Abstract
Purpose :
Tyrosinase (Tyr) and Tyrosinase Related Protein 1 (Tyrp1) are melanocyte-specific enzymes involved in melanin biosynthesis. Mutations in their genes cause different types of oculocutaneous albinism, the autosomal recessive disorders associated with reduced or altered pigmentation of skin, hair, and eyes. Both enzymes are transmembrane glycoproteins share the same signaling sequence and two metal (copper or zinc) binding sites responsible for their catalytic activities. It was shown in vivo that in cells expressing both, Tyr and Tyrp1, a total amount of melanin increased compare to that of expressed by Tyr alone. Moreover, data from the cell culture demonstrated their hetero-dimerization. Here, we study in vitro the intra-melanosomal domains of human recombinant Tyr and Tyrp1, and the potential role of Tyrp1 in stabilization of tyrosinase.
Methods :
Proteins expressed separately or co-expressed in baculovirus and produced in whole T.ni larvae, then purified using the IMAC and size exclusion chromatography (SEC). Homo-and hetero-associations of Tyr and Tyrp1 obtained using SEC and sedimentation equilibrium (SE). Tyr diphenol oxidase activities measured by the dopachrome absorption at 475 nm in the presence or the absence of Tyrp1. The measurement repeated at physiological (37°C) and stress (43°C) conditions at pH 5.5 and pH 7.2 each mimicking pH at melanosome and ER, respectively.
Results :
Both proteins showed broad bands due to glycosylation with molecular masses of ~ 60 kDa. SEC and SE revealed that Tyr and Tyrp1 are the monomers, which do not form stable homo- and hetero-oligomers. The initial reaction rate of Tyr diphenol oxidase activity was suppressed by the excessive amount of Tyrp1. Maximum of dopachrome formation increased in presence of Tyrp1 mostly at pH 5.5 and was delayed. In presence of Tyrp1 the melanin-like precipitate appears much later and in significantly lower amount.
Conclusions :
Our data shows that incubation of excessive amount of Tyrp1 with Tyr display protection of Tyr stability over the time. However, this mechanism does not involve the formation of stable hetero-oligomeric complexes to maintain the protective function.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.