Subject(s)
Pemphigoid, Benign Mucous Membrane , Pemphigoid, Bullous , Humans , Autoantibodies , Retrospective Studies , Integrin beta4 , Italy , Mucous Membrane , AutoantigensABSTRACT
Plectin is a linker protein that interacts with intermediate filaments and ß4 integrin in hemidesmosomes of the epidermal basement membrane zone (BMZ). Type XVII collagen (COL17) has been suggested as another candidate plectin binding partner in hemidesmosomes. Here, we demonstrate that plectin-COL17 binding helps to maintain epidermal BMZ organization. We identified an epidermolysis bullosa (EB) simplex patient as having markedly diminished expression of plectin and COL17 in skin. The patient is compound heterozygous for sequence variants in the plectin gene (PLEC); one is a truncation and the other is a small in-frame deletion sequence variant. The in-frame deletion is located in the putative COL17-binding domain of plectin and abolishes the plectin-COL17 interaction in vitro. These results imply that disrupted interaction between plectin and COL17 is involved in the development of EB. Our study suggests that protein-protein binding defects may underlie EB in patients with unidentified disease-causing sequence variants.
Subject(s)
Autoantigens/metabolism , Epidermolysis Bullosa Simplex/genetics , Non-Fibrillar Collagens/metabolism , Plectin/genetics , Autoantigens/genetics , Basement Membrane/metabolism , Epidermolysis Bullosa Simplex/diagnosis , Epidermolysis Bullosa Simplex/pathology , Genetic Variation , Hemidesmosomes/metabolism , Humans , Infant, Newborn , Keratinocytes/metabolism , Male , Non-Fibrillar Collagens/genetics , Plectin/metabolism , Protein Binding , Protein Domains , Sequence Deletion , Skin/pathology , Collagen Type XVIIABSTRACT
In this study, we generated a new set of monoclonal antibodies (mAbs) to bovine and human type VII collagen (COL7) by immunizing mice with bovine cornea-derived basement membrane zone (BMZ) fraction. The four mAbs, tentatively named as COL7-like mAbs, showed speckled subepidermal staining in addition to linear BMZ staining of normal human skin and bovine cornea, a characteristic immunofluorescence feature of COL7, but showed no reactivity with COL7 by in vitro biochemical analyses. Taking advantage of the phenomenon that COL7-like mAbs did not react with mouse BMZ, we compared immunofluorescence reactivity between wild-type and COL7-rescued humanized mice and found that COL7-like mAbs reacted with BMZ of COL7-rescued humanized mice. In ELISAs, COL7-like mAbs reacted with intact triple-helical mammalian recombinant protein (RP) of COL7 but not with bacterial RP. Furthermore, COL7-like mAbs did not react with COL7 within either cultured DJM-1 cells or basal cells of skin of a bullous dermolysis of the newborn patient. These results confirmed that COL7-like mAbs reacted with human and bovine COL7. The epitopes for COL7-like mAbs were considered to be present only on mature COL7 after secretion from keratinocytes and deposition to BMZ and to be easily destroyed during immunoblotting procedure. Additional studies indicated association of the speckled subepidermal staining with both type IV collagen and elastin. These unique anti-COL7 mAbs should be useful in studies of both normal and diseased conditions, particularly dystrophic epidermolysis bullosa, which produces only immature COL7.
Subject(s)
Basement Membrane/metabolism , Collagen Type VII/immunology , Collagen Type VII/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Cattle , HEK293 Cells , Humans , MiceABSTRACT
Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and ß4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.
