Introduction

It is now well established that the proopiomelanocortin (POMC)-processing machinery is present in human skin (Slominski and Wortsman, 2000). The involvement of POMC-derived peptides such as ACTH, -, and -melanocyte-stimulating hormone (MSH) in human skin pigmentation was first recognized upon systemic application into human volunteers, where these peptides induced noticeable skin darkening especially in the sun-exposed body region (Lerner and McGuire, 1961, 1964; Geschwind et al., 1972). Today, there is compelling evidence that POMC-derived peptides are implicated in the regulation of skin color in humans (Wakamatsu et al., 1997; Slominski et al., 2004). It has been shown that the skin and the hair follicle are local sources and targets for POMC-derived peptides including ACTH, -, and -MSH as well as -endorphin (Wakamatsu et al., 1997; Thody and Graham, 1998; Kauser et al., 2003; Spencer et al., 2005). Only recently, it was recognized that -endorphin can also induce pigmentation and proliferation via the -opiate receptor of epidermal and hair follicle melanocytes and that this peptide modulates cell dendricity (Kauser et al., 2003, 2005). Earlier, it was shown that -MSH stimulates changes in melanocyte morphology, growth rates, and melanin production (McLane and Pawelek, 1988; Chakraborty et al., 1991). Moreover, the presence of ACTH, -, -MSH, and -endorphin together with the complete POMC-processing machinery, including prohormone convertases 1/2 (PC1 and PC2) and the regulatory protein 7B2, has been demonstrated inside the melanosome of epidermal and hair follicle melanocytes (Peters et al., 2000; Kauser et al., 2003; Spencer et al., 2005). In this context, a receptor-independent regulation of tyrosinase (EC 1.14.18.1), the key enzyme for pigmentation, by - and -MSH via the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH₄) and its 7-isomer has been put forward (Schallreuter et al., 1999a; Peters et al., 2000; Spencer et al., 2005).

Besides the function to regulate pigmentation vis a vis POMC-derived peptides, a wide range of pleiotropic functions for -MSH has been discovered and documented in the skin including the modulation of inflammatory stimuli such as regulation of expression and secretion of chemokines, downregulation of proinflammatory signal-induced NF-B activation and adhesion molecule expression, prostaglandin E2 synthesis, as well as induction of IL-10 (reviewed by Luger et al., 1999). In dermal fibroblasts, -MSH modulates collagen metabolism by upregulating interstitial collagenase as well as by attenuating the inductive effect of transforming growth factor-1 on collagen synthesis and fibrosis (Graham et al., 1999; Thody, 1999; Bohm and Luger, 2004). Moreover, an antioxidant capacity was proposed and demonstrated for -MSH by several investigators (Valverde et al., 1996; Haycock et al., 2000; Moustafa et al., 2002).

Previously, reduced POMC processing has been reported in the epidermis of patients with the pigmentation disorder vitiligo (Graham et al., 1999). To date, there are several lines of in vivo and in vitro evidence for the accumulation of hydrogen peroxide (H₂O₂) in the 10^-3 M range in the epidermal compartment of acute vitiligo (Schallreuter et al., 1999b, 2001), and therefore it was suggested that the reduced expression of -MSH as well as the PC1 and PC2 in vitiligo epidermis possibly contributes to an impaired antioxidant defense capacity in this disorder (Graham et al., 1999). The accumulation of H₂O₂ leads to a plethora of events including oxidation of methionine (Met) and tryptophan (Trp) residues in epidermal enzyme and protein structures; for instance, catalase (Maresca et al., 2006), dihydropteridine reductase (Hasse et al., 2004), acetylcholinesterase (Schallreuter et al., 2004), pterin 4a-carbinolamine dehydratase (Schallreuter et al., 2001), and albumin (Rokos et al., 2004; Hasse et al., 2005) as well as oxidation of the essential cofactor 6BH₄ in both melanocytes and keratinocytes (Moore et al., 2002; Rokos et al., 2002). The latter pathway and the cofactor itself are greatly affected by H₂O₂-mediated oxidation in vitiligo (Schallreuter et al., 1994; Schallreuter, 2005).

