Molecular Mechanisms Underlying Maintenance Of Immune Hyporesponsiveness To Melanosomal Proteins Under Oxidative Stress

Backgrounds and ObjetivesEumelanin consists of one group of monomer indoles (DHI-Eumelanin and DHICA-Eumelanin) connecting through covalent bonds and highly polymerized with quinones and proteins while formed a kind of heterogeneous copolymers. Two key indole precursors, DHI and DHICA, cross-linked in various proportion, copolymerize to build aπ-stacked layered macromolecular network system. Mutiple proteins participate in melanogenesis to regulate the quality and quantity of melanin. Among them, tyrosinase is the key enzyme required for melanin production, which catalyzes the hydroxylation of L-tyrosine to L-Dopa and the oxidation of L-Dopa to L-Dopaquninone. Dct is another critical enzyme in the melanogenesis pathway that isomerizes dopachrome to dihydroxy indole carboxylic acid (DHICA). However, DHICA could not auto-polymerize, only in the presence of DHI and Tyr can it incorporate into melanin polymer, otherwise dopachrome will be rapidly decarboxylated to DHI accompanied with mass ROS. It has been revealed that the three melanogenic proteins (Tyr, Tyrpl and Dct) compose a multi-enzyme complex in melanosomes in order to raise the efficiency of melanogenesis, maintain the proteins stability and reduce the cytotoxicity of intermediate products maximatily.In the multienzyme complex, Dct is deemed to be a kind of in-situ real-time scavenger, which can immediately scavenge ROS induced by intermediate products, and dynamically change the rate of melanogenesis and biopolymerization of indole molecules by modulating the proportion of DHI/DHICA to improve the protection capability of melanocytes against UVR, and finally increase melanogenesis of skin. Furthermore, in the process of melanin biochemical reaction, the nearly-closed structures inside the melanosomes are considered as a protection strategy of melanocyte to minimize the cytotoxicity of precursors. Melanosomal proteins are very likely to be embedded in the core of each entity in melanosomes, and eumelanin then functions as a shield to protect the entrapped proteins from potential oxidative insults. Meanwhile, melanocytes also provide with powerful enzyme and non-enzyme anti-oxidative defense mechanisms (such as glutathione peroxidase, catalase and Fenton reaction and so on) to immediately scavenge ROS induced by intermediate products and therein protect themselves. However, we still know little about how melanosomal proteins interact with melanin polymers and how melanosomal proteins are protected from oxidative stress damage even under the conditions of high and/or continuously oxidative stress for maintaining mechanism of immune hyporesponsiveness.It is well documented that unusual accumulation of H2O2 is present in the epidermis of active vitiligo, even up to micromolar level. In the skin lesions and/or serum of progressive vitiligo patients, multiple auto-antibodies to melanosomal proteins and expression abnormalities of T lymphocyte are both detected. The functional disorder or missing of melanocytes is regarded as a consequence of an immune response mediated by self-responsive T cells or auto-antibodies against melanosomal proteins. Up to now, how functional dysregulation and molecular injury for Dct gene of melanocytes in vitiligo occur, how the cryptic epitopes of entrapped melanosomal proteins are recognized by immune system and how imumune tolerance of melanosomal proteins is broken remain unclear. Lately, researches discovered that, under certain concentration of hydrogen peroxide (H2O2), thyroglobulin may crack and generate polypeptide with immunoreactivity, which may be identified by antithyroglobulin antibodies in serum of Hashimoto's thyroiditis. Therefore, we assume that the initial causes inducing immune destruction of melanocyte are 1) There are some defects vitiligo melanocytes for eliminating intermediate products of melanogenesis and reactive oxygen species, the latter of which (especially H2O2) may intensively attack melanin polymer, resulting in oxidative fissure of the indole unit within to expose cryptic epitopes to the immune system; 2) Melanosomal protein fragments bound to indole molecules cleave to polypeptides, which are oxidative modificated, if not eliminated instantly, to haptens with high