Subject(s)
Carcinoma, Squamous Cell/ultrastructure , Hemidesmosomes/ultrastructure , Skin Neoplasms/ultrastructure , Autoantigens/isolation & purification , Autoantigens/metabolism , Carrier Proteins , Cell Adhesion Molecules/isolation & purification , Cell Adhesion Molecules/metabolism , Cell Fractionation , Cell Line, Tumor , Cytoskeletal Proteins , Dystonin , Hemidesmosomes/chemistry , Humans , Integrin alpha6/isolation & purification , Integrin alpha6/metabolism , Integrin beta4/isolation & purification , Integrin beta4/metabolism , Membrane Glycoproteins/isolation & purification , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins , Non-Fibrillar Collagens/isolation & purification , Non-Fibrillar Collagens/metabolism , Plectin/isolation & purification , Plectin/metabolism , Subcellular Fractions , Kalinin , Collagen Type XVIIABSTRACT
We report a 68-year-old Japanese female patient with subepidermal blistering disease with autoantibodies to multiple laminins, who subsequently developed membranous glomerulonephropathy. At skin disease stage, immunofluorescence demonstrated IgG anti-basement membrane zone antibodies reactive with dermal side of NaCl-split skin. Immunoblotting of human dermal extract, purified laminin-332, hemidesmosome-rich fraction and laminin-521 trimer recombinant protein (RP) detected laminin γ-1 and α-3 and γ-2 subunits of laminin-332. Three years after skin lesions disappeared, nephrotic symptoms developed. Antibodies to α-3 chain of type IV collagen (COL4A3) were negative, thus excluding the diagnosis of Goodpasture syndrome. All anti-laminin antibodies disappeared. Additional IB and ELISA studies of RPs of various COL4 chains revealed reactivity with COL4A5, but not with COL4A6 or COL4A3. Although diagnosis of anti-laminin γ-1 (p200) pemphigoid or anti-laminin-332-type mucous membrane pemphigoid could not be made, this case was similar to previous cases with autoantibodies to COL4A5 and/or COL4A6.
Subject(s)
Autoantibodies/analysis , Autoimmune Diseases/immunology , Blister/immunology , Collagen Type IV/immunology , Glomerulonephritis, Membranous/immunology , Kidney/immunology , Laminin/immunology , Skin/immunology , Aged , Autoantibodies/blood , Autoimmune Diseases/blood , Autoimmune Diseases/diagnosis , Autoimmune Diseases/therapy , Biopsy , Blister/blood , Blister/diagnosis , Blister/therapy , Female , Fluorescent Antibody Technique , Glomerulonephritis, Membranous/blood , Glomerulonephritis, Membranous/diagnosis , Glucocorticoids/therapeutic use , Humans , Kidney/ultrastructure , Plasma Exchange , Predictive Value of Tests , Protein Subunits , Skin/drug effects , Skin/pathology , Time FactorsABSTRACT
Plectin is a cytoskeletal linker protein which has a long central rod and N- and C-terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS-MD), and EBS with pyloric atresia (EBS-PA). Previous studies have demonstrated that loss of full-length plectin with residual expression of the rodless isoform leads to EBS-MD, whereas complete loss or marked attenuation of expression of full-length and rodless plectin underlies the more severe EBS-PA phenotype. However, muscular dystrophy has never been identified in EBS-PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon-causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient.
Subject(s)
Digestive System Abnormalities/genetics , Epidermolysis Bullosa Simplex/complications , Epidermolysis Bullosa Simplex/genetics , Muscular Dystrophies/genetics , Mutation , Plectin/deficiency , Pylorus/abnormalities , Base Sequence , Cells, Cultured , Digestive System Abnormalities/complications , Epidermolysis Bullosa Simplex/physiopathology , Exons/genetics , Fatal Outcome , Female , Fibroblasts/metabolism , Fluorescent Antibody Technique , Genotype , Humans , Immunoblotting , Infant, Newborn , Male , Molecular Sequence Data , Muscular Dystrophies/complications , Phenotype , Plectin/chemistry , Plectin/genetics , Plectin/metabolism , Skin/metabolismABSTRACT
Plectin is a cytoskeletal linker protein that has a dumbbell-like structure with a long central rod and N- and C-terminal globular domains. Mutations in the gene encoding plectin (PLEC1) cause two distinct autosomal recessive subtypes of epidermolysis bullosa (EB): EB simplex with muscular dystrophy (EBS-MD), and EB simplex with pyloric atresia (EBS-PA). Here, we demonstrate that normal human fibroblasts express two different plectin isoforms including full-length and rodless forms of plectin. We performed detailed analysis of plectin expression patterns in six EBS-MD and three EBS-PA patients. In EBS-PA, expression of all plectin domains was found to be markedly attenuated or completely lost; in EBS-MD, the expression of the N- and C-terminal domains of plectin remained detectable, although the expression of rod domains was absent or markedly reduced. Our data suggest that loss of the full-length plectin isoform with residual expression of the rodless plectin isoform leads to EBS-MD, and that complete loss or marked attenuation of full-length and rodless plectin expression underlies the more severe EBS-PA phenotype. These results also clearly account for the majority of EBS-MD PLEC1 mutation restriction within the large exon 31 that encodes the plectin rod domain, whereas EBS-PA PLEC1 mutations are generally outside exon 31.
