DOI:10.1111/j.1600-0625.2008.00813.x www.blackwellpublishing.com/EXD Original Article Mapping of melanin-concentrating hormone receptor B cell epitopes predicts two major binding sites for vitiligo patient autoantibodies Nikos G Gavalas1, Raju V S R K Gottumukkala1, David J Gawkrodger2, Philip F Watson1, Anthony P Weetman1 and E Helen Kemp1 School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK; Department of Dermatology, Royal Hallamshire Hospital, Sheffield, UK Correspondence: Dr E Helen Kemp, School of Medicine and Biomedical Sciences, University of Sheffield, Royal Hallamshire Hospital, Glossop Road, Sheffield, S10 2JF, UK, Tel.: +44 114 271 2844; Fax: +44 114 271 3960, e-mail: e.h.kemp@sheffield.ac.uk Accepted for publication 15 October 2008 Abstract: The melanin-concentrating hormone receptor (MCHR1) has been identified as a B cell autoantigen in vitiligo with antibodies to the receptor detectable in binding and function-blocking assays Two epitope domains (amino acids 1–138 and 139–298) have been previously identified In this study, we aimed to further define the epitope specificity of MCHR1 antibodies using phage-display technology and to identify the epitopes recognised by receptor antibodies detected in MCHR1 function-blocking assays Antibody reactivity to MCHR1 peptides 51–80, 85–98, 154–158 and 254–260 was identified by phagedisplay and subsequently confirmed in phage ELISA in ⁄ 12, ⁄ 12, ⁄ 12 and ⁄ 12 of vitiligo patients, respectively The results suggest that major autoantibody epitopes are localised in the 85– 98 and 254–260 amino acid regions of MCHR1 with minor epitopes in amino acid sequences 51–80 and 154–158 Antibodies with MCHR1 function-blocking activity were determined to recognise epitope 254–260, this being the first epitope to be reported as a target site for antibodies that block the function of the receptor Key words: autoantibody – autoantigen – epitope – melaninconcentrating hormone receptor – vitiligo Please cite this paper as: Mapping of melanin-concentrating hormone receptor B cell epitopes predicts two major binding sites for vitiligo patient autoantibodies Experimental Dermatology 2009; 18: 454–463 Introduction Vitiligo is an acquired hypomelanotic disorder characterised by circumscribed depigmented macules resulting from the loss of functional melanocytes from the cutaneous epidermis An autoimmune aetiology has been proposed for the disease due to the frequent association of vitiligo with autoimmune disorders including Graves’ disease and autoimmune polyglandular syndromes (1–3) Studies demonstrating the presence of autoantibodies and autoreactive T cells against pigment cells in individuals with vitiligo also Abbreviations: Ab, antibody; bp, base pair; BSA, bovine serum albumin; BSS, balanced salt solution; cfu, colony-forming units; CHO-K1, Chinese hamster ovary; IgG, immunoglobulin G; LB, Luria Bertani; MCH, melanin-concentrating hormone; MCHR1, melanin-concentrating hormone receptor 1; a-MSH, a-melanocyte-stimulating hormone; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PIA, N6-(l-2-phenylisopropyl)-adenosine; TSH, thyrotropin 454 support the involvement of immune factors in the development of the disease (4,5) Furthermore, susceptibility to vitiligo has been associated with polymorphisms in several immune-regulatory genes that also confer a predisposition to other autoimmune endocrine diseases (6,7) Much research has been undertaken to identify vitiligoassociated autoantigens for the purpose of disease diagnosis and to uncover the immune-mechanisms involved in vitiligo pathogenesis Recently, the G-protein coupled melanin-concentrating hormone receptor (MCHR1) was identified as a B cell autoantigen in vitiligo using radiobinding assays (8– 12) Further study has revealed that MCHR1 antibodies can also block the response of the receptor to melanin-concentrating hormone (MCH) (13) Interestingly, when stimulated with MCH, the receptor can down-regulate a-melanocytestimulating hormone (a-MSH)-induced melanogenesis in pigment cells, suggesting that the MCHR1 ⁄ MCH signalling pathway might have a role with the melanocortins in regulating melanocyte function (14) It remains to be determined, however, whether or not MCHR1 antibodies can alter the ª 2009 The Authors Journal compilation ª 2009 Blackwell Munksgaard, Experimental Dermatology, 18, 454–463 Epitopes on the melanin-concentrating hormone receptor behaviour of melanocytes in a manner that could lead to depigmentation and the development of vitiligo Previously, two large epitope domains were identified on MCHR1 for vitiligo patient MCHR1 antibodies (15) In this study, we