Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic ActivityReport as inadecuate




Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity - Download this document for free, or read online. Document in PDF available to download.

Background

Tyrosinase TYR catalyzes the rate-limiting, first step in melanin production and its gene TYR is mutated in many cases of oculocutaneous albinism OCA1, an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.

Methodology-Principal Findings

The intra-melanosomal domain of human tyrosinase residues 19–469 and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.

Conclusions-Significance

The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.



Author: Monika B. Dolinska, Elena Kovaleva, Peter Backlund, Paul T. Wingfield, Brian P. Brooks , Yuri V. Sergeev

Source: http://plos.srce.hr/



DOWNLOAD PDF




Related documents