The Autoantigen of Anti-p200 Pemphigoid Is an Acidic Noncollagenous N-Linked Glycoprotein of the Cutaneous Basement Membrane
2003; Elsevier BV; Volume: 121; Issue: 6 Linguagem: Inglês
10.1111/j.1523-1747.2003.12609.x
ISSN1523-1747
AutoresIakov Shimanovich, Yoshiaki Hirako, Cassian Sitaru, Takashi Hashimoto, Eva‐B. Bröcker, Elke Butt, Detlef Zillikens,
Tópico(s)Urticaria and Related Conditions
ResumoAnti-p200 pemphigoid is an autoimmune subepidermal blistering disease characterized by autoantibodies to a 200-kDa protein (p200) of the dermal–epidermal junction (DEJ). p200 has been demonstrated to be distinct from all major DEJ autoantigens and is thought to be important for cell-matrix adhesion. This study provides the first biochemical characterization of p200. Differential extraction experiments demonstrated that efficient recovery of p200 from the dermis was strongly dependent on the presence of reducing agents, suggesting that it forms highly insoluble oligomers and/or is extensively cross-linked to other extracellular matrix components by disulfide bonding. p200 was resistant to digestion with bacterial collagenase, whereas this treatment did degrade major collagenous proteins of the dermis, including type I, VI, and VII collagen. This finding firmly established the noncollagenous nature of p200. N-Glycosidase F reduced the molecular size of the p200 autoantigen from 200 to 190 kDa without decreasing its immunoreactivity. In contrast, digestion of p200 with neuraminidase, O-glycosidase, chondroitinase ABC, and heparitinase I had no effect on its electrophoretic mobility. These data suggest that the p200 molecule contains N-glycans but lacks O-linked oligosaccharides and chondroitin/heparan sulfate side chains. Two-dimensional gel electrophoresis demonstrated that p200 is an acidic protein with an isoelectric point of 5.4 to 5.6. Six different p200-specific sera recognized an identical protein spot of two-dimensionally separated dermal extracts, confirming that patients with this novel autoimmune disease indeed form a single pathobiochemical entity. Anti-p200 pemphigoid is an autoimmune subepidermal blistering disease characterized by autoantibodies to a 200-kDa protein (p200) of the dermal–epidermal junction (DEJ). p200 has been demonstrated to be distinct from all major DEJ autoantigens and is thought to be important for cell-matrix adhesion. This study provides the first biochemical characterization of p200. Differential extraction experiments demonstrated that efficient recovery of p200 from the dermis was strongly dependent on the presence of reducing agents, suggesting that it forms highly insoluble oligomers and/or is extensively cross-linked to other extracellular matrix components by disulfide bonding. p200 was resistant to digestion with bacterial collagenase, whereas this treatment did degrade major collagenous proteins of the dermis, including type I, VI, and VII collagen. This finding firmly established the noncollagenous nature of p200. N-Glycosidase F reduced the molecular size of the p200 autoantigen from 200 to 190 kDa without decreasing its immunoreactivity. In contrast, digestion of p200 with neuraminidase, O-glycosidase, chondroitinase ABC, and heparitinase I had no effect on its electrophoretic mobility. These data suggest that the p200 molecule contains N-glycans but lacks O-linked oligosaccharides and chondroitin/heparan sulfate side chains. Two-dimensional gel electrophoresis demonstrated that p200 is an acidic protein with an isoelectric point of 5.4 to 5.6. Six different p200-specific sera recognized an identical protein spot of two-dimensionally separated dermal extracts, confirming that patients with this novel autoimmune disease indeed form a single pathobiochemical entity. bullous pemphigoid dermal–epidermal junction extracellular matrix ethylenediaminetetraacetic acid phenylmethylsulfonyl fluoride Anti-p200 pemphigoid is an autoimmune subepidermal blistering disease first described in 1996 (Zillikens et al., 1996Zillikens D. Kawahara Y. Ishiko A. et al.A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone.J Invest Dermatol. 