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compounds, TGH11, TGD10, TGD39 and TGJ29, as potent pigmentation inhibitors with IC(50)
values in the range of 10 muM. These newly identified depigmenting compounds were found to
function as reversible inhibitors of tyrosinase, the key enzyme involved in melanin synthesis.
Tyrosinase was further confirmed as the cellular target of these compounds by affinity
chromatography. Kinetic data suggest that all four compounds act as competitive inhibitors of
tyrosinase, most likely competing with l-3,4-dihydroxyphenylalanine (l-DOPA) for binding to the
DOPA-binding site of the enzyme. No effect on levels of tyrosinase protein, processing or
trafficking was observed upon treatment of melanocytes with these compounds. Cytotoxicity was
not observed with these compounds at concentrations up to 20 muM. Our data suggest that
TGH11, TGD10, TGD39 and TGJ29 are novel potent tyrosinase inhibitors with potential
beneficial effects in the treatment of cutaneous hyperpigmentation.
- Ni-Komatsu L, Orlow SJ.
Heterologous expression of tyrosinase recapitulates the misprocessing and mistrafficking in
oculocutaneous albinism type 2: Effects of altering intracellular pH and pink-eyed dilution
gene expression. Exp Eye Res. 2005 Sep 29; [Epub ahead of print]
The processing and trafficking of tyrosinase, a melanosomal protein essential for pigmentation,
was investigated in a human epithelial 293 cell line that stably expresses the protein. The effects of
the pink-eyed dilution (p) gene product, in which mutations result in oculocutaneous albinism type
2 (OCA2), on the processing and trafficking of tyrosinase in this cell line were studied. The
majority of tyrosinase was retained in the endoplasmic reticulum-Golgi intermediate compartment
and the early Golgi compartment in the 293 cells expressing the protein. Coexpression of p could
partially correct the mistrafficking of tyrosinase in 293 cells. Tyrosinase was targeted to the late
endosomal and lysosomal compartments after treatment of the cells with compounds that correct
the tyrosinase mistrafficking in albino melanocytes, most likely through altering intracellular pH,
while the substrate tyrosine had no effect on the processing of tyrosinase. Remarkably, this
heterologous expression system recapitulates the defective processing and mistrafficking of
tyrosinase observed in OCA2 albino melanocytes and certain amelanotic melanoma cells.
Coexpression of other melanosomal proteins in this heterologous system may further aid our
understanding of the details of normal and pathologic processing of melanosomal proteins.
- Obata H, Ishida H, Hata Y, Kawato A, Abe Y, Akao T, Akita O, Ichishima E.
Cloning of a novel tyrosinase-encoding gene (melB) from Aspergillus oryzae and its
overexpression in solid-state culture (Rice Koji). J Biosci Bioeng. 97(6):400-5, 2004.
We have cloned a novel tyrosinase-encoding gene (melB) specifically expressed in solid-state
culture of Aspergillus oryzae. A tyrosinase-encoding gene (melO) from A. oryzae was already
cloned and the protein structures of its catalytic and copper binding domains were investigated.
However, our recent results revealed that the melO gene was highly expressed in submerged
culture but not in solid-state culture. Because tyrosinase activity was also detected in solid-state
culture, we assumed that another tyrosinase gene other than melO is expressed in solid-state
culture. Another tyrosinase gene was screened using the expressed sequence tag (EST) library. One
redundant cDNA clone homologous with the tyrosinase gene was found in the collection of wheat
bran culture. Northern blot analysis revealed that the gene corresponding to the cDNA clone was
specifically expressed in solid-state culture (koji making), but not in submerged culture. Molecular
cloning showed that the gene carried six exons interrupted by five introns and had an open reading
frame encoding 616 amino acid residues. This gene was designated as melB. The deduced amino
acid sequence of the gene had weak homology (24%-33%) with MelO and other fungal tyrosinases
but the sequences of the copper binding domains were highly conserved. When the melB gene was
expressed under the control of the glaB promoter in solid-state culture, tyrosinase activity was
markedly enhanced and the culture mass was browned with the melanization by MelB tyrosinase.
These results indicated that the melB gene encodes a novel tyrosinase associated with melanization
in solid-state culture.
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