Assembly, target-signaling and intracellular transport of tyrosinase gene family proteins in the initial stage of melanosome biogenesis

Assembly, target-signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein-3 (AP-3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di-leucine motif in their cytoplasmic tail, to which AP-3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Assembly,Target‐Signaling and Intracellular Transport of Tyrosinase Gene Family Proteins in the Initial Stage of Melanosome Biogenesis

Assembly, target‐signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein‐3 (AP‐3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di‐leucine motif in their cytoplasmic tail, to which AP‐3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Inulavosin and its benzo‐derivatives,melanogenesis inhibitors,target the copper loading mechanism to the active site of tyrosinase

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure–activity relationship of inulavosin and its benzo‐derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper‐binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo‐derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo‐tyrosinase that has a conformational defect.     

Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in Mammalian tyrosinase. Implications to the catalytic cycle.

Tyrosinase (Tyr) and tyrosinase-related proteins (Tyrps) 1 and 2 are the enzymes responsible for mammalian melanogenesis. They display high similarity but different substrate and reaction specificities. Loss-of-function mutations lead to several forms of albinism or other pigmentation disorders. They share two conserved metal binding sites (CuA and CuB) which, in Tyr, bind copper. To define some structural determinants for these differences, we mutated Tyr at selected residues on the basis of (i) conservation of the original residues in most tyrosinases, (ii) their nonconservative substitution in the Tyrps, and (iii) their possible involvement as an endogenous bridge between the copper pair. Two mutations at the CuA site, S192A and E193Q, did not affect Tyr activities, thus excluding S192 and E193 as endogenous ligands of the copper pair. Concerning CuB, the H390Q mutation completely abolished Tyr activity, whereas Q378H and H389L mutants showed 10-20% residual specific activities. Their kinetic behavior suggests that (i) H390 is the actual third ligand for CuB, (ii) H389 is critical for stereospecific recognition of o-diphenols but not monophenols, and (iii) the involvement in metal binding of the central extra H residue at the Tyrps CuB site is unlikely. However, replacement of Q (in Tyr) by H (in Tyrps) greatly diminished the affinity for L-dopa, consistent with the low/null tyrosinase activity of the Tyrps. These are the first data showing a physical difference in docking of mono- and o-diphenols to the Tyr active site, and they are used to propose a revised scheme of the catalytic cycle.     

Molecular basis of the extreme dilution mottled mouse mutation: a combination of coding and noncoding genomic alterations

Tyrosinase is the rate-limiting enzyme in melanin biosynthesis. It is an N-glycosylated, copper-containing transmembrane protein, whose post-translational processing involves intracytoplasmic movement from the endoplasmic reticulum to the Golgi and, eventually, to the melanosome. The expression of the tyrosinase (Tyr) gene is controlled by several regulatory regions including a locus control region (LCR) located 15 kb upstream from the promoter region. The extreme dilution mottled mutant mice (Tyrc-em) arose spontaneously at the MRC Institute in Harwell (United Kingdom) from a chinchilla-mottled mutant (Tyrc-m) stock, whose molecular basis corresponds to a rearrangement of 5'-upstream regulatory sequences including the LCR of the Tyr gene. Tyrc-em mice display a variegated pigmentation pattern in coat and eyes, in agreement with the LCR translocation, but also show a generalized hypopigmented phenotype, not seen in Tyrc-m mice. Genomic analyses of Tyrc-em mice showed a C1220T nucleotide substitution within the Tyr encoding region, resulting in a T373I amino acid change, which abolishes an N-glycosylation sequon located in the second metal ion binding site of the enzyme. Tyrosinase from Tyrc-em displayed a reduced enzymatic activity in vivo and in vitro, compared with wild-type enzyme. Deglycosylation studies showed that the mutant protein has an abnormal glycosylation pattern and is partially retained in the endoplasmic reticulum. We conclude that the phenotype of the extreme dilution mottled mouse mutant is caused by a combination of coding and noncoding genomic alterations resulting in several abnormalities that include suboptimal gene expression, abnormal protein processing, and reduced enzymatic activity.     

Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity

Pigmentation of the hair, skin, and eyes of mammals results from a number of melanocyte-specific proteins that are required for the biosynthesis of melanin. Those proteins comprise the structural and enzymatic components of melanosomes, the membrane-bound organelles in which melanin is synthesized and deposited. Tyrosinase (TYR) is absolutely required for melanogenesis, but other melanosomal proteins, such as TYRP1, DCT, and gp100, also play important roles in regulating mammalian pigmentation. However, pigmentation does not always correlate with the expression of TYR mRNA/protein, and thus its function is also regulated at the post-translational level. Thus, TYR does not necessarily exist in a catalytically active state, and its post-translational activation could be an important control point for regulating melanin synthesis. In this study, we used a multidisciplinary approach to examine the processing and sorting of TYR through the endoplasmic reticulum (ER), Golgi apparatus, coated vesicles, endosomes and early melanosomes because those organelles hold the key to understanding the trafficking of TYR to melanosomes and thus the regulation of melanogenesis. In pigmented cells, TYR is trafficked through those organelles rapidly, but in amelanotic cells, TYR is retained within the ER and is eventually degraded by proteasomes. We now show that TYR can be released from the ER in the presence of protonophore or proton pump inhibitors which increase the pH of intracellular organelles, after which TYR is transported correctly to the Golgi, and then to melanosomes via the endosomal sorting system. The expression of TYRP1, which facilitates TYR processing in the ER, is down-regulated in the amelanotic cells; this is analogous to a hypopigmentary disease known as oculocutaneous albinism type 3 and further impairs melanin production. The sum of these results shows that organellar pH, proteasome activity, and down-regulation of TYRP1 expression all contribute to the lack of pigmentation in TYR-positive amelanotic melanoma cells.     

C-terminus glycans with critical functional role in the maturation of secretory glycoproteins

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.     

Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.     

Sorting and targeting of melanosomal membrane proteins: signals, pathways, and mechanisms

Newly synthesized melanosomal proteins, like many other cellular proteins, traverse through a series of intracellular compartments en route to melanosomes. Entry and exit of proteins through these compartments is orchestrated by cellular sorting machinery that recognize specific sorting signals. Melanosomal membrane proteins begin their intracellular journey upon co-translational importation into the endoplasmic reticulum (ER). The biosynthetic output of tyrosinase, the key melanogenic enzyme, appears to be regulated by quality-control events at the ER, the 'port of entry' to the secretory pathway. Following maturation in the ER and through the Golgi, the sorting of these proteins in the trans-Golgi network for intracellular retention and transport along endosome/lysosome pathway requires cytoplasmically exposed signals. A di-leucine motif, present in the cytoplasmic tails of most melanosomal proteins, and its interaction with adaptor protein (AP) complexes, specifically AP-3, are critical for these events. Defects in sorting signals and the cytosolic components that interact with these signals result in a number of murine coat color phenotypes and cause human pigmentary disorders. Thus, missense or frame-shift mutations that produce truncated tyrosinase lacking the melanosomal sorting signal(s) appear to be responsible for murine platinum coat color phenotypes and a proportion of human oculocutaneous albinism-1; mutations in AP-3 appear to be responsible for the mocha phenotype in mice and Hermansky-Pudlak-like syndrome in man. Additional signals and sorting steps downstream of AP-3 appear to be required for endosomal sorting and targeting proteins to melanosomes. Signals and mechanisms that sequester melanosomal proteins from endosomes/lysosomes are not understood. Potential candidates that mediate such processes include proteins encoded by lyst and pallid genes. The common occurrence of abnormalities in melanosomes in many storage-pool disorders suggests that melanocytes utilize signals, pathways, and mechanisms shared by other proteins and cell types to assemble a number of specialized proteins and produce unique cell-type-specific organelles.     

Low temperature blocks exit of pro-opiomelanocortin from the endoplasmic reticulum but not subsequent delivery to the site of prohormone processing

The effect of reduced temperature on the delivery of the prohormone pro-opiomelanocortin (POMC) to the site of prohormone processing was investigated in the mouse anterior pituitary cell line AtT20. At 20 degrees C processing was substantially inhibited and was almost completely arrested at 18 degrees C. Earlier studies with membrane glycoproteins indicated that at these temperatures protein movement was blocked at the level of exit from the Golgi apparatus. In contrast it was found here that the inhibition of processing at reduced temperature was due to the retention of POMC in the endoplasmic reticulum. When POMC was allowed to progress to the Golgi before temperature was reduced, subsequent processing was only slightly retarded by incubation at 18 degrees C. This indicates either that Golgi exit is not inhibited at this temperature, or that the processing apparatus exists in the Golgi. A surprising incidental result was that when held in the endoplasmic reticulum at low temperature POMC is apparently subject to post-translational N-linked glycosylation.     