1996; 106: 465-470Crossref PubMed Scopus (44) Google Scholar;Chen et al., 1996Chen K.R. Shimizu S. Miyakawa S. Ishiko A. Shimizu H. Hashimoto T. Coexistence of psoriasis and an unusual IgG-mediated subepidermal bullous dermatosis: Identification of a novel 200-kDa lower lamina lucida target antigen.Br J Dermatol. 1996; 134: 340-346Crossref PubMed Scopus (85) Google Scholar). Immuno-pathologically, it is characterized by linear deposits of IgG and C3 along the dermal–epidermal junction (DEJ) as detected by direct immunofluorescence microscopy of perilesional skin biopsies. Indirect immunofluorescence of patients' sera demonstrates circulating IgG autoantibodies labeling the dermal side of NaCl-split normal skin. The antigenic target of these antibodies is a 200-kDa protein (p200) of the human dermis that is thought to be important for cell-matrix adhesion (Zillikens et al., 1996Zillikens D. Kawahara Y. Ishiko A. et al.A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone.J Invest Dermatol. 1996; 106: 465-470Crossref PubMed Scopus (44) Google Scholar;Kawahara et al., 2000Kawahara Y. Zillikens D. Yancey K.B. Marinkovich M.P. Nie Z. Hashimoto T. Nishikawa T. Subepidermal blistering disease with autoantibodies against a novel dermal 200 kDa antigen.J Dermatol Sci. 2000; 23: 93-102Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Ultrastructural studies localized p200 to the lower portion of the lamina lucida of the cutaneous basement membrane (Zillikens et al., 1996Zillikens D. Kawahara Y. Ishiko A. et al.A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone.J Invest Dermatol. 1996; 106: 465-470Crossref PubMed Scopus (44) Google Scholar;Chen et al., 1996Chen K.R. Shimizu S. Miyakawa S. Ishiko A. Shimizu H. Hashimoto T. Coexistence of psoriasis and an unusual IgG-mediated subepidermal bullous dermatosis: Identification of a novel 200-kDa lower lamina lucida target antigen.Br J Dermatol. 1996; 134: 340-346Crossref PubMed Scopus (85) Google Scholar;Egan et al., 2002Egan C.A. Yee C. Zillikens D. Yancey K.B. Anti-p200 pemphigoid: Diagnosis and treatment of a case presenting as an inflammatory subepidermal blistering disease.J Am Acad Dermatol. 2002; 46: 786-789Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). While p200 was demonstrated to be immunologically distinct from all major autoantigens of the DEJ, including bullous pemphigoid (BP) antigens 180 and 230, α6β4 integrin, laminin 5 and 6, and type VII collagen (Zillikens et al., 1996Zillikens D. Kawahara Y. Ishiko A. et al.A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone.J Invest Dermatol. 1996; 106: 465-470Crossref PubMed Scopus (44) Google Scholar,Zillikens et al., 2000Zillikens D. Ishiko A. Jonkman M.F. Chimanovitch I. Shimizu H. Hashimoto T. Bröcker E.B. Autoantibodies in anti-p200 pemphigoid stain skin lacking laminin 5 and type VII collagen.Br J Dermatol. 2000; 143: 1043-1049Crossref PubMed Scopus (50) Google Scholar;Kawahara et al., 2000Kawahara Y. Zillikens D. Yancey K.B. Marinkovich M.P. Nie Z. Hashimoto T. Nishikawa T. Subepidermal blistering disease with autoantibodies against a novel dermal 200 kDa antigen.J Dermatol Sci. 2000; 23: 93-102Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), little is known about its structure and biochemical properties. In this study, we investigate the association of p200 with the extracellular matrix (ECM) and the presence of collagenous domains and carbohydrate moieties within this molecule. In addition, we demonstrate that IgG4 autoantibodies from six patients with anti-p200 pemphigoid recognize the same unique protein of two-dimensionally separated dermal extracts. In this study, we used sera from six patients with anti-p200 pemphigoid. Four of these patients have been described in detail elsewhere (Zillikens et al., 1996Zillikens D. Kawahara Y. Ishiko A. et al.A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone.J Invest Dermatol. 1996; 106: 465-470Crossref PubMed Scopus (44) Google Scholar,Zillikens et al., 2000Zillikens D. Ishiko A. Jonkman M.F. Chimanovitch I. Shimizu H. Hashimoto T. Bröcker E.B. Autoantibodies in anti-p200 pemphigoid stain skin lacking laminin 5 and type VII collagen.Br J Dermatol. 