Subject(s)
Epidermolysis Bullosa Simplex/metabolism , Plectin/metabolism , Algorithms , DNA Mutational Analysis , Epidermolysis Bullosa Simplex/diagnosis , Exons , Fibroblasts/metabolism , Humans , Keratinocytes/cytology , Microscopy, Fluorescence/methods , Models, Genetic , Muscle, Skeletal/metabolism , Mutation , Plectin/chemistry , Protein Isoforms , Protein Structure, Tertiary , RNA, Messenger/metabolismABSTRACT
Transmembrane collagen XVII (COL17) is a hemidesmosomal component of basal keratinocytes that can be targeted by autoantibodies in autoimmune blistering disorders, including linear IgA dermatosis (LAD). COL17 can be physiologically cleaved within the juxtamembranous extracellular NC16A domain, and LAD autoantibodies preferentially react with the processed ectodomains, indicating that the processing induces neoepitopes. However, the details of how neoepitopes develop have not been elucidated. In this study, we show that C-terminal processing of COL17 also plays a role in inducing neoepitopes for LAD autoantibodies. First, the mAb hC17-ect15 targeting the 15th collagenous domain of COL17 was produced, which showed characteristics similar to LAD autoantibodies. The mAbs preferentially reacted with C-terminally deleted (up to 682 amino acids) recombinant COL17, suggesting that C-terminal processing shows neoepitopes on the 15th collagenous domain. The LAD autoantibodies also react with C-terminal deleted COL17. Therefore, neoepitopes for LAD autoantibodies also develop after C-terminal processing. Finally, the passive transfer of the mAb hC17-ect15 into human COL17-expressing transgenic mice failed to induce blistering disease, suggesting that neoepitope-targeting antibodies are not always pathogenic. In summary, this study shows that C-terminal processing induces dynamic structural changes and neoepitopes for LAD autoantibodies on COL17.
Subject(s)
Autoantigens/chemistry , Epitopes/chemistry , Linear IgA Bullous Dermatosis/immunology , Non-Fibrillar Collagens/chemistry , Animals , Antibodies, Monoclonal/chemistry , Autoantibodies/chemistry , Autoantibodies/immunology , Autoimmune Diseases/immunology , Gene Deletion , Humans , Immunoglobulin A/immunology , Immunoglobulin G/chemistry , Keratinocytes/chemistry , Linear IgA Bullous Dermatosis/therapy , Mice , Mice, SCID , Mice, Transgenic , Pemphigoid, Bullous/immunology , Protein Domains , Recombinant Proteins/chemistry , Skin/chemistry , Collagen Type XVIIABSTRACT
Previous studies of ocular mucous membrane pemphigoid (OMMP) have identified several components of the basement membrane zone to be autoantigens, including integrin ß4. However, there are no extensive or definitive reported studies that address this, particularly in pure OMMP. To clarify the major autoantigens in pure OMMP. In this study, we examined sera from 43 pure OMMP patients for both IgG and IgA antibodies using newly developed immunoblotting analyses with a hemidesmosome-rich fraction and various recombinant proteins of integrin α6ß4, in addition to our routine immune-serological tests. Using a hemidesmosome-rich fraction, sera from patients with pure OMMP demonstrated reactivity of IgG and/or IgA antibodies to integrin ß4, BP180 and laminin-332. The reactivity of pure OMMP sera to integrin ß4 was further confirmed by immunoblotting using integrin ß4 recombinant proteins. Using concentrated supernatant of HaCaT cells, only one serum sample showed positive IgG and IgA reactivity to LAD-1, the ectodomain of BP180. None of the pure OMMP sera reacted with any autoantigens on immunoblotting using normal human epidermal or dermal extracts, or purified human laminin-332. Integrin ß4 was considered to be the major and specific autoantigen for pure OMMP. The new methods established in this study are useful for detection of various autoantigens, particularly integrin ß4.