immunogenicity; 3) The disruption of DHICA/DHI indole units due to Dopachrome tautomerase(Dct) inactivation diminishes their anti-oxidation, even showing a pro-oxidant behavior.However, in vivo researches on early upstream events that Dct mediating oxidative stress changes influenced the immunogenicity of melanosomal proteins were hampered, partially due to the multienzyme complex structure inside melanosome constituted by Dct close combining with other melanogenesis proteins. In this study, melanosomal proteins at different stages from Dct-mutant salty MCs (amino acid substitution (R194Q(Arg194→Gln)) that occurred spontaneously in the first metal-binding domain of the Dct protein) and wild-type melan-a MCs were purified with a sucrose gradient centrifugation, and immunized the hind footpad and the base of the tail of each mouse. To further elucidate the molecular mechanism of loss of immune tolerance toward autoantigens in the melanosome membranes and explore the early upstream initiating event involved in the autoimmune response to melanosomal proteins, in vivo assay was performed to determine the immunogenicity of melanosomal proteins derived from these two kinds of melanocyte under different oxidative stress.Methods and Results1. Measurement of melanogenic proteases activities and ROS levels in Dct mutant slaty MCs and wildtype melan-a MCs Tyrosinase, mainly as dopa oxidase, Dct and catalase activities in Dct mutant slaty MCs and wildtype melan-a MCs were measured with a spectrophotometry. The melanocytes were incubated with 100μM H2O2 and the level of intracellular ROS was monitored by 2,7-dichlorofluorescin diacetate (H2DCF-DA) labeling. The results showed that the Dct activity of slaty MCs was significantly lower than that of melan-a MCs, but there was no statistically significant difference in the activity of tyrosinase and catalase in these two groups of MCs (P>0.05). The analysis results of intra-cellular ROS level showed that the relative fluorescence intensity of slaty MCs and melan-a MCs before being treated with H2O2 was 6.33±0.17 and 5.42±0.14, respectively. However, the relative fluorescence intensity of slaty MCs being treated with H2O2 increased abruptly to 18.29±0.54 and there was a statistically significant difference (P<0.05) comparing to that of melan-a MCs (9.14±0.28). 2. Observation of ultrastructure and protein expression of sucrose density gradient ultracentruifugation purified melanosomes in Dct mutant slaty MCs and wildtype melan-a MCs The cells were harvested with 0.25% trypsin/0.02% EDTA and then homogenized on ice using 20 stokes of a Dounce glass-glass homogenizer. Melano-somes in the cellular homogenate were isolated with a sucrose density-gradient ultracentrifugation, the density gradients of sucrose were:1.0,1.2,1.4,1.5,1.6,1.8 and 2.0 M, respectively. Sucrose interfaces between the 1.0 M to 1.2 M and the 1.6 M to 1.8 M were collected for a transmission electron microscopy (TEM) observation to examine the development and maturation of melanosomes. The protease activity of this family of three tyrosinases and their protein expression levels were assayed by spectrometry and Western Blot. The results showed that the color of homogenated cell aggregates of these two MCs was brown and milk yellow, respectively. Isolated by sucrose gradient ultracentrifugation, melanosomes were localized at various layers of the gradient. The TEM observation indicated that the 1.2-1.4 M sucrose fragment was mainly spherical or oval melanosomes in StageⅠ-Ⅱ(early stage melanosomes), while the 1.6-1.8 M melanosomal fraction contained mainly melanosomes in Stage III-IV (late stage melanosomes) with melanin depositions in the fibrous transverse ridges. Comparing to that of melan-a MCs, the 1.6-1.8 M sucrose fraction of slaty MCs was primarily filled with Stage III melanosomes. The Western Blot Analysis showed that the Dct activity of later stage melanosomal proteins of slaty MCs was decreased significantly and also there was a statistically significant difference comparing to that of melan-a MCs. However, the change of the activity of tyrosinase in these two groups of melanosomal proteins was not obvious.3. Groups and immune challenge of experimental mice CB6F1 mice (BABL/C×C57BL/6 hybridization F1 generation, HLA haplotype is H2d×2b) were housed in pathogen-free microisolator cages in our animal