2000; 143: 1043-1049Crossref PubMed Scopus (50) Google Scholar;Mascaro et al., 2000Mascaro J.M. Zillikens D. Giudice G.J. Caux F. Fleming M.G. Katz H.M. Diaz L.A. A subepidermal bullous eruption associated with IgG autoantibodies to a 200 kd dermal antigen: The first case report from the United States.J Am Acad Dermatol. 2000; 42: 309-315Abstract Full Text Full Text PDF PubMed Google Scholar;Egan et al., 2002Egan C.A. Yee C. Zillikens D. Yancey K.B. Anti-p200 pemphigoid: Diagnosis and treatment of a case presenting as an inflammatory subepidermal blistering disease.J Am Acad Dermatol. 2002; 46: 786-789Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). All patients fulfilled the following criteria: (1) linear deposits of IgG and C3 along the DEJ as detected by direct immunofluorescence of perilesional skin biopsies; (2) linear deposition of IgG along the dermal side of NaCl-split normal human skin by indirect immunofluorescence microscopy; and (3) circulating antibodies against a 200-kDa protein by immunoblotting of extracts of normal human dermis. Rabbit antibodies to human type I and type VI collagen were purchased from Rockland (Gilbertsville, PA). Rabbit serum to human proteoglycan I (biglycan) was provided by H. Kresse (Münster, Germany). Rabbit serum SA8010 was raised against recombinant BP180 NC16A (Sitaru et al., 2002Sitaru C. Schmidt E. Petermann S. Munteanu L.S. Bröcker E.B. Zillikens D. Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin.J Invest Dermatol. 2002; 118: 664-671Crossref PubMed Scopus (158) Google Scholar). Monoclonal antibodies 1A8c and BM515 were raised against BP180 and the α3 chain of laminin 5, respectively (Hirako et al., 1996Hirako Y. Usukura J. Nishizawa Y. Owaribe K. Demonstration of the molecular shape of BP180, a 180 kDa bullous pemphigoid antigen and its potential for trimer formation.J Biol Chem. 1996; 271: 13739-13745Crossref PubMed Scopus (106) Google Scholar,Hirako et al., 2003Hirako Y. Yoshino K. Zillikens D. Owaribe K. Extracellular cleavage of bullous pemphigoid antigen 180/type XVII collagen and its involvement in hemidesmosomal assembly.J Biochem. 2003; 133: 197-206Crossref PubMed Scopus (21) Google Scholar). Monoclonal antibody LH7.2 against the NC1 domain of type VII collagen was purchased from Sigma (St. Louis, MO) and monoclonal antibody A1 against the γ1 chain of laminin 1 was from Neomarkers (Fremont, CA). For the experiments conducted, we obtained institutional approval (Institutional Board Project # 37/98) issued by the director of the ethics committee at the Medical Faculty of the University of Wuerzburg. We obtained informed consent from all patients whose material was used in the study, in adherence to the Helsinki Principles. Immunoblotting of dermal extracts and other substrates was performed as described previously (Zillikens et al., 1996Zillikens D. Kawahara Y. Ishiko A. et al.A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone.J Invest Dermatol. 1996; 106: 465-470Crossref PubMed Scopus (44) Google Scholar). Unless otherwise specified, all immunoblotting studies with serum samples from patients with anti-p200 pemphigoid were performed using anti-IgG4 secondary antibodies (see below). Subclass distribution of p200-specific autoantibodies was determined using peroxidase-conjugated mouse anti-human IgG1 (clone 8c/6-39), antihuman IgG2 (clone HP6014), anti-human IgG3 (clone HP6050), and anti-human IgG4 (clone HP6023) (all from The Binding Site, Birmingham, UK) following a previously published protocol (Chimanovitch et al., 1999Chimanovitch I. Schmidt E. Messer G. Döpp R. Giudice G.J. Bröcker E.B. Zillikens D. IgG1 and IgG3 are the major immunoglobulin subclasses targeting epitopes within BP180 NC16A in pemphigoid gestationis.J Invest Dermatol. 1999; 113: 140-142Crossref PubMed Scopus (61) Google Scholar). Sensitivities of these secondary monoclonal antibodies, used at their working dilutions, had been shown to be comparable by immunoblot analysis with equal amounts of human myeloma proteins (Chimanovitch et al., 1999Chimanovitch I. Schmidt E. Messer G. Döpp R. Giudice G.J. Bröcker E.B. Zillikens D. IgG1 and IgG3 are the major immunoglobulin subclasses targeting epitopes within BP180 NC16A in pemphigoid gestationis.J Invest Dermatol. 