aimed to use the pComb3 phage-display system (16,17) to further define the epitope specificity of MCHR1 antibodies A second objective was to address the question of the identity of the epitopes recognised by MCHR1 antibodies detected in MCHR1 function-blocking assays (13) More generally, mapping of autoepitopes is invaluable in providing an understanding of the association of an autoantigen with a particular autoimmune disorder (18), affording an insight into the initiation of an autoimmune disease (19), defining the heterogeneity of the humoral immune response against a particular autoantigen and allowing the development of specific diagnostic assays if disease-defining epitopes can be demonstrated (20) Materials and methods Patients and controls Sera from 12 vitiligo patients (V1–V12) (six male, six female; mean age: 48 years with range 23–77 years) were used in this study Vitiligo was classified as: peri-orificial, 1; symmetrical ⁄ peri-orificial, 1; symmetrical, 9; symmetrical and segmental Nine patients had no personal or family history of autoimmune disease and three patients had autoimmune thyroid disease Seven patients (V1–V7) were positive for MCHR1 antibodies in binding and functionblocking assays (8,13) In two patients (V8,V9), MCHR1 antibodies could only be detected in binding assays and in three patients (V10–V12), MCHR1 antibodies were only apparent in function-blocking assays (8,13) None of the patients had undergone any immunosuppressive treatment prior to their blood sample being collected for use in this study Sera from 20 healthy individuals (nine males, 11 females; mean age: 32 years with range 24–48 years) with no history of either vitiligo or autoimmune disorders were also included as controls No controls were positive for MCHR1 antibodies (8,13) All sera were stored at )20°C prior to use The South Sheffield Ethics Committee, Sheffield, UK approved this work and all subjects gave informed consent Isolation and biotinylation immunoglobulin G Immunoglobulin G (IgG) was isolated from sera by protein G Sepharose Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) affinity column chromatography as previously detailed (8) Samples at 10 mg ⁄ ml were stored at )20°C Biotinylation of IgG was performed using EZLinkÔ Sulfo-NHS-LC-LC-Biotin (Pierce, Rockford, IL, USA), according to the protocol of the manufacturer and samples at mg ⁄ ml were stored at 4°C Anti-MCHR1 antibody Rabbit polyclonal anti-MCHR1 antibody C11CB (21) was a gift from Professor G Hervieu (Neurology and GastroIntestinal Centre for Drug Discovery, GlaxoSmithKline Beecham, Harlow, UK) and was raised against the amino acid residues 387–402 (SNAQTADEERTESKGT) at the C-terminal intracellular tail of the receptor (21) Phage-display library construction To construct a MCHR1 cDNA fragment library in pComb3 (17), full-length MCHR1 cDNA [1206 base pairs (bp) encoding amino acid residues 1–402] was firstly prepared from pcMCHR1 (8) by restriction of the plasmid with EcoRI and XbaI The MCHR1 cDNA fragment was separated by agarose gel electrophoresis (22) and purified using a Wizard PCR Preps DNA Purification System (Promega, Southampton, UK) Random 100–300-bp fragments of MCHR1 cDNA were generated by digestion with DNAseI in a reaction containing: lg of MCHR1 cDNA, U of DNAseI (Promega) and DNAseI buffer (Promega) The reaction was left at room temperature for 15 and then terminated by the addition of 50 mm EDTA (pH 8.0) The DNA fragments were purified using a Wizard PCR Preps DNA Purification System (Promega) and treated with T4 DNA polymerase (Promega) according to the manufacturer’s protocol to create blunt-ends After further purification, the fragments were ligated into the EcoRV restriction site of pComb3 (17) using standard methods (22) The MCHR1 cDNA fragment library was recovered by electroporation of Escherichia coli XL1-Blue cells (Stratagene, La Jolla, CA, USA), as described by the manufacturer, and the library size estimated by plating out samples onto Luria Bertani (LB) agar (22) containing 100 lg ⁄ ml ampicillin and 10 lg ⁄ ml tetracycline To prepare the MCHR1 peptide phage-display library, the electroporated cells were incubated for h at 37°C before superinfection with · 1012 plaque-forming units of VCMS13 helper phage (Stratagene) at room temperature for 30 The culture was subsequently transferred to 100 ml of LB medium (22) supplemented with 100 lg ⁄ ml ampicillin, 10 lg ⁄ ml tetracycline and 10 lg ⁄ ml kanamycin After overnight incubation at 37°C, the culture was centrifuged and phage precipitated from the supernatant with 0.2 volumes of 20% (w ⁄ v) polyethylene glycol 4000 ⁄ 2.5 m