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Monika B Dolinska, Peter Backlund, Paul T Wingfield, Elena Kovaleva, Ewa Grajkowska, Brian Patrick Brooks, Yuri V Sergeev; The consequences of deglycosylation of intra-melanosomal domain of human tyrosinase.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):411.
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© ARVO (1962-2015); The Authors (2016-present)
Oculocutaneous albinism type 1 is an autosomal recessive disease associated with decreased melanin pigment production, decreased best-corrected visual acuity, and mutation in the tyrosinase (TYR) gene. Tyrosinase is a type 1 trans-membrane and copper-containing glycoenzyme that catalyzes the initial steps of melanin pigment production in melanosomes. Human tyrosinase is modified post-translationally by N-linked glycosylation on asparagine (Asn, N) residues, which is important for enzymatic activity. Recently we characterized in vitro 5 N-glycosylation sites in the recombinant intra-melanosomal domain of human tyrosinase, hTyrCtr (Dolinska et al,). To explore this study we expressed, purified and characterize enzymatic activity of 2 N-deglycosylated mutant variants.
The 5-sites and fully (7-sites) deglycosylated recombinant mutant variants were prepared using the site-directed mutagenesis where Asn were replaced with Asp residues to remove the N-glycosylation sites, respectively. Recombinant human tyrosinase (residues 19 - 469 of the native protein) and deglycosylated mutants were produced in larvae (C-PERL, MD) and purified by immobilized metal affinity and size-exclusion chromatography. N-glycosylation sites in intra-melanosomal domain were confirmed using mass spectroscopy. Enzyme activities were measured using L-DOPA color reaction.
The 5- and 7 residues mutagenesis of the hTyrCtr glycosylation sites resulted in decreased protein expression and increased difficulty with protein purification, respectively. Specific L-DOPA activity measured for the wild type, 5-and 7-deglycosylated mutants show decrease of enzymes activities with a dramatic change for the fully deglycosylated protein (50033, 37879, and 95 U/mg, respectively).
Removal of 5 N-glycosylation sites from the hTyrCtr causes modest decrease of activity, which is consistent with our molecular modeling of human tyrosinase which suggests that those sites are located at the protein surface and do not directly affect the 4-helix bundle structure maintaining the copper-binding sites. The complete removal of N-glycans affects the protein expression and diminishes the enzymatic activity, in agreement with previous data in cell culture.
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