Characterization and downstream mannose phosphorylation of human recombinant α‐ L ‐iduronidase produced in A rabidopsis complex glycan‐deficient ( cgl ) seeds

Summary Mucopolysaccharidosis ( MPS ) I is a lysosomal storage disease caused by a deficiency of α‐ L ‐iduronidase ( IDUA ) ( EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. A rabidopsis cgl mutants are characterized by a deficiency of the activity of N ‐acetylglucosaminyl transferase I ( EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N ‐glycan biosynthesis. To develop a seed‐based platform for the production of recombinant IDUA for potential treatment of MPS I , cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N ‐linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl ‐ IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured C hinese hamster ovary ( CHO ) cells ( A ldurazyme TM ). The N ‐glycan profile showed that cgl ‐derived IDUA contained predominantly high‐mannose‐type N ‐glycans (94.5%), and the residual complex/hybrid N ‐glycan‐containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl ‐ IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose‐6‐phosphate ( M 6 P ) tag in mammalian cells— UDP ‐Glc NA c:lysosomal enzyme N −acetylglucosamine ( G lc NA c)−1−phosphotransferase—and Glc NA c−1−phosphodiester α− N −acetylglucosaminidase (the ‘uncovering enzyme’). A rabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M 6 P , a recognition marker essential for efficient receptor‐mediated uptake into lysosomes of human cells.