Preparation and uses of multiple mannose-6-phosphates conjugated β-N-Acetylhexosaminidases

Lysosomal acid hydrolases are crucial for degradation and recycling of various biomolecules. They are mostly produced in a form of glycoproteins with oligosaccharides containing terminal mannose-6-phosphate (M6P). Genetic deficiencies of lysosomal enzymes lead to the inability of the lysosome to digest biomolecular substrates and cause the progressive accumulation of non-degraded substrates in the lysosome. This event causes the onset of lysosomal storage disorders (LSDs). Tay-Sachs and Sandhoff diseases, a class of LSDs, are caused by deficiency of β-N-acetylhexosaminidases, thereby leading to accumulation of GM2-ganglioside in lysosomes. To remove GM2-ganglioside in diseased cells, multiple mannose-6-phosphate (M6P)-appended β-N-acetylhexosaminidases were prepared by using combined genetic code expansion and click chemistry techniques. It was found that the multiple M6P-appended β-N-acetylhexosaminidase efficiently is internalizes into cells via M6P receptor-mediated endocytosis and then reaches the lysosome to regain its enzyme activity. Furthermore, the glycoengineered enzyme was able to efficiently cleave GM2-ganglioside in primary diseased cells, indicating the restoration of its activity in cells. The present strategy using the coupled genetic code expansion and biorthogonal ligation techniques is highly attractive to construct multiple M6P-containing enzymes which can be used to study LSDs.