Production of recombinant human acid α-glucosidase with mannosidic N-glycans in α-mannosidase I mutant rice cell suspension culture

Abstract

High-mannose glycans, containing 6-9 mannose residues, are favorable for in vitro mannose phosphorylation to produce Mannose-6-phosphate (M6P) residues. These are important for lysosomal enzyme targeting and essential for uptake. Golgi -mannosidase-I mediates mannose trimming in the N-glycosylation pathway. In this study, I used mutant rice with a T-DNA insertion in the Os04g0606400 ({Delta}-manI) gene to produce recombinant human acid -glucosidase (rhGAA) for enzyme replacement therapy to treat Pompe disease. After characterization of {Delta}-manI mutant rice, the gene encoding rhGAA was introduced into mutant calli by Agrobacterium-mediated transformation. Integration of the target sequence into the rice genome was detected by genomic DNA PCR. Expression of rhGAA from {Delta}-manI mutant ({Delta}-manI-GAA) and its secretion into culture media were confirmed by western blot analysis. The N-glycosylation pattern of purified {Delta}-manI-GAA was analysed by MALDI-TOF mass spectrometry. This showed that the high-mannose type N-glycan with man8 (63.8%) was the most abundant form, followed by Man7 (22.5%), GnGnXF3 (6.7%), Man6 (5.4%), and Man5 (1.6%). The results suggest the successful production of rhGAA with N-glycan containing 6-8 mannose residue in the {Delta}-manI mutant rice cell suspension culture.