facility. Melanosomal proteins were treated with 3 different factors for subsequent immune challenge in mice, then compared their immunogenicity changes:1) The two MCs were pulsed with 100μM H2O2 for 1 hour, then early stage and late stage melanosomal proteins were purified with the sucrose density-gradient ultracentrifugation to evaluate the maintenance of immune hyporesponsiveness to melanosomal proteins.2) Synthetic DHI-eumelanin and DHICA-eumelanin were incubated with that two late stage melanosomal proteins respectively and then treated with H2O2 for antioxidative protection determination.3) Late stage melanosomal proteins were treated with H2O2 respectively to validate if melanosomal proteins at late stage of Dct mutant salty MC more sensitive to oxidative stress. In each assay, chicken ovalbumin, a well-defined model antigen containing an immunodominant epitope in H-2b mice, was used as positive control. The results showed that gradual swellings of the immunized-side footpad and ankle of the mice were seen after the mice had been immunized with 50μg (microgram) of melanosomal proteins emulsified in an equal volume of complete Freund's adjuvant (CFA) for 1 week and there were significant swellings of the groin, the rib draining lymph nodes and the spleen when the mice were sacrificed and the regional draining lymph nodes were stripped 3 weeks later.4. Effects of oxidative stress and synthetic DHI/DHICA(1:1)-eumelanin on the immunogenicity of melanosomal proteins Primed T lymphocytes were isolated and counted with Trypan Blue Staining then cultured in 96-well plates which were added with the same proteins in a final concentration of 0.3,1,3,10 and 30μg/mL, respectively. After 48 h at 37℃, [3H] thymidinewas added and the cultures were harvested 16 h later using a multichannel cell harvester. The radioactivity of each sample was counted in a liquid scintillation counter. After the microtiter plates were coated with the same melanosomal proteins, the titers of antimelanosomal protein antibodies were calculated as the dilution of the test serum to the end point. Enlarged T-lymphocyte colonies of late stage melanosomes isolated from H2O2-treated slaty MCs were more than those of the early stage melanosomes under microscopy. Large amounts of darkly stained cytoplasm were seen in those spherical or oval cells. The T-lymphocyte colonies were less proliferated and smaller in DHI/DHICA(1:1)-eumelanin incubated slaty MC late stage melanosomal proteins group than DHI-eumelanin incubated group. H2O2 pulsed late stage melanosomal protein from staty MC stimulated significant proliferation and enlargement of T-lymphocyte colonies comparing to the single protein group, yet this phenomenon was not displayed in the group of late stage melanosoml protein from melan-a MC even in presence of H2O2. The 3H-TdR and ELISA assays showed that mice exhibited hyperresponsiveness to a challenge with late-stage melanosomal proteins, especially to late-stage melanosomal proteins derived from Dct-mutant melanocytes. T cell proliferation and IgG antibody responses were significantly induced by late-stage melanosomal proteins incubated with DHI-melanin, but not with DHI/DHICA-melanin, and the same results were showed in slaty MC late-stage melanosomal protein group rather than in melan-a MC late-stage melanosomal protein group, which revealed late-stage melanosomal proteins from slaty MC were more sensitive in immunogenic changes induced by oxidative stess.ConclusionsDct regulates the antioxidative capacity of eumelanin by changing the proportion of DHICA monomer incorporated into the DHI polymer backbone, which plays a ctritical role in the maintenance of immune hyporesponsiveness to melanosoml proteins. Mutations in Dct seriously affected the development and maturation of later stage melanosomes, decreased DHICA formation and weakened ROS scavenging, especially under oxidative stess. Mouse slaty mutation, a single amino acid substitution (R194Q(Arg194→Gln)) that occurred spontaneously in the first metal-binding domain of the Dct protein, seriously affects the steric configuration of the mutant Dct protein and the stability of the melanogenic protein complex. Under high and/or sustained oxidation stress, the oxidative modificated melanomal proteins expose partial cryptic epitopes, which enhances the immunogenicity of melanosomal proteins.