Taken together, this disease provides an excellent model to follow the influence of oxidative stress in vivo and in vitro. As Met and/or Trp residues in the sequence of POMC-derived peptides are possible targets for H₂O₂ oxidation, it was tempting to ask the question whether these amino acids are indeed oxidized in these peptides by this reactive oxygen species. In order to address this question, we employed different techniques including in situ immunofluorescence labelling and image analysis, dot blot analysis, Fourier transform (FT)-Raman spectroscopy, functional studies, and computer simulation of the peptide structures. The immunoreactivity of the in situ study confirmed a significantly lower expression of -MSH in untreated vitiligo as reported earlier by Graham et al. (1999). In addition, a significantly reduced expression was also observed for -endorphin, whereas -MSH and ACTH expression was higher. After removal of epidermal H₂O₂ by a narrowband-activated pseudocatalase PC-KUS (Schallreuter et al., 1999b), both -MSH and -endorphin expression returned to normal. The direct effect of H₂O₂ on the peptides revealed -endorphin as the most sensitive peptide followed by -MSH and ACTH, whereas -MSH was only affected by extremely high concentrations of this reactive oxygen species. These findings were in agreement with the formation of Met sulfoxide from Met as shown by FT-Raman spectroscopy and computer simulation in the presence of H₂O₂. Oxidation of the peptides also affected the functionality because oxidized -endorphin could not promote melanogenesis in primary human melanocytes compared to the native peptide. In addition, we provide evidence that complexation of 6BH₄ to -MSH in a 1:1 stoichiometry protects both -MSH and the cofactor against oxidation by H₂O₂, offering a novel mechanistic basis for the proposed antioxidant property of -MSH.

Our results demonstrate that epidermal POMC peptides can be directly targeted by H₂O₂ in vitro but also in vivo as shown in the skin of patients with vitiligo. This oxidation affects the peptide structure and consequently their functionality. However, these changes are reversible after reduction of H₂O₂ levels by a pseudocatalase PC-KUS. Based on these results, we propose an important role for H₂O₂ in control of the redox balance of POMC peptides and consequently their downstream signals including pigmentation.

Results

Decreased epidermal -MSH and -endorphin peptide expression in vitiligo recovers after H₂O₂ removal with pseudocatalase PC-KUS

Earlier, it was shown that -MSH, PC1, PC2, and 7B2 expression was decreased in the epidermis of patients with vitiligo, but a mechanism for this loss escaped definition so far (Graham et al., 1999). As the sequences of all peptides as well as the PCs contain Met and/or Trp residues and these amino acids can be targets for oxidation by H₂O₂ as shown recently by us and others, it was tempting to investigate whether epidermal POMC peptide expression was possibly affected by H₂O₂. For this purpose, we evaluated the immunoreactivity of ACTH, -, -MSH, and -endorphin in skin biopsies of untreated and treated patients in situ compared with healthy controls. The immunoreactivity was subject to image analysis as described in the Materials and Methods section.

The results showed indeed that both epidermal -MSH and -endorphin peptide expression is significantly reduced in untreated patients with active disease compared to healthy controls (Figure 1a, b, e, and f). After removal/reduction of epidermal H₂O₂ with narrowband-activated pseudocatalase PC-KUS, peptide expression was significantly higher compared to controls (Figure 1a, c, e, and g). As accumulation of H₂O₂ in the 10^-3 M range had already been demonstrated earlier by in vivo FT-Raman spectroscopy (Schallreuter et al., 1999b), and this process is reversible after reduction of H₂O₂ levels below 10^-3 M concentrations with a pseudocatalase PC-KUS (Schallreuter, 1999), these in situ results strongly suggested that H₂O₂ alters the structure yielding to a loss of antigenic epitope recognition of both POMC peptides.