NaCl The phage was resuspended in 2–3 ml of phosphatebuffered saline (pH 7.4; PBS; Sigma, Poole, UK) and stored at )20°C Phage titres were determined by infecting log-phase E coli XL1-Blue with an aliquot of the phagedisplay library and then plating out samples onto selective LB agar ª 2009 The Authors Journal compilation ª 2009 Blackwell Munksgaard, Experimental Dermatology, 18, 454–463 455 Gavalas et al Biopanning experiments For each biopanning experiment, a 15-ll aliquot of biotinylated IgG was incubated with 200 lg of DynabeadsÒ M280 Streptavidin (Dynal Biotech, Oslo, Norway), prepared according to the manufacturer in 235 ll of sterile water, and incubated at 4°C for 30 on a rotating platform to permit antibody-bead binding To block any non-specific phage binding to the beads when phage are added later in the procedure, 300 ll of 2% (w ⁄ v) dried milk in PBS containing 10% (w ⁄ v) glycerol were then added to the beadIgG suspension and incubation at 4°C continued for h The bead-IgG complexes were separated from the blocking buffer using a Dynal Magnetic Particle Concentrator (Dynal Biotech), washed twice and finally resuspended in 150 ll of PBS ⁄ 0.05% (w ⁄ v) Tween 20 (PBS ⁄ Tween) before the addition of a 100-ll sample of phage-display library containing · 1010 colony-forming units (cfu) The suspension was then incubated overnight at 4°C to allow interaction of the antibody-bead complexes with peptides displayed on the surface of the phage particles The beadIgG complexes were washed extensively with PBS ⁄ Tween to remove any unbound phage Bound phage were eluted from the bead-IgG complexes with 150 ll of 100 mm HCl (adjusted to pH 2.2 with solid glycine) and the beads magnetically separated from the supernatant which was neutralised by ll of m Tris–HCl (pH 7.6) The phage suspension was subsequently used to infect ml of exponentially growing E coli XL1-Blue for 15 at room temperature Aliquots of the infected cells were then plated onto selective medium to allow the recovery of individual bacterial clones for analysis To generate a phage-display library for a further round of selection, the infected E coli XL1-Blue culture was superinfected with helper phage and phage prepared and titred as described above This first round library enriched in phage displaying IgG-binding peptides was used in a second round of selective enrichment A total of five rounds of biopanning were undertaken For analysis, individual bacterial clones were cultured and phagemid DNA prepared using a Wizard Minipreps DNA Purification System (Promega) To confirm the presence of a cDNA insert, phagemid DNA (50 ng samples) was subjected to 36 cycles of polymerase chain reaction (PCR) amplification in a DNA Thermal Cycler (PerkinElmer Cetus, Norwalk, CT, USA) with primers 5¢-GGTGGCGGCCGCAAATTC-3¢ (forward) and 5¢-GCCGCCAGCATTGACAGG-3¢ (reverse) (MWG Biotech, Munich, Germany) using previously detailed reaction conditions (23) The primers used flank the EcoRV cloning site in pComb3 (17) The PCR amplification products were analysed by agarose gel electrophoresis (22) and purified according to a Wizard PCR Preps DNA Purification System (Promega) Sequencing with primer 5¢-GGTGGCGGCCGC- 456 AAATTC-3¢ was carried using a BigDyeÒ Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3730 capillary sequencer (Applied Biosystems) Insert DNA sequences were compared with the full-length MCHR1 cDNA sequence using the BLAST network service of the National Centre for Biotechnology Information (Bethesda, MD, USA) Phage ELISA Phage displaying MCHR1 peptides required for analysis in phage ELISA were prepared from individual bacterial clones as described above after electroporating the appropriate phagemid DNA into E coli XL1-Blue and superinfecting with helper phage Corning polystyrene 96-well microtitre plates (Bibby Sterilin Ltd, Mid Glamorgan, UK) were coated with 100-ll aliquots (1010 cfu) of phage in coating buffer containing 1.5 mm Na2CO3, 3.5 mm NaHCO3 and 3.0 mm NaN3 (pH 9.2), and incubated overnight at 4°C Helper phage was included in each assay as a control for background antibody reactivity Wells were washed with PBS ⁄ 0.1% (w ⁄ v) Tween 20, blocked with 3% (w ⁄ v) bovine serum albumin (BSA) in PBS at room temperature for h and then washed with PBS ⁄ 0.1% Tween 20 IgG samples and C11CB antibody were preabsorbed with E coli cell extract and helper phage and then tested in phage ELISA at dilutions of 1:100 and 1:1000, respectively Aliquots (100-ll) of IgG ⁄ antibody were added to wells and PBS was applied as a control The plates were incubated at room temperature for h and then washed six times with PBS ⁄ 0.1% Tween 