enzyme for hydrolyzing glycogen

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