June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Oculocutaneous albinism Type 1 mutations indicate changes in the recombinant tyrosinase folding and activity.
Author Affiliations & Notes
  • Monika B Dolinska
    OGVFB, National Eye Institute, Bethesda, MD
  • Paul T Wingfield
    NIAMS, Bethesda, MD
  • Sara Farney
    OGVFB, National Eye Institute, Bethesda, MD
  • Brian Patrick Brooks
    OGVFB, National Eye Institute, Bethesda, MD
  • Yuri V Sergeev
    OGVFB, National Eye Institute, Bethesda, MD
  • Footnotes
    Commercial Relationships Monika Dolinska, None; Paul Wingfield, None; Sara Farney, None; Brian Brooks, None; Yuri Sergeev, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4665. doi:
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      Monika B Dolinska, Paul T Wingfield, Sara Farney, Brian Patrick Brooks, Yuri V Sergeev; Oculocutaneous albinism Type 1 mutations indicate changes in the recombinant tyrosinase folding and activity.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4665.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: 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 characterized the recombinant mutant variants of human tyrosinase intra-melanosomal domain mimicking OCA1 genetic changes.

Methods: Recombinant human tyrosinase (residues 19 - 469 of the native protein) and mutant variants, P406L, R402Q, R422Q, R422W, and T373K were prepared using the site-directed mutagenesis, produced in larvae and purified by IMAC and size-exclusion chromatographies. Specific L-DOPA enzyme activities were obtained by the dopachrome absorption at 475 nm. Trp fluorescence ratio (F360 nm/F320 nm) was measured as a function of urea concentration (1-8 M) for the wild type protein and mutant variants using SpectraMax i3 multimode detection platform. Denaturation curves show a sigmoidal, cooperative transition from the native to the denatured state. From these curves we calculated the denaturation mid-points and the standard free energies of unfolding for each protein unfolding reaction.

Results: Mutants mimicking OCA1B show similar expression and protein yield as the wild type. The specific activity present 29, 49, and 47% of the wild type activity, respectively. The OCA1A mutant shows very low expression and protein yield and no enzymatic activity. The analysis of denaturation curves shown that wild type protein and R402Q were the most stable proteins with the midpoint urea concentration of 5.0 M and 5.2M, respectively. Other mutant proteins show lesser stabilities in the range 4.5M-3.2M in a following decreasing order P406L>T373K>R422Q>R422W. The T373K change of unfolding free energy was 2.4 kcal/mol. Changes for OCA1B mutant variants were smaller values ranging from 0.8 to 1.6 kcal/mol.

Conclusions: Results are consistent with current literature data on a severe role of T373K mutation causing OCA1A and residual protein activity for the mutations involved in OCA1B albinism. OCA1 causing mutations might be associated with a full or partial misfolding of tyrosinase intra-melanosomal domain.


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