Engineering Of HEK293 Cells Producing Lysosomal Protein With High-mannose-type N-glycans

Lysosomes contain many hydrolytic enzymes,such as nucleases,proteases,lipases and glycosidases.These enzymes break down cellular metabolites in lysosomes.Lysosomal storage diseases(LSDs)are inherited disorders caused by deficiency of specific lysosomal enzymes that metabolize substrates in lysosomes.Enzyme replacement therapy is normally performed by giving the patient an intravenous injection of recombinant lysosomal enzymes.The enzymes are incorporated into the cells through cation-independent M6 P receptors or other receptors such as Man receptors on the cell surface.Therefore,enzymes require high Man-type glycans containing M6 P for their efficient incorporation into cells.In this study,we established cell lines having asparagine(N)-linked glycans with high mannose types,but less complex types.We first knocked out two Golgi mannosidases(MAN1A1 and MAN1A2)in HEK293 cells.Single knockout(KO)cells did not change the N-glycan structures.When we established a doubleKO the complex types of N-glycans were significantly decreased,whereas the high mannose types were increased.To eliminate the remaining complex types of glycans,we checked effects of other a-1,2-mannosidases.Among them,KO of a gene encoding the ER mannosidase I(MAN1B1)in the double-KO cells was further decreased the complex types of glycans.In the triple-KO cells,Man9,Man8 and Man7 structures of glycans became major.Two lysosomal enzymes,alpha-galactoside A and lysosomal acid lipase,were expressed in the double-and triple-KO(MAN1A1,MAN1A2 and MAN1B1)cells.The glycans on recombinant lysosomal enzymes released to medium were completely sensitive to endoglycosidase H treatment in the double-and triple-KO cells,suggesting that majority of glycans on the proteins become high mannose types of glycans.Also,the glycan on recombinant EGFP-FLAG-HyHEL10 released to medium were detected by MALDI-TOF MS,comfirmed the glycan structure on the IgG protein are high Man-type.Our results indicate that the double-and triple-KO cells are suitable for the production of recombinant proteins including lysosomal enzymes with high mannose types of glycans.