Protein Stability and Functional Characterization of Intra-Melanosomal Domain of Human Recombinant Tyrosinase-Related Protein 1

Abstract

Pigmentation is the result of a complex process by which the biopolymer melanin is synthesized and packed into melanosomes of melanocytes. Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to genetic mutations of proteins involved in melanogenesis. Human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) drives the enzymatic process of pigment bio-polymerization. However, within the melanogenic pathway, Tyrp1 has catalytic functions not clearly defined and distinct from Tyr. Here, we characterize the biochemical and biophysical properties of recombinant human Tyrp1. For this purpose, we purified and analyzed the intra-melanosomal domain (Tyrp1tr) for protein stability and enzymatic function in conditions mimicking the environment within melanosomes and the endoplasmic reticulum. The study suggests that Tyrp1tr is a monomeric molecule at ambient temperatures and below (<25 °C). At higher temperatures, >31 °C, higher protein aggregates form with a concurrent decrease of monomers in solution. Also, Tyrp1tr diphenol oxidase activity at pH 5.5 rises as both the pre-incubation temperature and the higher molecular weight protein aggregates formation increases. The enhanced protein activity is consistent with the volume exclusion change caused by protein aggregates.

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

“…However, only the variant, which was truncated similarly to the Tyr intra-melanosomal domain (just several amino acids at the C-terminus before the transmembrane domain) was stable and properly folded (Figure 1 and Figure S1). The purified Tyrp2 was a monomeric ≈54 kDa glycoprotein (Figure 2, Table 1), similarly to our previously characterized intra-melanosome domains of Tyr and Tyrp1 [14,22]. The metal-binding analysis using ICP-MS showed that Tyrp2 contains zinc atoms in its active site instead of coppers making Tyrp2 the catalyst in the tautomerization versus the copper-mediated oxidation catalyzed by Tyr [23].…”

Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

“…Their surface exhibited a rough, “bumpy” morphology with features measuring ~5–10 nm in height and ~15–20 nm at their base. Before performing AFM on Tyr-bound MB, DLS measurements revealed the hydrodynamic diameter of Tyr to be 8.30 nm, which was similar to that of measurements published previously [ 16 ]. Additionally, the isolated dopachrome products had hydrodynamic diameters of 0.87, 63.0, and 1200.0 nm.…”

Phenylthiourea Binding to Human Tyrosinase-Related Protein 1

“…Because zinc ions, in contrast to copper ions, are not redox-active, this implies that TYRP1 must have a different activity from TYR, as indeed corroborated experimentally [4]. In this respect, the 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase activity of the recombinant melanosomal domain of human TYRP1, reported recently [5], may result from the presence of (trace amounts of) copper ions, and further research is needed to establish the physiological significance of this observation.…”