Purchase this article with an account.
Monika B. Dolinska, Elena Kovaleva, Peter Backlundt, Grzegorz Piszczek, Paul T. Wingfield, Brian P. Brooks, Yuri V. Sergeev; Human Modified Tyrosinase And Two Temperature-Sensitive Mutants Are Soluble Active Enzymes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1605.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Mutations in tyrosinase gene cause an autosomal recessive disorder, oculocutaneous albinism Type 1 (OCA1). OCA1 is classified into a disorder with complete absence of tyrosinase activity (OCA1A) and with residual tyrosinase activity (OCA1B). Tyrosinase is a Type I membrane protein with a single trans-membrane fragment located at C-terminus, which catalyzes the rate-limiting conversions of tyrosine to L-DOPA and dopachrome in melanin production in the skin and eye. Here we designed, expressed and purified human tyrosinase and two temperature-sensitive mutants which were modified to remove a trans-membrane fragment to avoid potential protein insolubility but to preserve all other functional features of the enzyme.
Truncated human tyrosinase gene, hTyrCtr (residues 19 - 469 of the native protein) and two mutants R404Q and R404W (subtype OCA1B in position 422) were engineered as synthetic codon optimized DNA comprising insect signal sequence and C-terminal 6His-tag and cloned into baculovirus. Recombinant proteins were produced in whole insect Trichoplusia ni and purified by immobilized metal affinity and size-exclusion chromatographies.
By using L-DOPA enzymatic reaction, SEC, sedimentation equilibrium and dynamic light scattering we demonstrated that hTyrCtr is an active monomeric enzyme with molecular weight of about 56 kDa and Km=0.88±0.13 mM. hTyrCtr is N-glycosylated as shown by the PNGase de-glycosylation. Both temperature-sensitive mutants are enzymatically active at 37 oC: Km=0.24±0.03 mM (R404Q) and Km=1.04±0.27 mM (R404W).
The hTyrCtr and temperature sensitive mutants are expressed and purified as soluble active enzymes suggesting that they could be used as an in vitro model for the characterization of the human tyrosinase function and molecular perturbations caused by the OCA1B pathogenic changes. These observation suggest that the defect in melanin production associated with these two temperature-sensitive mutations are likely not due to intrinsic enzymatic activity of tyrosinase.
This PDF is available to Subscribers Only