Figure 1.

In situ expression of POMC peptides in healthy controls and in patients with vitiligo. Epidermal expression of the POMC peptides (a–d) -MSH and (e–h) -endorphin, (i–l) -MSH, and (m–p) ACTH, is decreased in active patients with vitiligo compared to healthy controls. After reduction/removal of epidermal H₂O₂ by a narrowband UVB-activated pseudocatalase PC-KUS, the peptides recover (a, e, i, m) healthy control, (b, f, j, n) untreated skin, (c, g, k, o) treated repigmenting skin, (d, h, l, p) antibody neutralization with blocking peptide, and (q–t) image analysis of staining intensities. Bar=100 M. Note the strong expression of -endorphin in basal melanocytes (e, g).

Full figure and legend (365K)

Interestingly, epidermal -MSH expression appeared to be unaffected by H₂O₂. Figure 1i, k, and q–t shows a small increase, although significant compared to control skin after reduction of epidermal H₂O₂ (P<0.005). Expression of ACTH is significantly increased in situ compared to controls (Figure 1m–o and q–t) and peptide expression is even higher following removal of epidermal H₂O₂. This result suggests that PC1 must be more active in vitiligo to produce ACTH.

H₂O₂ alters the structure of ACTH, -MSH, and -endorphin but not -MSH

The structures of all peptides studied highlighting the position of H₂O₂-sensitive Met residues are presented in Table 1. In order to examine the structural stability of ACTH, -, -MSH, and -endorphin to direct oxidation by H₂O₂, we employed dot blot analysis in the absence and presence of increasing H₂O₂ concentrations as outlined in Materials and Methods. The results showed that -endorphin is the most sensitive peptide to H₂O₂ oxidation followed by -MSH and ACTH, whereas -MSH is significantly more stable under the same experimental conditions. (Figure 2a–d).

Figure 2.

The direct effect of H₂O₂ on POMC peptides. Dot blot analysis in the absence and presence of H₂O₂ (0–100 10^-3 M). (a) -MSH, (b) -endorphin, (c) -MSH, and (d) ACTH. The result indicates structural alteration with a possible loss of antigenic epitope of -MSH, -endorphin, and ACTH with increasing H₂O₂ concentrations, whereas -MSH is much more stable to oxidation. Data shown are meanSEM (n=3).

Full figure and legend (114K)

Table 1 - The position of Met and Trp residues in the primary sequences of POMC-derived peptides as potential targets for H₂O₂ oxidation.

Full table

H₂O₂-oxidized -endorphin looses its function in the promotion of melanogenesis

As structural alteration could be a prerequisite for the lack of epitope recognition by the antibodies, we tested the functionality of -endorphin as one representative peptide following induction of melanogenesis in primary human epidermal melanocytes in the presence of the oxidized and native peptide. The result of this experiment is presented in Figure 3, showing that -endorphin after oxidation with 10^-3 M H₂O₂ cannot induce melanogenesis compared to the native peptide. Therefore, we can conclude that oxidation of the peptide severely affects downstream signalling.

Figure 3.

H₂O₂-oxidized -endorphin stops the induction of pigmentation in melanocytes in vitro. Addition of native -endorphin (10^-8 M) shows a statistically significant increase of melanin formation compared to the untreated control cells (^***P<0.001). After oxidation of -endorphin with 10^-3 M H₂O₂, this effect is significantly abrogated proving that the oxidation of the peptide affects the function/downstream signal of the native peptide (^*P<0.05). Melanin production is standardized to 1 mg of melanocyte protein. Results are presented as the mean of three determinationsSEM. (ns P>0.05)

Full figure and legend (30K)

-MSH/6BH₄ 1:1 complexation protects both the peptide and the cofactor against H₂O₂-mediated oxidation

