Tyrosinase related
protein 1 (TYRP1) is the most abundant melanosomal
protein of the melanocyte, where plays an important role in the synthesis of
eumelanin, possibly catalyzing the oxidation of
5,6-dihydroxyindole-2-carboxylic acid to 5,6-quinone-2-carboxylic
acid. Mutations to the TYRP1 gene can result in
oculocutaneous albinism type 3 (OCA3), a
rare disease characterized by reduced synthesis of
melanin in skin, hair, and eyes. To investigate the effect of genetic mutations on the TYRP1 structure, function, and stability, we engineered the intramelanosomal domain of TYRP1 and its mutant variants mimicking either OCA3-related changes, C30R, H215Y, D308N, and R326H or R87G mutant variant, analogous to OCA1-related pathogenic effect in
tyrosinase.
Proteins were produced in Trichoplusia Ni larvae, then purified, and analyzed by biochemical methods. Data shows that D308N and R326H mutants keep the native conformations and demonstrate no change in their stability and enzymatic activity. In contrast, mutations C30R and R87G localized in the Cys-rich domain show the variants misfolding during the purification process. The H215Y variant disrupts the binding of Zn2+ in the active site and thus reduces the strength of the
enzyme/substrate interactions. Our results, consistent with the clinical and in silico studies, show that mutations at the
protein surface are expected to have a negligible phenotype change compared to that of TYRP1. For the mutations with severe phenotype changes, which were localized in the Cys-rich domain or the active site, we confirmed a complete or partial
protein misfolding as the possible mechanism of
protein malfunction caused by OCA3 inherited mutations.