Subject(s)
Immunoglobulin A/blood , Immunoglobulin G/blood , Integrin beta4/immunology , Pemphigoid, Benign Mucous Membrane/blood , Autoantibodies/blood , Autoantigens/immunology , Case-Control Studies , Cell Adhesion Molecules/immunology , Fluorescent Antibody Technique, Indirect , Hemidesmosomes , Humans , Immunoblotting/methods , Non-Fibrillar Collagens/immunology , Pemphigoid, Benign Mucous Membrane/immunology , Recombinant Proteins/immunology , Kalinin , Collagen Type XVIIABSTRACT
Myoepithelial cells present in exocrine glands cause secretion from the glands by contraction. They have mixed characteristics with regard to cytoskeletal elements, containing both epithelial-type intermediate filaments and smooth muscle-type myofilaments. For further characterization, myoepithelial cells from bovine apocrine sweat glands and tracheal glands were here examined with special attention to the cell-substratum adhesion system. Immunofluorescence microscopy using a panel of antibodies against adherens-type junctional and hemidesmosomal proteins demonstrated two types of cell-substratum junctions in myoepithelial cells from both glands. Type-I hemidesmosomes (HDs) consisting of plectin, BP230, integrin alpha6beta4, and BP180 were thus observed as punctate arrays longitudinally arranged along myoepithelial cell surfaces, while adherens-type junctions were similarly evident as linear rib-like structures. Double-label immunofluoresence revealed the two junctions to be distributed in a mutually exclusive or independent manner. Electron microscopy further demonstrated that apocrine myoepithelial cells surround secretory epithelial cells completely, without any gaps, HDs being abundant along the basement membrane, but with no distinct structures in the inter-hemidesmosomal regions. Immunoelectron microscopy, however, revealed an interhemidesmosomal localization of vinculin, pointing to the existence of adherens-type junctions. Secretory epithelial cells in tracheal glands were found not to be completely covered with myoepithelial cells, so that more than half of them are directly attached to the basement membrane, where they form type II-HDs lacking BP230 and BP180, but no detectable adherens junctions, like epidermal basal cells and sebaceous gland cells. These observations demonstrate that, in addition to their cytoskeleton, myoepithelial cells have both epithelial- and smooth muscle-type cell-substratum adhesion structures, i.e. HDs and dense plaque-like adherens junctions.
Subject(s)
Adherens Junctions/physiology , Apocrine Glands/physiology , Hemidesmosomes/physiology , Trachea/physiology , Animals , Apocrine Glands/ultrastructure , Cattle , Cell Adhesion/physiology , Epithelium/physiology , Fluorescent Antibody Technique , Microscopy, ElectronABSTRACT
We describe a patient with widespread skin lesions and circulating IgG autoantibodies to both type VII collagen and laminin 5. Although autoantibodies to type VII collagen belonged to IgG2, IgG3, and IgG4 subclasses, laminin 5 was targeted exclusively by IgG3 autoantibodies. Interestingly, despite the presence of IgG3 autoantibodies, the patient's serum failed to fix complement to the dermoepidermal junction. In addition, these autoantibodies did not recruit and activate leukocytes or induce dermoepidermal separation in skin sectioned by cryostat. We report a most unusual case of an autoimmune subepidermal blistering with an exclusive IgG3 reactivity to laminin 5.
Subject(s)
Autoimmune Diseases/immunology , Blister/immunology , Collagen Type VII/immunology , Immunoglobulin G/immunology , Laminin/immunology , Autoimmune Diseases/pathology , Blister/pathology , Cell Adhesion Molecules/immunology , Erythema/immunology , Humans , Male , Microscopy, Fluorescence , Middle Aged , Skin/pathology , KalininABSTRACT
BACKGROUND: Since the original description by Zone et al in 1994, the disease entity and target antigens in linear IgA/IgG bullous dermatosis (LAGBD) have not been clarified in 20 years. OBJECTIVES: To determine autoantibodies and autoantigens in a new LAGBD case which showed atypical clinical and histopathological findings without apparent mucosal involvement. MATERIALS AND METHODS: We performed various indirect immunofluorescence and immunoblotting studies. RESULTS: Indirect immunofluorescence of 1M NaCl-split skin showed IgG and IgA reactivity with both epidermal and dermal sides. Immunoblotting studies using various antigen sources revealed circulating IgG and IgA antibodies reactive with laminin-332, laminin-γ1 and integrin α6ß4 in various patterns. Absorption study using recombinant proteins of laminin-γ1 indicated that the patient serum reacted with different epitopes between laminin-γ1 and laminin-γ2. CONCLUSIONS: This study presented for the first time a LAGBD patient with IgG and IgA antibodies to various laminins and integrins.
Subject(s)
Autoantibodies/blood , Integrins/metabolism , Laminin/metabolism , Linear IgA Bullous Dermatosis/immunology , Linear IgA Bullous Dermatosis/pathology , Administration, Oral , Administration, Topical , Adrenal Cortex Hormones/therapeutic use , Aged, 80 and over , Biopsy, Needle , Fluorescent Antibody Technique, Indirect , Follow-Up Studies , Humans , Immunohistochemistry , Integrins/immunology , Laminin/immunology , Linear IgA Bullous Dermatosis/drug therapy , Male , Risk Assessment , Severity of Illness Index , Treatment OutcomeABSTRACT
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.