1999; 113: 140-142Crossref PubMed Scopus (61) Google Scholar). Fresh normal human skin was obtained from plastic surgery and incubated in phosphate-buffered saline, pH 7.2, supplemented with 1 M NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 mM ethylenediaminetetraacetic acid (EDTA) at 4°C for 96 h. After dermal–epidermal separation, the epidermis was peeled off and discarded. The epidermal surface of the dermis was extensively washed with phosphate-buffered saline and extracted with buffer A (12.5 mM Tris-HCl, pH 7.0, 8 M urea, 6%β-mercaptoethanol, 1 mM PMSF, and 5 mM EDTA) at room temperature. After 1 h, the dermal extract was collected and centrifuged, and the supernatant was stored at –80°C. For differential extraction experiments, deepidermized dermis was prepared as described above and then extracted with buffer B (same as buffer A but without β-mercaptoethanol) followed by buffer A. For some experiments, the epidermal surface of the dermis was digested with bacterial collagenase as described below and subsequently extracted with buffers B and A. To determine the solubility of extracted protein, dermal extracts were alkylated by addition of 100 mM iodoacetamide for 1 h in the dark and subsequently dialyzed against 4, 2, or 1 M urea (all solutions buffered with 50 mM Tris-HCl, pH 7.5), RIPA buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), or Tris-buffered saline (same as RIPA but without detergents), respectively. Dialyzed extracts were centrifuged at 100,000×g for 2 h, and both supernatants and pellets were analyzed for reactivity with anti-p200 sera by immunoblotting. Normal keratinocytes and fibroblasts were cultured from outgrowing cells of human neonatal foreskins as described previously (Zillikens et al., 1996Zillikens D. Kawahara Y. Ishiko A. et al.A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone.J Invest Dermatol. 1996; 106: 465-470Crossref PubMed Scopus (44) Google Scholar). HaCaT keratinocytes were provided by N.E. Fusenig (Heidelberg, Germany) and grown as described byBoukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreuz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3276) Google Scholar. Confluent monolayers of keratinocytes or fibroblasts were extracted with Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 6%β-mercaptoethanol) supplemented with 5 mM EDTA and 1 mM PMSF. Extracts were pulse-sonicated on ice and centrifuged, and the supernatant was collected. Proteins in the medium conditioned by HaCaT keratinocytes or fibroblasts were concentrated by ammonium sulfate precipitation as described (Kromminga et al., 2000Kromminga A. Scheckenbach C. Georgi M. et al.Patients with bullous pemphigoid and linear IgA disease show a dual IgA and IgG autoimmune response to BP180.J Autoimmun. 2000; 15: 293-300Crossref PubMed Scopus (69) Google Scholar). Cryosections of organotypic cocultures of human keratinocytes and fibroblasts harvested at 3 wk (Smola et al., 1998Smola H. Stark H.J. Thiekötter G. Mirancea N. Krieg T. Fusenig N.E. Dynamics of basement membrane formation by keratinocyte–fibroblast interactions in organotypic skin culture.Exp Cell Res. 1998; 239: 399-410Crossref PubMed Scopus (211) Google Scholar) were provided by N.E. Fusenig. Collagenase digestion was performed as described byGiudice et al., 1992Giudice G.J. Emery D. Diaz L.A. Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180.J Invest Dermatol. 1992; 99: 243-250Crossref PubMed Scopus (469) Google Scholar with some modification. Dermal extracts were dialyzed against collagenase buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM CaCl2). Protease inhibitors (1 mM PMSF, 1 mM AEBSF, 10 μg/mL leupeptin, 10 μg/mL antipain, 15 μg/mL pepstatin A, 15 μg/mL chymostatin) and 1 Mandl U/mL of collagenase from Clostridium histolyticum (Amano Pharmaceuticals, Japan) were added to the dialysate, and after incubation at 37°C for various periods of time, the reaction was stopped by addition of 5×Laemmli sample buffer, containing 100 mM EDTA, and boiling for 5 min. Negative controls included dermal extracts incubated at 37°C without collagenase and collagenase digestion of bovine serum albumin (Sigma). For tissue digestion, collagenase buffer containing protease inhibitors and 100 Mandl U per mL collagenase was applied to