Elucidating the structure and improving the cellular uptake of HexM, a candidate for enzyme replacement therapy of GM2 gangliosidosis

Thumbnail Image
Graeme, Benzie
Journal Title
Journal ISSN
Volume Title
Tay-Sachs disease and Sandhoff disease are genetic disorders resulting from mutations in genes HEXA or HEXB, which code for the α- and β-subunits of the heterodimer β-hexosaminidase A (HexA), respectively. HexA is a lysosomal glycosidase responsible for degrading GM2 ganglioside (GM2), a glycolipid found in the plasma membrane of neurons. Loss of HexA causes GM2 accumulation in neurons and deterioration of motor and cerebral function, typically resulting in death by age 4. Previously, the critical features of the α- and β-subunits of HexA were combined into a single engineered subunit (µ) to create a stable homodimeric version of HexA, known as HexM. HexM is twice as active as HexA, degrades GM2 both in cellulo and in vivo. Two major issues that often impede ERT development include the inability to produce therapeutic enzyme in sufficient quantities and inadequate enzyme phosphorylation, which is necessary for uptake into lysosomes of affected cells by the manose-6-phophate pathway). Here I demonstrate that HexM can be produced in gram-scale quantities, making it a promising candidate for enzyme replacement therapy (ERT) and that lysosomal uptake of HexM via the mannose-6-phosphate receptor can be enhanced (3- to 4- fold) by co-expressing HexM with an engineered Glc-NAc-1-phosphotransferase that hyper-phosphorylates lysosomal proteins. I also produced a HexM mutant incapable of hydrolyzing substrate and investigated methods of binding HexM to GM2AP to form a long-lived complex capable of being assessed by either transmission electron microscopy or x-ray crystallography. A crystal structure of HexM, combined with mass spectrometry-based glycoform analysis also revealed that HexM retains a fold and glycosylation pattern consistent with HexA. My studies further advance HexM as a potential ERT for Tay-Sachs disease and Sandhoff disease
Enzyme Replacement Therapy, Tay-Sachs Disease, Beta-hexosaminidase, Recombinant Protein