Subject(s)
Autoantigens/chemistry , Autoantigens/immunology , Dermis/immunology , Epidermis/immunology , Pemphigoid, Bullous/immunology , Acids , Autoantigens/metabolism , Basement Membrane/chemistry , Basement Membrane/immunology , Buffers , Cells, Cultured , Chondroitin Sulfates/metabolism , Collagen , Dermis/chemistry , Disulfides/chemistry , Disulfides/immunology , Electrophoresis, Gel, Two-Dimensional , Epidermis/chemistry , Fibroblasts/cytology , Glycoproteins/chemistry , Glycoproteins/immunology , Glycoproteins/metabolism , Glycosylation , Heparitin Sulfate/metabolism , Humans , Keratinocytes/cytology , Mass Spectrometry , Molecular Weight , Pemphigoid, Bullous/metabolism , Sequence Analysis, Protein , Solubility , UreaABSTRACT
Bullous pemphigoid antigen 180 (BP180)/type XVII collagen is a transmembrane hemidesmosomal protein. Previously, we demonstrated that the collagenous ectodomain of BP180 can be cleaved within the extracellular non-collagenous (NC) 16A domain adjacent to the cell membrane and released from the cell surface. Here, we report that the BP180 cleavage is mediated by a membrane-associated metalloprotease expressed in epithelial cells. A tissue inhibitor of metalloprotease 1 (TIMP-1), but not TIMP-2, like the synthetic metalloprotease inhibitor KB-R8301, significantly reduced the cleavage. Within epithelial cells cultured for more than 36 h past confluency, antibodies to BP180 showed a reduced hemidesmosomal staining. Observed for the first time, addition of KB-R8301 to the cell culture preserved this staining. To examine the effect of the extracellular cleavage of BP180 on molecular interactions among hemidesmosomal components, we eliminated its collagenous extracellular portion, except for the NC16A domain, by collagenase digestion. Interestingly, this collagenase treatment caused partial disassembly of hemidesmosomal components in cultured human keratinocytes. Moreover, a monoclonal antibody specific for the cleaved extracellular fragment detected a unique tissue distribution of the fragment that might reflect an association of the cleavage process with the mitotic activity of epithelial tissues. Our observations demonstrate that the cleavage of BP180 occurring within the NC16A domain is mediated by a membrane-associated metalloprotease and suggest a possible involvement of the cleavage in hemidesmosomal disassembly.
Subject(s)
Autoantigens/metabolism , Autoantigens/physiology , Hemidesmosomes/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/pharmacology , Carrier Proteins , Cattle , Cell Line , Collagenases/pharmacology , Cytoskeletal Proteins , Dystonin , Epithelial Cells/chemistry , Hemidesmosomes/immunology , Metalloproteases/metabolism , Metalloproteases/physiology , Nerve Tissue Proteins , Non-Fibrillar Collagens , Protease Inhibitors/pharmacology , Protein Structure, Tertiary , Tissue Distribution , Tissue Inhibitor of Metalloproteinase-1/pharmacology , Collagen Type XVIIABSTRACT
BACKGROUND: The autoantigen for the major type of linear IgA bullous dermatosis (LAD, lamina lucida type) is the shed ectodomain of BP180. However, it is unknown why most LAD sera react with the shed ectodomain but not with the intact BP180/type XVII collagen. OBJECTIVE: The aim of this study was to characterize the unique cleavage-dependent epitope(s) in the shed ectodomain. METHODS: We used a monoclonal antibody (MAb-1337) and six LAD sera, which reacted preferentially with the shed ectodomain of BP180. The location and characteristics of the epitopes for these antibodies were analyzed mainly by immunoblotting using chimeric bovine-human BP180 mammalian recombinant proteins and variously truncated bacterial recombinant proteins. RESULTS: LAD sera and MAb-1337 reacted with the plasmin-digested products of full-length BP180. Four of the six LAD sera reacted to a bacterial recombinant protein consisting of the human non-collagenous 16th A (NC16A) and the collagenous 15th (C15) domains, while these sera were negative or only faintly reactive with the NC16A and C15 recombinants. The epitope for MAb-1337 was localized to the COOH-terminal 21 amino acid region within the NC16A domain. CONCLUSION: The results in this study indicate that the major epitopes for LAD sera are formed or exposed by a cleavage-induced conformational change, but not by a post-translational modification that occurs only in the shed ectodomain, and are located at the boundary between the NC16A and C15 domains.
