1. Membrane-associated human tyrosinase is an enzymatically active monomeric glycoprotein.
- Author
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Kus NJ, Dolinska MB, Young KL 2nd, Dimitriadis EK, Wingfield PT, and Sergeev YV
- Subjects
- Albinism, Oculocutaneous enzymology, Albinism, Oculocutaneous genetics, Albinism, Oculocutaneous therapy, Enzyme Replacement Therapy, Humans, Monophenol Monooxygenase genetics, Monophenol Monooxygenase therapeutic use, Protein Domains, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins therapeutic use, Monophenol Monooxygenase chemistry
- Abstract
Human tyrosinase (hTyr) is a Type 1 membrane bound glycoenzyme that catalyzes the initial and rate-limiting steps of melanin production in the melanosome. Mutations in the Tyr gene are linked to oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder. Currently, the application of enzyme replacement therapy for a treatment of OCA1 is hampered by the absence of pure hTyr. Here, full-length hTyr (residues 1-529) was overexpressed in Trichoplusia ni larvae infected with a baculovirus, solubilized with detergent and purified using chromatography. Michaelis-Menten kinetics, enzymatic specific activity, and analytical ultracentrifugation were used to compare the hTyr in detergent with the soluble recombinant intra-melanosomal domain, hTyrCtr (residues 19-469). Active hTyr is monomeric in detergent micelles suggesting no stable interactions between protein molecules. Both, hTyr and hTyrCtr, exhibited similar enzymatic activity and ligand affinity in L-DOPA and L-Tyrosine reactions. In addition, expression in larvae is a scalable process that will allow high yield protein production. Thus, larval production of enzymatically active human tyrosinase potentially could be a useful tool in developing a cure for OCA1., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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