20 Aliquots (100-ll) of either anti-human or anti-rabbit IgG alkaline phosphatase conjugate (Sigma), diluted 1:1 000 in PBS ⁄ 0.1% Tween 20, were applied to the wells for h at room temperature After washing six times with PBS ⁄ 0.1% Tween 20, 100 ll of alkaline phosphatase substrate (Sigma Fast p-Nitrophenyl Phosphate Tablet Set, Sigma) were applied to each well and plates incubated at room temperature for 30 A LabSystems Integrated EIA Management System (Life Sciences International, Hampshire, UK) was used to read absorption of the wells at 405 nm All IgG samples were tested in triplicate and the average OD405 value taken OD405 values were corrected for background reactivity to helper phage An antibody (Ab) index for each sample was calculated as: OD405 of tested IgG ⁄ mean OD405 of 20 healthy control IgG Each IgG was tested in at least two experiments and the mean Ab index was calculated The upper level of normal for the assay was calculated using the mean Ab index + 3SD of the population of 20 healthy individuals Any sample with an Ab index above the upper level of normal was designated as positive for antibody reactivity to the phage-displayed MCHR1 peptide Synthetic peptides corresponding to MCHR1 amino acid consensus sequences 51–80, 85–98, 154–158, 254–260 and ª 2009 The Authors Journal compilation ª 2009 Blackwell Munksgaard, Experimental Dermatology, 18, 454–463 Epitopes on the melanin-concentrating hormone receptor peptide C11CB (amino acid residues 387–402) were purchased from Severn Biotech Ltd (Kidderminster, UK) In absorption experiments, peptides were incubated with IgG at 4°C for h at mg ⁄ ml prior to testing the IgG sample in phage ELISA Ab indices were then calculated and the percentage Ab binding determined as: Ab index in presence of peptide ⁄ Ab index in absence of peptide · 100 Samples were tested in three experiments and the mean percentage Ab binding calculated MCHR1 function-blocking antibody assay The presence of MCHR1 function-blocking antibodies in IgG samples was assessed as previously described (24) Chinese hamster ovary (CHO-K1) cells were obtained from the European Collection of Animal Cell Cultures (Salisbury, UK) A stable CHO-K1 cell line, expressing MCHR1 (CHO-MCHR1), was kindly given by Dr Emmanuel Burgeon (Euroscreen, Brussels, Belgium) Cells were cultured in Ham’s F12 medium with 10% (v ⁄ v) fetal calf serum, mm l-glutamine, 50 U ⁄ ml penicillin G and 50 lg ⁄ ml streptomycin (All from Invitrogen, Paisley, UK) at 37°C in a 5% CO2 incubator For culturing MCHR1 cells, geneticin sulphate (Invitrogen) was also included in the growth medium at 400 lg ⁄ ml Subsequently, CHO-K1 and CHO-MCHR1 cells were plated out on glass coverslips in six-well plates at 5.0 · 105 cells ⁄ well in 2.5 ml of culture medium and incubated at 37°C for two days Cell monolayers, at 80% confluence, were incubated for 30 at 37°C with the fluorescent dye FURA-2 ⁄ AM (Merck Biosciences, Nottingham, UK) at lm in balanced salt solution (BSS) containing 135 mm NaCl, 4.5 mm KCl, 1.5 mm CaCl2, 0.5 mm MgCl2, 5.6 mm glucose and 10 mm HEPES (pH 7.4) and before rinsing twice with BSS Subsequently, CHO-MCHR1 cells were incubated with vitiligo patient or control IgG samples at mg ⁄ ml in duplicate, with or without synthetic peptides at 0.1 mg ⁄ ml, for h at 37°C in BSS As positive and negative controls, respectively, CHO-MCHR1 and CHO-K1 cells were incubated with BSS alone For stimulation with MCH, the coverslips of cells were placed in a Kontron SFM 25 Fluorimeter (Kontron Instruments Ltd, Watford, UK) across the diagonal of a 1-cm quartz cuvette being supported on a plastic bridge within the cuvette that allowed a small magnetic stirrer to be used to give a continuous mixing of the cuvette contents A stable baseline of fluorescence was obtained by adding BSS to the cells Additions of MCH (Alpha Diagnostics, San Antonio, TX, USA) were then made to 10)12 to 10)7 m along with the adenosine agonist N6-(l-2-phenylisopropyl)-adenosine (PIA; Sigma) to lm Changes in intracellular calcium levels following MCH-stimulation were detected by measuring changes in relative fluorescence intensity versus time by exciting cells at 335 nm and collecting emissions at 490 nm Each IgG sample was tested in three separate experiments Statistical analyses The prevalence of antibody reactivity to phage-displayed MCHR1 peptides was compared between patient and control groups using Fisher’s exact test for · contingency tables Intra- and inter-assay variations were calculated as percentage coefficients of variation Differences in antibody titres were analysed by the Wilcoxon matched pairs test P values