Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity

Abstract

Summary Oculocutaneous albinism Type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intra-melanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities- tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants show very low protein expression, protein yield, and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactive tyrosinase and results in OCA1B albinism.

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

“…These observations are consistent with our molecular modeling of human tyrosinase, which suggests that the six sites are located at the protein surface and have less influence on the 4-helix bundle structure maintaining the copper-binding sites (Dolinska et al, 2014, 2017). However, the conformation of the 4-helix bundle in a fully deglycosylated tyrosinase (7D) could be affected by N371D mutation due to an additional negative charge introduced in the protein cavity surrounding this residue.…”

“…In the T373K mutation, which is very common in Northern Europeans, the threonine residue is replaced with positively charged lysine, which disrupts N-X-S/T sequence motif. Results from genetic studies agree with our in vitro data suggesting that T373K mutation causes the proteolytic degradation of expressed protein, destabilization of the tyrosinase structure, and abolishs protein activity (Dolinska et al, 2017). Previously shown in vivo these mutations could prevent tyrosinase from leaving the ER and initiate a severe disease phenotype in OCA1 albinism causing the loss of pigment (Branza-Nichita et al, 2000).…”

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

“…Although this was reasonable (and standard practice) we generated human full-length tyrosinase and showed that it had similar enzymic properties as the truncated variant (Dolinska et al, 2017). However, compare to full-length protein, enzymatically active melanosomal domain shows better protein expression and purification, robust laboratory handling, and should be a first choice for future drug screens.…”

“…The strategy of deleting membrane-anchoring helix is standard to simplify handling of protein. Recently, we expressed the full-length tyrosinase and demonstrated that the deletion of the anchoring helix does not affect the tyrosinase activity (Dolinska et al, 2017). …”

“…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.…”