Abstract
Purpose:
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 an enzyme which catalyzes the rate-limiting conversions of tyrosine to L-DOPA and dopachrome in melanin production in the skin and eye. Here we characterized intra-melanosomal domain of human tyrosinase, hTyrCtr (residues 19 - 469 of the native protein).
Methods:
Recombinant hTyrCtr was produced in larvae (C-PERL, MD) and purified from the soluble fraction by immobilized metal affinity and size-exclusion chromatography. The effect of various inhibitors and activators, kinetics parameters and optimum temperature and pH were examined by using L-DOPA and L-tyrosine enzymatic reactions. N-glycosylation sites and a composition of sugar components in hTyrCtr were determined using MS/MS, Maldi-Tof MS, and Q-Tof MS/MS mass spectroscopy combined with trypsin, asp-proteinase (peptide sequencing) and PNGase-F proteinase digests (sugar composition).
Results:
Recombinant hTyrCtr show maximum enzymatic activity at optimal temperature of 37°C and neutral pH. The hTyrCtr is N-glycosylated as shown by the PNGase de-glycosylation. Five N-glycosylation sites are either modified (N86, N337) or partially modified (N111, N230, N290) by carbohydrates. Expected asparagins N161 and N371 are not glycosylated. Molecular modeling of human tyrosinase suggests that all observed N-glycosylation sites are located at the surface of intra-melanosomal domain with sugar chains accessible for inter-molecular interactions within the melanosome.
Conclusions:
Recombinant hTyrCtr is a functional N-glycosylated enzyme. In contrast to current observation that genetic mutations affect just a few N-glycosylation sites our work suggests that a wider spectrum of sequence positions associated with N- glycosylation sequons, 84-91, 109-116, 228-235, 288-295, and 335-342, and might be associated with genetic defects. The recombinant hTyrCtr could be used to search for small molecule activators of tyrosinase mutant variants as a potential treatment of OCA1 type of albinism.
Keywords: 658 protein purification and characterization •
657 protein modifications-post translational •
659 protein structure/function