Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants.

Monika B Dolinska,Yuri V Sergeev,Taariq Woods,Isabella Osuna

doi:10.3390/ijms23031305

Monika B Dolinska, Yuri V Sergeev + Show 2 more

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https://doi.org/10.3390/ijms23031305

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Abstract

Tyrosinase-related protein 2 (Tyrp2) is involved in the melanogenesis pathway, catalyzing the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Recently, a new type of albinism was discovered with disease-causing mutations in the TYRP2 gene. Here, for the first time, we characterized the intra-melanosomal protein domain of Tyrp2 (residues 1-474) and missense variants C40S and C61W, which mimic the alterations found in genetic studies. Recombinant proteins were produced in the Trichoplusia Ni (Ti. Ni) larvae, purified by a combination of immobilized metal affinity (IMAC) and gel-filtration (GF) chromatography, and biochemically characterized. The mutants showed the protein expression in the lysates such as the wild type; however, undetectable protein yield after two steps of purification exhibited their misfolding and instability. In addition, the misfolding effect of the mutations was confirmed computationally using homology modeling and molecular docking. Together, experiments in vitro and computer simulations indicated the critical role of the Cys-rich domain in the Tyrp2 protein stability. The results are consistent with molecular modeling, global computational mutagenesis, and clinical data, proving the significance of genetic alterations in cysteine residues, which could cause oculocutaneous albinism type 8.

Highlights

Oculocutaneous albinism (OCA) is a heterogeneous group of rare inherited disorders affecting the biosynthesis of melanin
Pennamen et al showed that mutations in the dopachrome tautomerase (DCT) gene, C40S, and OCA8-related mutant variants (C61W) can cause a new type of oculocutaneous albinism, which they proposed to name oculocutaneous albinism type 8 (OCA8)
Tyrosinase-related protein 2 (Tyrp2) is involved in the melanogenesis pathway, catalyzing the tautomerization of dopachrome to dihydroxyindole-2-carboxylic acid (DHICA)

Summary

Introduction

Oculocutaneous albinism (OCA) is a heterogeneous group of rare inherited disorders affecting the biosynthesis of melanin. The new mutations in the TYRP2 gene ( known as the DCT gene), involved in albinism, were identified in the genetic study [1]. Pennamen et al showed that mutations in the DCT gene, C40S, and C61W can cause a new type of oculocutaneous albinism, which they proposed to name OCA8. Volk et al identified the new mutation in the TYRP2 gene, G59V [2]. Loss of function of Tyrp protein was investigated in mice and zebrafish [1]. Tyrp insufficiency impairs melanin metabolism in both melanophores and retinal pigment epithelium (RPE) cells with possible consequences on eye development and/or function

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