Taking into consideration that -MSH has antioxidant properties (Valverde et al., 1996; Haycock et al., 2000; Moustafa et al., 2002), and that the results of this study show that this peptide is sensitive to direct oxidation by H₂O₂ (Figure 2a), we asked the question, how can -MSH exert antioxidant properties if it is sensitive to H₂O₂ oxidation itself? In this context, it was reconciled that -MSH reacts with the cofactor 6BH₄ by forming a 1:1 complex and that this complex can protect 6BH₄ against H₂O₂ oxidation (Moore et al., 1999; Schallreuter et al., 1999a). Based on those earlier observations, it was tempting to explore whether the -MSH/6BH₄ 1:1 complex could possibly also protect -MSH from oxidation by H₂O₂. The results of this study proved that -MSH is indeed protected against H₂O₂ oxidation up to concentrations of 5 10^-3 M, whereas -MSH alone is oxidized (Figure 4). Based on these results, we can conclude that the complex is an efficient scavenger for H₂O₂.

Figure 4.

Oxidation of -MSH and 6BH₄ is proteceted against oxidation by H₂O₂ in the -MSH/6BH₄ 1:1 stoichiometry complex. Dot blot analysis shows that oxidation of -MSH in the complex together with 6BH₄ is indeed protected. Data shown are meanSEM (n=3) oxidation up to 5 10^-3 M H₂O₂, supporting a significant antioxidant capacity for -MSH and 6BH₄.

Full figure and legend (45K)

FT-Raman spectroscopy confirms oxidation of Met to Met sulfoxide in ACTH, -, -MSH, and -endorphin

As all four peptide hormones contain a single Met residue in their sequence (Figure 5) and this residue is a sensitive target of H₂O₂ oxidation, we employed FT-Raman spectroscopy to follow the oxidation of the Met residues to Met sulfoxide. The result of this analysis proved the oxidation of Met by H₂O₂ in all four peptides yielding the expected S=O-stretch at 1026 cm^-1. The correct assignment was confirmed using a Met sulfoxide standard (Figure 5).

Figure 5.

FT-Raman spectroscopy confirms oxidation of Met to Met sulfoxide in ACTH, -, -MSH, and -endorphin, yielding the S=O-stretch at 1026 cm^-1 (

). The correct assignment confirmed using a Met sulfoxide standard (

). (a) Native -endorphin (—) compared to oxidized -endorphin (- - -). (b) Native -MSH (—) compared to oxidized -MSH (- - -). (c) Native -MSH (—) compared to oxidized -MSH (- - -). (d) Native ACTH (—) compared to oxidized ACTH (- - -). (e) Met (—) compared to Met sulfoxide standard (- - -). The second peak (

) can be attributed to Met sulfone (......). The peak at 1004 cm^-1 in (a–d) refers to phenylalanine.

Full figure and legend (84K)

Computer simulation supports structural alteration by H₂O₂-mediated oxidation for ACTH and -endorphin, whereas -MSH is only weakly affected