Tyrosinase expression during black truffle development: from free living mycelium to ripe fruit body

The present work studies the expression of tyrosinase (monophenol:diphenol oxygen oxidoreductase, EC 1.14.18.1) during the development of the black truffle Tuber melanosporum Vittad., an ectomycorrhizal fungus of great biological and economic interest. As widely reported in the literature, melanins and the enzymes that synthesize them, are of paramount importance in fungal development and sexual differentiation. Tyrosinase and laccase are the enzymes that produce melanins from monophenols and diphenols. We have detected tyrosinase expression from the stage of free living mycelium, through the mychorrizal stage and the six fruit body developmental stages by measuring the levels of tyrosinase mRNA by quantitative PCR (q-PCR), spectrophotometry, histochemistry, immunohistochemistry and electrophoresis. Tyrosinase is always expressed, from the free living mycelium to the ripe fruit body developmental stages, when it is very low. The switching off of the tyrosinase gene during T. melanosporum development when the fruit body is ripe and no more cell walls are to be built is discussed in relation of thioflavour production. Specific primers, prepared from the cloned T. melanosporum tyrosinase cDNA were used for the q-PCR and the deduced aminoacid sequences of the CuA and CuB binding sites were compared to those of various ascomycetes and basidiomycetes.     

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.     

Identification of a Site in Sar1 Involved in the Interaction with the Cytoplasmic Tail of Glycolipid Glycosyltransferases

Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1^D198A) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1^D189A also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.     

Tyrosinase of hamster melanoma: its purification and estimation by radioimmunoassay

1. Tyrosinase was purified from melanosomal fraction of hamster melanoma. 2. A radioimmunoassay was developed to quantitate the tyrosinase protein in hamster serum and hamster melanoma tissue using polyclonal anti-tyrosinase antibodies and 125I-labeled enzyme. 3. The serum tyrosinase levels were found to be about 0.24 micrograms and 1.14-4.48 micrograms/ml in normal hamsters and melanoma-bearing hamsters, respectively. 4. Tyrosinase protein in serum correlated significantly with the enzyme activity in hamsters with melanoma (r = 0.733). 5. In the cytosol fraction of hamster melanoma, a level of 2.2 micrograms of tyrosinase/mg protein was determined. 6. The usefulness and possible applications of the tyrosinase radioimmunoassay are discussed.     

Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity

The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH.     

Degradation of tyrosinase induced by phenylthiourea occurs following Golgi maturation

Tyrosinase, the rate-limiting enzyme of melanin synthesis, is a di-copper metalloprotein that catalyzes the conversion of L-tyrosine to L-DOPAquinone. Phenylthiourea (PTU) is a well-known inhibitor of tyrosinase and melanin synthesis and is known to interact with sweet potato catechol oxidase, an enzyme possessing copper binding domain homology to tyrosinase. While PTU is frequently used to induce hypopigmentation in biological systems, little is known about its effects on tyrosinase and other melanogenic proteins. We have found that PTU induces degradation of tyrosinase but not of other melanogenic proteins including the tyrosinase-related metalloproteins tyrosinase-related protein (Tyrp)1 and Tyrp2. Using pulse-chase analysis coupled with glycosidase digestion, we observed that tyrosinase degradation occurs following complete maturation of the protein and that degradation was reversed by cysteine protease inhibitor E64 but not proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. We conclude that PTU specifically induces tyrosinse degradation following Golgi maturation. Our data suggest that in addition to well-known ER-directed quality control, tyrosinase is also subject to post-Golgi quality control.     

Neurospora tyrosinase: structural,spectroscopic and catalytic properties

Summary Tyrosinase is a copper containing monooxygenase catalyzing the formation of melanin pigments and other polyphenolic compounds from various phenols. This review deals with the recent progress on the molecular structure of the enzyme from Neurospora crassa and the unique features of the binuclear active site copper complex involved in the activation of molecular oxygen and the binding of substrates. The results of the spectroscopic properties of Neurospora tyrosinase will also be discussed in the light of the structural similarity of the copper complex in the oxygen binding hemocyanins.     

Translation rate of human tyrosinase determines its N-linked glycosylation level

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or seven N-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential cotranslational maturation events.     

Analysis of Tyrosinase Mutations Associated With Tyrosinase-Related Oculocutaneous Albinism (OCAI)

Mutations of the tyrosinase gene associated with a partial or complete loss of enzymatic activity are responsible for tyrosinase related oculocutaneous albinism (OCA1). A large number of mutations have been identified and their analysis has provided in-sight into the biology of tyrosinase and the pathogenesis of these different mutations. Missense mutations produce their effect on the activity of an enzyme by altering an amino acid at a specific site. The location of these mutations in the peptide can be used to indicate potential domains important for enzymatic activity. Missense mutations of the tyrosinase polypeptide cluster in four regions, suggesting that these are important functional domains. Two of the potential domains involve the copper binding sites while the others are likely involved in substrate binding. More critical analysis of the copper binding domain of tyrosinase can be gained by analyzing the structure of hemocyanin, a copper-binding protein with a high degree of homology to tyrosinase in the copper binding region. This analysis indicates a single catalytic site in tyrosinase for all enzymatic activities.