the epidermal surface of deepidermized dermis and incubated at 37°C 4 h. For digestion with bacterial protease V8 (Glu-C), dermal extracts were dialyzed against 0.01% SDS in 25 mM NH4HCO3 (pH 7.8) and protease V8 from Staphylococcus aureus (Roche, Mannheim, Germany) was added at an enzyme to substrate ratio of 1:100. After incubation at room temperature for various periods of time, the reaction was stopped by addition of 5×Laemmli sample buffer and boiling. The products of V8 digestion were separated in 4% to 20% gradient gels (Bio-Rad, Hercules, CA) and studied by immunoblotting. To investigate the presence of N-linked carbohydrate moieties within the p200 molecule, dermal extracts were dialyzed against 20 mM Na3PO4, pH 7.2, supplemented with 0.2% SDS, 1%β-mercaptoethanol, 15 mM EDTA and then denatured at 100°C for 5 min. After addition of 1% Nonidet P-40 and 300 U per mL recombinant N-glycosidase F cloned from Flavobacterium meningosepticum (Roche), the sample was incubated at 37°C overnight. Bovine fetuin (Sigma) and human transferrin (Roche) were used as positive controls. In contrast to N-linked carbohydrates, O-linked oligosaccharides may be difficult to remove from a protein in one step. O-deglycosylation studies were therefore performed by sequential treatment of dermal extracts first with neuraminidase from Arthrobacter ureafaciens (400 mU/mL) and then O-glycosidase from Diplococcus pneumoniae (200 mU/mL) at 37°C for 12 h, respectively (both enzymes from Roche). Deglycosylation of bovine fetuin (Sigma), known to contain neuraminic acid and O-linked sugars, was used as a positive control and monitored by Coomassie staining. Finally, to assess the presence of chrondroitin sulfate and heparan sulfate side chains in the p200 molecule, dermal extracts were digested with 4 U per mL chondroitinase ABC from Proteus vulgaris (in 0.1 M Tris-HCl, pH 8.0, containing 30 mM sodium acetate) or 100 mU per mL heparitinase I from Flavobacterium heparinum (in 20 mM sodium acetate, pH 7.0, containing 1 mM calcium acetate) at 37°C overnight. Heparitinase was from Seikagaku (Tokyo, Japan) and chondroitinase from Calbiochem (Schwalbach, Germany). Heparan sulfate from bovine kidney (Sigma) was used as a positive control for heparitinase digestion and the reaction was monitored at 232 nm. Negative controls for all deglycosylation experiments were incubated under identical conditions without addition of glycosidases. All enzymes used were of protease-free quality. Two-dimensional gel electrophoresis was performed as described byO'Farrel, 1975O'Farrel P.H. High resolution two-dimensional electrophoresis of proteins.J Biol Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar with some modification. Dermal extracts were exhaustively dialyzed against double-distilled water and precipitated protein pelleted by centrifugation at 100,000×g for 1 h. The pellet was resuspended in 1/10 original volume of 2D sample buffer (9 M urea, 4% Triton X-100, 10%β-mercaptoethanol, 2% ampholines 3.5–10 (Sigma)) and separated by isoelectric focusing (first dimension) using 125-mm 3% polyacrylamide tube gels over 5000 Vh. After equilibration of the focused gels in Laemmli sample buffer, they were loaded on 6% polyacrylamide gels and subjected to SDS-PAGE (second dimension) in a xi electrophoresis cell (Bio-Rad). The two-dimensionally separated dermal proteins were then electrophoretically transferred to nitrocellulose membrane and probed with p200-specific antibodies. For N-terminal sequencing, two-dimensionally separated dermal extracts were transferred to polyvinylidenedifluoride membrane and the immunoreactive spot was analyzed using an ABI 492 CLC sequencing system (Applied Biosystems, Foster City, CA) following the instructions of the manufacturer. Mass spectrometry analysis was performed as described byButt et al., 2000Butt E. Bernhardt M. Smolenski A. et al.Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases.J Biol Chem. 2000; 275: 5179-5187Crossref PubMed Scopus (248) Google Scholar. Briefly, the protein spot was excised from the gel and transferred to a quartz vessel. Gel pieces were dehydrated under vacuum and soaked with trypsin solution (0.05 μg/μL trypsin; Promega, Madison, WI). After digestion, the peptides were extracted with 8 μL of 5% formic acid for 15 min in the sonication bath and analyzed using an ESI-ion trap mass spectrometer (LCQ Classic, Thermo Finnigan, San Jose, CA) directly coupled to a nano-HPLC system (LC-Packings, Dionex, Idstein, Germany). For fully automated interpretation of the mass spectra, the NCBI protein database and the EST database were searched using the SEQUEST algorithm. Sera from six patients with anti-p200 pemphigoid were assessed for IgG subclass distribution of p200-specific autoantibodies. Representative results of this analysis are shown in Figure 1. In all six sera anti-p200 reactivity was mediated by IgG4 autoantibodies. Only one serum demonstrated weak additional IgG1 and IgG2 reactivity. As shown in Figure 2(a), under nonreducing conditions, a buffer containing 8 M urea can solubilize only a trace amount of p200, whereas after incubation with 4 M urea, no extractable protein can be detected by immunoblotting. In contrast, reduction of disulfide bonds with β-mercaptoethanol enables significant amounts of p200 to be solubilized by both urea concentrations. Pretreatment of the dermis with bacterial collagenase fails to improve the extractability of p200 under nonreducing conditions. The solubility of p200 obtained by extraction with 8 M urea plus β-mercaptoethanol was determined in decreasing concentrations of urea, a urea-free buffer containing a mixture of three detergents (RIPA), and a high-salt buffer without urea or detergents (TBS). The results of this analysis are presented in Figure 2(b). Reduction of the urea concentration by 50% caused roughly 50% of p200 to precipitate. Approximately 60% of originally extracted protein remained soluble in 4 M, 20% in 2 M, and 10% in 1 M urea. p200 was completely insoluble in nonurea buffers. Addition of detergents did not improve the solubility of the protein. Total cellular extracts and concentrated conditioned medium of both cultured human keratinocytes and fibroblasts were separated by SDS-PAGE and studied by immunoblotting. The results of this analysis are presented in Figure 2(c). Although cultured cells were demonstrated to produce various basement membrane proteins, including BP180, the unprocessed (200-kDa) and processed (165-kDa) forms of the α3 chain of laminin 5, and type VII collagen, sera from patients with anti-p200 pemphigoid did not react with any protein derived from the cellular extract or medium. To investigate the possibility that p200 requires the presence of both keratinocytes and fibroblasts for its expression in vitro, organotypic cocultures of human keratinocytes and fibroblasts were studied by indirect immunofluorescence microscopy using p200-specific sera. While type VII collagen was revealed as continuous lining at the epithelial–mesenchymal junction of cocultures, no deposition of p200 was detected (Figure 2d). To determine whether p200 is a member of the collagen family, dermal extracts were digested with bacterial collagenase and assayed for reactivity with sera from patients with anti-p200 pemphigoid and monoclonal antibody LH7.2 against human type VII collagen. Even prolonged incubation with bacterial collagenase did not result in degradation of p200 (Figure 3). In contrast, full-length type VII collagen (290 kDa) was digested to its collagenase-resistant NC1 domain (145 kDa) within 1 h of collagenase treatment. With longer incubation times, the 145-kDa band increased in sharpness but was not degraded any further, confirming the specificity of collagenolytic activity. Similar results were obtained using dermal type I and type VI collagens visualized by immunoblotting with specific rabbit antisera as controls (not shown). When dermal extracts were incubated at 37°C without collagenase, p200 and type VII collagen remained intact, as did bovine serum albumin subjected to collagenase digestion. Treatment of dermal extracts with bacterial protease V8 resulted in complete loss of p200 reactivity within 30 min after addition of the protease. No immunoreactive fragments could be detected even after separation of digestion products in a 4% to 20% gradient gel (not shown). N-Glycosidase F (PNGase) is capable of selectively removing all classes of asparagine-bound glycans from the protein backbone (Tarentino and Plummer, 1987Tarentino A.L. Plummer T.H. Peptide-N-(N-acetyl-β-glucosaminyl) asparagine amidase and endo-β-N-acetylglucosaminidase from Flavobacterium meningosepticum.Methods Enzymol. 