As it has been established in vivo that epidermal H₂O₂ accumulation in patients with vitiligo reaches concentrations in the 10^-3 M range (Schallreuter et al., 1999b; Schallreuter, 2005) and these concentrations are sufficient to oxidize Met and Trp residues to Met sulfoxide and to 5-OH-Trp, respectively (Schallreuter et al., 2001; Hasse et al., 2004; Schallreuter et al., 2004; Schallreuter, 2006; Wood, 2006), we employed Hyperchem™ software to ascertain structural changes for the melanocortins ACTH, -MSH, and -endorphin before and after oxidation of Met and Trp residues with H₂O₂. Unfortunately, the structure of -MSH in solution in crystals and upon association with phospholipids reveals that secondary structural features (e.g. helices and -pleated sheets) only occur when -MSH associates with phospholipids (Biaggi et al., 1996, 1997; Contreras et al., 2001). Therefore, it was concluded that -MSH has no secondary structure in solution (Admiraal and Vos, 1984; Biaggi et al., 1996, 1997; Prabhu et al., 1999). Using Hyperchem™, we were unable to find any defined structure for -MSH. Therefore, we examined its structure in water using Circular Dichroism, Intrinsic Fluorescence of Trp, and Isothermal Calorimetry. These methods confirmed that -MSH is a random structure in solution (Pey et al., 2006, unpublished results). The structural changes of the other peptides after H₂O₂ exposure and the structures of the native peptides were superimposed and are presented in Figure 6a–c. As shown in Table 1, ACTH and -MSH have single Met and Trp residues in their sequence, whereas -endorphin contains only one Met residue. Computer simulation supports a significantly altered structure in the case of ACTH and -endorphin, whereas the -MSH structure is only slightly affected after H₂O₂ oxidation. The latter result is in agreement with the in situ results (Figure 1e–h and i–l) and dot blot analyses (Figure 2b and c), favoring a possible loss of the antigenic epitope. However, despite the computer and dot blot analyses indicate instability for ACTH to H₂O₂, we found increased in situ peptide expression (Figure 1m–p). As pointed out above, this result may suggest that the processing of ACTH by PC1 could be altered.

Figure 6.

Comparison of computer-simulated native and H₂O₂-oxidized POMC peptide structures. (a) -MSH. Met¹¹ and Trp¹⁶ of native -MSH are shown in green, MetSO¹¹ and 5-OH-Trp¹⁶ of H₂O₂-oxidized -MSH are shown in orange. N.B. The structural changes are very small after oxidation of the Met and Trp residues in this POMC peptide (native peptide in yellow, oxidized in light blue). (b) -Endorphin. Met⁵ of native -endorphin is shown in green, MetSO⁵ of H₂O₂-oxidized -endorphin is shown in orange. This oxidation yields a significant change in the secondary structure of the peptide (native peptide in yellow, oxidized in light blue). (c) ACTH. Met⁴ and Trp⁹ of native ACTH are shown in green, MetSO⁴ and 5-OH-Trp⁹ of H₂O₂ oxidized ACTH are shown in orange. Oxidation of those residues leads to major changes in the secondary structure in this peptide (native peptide in yellow, oxidized in light blue).

Full figure and legend (264K)

Discussion

Vitiligo is a depigmentation disorder characterized by the sudden loss of inherited skin color. The cause of the disease is still unknown. One dogma is that the pigment-forming melanocyte is completely absent in affected areas (Le Poole et al., 1993). However, there is evidence that even in long-lasting disease melanocytes are still present but these cells have lost their integrity and functionality (Tobin et al., 2000). This loss is associated with severe epidermal oxidative stress caused by the continuous accumulation of H₂O₂ up to concentrations in 10^-3 M range as shown in vivo (Schallreuter et al., 1999b; Schallreuter, 2005). One consequence of this H₂O₂-mediated stress is the oxidation and deactivation of several important enzymes and proteins in the epidermal compartment of these patients owing to oxidation of Met and Trp residues in their sequences causing pronounced structural changes (Schallreuter et al., 2001, 2004; Hasse et al., 2004; Maresca et al., 2006). Structural analysis of POMC-derived peptides revealed the presence of Met and Trp residues as potential targets for H₂O₂-mediated oxidation. Moreover, an earlier study by Graham et al. (1999) showed that -MSH expression was decreased in the epidermis and in melanocytes of patients with vitiligo. Therefore, we investigated the fate of ACTH, -, -MSH, and -endorphin expression in situ in these patients before and after reduction/removal of epidermal H₂O₂ with a narrowband UVB-activated pseudocatalase PC-KUS (Schallreuter et al., 1999b). Moreover, we followed the direct susceptibility of these melanocortins to H₂O₂-mediated oxidation. The results revealed that -endorphin and -MSH but not ACTH and -MSH show a significantly reduced expression in patients with untreated vitiligo compared to controls. Moreover, we showed that the most sensitive peptide -endorphin lost its functionality after oxidation by 10^-3 M H₂O₂ (Figure 3). These in situ results were supported by H₂O₂ oxidation/stability studies, where sensitivities are -endorphin>-MSH >ACTH>-MSH. The structural simulation study on -MSH implies that this peptide is not an easy target to oxidation owing to the position of Met and Trp in the sequence, whereas Met in -endorphin is very exposed. -MSH also contains its Met residue in a very exposed position. However, owing to its small size, it is not possible to study the effect of H₂O₂ on its structure in solution by computer simulation (Prabhu et al., 1999). Although native ACTH is susceptible to oxidative damage owing to the position of both Met and Trp residues, the in situ results do not show a loss of this peptide (Figure 1d). It could be possible that the significant increase of ACTH compared to controls is based on low PC2 levels in these patients as reported previously (Graham et al., 1999), or on increased PC1 activity. As this PC is coupled to the regulatory protein 7B2, it would be most tempting to follow the effect of H₂O₂ on both PCs as well as the regulatory protein. Unfortunately at this time, we do not have access to native enzymes and to the regulatory protein, although computer simulation suggests that this processing machinery is structurally affected (data not shown). As ACTH, -, -MSH, and -endorphin have been implicated in melanocyte growth, dendricity, and pigmentation and these peptides, except -MSH can be directly affected by H₂O₂ stress, our results support these peptides as major players in the loss of melanocyte integrity and function in vitiligo. Moreover, these results suggest a role for H₂O₂ in the regulation of POMC peptide processing as a general mechanism.

