Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

Abstract

BackgroundTyrosinase (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 FindingsThe 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/SignificanceThe 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.

The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

“…We have previously shown that the recombinant intramelanosomal domain of human tyrosinase, hTyrC tr , is a soluble monomeric protein with both, monophenolase and diphenol oxidase enzymatic activities (Dolinska et al, 2014). Additionally, mass-spectroscopy analysis demonstrated, that hTyrC tr contains at least five N-glycosylation sites, which were completely (N86, N337) or partially (N111, N230, N290) modified.…”

Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag

“…Another important problem in this regard is the contamination risk of proteins with disease-causing agents (Houdebine, 2000;Swartz, 2001;Ma et al, 2003). Recombinantly produced proteins are very close to their natural state; thus they can have various features compared with nonrecombinants such as they can be used without triggering an immune rejection, there is no contamination risk with human or animal disease agents because they are obtained using isolated hosts, and their activities are alterable using molecular techniques (Demain and Vaishnav, 2009;Dolinska et al, 2014). Besides the advantages mentioned above there are also some restrictions.…”

Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector

“…The intra-melanosomal domain of human tyrosinase was expressed and purified from larval biomass and this work forms the basis of this unit (Dolinska et al, 2014, 2017). To simplify expression and purification we implemented the commonly used approach of deleting the trans-membrane- helix shown in red in Figure 1, which only serves to anchor protein to the cell membrane playing no part in enzyme function.…”