subsequent treatment of IDUA (acceptor) with Endo-D at pH5.0 for retaining the catalytic activity, and then Endo-M N175Q at pH6.0 or Endo-CC N180H at pH7.5 in the presence of M5(6P2)GN2 (donor) (acceptor : donor = 1 : 1,000 molar ratio)(Figure 3A, B). The terminal M6P addition was confirmed by lectin blotting using the recombinant M6P receptor domain 9 (M6PR-Dom9)9).Dr. T. Oishi et al. identified a Japanese macaque (Macaca fuscata) exhibiting MPS-like phenotypes, including skin thickening, gingival growth, tongue enlargement, bone deformity, joint contracture, valvular heart disease, and urinal excretion of mucopolysaccharides, in 2017 in the former Primate Res. Inst. (current Center for the Evolutionary Origins of Human Behavior, Kyoto Univ. (EHUB)(Figure 3C). We diagnosed the monkey with a mild form of MPS1, IDUA deficiency caused by homozygous amino acid substitution, which does not show neurological symptoms.We evaluated the replacement effects of the M6P-IDUA by assessing the incorporation through binding with cell surface M6P receptor (M6PR) to be transported to lysosomes and restoring IDUA activity in the fibroblasts derived from Japanese macaque with MPS1 (Figure 3D, E). The restoration of IDUA activity in MPS1 monkey fibroblasts was inhibited in the presence of M6P as competitive inhibition in a cultured medium, indicating that the incorporation of M6P-IDUA was dependent on the binding with the cell surface M6P receptor.Development of neoglycobiologics using transglycosylation technologyKohji Itohorgans and develops neurovisceral symptoms, including mental retardation, hepatosplenomegaly, bone deformity, joint contracture, valvular heart disease, gargoyle-like face8). The intravenous (iv) ERT using the M6P-glycan carrying enzyme drug laronidase, a recombinant human IDUA produced by the CHO cell line, has been clinically applied globally8). However, the symptoms involving the central nervous system, bone, joint, and heart valve are intractable by the iv ERT because of the blood-brain barrier (BBB) and lack of blood vessels in the tissues.We successfully produced and purified the recombinant enzymatically active IDUA from the cocoons (0.2 mg IDUA/cocoon) derived from Tg silkworm overexpressing the IDUA gene in the middle or posterior silk glands. The cocoon-derived IDUA carried the high mannose-, hybrid, complex, or pauci-mannose-type N-glycans but neither contained the insect-specific sugar residues, including α1,3-linked fucose nor the mammalian-type M6P-glycans (Figure 1). Therefore, we produced chemoenzymatically the neoglycoIDUAs utilizing ENGases, including Endo-D, Endo-M N175Q or Endo-CC N180H, to transglycosylate the N-glycans attached to the IDUA to synthetic branched M5(6P2)GN2 glycans with terminal M6P residues and non-oxazoline methoxyphenyl derivative (Figure 2). The enzymatically active neoglycoIDUA (M6P-IDUA) was successfully produced by Figure 2. Production of M6P-type neoglycoIDUATg silkworm cocoon-derived human IDUAs were treated with Endo-D for glycan trimming except for ones necessary for catalytic activity and then treated with ENGases (Endo-M N175Q or Enco-CC N180H) and the synthetic Man5(6P2)GN2–methoxyphenyl (MP) derivative for transglycosylation.88
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