The results of the study presented herein also demonstrated that -MSH alone has very low antioxidant properties per se, whereas the -MSH/6BH₄ 1:1 complex is an efficient antioxidant against H₂O₂ where 6BH₄ protects -MSH and vice versa supporting a close symbiotic relationship between hormone and cofactor. These novel results are supported by an earlier study by Moore et al. (1999), who showed the protection of 6BH₄ oxidation by -MSH. As the cofactor 6BH₄ is present in concentrations of 10^-6 M in epidermal cells as well as in many other cells, this mechanism could be important in general for protection of -MSH (Schallreuter et al., 1994). None of the other POMC peptides have this capacity, because of the lack of the specific binding domain for 6BH₄. As the human epidermis holds the capacity for Met sulfoxide repair, it is expected that the oxidation of Met residues in POMC-derived peptides are reversible in normal healthy skin (Ogawa et al., 2006; Schallreuter, 2006). However, preliminary results from our laboratory indicate that this repair mechanism can also be affected in untreated active vitiligo (data not shown). Future work is underway to elucidate this outcome in more detail.

In summary, we show for the first time that the melanocortins with the exception of -MSH are directly subjected to oxidative damage by H₂O₂ as shown in the skin of patients with vitiligo and by direct exposure of the peptides to this reactive oxygen species. This oxidation can severely affect also the functionality/downstream signal. Importantly, oxidation of -MSH can be prevented by -MSH/6BH₄ 1:1 complexation, which in turn identified a novel effective antioxidant mechanism.

Materials and Methods

Human tissue samples

Full skin was obtained under local anesthesia from sun-unexposed sites of each donor as 3-mm punch biopsies from healthy donors (n=6) and from patients with vitiligo before (n=9) and after (n=9) treatment with narrowband UVB-activated pseudocatalase PC-KUS (Schallreuter et al., 1999b). Patients were otherwise healthy. All probands had skin phototype III, Fitzpatrick classification (Fitzpatrick et al., 1967), and were age matched. Biopsies from the patients were taken from the center of untreated lesional skin and from repigmenting skin. This study was conducted after signed consent of each participant in accordance with the guidelines in the Declaration of Helsinki Principles and was approved by the local Ethics Committees.