1987; 138: 770-778Crossref PubMed Scopus (59) Google Scholar). Incubation of dermal extracts with N-glycosidase F resulted in an increase of electrophoretic mobility of the p200 autoantigen (Figure 4, left). The deglycosylated protein migrated almost exactly midway between the intact p200 in dermal extracts and BP180 recognized by rabbit serum SA8010 in extracts of cultured human keratinocytes (not shown). Therefore, the extent of N-glycosylation of p200 was estimated at approximately 10 kDa. Negative controls incubated without enzyme did not show any nonspecific degradation of p200, whereas digestion of bovine fetuin and human transferrin resulted in reduction of their apparent molecular weights to the expected sizes of N-deglycosylated proteins. The most common O-linked structure found in glycoproteins is Gal-β-1,3 GalNAc that can be successfully removed by O-glycosidase (Endo and Kobata, 1976Endo Y. Kobata A. Partial characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Diplococcus pneumoniae.J Biochem. 1976; 80: 1-8Crossref PubMed Scopus (87) Google Scholar). Nevertheless, modification of this core structure, usually by sialylation, can block the action of the enzyme and needs to be removed by a suitable neuraminidase (sialidase). Therefore, to investigate the presence of O-linked oligosaccharides on the polypeptide backbone of p200, dermal extracts were sequentially treated first with neuraminidase and then with O-glycosidase. This treatment did not result in a reduction of apparent molecular weight of the p200 molecule, whereas bovine fetuin showed an expected increase in electrophoretic mobility after each deglycosylation step (Figure 4, right). Hence, sialic acid and O-glycans appear to be absent in p200. For further characterization of the glycosylation status of p200, dermal extracts were treated with bacterial chondroitinase ABC and heparitinase I. After digestion with chondroitinase, no shift in electrophoretic mobility of p200 was observed. In contrast, proteoglycan I (biglycan), also present in dermal extract, was efficiently degraded to its 47-kDa core protein as detected by immunoblotting with a biglycan-specific rabbit antibody. Similarly, heparitinase I had no effect on the apparent molecular weight of the p200 molecule, while it rapidly hydrolyzed bovine heparan sulfate as evidenced by an increased adsorption at 232 nm (not shown). To demonstrate that all six anti-p200 sera bind the same molecule, we electrophoresed extracts of normal human dermis in two dimensions (isoelectric focusing followed by SDS-PAGE). Separated proteins were transferred to nitrocellulose for immunoperoxidase staining. Figure 5 shows that different p200 sera identify the same major acidic spot at pI 5.4 to 5.6 and molecular weight of 200 kDa. The spots recognized by different patient sera comigrate with each other and with the 200-kDa protein stained by the same sera in dermal extracts separated only by SDS-PAGE. Normal human sera did not label this spot. In an attempt to determine the sequence of the p200 autoantigen, the immunoreactive spot of two-dimensionally separated dermal extracts was subjected to N-terminal sequencing. However, the protein concentration of the spot proved to be too low for this analysis. Alternatively, the immunoreactive spot was studied by mass spectrometry directly coupled to a nano-HPLC system. Mass spectrometry repeatedly identified the α3 chain of type VI collagen and filamin A as the major components of the spot. No other proteins could be detected even when using 10-fold concentrated dermal extract and narrow range high-resolution ampholines for isoelectric focusing. To determine whether p200 is identical to type VI collagen or filamin A, purified human type VI collagen (Rockland) and recombinant full-length human filamin A (gift of J.H. Hartwig, Boston, MA) were studied by immunoblotting using serum f
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