Abstract
Mucopolysaccharidosis (MPS) I is a severe lysosomal storage disease caused by α-L-iduronidase (IDUA) deficiency, which results in accumulation of non-degraded glycosaminoglycans in lysosomes. Costly enzyme replacement therapy (ERT) is the conventional treatment for MPS I. Toward producing a more cost-effective and safe alternative to the commercial mammalian cell-based production systems, we have produced recombinant human IDUA in seeds of an Arabidopsis mutant to generate the enzyme in a biologically active and non-immunogenic form containing predominantly high mannose N-linked glycans. Recombinant enzyme in ERT is generally thought to require a mannose 6-phosphate (M6P) targeting signal for endocytosis into patient cells and for intracellular delivery to the lysosome. Toward effecting in planta phosphorylation, the human M6P elaboration machinery was successfully co-expressed along with the recombinant human IDUA using a single multi-gene construct. Uptake studies using purified putative M6P-IDUA generated in planta on cultured MPS I primary fibroblasts indicated that the endocytosed recombinant lysosomal enzyme led to substantial reduction of glycosaminoglycans. However, the efficiency of the putative M6P-IDUA in reducing glycosaminoglycan storage was comparable with the efficiency of the purified plant mannose-terminated IDUA, suggesting a poor in planta M6P-elaboration by the expressed machinery. Although the in planta M6P-tagging process efficiency would need to be improved, an exciting outcome of our work was that the plant-derived mannose-terminated IDUA yielded results comparable to those obtained with the commercial IDUA (Aldurazyme® (Sanofi, Paris, France)), and a significant amount of the plant-IDUA is trafficked by a M6P receptor-independent pathway. Thus, a plant-based platform for generating lysosomal hydrolases may represent an alternative and cost-effective strategy to the conventional ERT, without the requirement for additional processing to create the M6P motif.
Highlights
Mucopolysaccharidosis (MPS) I is one of the over 70 lysosomal storage diseases that are caused by mutations in proteins critical for lysosomal function
A possible strategy to modify plant recombinant IDUA with the mannose 6-phosphate (M6P) tag—the motif required for the lysosomal trafficking of many recombinant enzymes used in enzyme replacement therapy (ERT)—is to treat a purified plant recombinant IDUA with a recombinant soluble PT α2β2 in vitro, lacking its transmembrane domains [4,6,16]), followed by treatment with a recombinant soluble uncovering enzyme’ (UCE) (Figure 1A)
The Vmax of the transfer of GlcNAc-phosphate to plant recombinant IDUA in vitro is relatively low, the Km values reflect that the PT binds with great tenacity to the plant-recombinant target hydrolase [6,33]
Summary
Mucopolysaccharidosis (MPS) I is one of the over 70 lysosomal storage diseases that are caused by mutations in proteins critical for lysosomal function. The deficiency of α-L-iduronidase (IDUA) that underlies MPS I disease results in the accumulation of non-degraded glycosaminoglycans (GAGs) and causes multisystem pathology in a progressive manner [1]. Toward producing a more cost-effective and safe alternative to the commercial mammalian cell-based production systems, we have established an efficient plant-based recombinant protein production platform resulting in high-level synthesis of active human IDUA in seeds of the complex-glycan-deficient (cgl) mutant of Arabidopsis thaliana. The seed-based platform accumulates human IDUA at ~5.7% total soluble protein and the enzyme is generated in a non-immunogenic form containing predominantly high mannose N-linked glycans (~94%) [3,4,5,6]. Purified cgl-recombinant IDUA was used to determine the 3-D structure of the human protein along with the essential details of its catalytic mechanism [7]
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