Human melanocyte cell cultures

Epidermal melanocytes (passage 3) established from normal healthy skin (skin phototype III, Fitzpatrick classification), obtained with informed consent after elective plastic surgery, were seeded at a density of 1 10⁵ cells per T-25 cell culture flask (Corning Costar Corporation, Cambridge, MA) in a commercially available M254 media (Cascade Biologics Inc., Nottinghamshire, UK) and allowed to attach overnight. Cells were subsequently grown without fetal bovine serum or 12-O-tetradecanoylphorbol-13-acetate for 48 hours before stimulation for 72 hours with -endorphin (Sigma, Poole, Dorset, UK), 10^-8 M, with or without prior exposure to 1 mM H₂O₂ (Sigma, Poole, Dorset, UK) for 45 minutes. Any excess H₂O₂ was removed by the addition of 5 l catalase (40 mM) (Sigma, Poole, Dorset, UK). As a negative control, melanocytes from the same donor were grown in the absence of the peptide.

Determination of melanin concentration

For the measurement of melanin content, the cells were trypsinized and pelleted by centrifugation (1,000 r.p.m., 4°C, 5 minutes) and solubilized in 0.5 ml 1 N sodium hydroxide, followed by boiling for 1 hour. Melanin content was measured spectrophotometrically at 475 nm using a standard curve of synthetic melanin (Sigma, Poole, Dorset, UK) 5–50 g/ml. The results were correlated to the protein content of each sample using the Bradford assay (BioRad Laboratories, Hercules, CA).

In situ immunofluorescence labelling of POMC peptides

Biopsies were mounted using optical coherence tomography-embedding medium (Raymond A. Lamb, Eastbourne, East Sussex, UK) and cryosections, (5 m) were cut onto poly-L-lysine- (Sigma, Poole, Dorset, UK) coated slides and were air-dried at room temperature followed by fixation in ice-cold acetone for 10 minutes at -20°C. Slides were rehydrated in phosphate-buffered saline (PBS) for 5 minutes and blocked with 10% normal donkey serum (Serotec, Oxford, UK), diluted in PBS for 90 minutes at room temperature, and washed with 4 PBS. Next, the slides were incubated for 2–3 hours at room temperature with the primary antibodies diluted in 1% normal donkey serum in PBS against -MSH (ICN Pharmaceuticals Inc., Aurora, IL; dilution 1:50), -MSH (Bachem Ltd, St Helens, Merseyside, UK; dilution 1:200), -endorphin (Bachem Ltd, St Helens, Merseyside, UK; dilution 1:10), and ACTH (Abcam Ltd, Cambridge, UK; dilution 1:400) followed by a wash for 20 minutes in 4 PBS containing 0.05% Tween 20™. In order to visualize the specific antigen, an FITC-conjugated donkey secondary antibody (Jackson Immunoresearch Laboratories Europe, Soham, Cambridgeshire, UK, anti-mouse for -endorphin and anti-rabbit for all others) was added to the sections at a 1:100 dilution in 1% normal donkey serum in PBS and incubated for 1 hour at room temperature. After a final wash of 4 PBS and once in 0.05% Tween 20™ in PBS, the slides were mounted in Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories Ltd, Peterborough, UK). For negative controls the primary antibody was omitted and for specific blocking, the antibodies were pre-incubated with a 5-fold excess of the respective peptide for 2 hours at room temperature before use.

Sections were viewed with a Leica DMIRB/E fluorescence microscope (Wetzlar, Germany) and photo-documented using a digital CCD Camera, C8484-05G (Hamamatsu Photonics UK Ltd, Welwyn Garden City, Herts, UK) and the IPLab for windows imaging software, version 3.6.4 (Scanalytics Inc., Fairfax, VA).

Image analysis

Image analysis was utilized to assess staining intensities for both the immunofluorescence labelling and the dot blot technique using the IPLab for windows imaging software (as above).

