April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Mouse Tyrosinase And Causing Oculocutaneous Albinism Type1 Missense Variants R77L And H420R: Effect Of Tyrosine On Protein Stability
Author Affiliations & Notes
  • Monika B. Dolinska
    OGVFB, National Eye Institute, Bethesda, Maryland
  • Yuri V. Sergeev
    OGVFB, National Eye Institute, Bethesda, Maryland
  • Ighovie F. Onojafe
    OGVFB, National Eye Institute, Bethesda, Maryland
  • Brian P. Brooks
    OGVFB, National Eye Institute, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  Monika B. Dolinska, None; Yuri V. Sergeev, None; Ighovie F. Onojafe, None; Brian P. Brooks, None
  • Footnotes
    Support  Z01-EY000476-01
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2358. doi:
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      Monika B. Dolinska, Yuri V. Sergeev, Ighovie F. Onojafe, Brian P. Brooks; Mouse Tyrosinase And Causing Oculocutaneous Albinism Type1 Missense Variants R77L And H420R: Effect Of Tyrosine On Protein Stability. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2358.

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

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Purpose: : Oculocutaneous albinism type1 (OCA1) is caused by mutations in the tyrosinase gene. There are two subtype of OCA1: OCA1A, characterized by absence of melanin pigment in eyes, hair and skin and OCA1B characterized by present, but reduced, amount of pigment. In order to understand better how specific tyrosinase mutations result in different phenotypes, we have studied the effects on enzymatic activity, protein structure and protein stability in mouse model mutations of OCA1A (Tyrc-2J/c-2J / R77L) and OCA1B (Tyrc-h/c- h / H420R). Because we have also shown that increase in plasma tyrosine improves pigmentation in Tyrc-h/c- h mice, we also tested the effect of extracellular tyrosine on tyrosinase functions.

Methods: : The wild type tyrosinase and missense R77L and H420R mutants were transiently expressed in chinise hamster ovary (CHO) cells using pcDNA3.1 vector. Transfected cells were harvested and microcentrifuged to obtain protein lysates for further functional and biochemical analysis. Stability of the proteins was analysed by cyclohexamide chase experiments in the presence or the absence of tyrosine. Cells were collected and analyzed by western bloting using GAPDH as an internal control. WT, R77L and H420R ratios to GAPDH were calculated for kinetic and statistic analysis. Molecular modeling of the protein structure of the wild type protein and two mutants was performed.

Results: : Statistical analysis of the cyclohexamide chase experiment demonstrated the increasing of the protein expression only for the H420R missense variant at 9 and 24 hours in the presence of tyrosine. The stabilizing role of tyrosine on H420R was in agreement with predictions of molecular modeling suggested that the binding of tyrosines in the hydrophobic cavity might improve the stability of the tyrosinase enzymatic domain. In contrast, the R77L mutant variant suggested decrease or eliminate a tyrosine binding.

Conclusions: : Increasing tyrosine concentration might stabilize mouse tyrosinase missense mutant H420R, which reflect OCA1B and appear to have no impact on R77L mutant reflecting OCA1A subtype of albinism.

Keywords: visual development • protein structure/function • enzymes/enzyme inhibitors 

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