Dot blot analysis

The manual spotting method used in this study followed the protocol recommended by the manufacturers of polyvinylidene difluoride membranes (Immobillon-P, Millipore, Bedford, UK). Briefly, 1 l of each sample was applied onto a pre-wetted polyvinylidene difluoride membrane, allowing the samples to be absorbed. The peptides were applied in concentrations of 0.45, 0.4, 0.44, and 0.38 g/l of ACTH (Bachem Ltd, St Helens, Merseyside, UK; dilution 1:10), -MSH (Bachem Ltd, St Helens, Merseyside, UK), -MSH (Bachem Ltd, St Helens, Merseyside, UK), and -endorphin (Sigma, Poole, Dorset, UK), respectively, following incubation for 2 hours with different concentrations of H₂O₂ (0–100 10^-3 M). The membrane was air dried before blocking with 3% BSA (Sigma, Poole, Dorset, UK) in Tris-buffered saline-T buffer (20 mM Tris-buffered saline with 0.047% Tween™ at pH 7.4) overnight at 4°C followed by a wash of 4 10 minutes with Tris-buffered saline-T buffer. Next, the membrane was incubated with the primary antibodies diluted in 1% BSA in PBS for 2 hours at room temperature in the case of ACTH (Abcam Ltd, Cambridge, UK; 1:5,000), -MSH (ICN Pharmaceuticals Inc., Aurora, IL; dilution 1:1,000), and -MSH (Bachem Ltd, St Helens, Merseyside, UK; dilution 1:2,000), whereas -endorphin (Bachem Ltd, St Helens, Merseyside, UK; dilution of 1:100) was incubated overnight at room temperature. This step was followed by a wash of 4 10 minutes in Tris-buffered saline-T buffer and a further incubation for 1 hour at room temperature with a horseradish peroxidase-conjugated secondary antibody (Sigma, Poole, Dorset, UK, anti-mouse for -endorphin and anti-rabbit for all others). Following a final wash, visualization of the "dots" was performed using modified enhanced chemiluminescence fixed on a film sheet (X-OMAT, Kodak, London, UK).

As it was shown earlier that -MSH protects 6BH₄ against oxidation by H₂O₂ (Moore et al., 1999), we decided to follow the possible antioxidant property of the 1:1 -MSH/6BH₄ complex compared to -MSH alone. For this purpose, we pre-incubated the peptide and 6BH₄ together for 15 minutes before incubating the peptide/6BH₄ complex for 2 hours with H₂O₂ following the same procedure as described above.

FT-Raman spectroscopy for detection of Met sulfoxide in H₂O₂-oxidized POMC peptides

FT-Raman spectra were acquired using a Bruker RFS 100/S spectrometer (Bruker, Karlsruhe, Germany) with a liquid-nitrogen-cooled Germanium detector. Near-infrared excitation was produced by a Nd³⁺:YAG laser operating at 1064 nm. Each spectrum was accumulated over 17 minutes with 1000 scans and a resolution of 4 cm^-1. All peptide samples were lyophilized after oxidation with H₂O₂ (100 10^-3 M) and measured as solids. The S=O-stretch was visualized as a peak at 1026 cm^-1. The assignment was confirmed with a solid Met sulfoxide standard (Sigma, Dorset, Poole, UK). The second peak detected in the standard was assigned to impurity owing to Met sulfone. This product was confirmed by amino-acid analysis (Hasse et al., 2004).

Molecular structural computer modelling of ACTH, -MSH, and -endorphin

Structural studies of the native and H₂O₂-oxidized Met and Trp residues in the POMC peptides ACTH, -MSH, and -endorphin were constructed using Hyperchem™ software (Gainsville, FL) to model the secondary structure minimized in water.

Statistical analysis

For statistical analysis of epidermal peptide expression the unpaired t-test (2-tail) was employed. Statistical analysis of the functional study with -endorphin was carried out with the one-tailed paired t-test.

Conflict of Interest

The authors state no conflict of interest.

References

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Acknowledgments

This research was kindly supported by Stiefel International with a grant to K.U.S. and by private donations. This work is part of the fulfillment towards a PhD thesis (J.D.S.).