Subject(s)
Collagen/chemistry , Epitopes/chemistry , Skin Diseases, Vesiculobullous/immunology , Amino Acid Sequence , Animals , Autoantigens/chemistry , Cattle , Cell Line, Tumor , Culture Media, Conditioned/chemistry , Epitope Mapping , Fibrinolysin/chemistry , HeLa Cells , Humans , Immunoglobulin A/chemistry , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Non-Fibrillar Collagens/chemistry , Protein Processing, Post-Translational , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Transfection , Collagen Type XVIISubject(s)
Autoantibodies/immunology , Laminin/immunology , Antibodies, Monoclonal/blood , Autoantibodies/blood , Epitopes/blood , Epitopes/immunology , Fluorescent Antibody Technique , Glycosylation , Humans , Immunoblotting , Immunologic Factors/blood , Immunologic Factors/immunology , Laminin/blood , Laminin/metabolism , Recombinant Proteins/blood , Recombinant Proteins/immunologySubject(s)
Autoantigens/chemistry , Autoantigens/genetics , Carrier Proteins , Collagen/chemistry , Collagen/genetics , Cytoskeletal Proteins , Nerve Tissue Proteins , Non-Fibrillar Collagens , Amino Acid Sequence , Dystonin , Humans , Molecular Sequence Data , Peptide Fragments/genetics , Protein Structure, Tertiary , Collagen Type XVIIABSTRACT
We have shown that binding of bullous pemphigoid (BP)-patient IgG (BP-IgG) causes the internalization of BP180 from the cell membrane. This study examined whether BP-IgG treatment can deplete cultured keratinocytes of BP180, how it affects cellular levels of alpha6 and beta4 integrins (by western blot analysis using monoclonal antibodies to these antigens), and whether it reduces adhesion of cells to the culture dish (by a vibration detachment assay). All BP-IgG or BP sera with high values of BP180-ELISA from 18 BP patients before and after oral corticosteroid treatment showed dramatically decreased BP180 in cells after 6 hours of BP-IgG stimulation, whereas alpha6 and beta4 integrin levels were not decreased. Even IgG from patients in whom oral corticosteroid had suppressed active blistering could deplete cells of BP180, as long as sera retained a high value of BP180-ELISA. On the other hand, reduction of cell BP180 content increased detachment of cells from the dish. These results suggest that BP-IgG reduces hemidesmosomal BP180 content, weakening the adhesion of hemidesmosomes to the lamina densa. In the presence of BP180 deficiency, inflammation generated by BP180 immune-complex formation might then tear the weakened lamina lucida, and this could lead to generation of the BP-specific split at the lamina lucida.
Subject(s)
Autoantibodies/physiology , Autoantigens/physiology , Immunoglobulin G/physiology , Keratinocytes/metabolism , Non-Fibrillar Collagens/physiology , Pemphigoid, Bullous/immunology , Adrenal Cortex Hormones/therapeutic use , Autoantigens/analysis , Autoantigens/immunology , Cell Adhesion , Cells, Cultured , Humans , Integrin alpha6/analysis , Integrin beta4/analysis , Non-Fibrillar Collagens/analysis , Non-Fibrillar Collagens/immunology , Pemphigoid, Bullous/drug therapy , Pemphigoid, Bullous/etiology , Collagen Type XVIIABSTRACT
Anti-p200 pemphigoid is an autoimmune skin disease characterized by tense blisters, subepidermal split formation, and mainly neutrophilic inflammatory infiltration of the dermal-epidermal junction (DEJ). Direct immunofluorescence microscopy of perilesional skin biopsies demonstrates linear deposits of IgG and C3 along the DEJ, while by indirect immunofluorescence microscopy on NaCI-split human skin, patients' IgG labels the dermal side. The antigenic target of the autoantibodies is a 200 kD protein (p200) of the lower lamina lucida that can be detected in human dermal extracts by immunoblotting. While p200 is thought to be important for cell-matrix adhesion, its exact identity is unknown. To date, the p200 autoantigen has been demonstrated to be distinct from bullous pemphigoid antigens 180 und 230, laminin 1, 5, and 6, alpha6beta4 integrin, and type VII collagen. Biochemical characterization of the p200 molecule revealed a noncollagenous N-glycosylated acidic protein with an isoelectric point of approximately 5.5. We provide an overview on pathogenesis, clinical features, diagnosis, and treatment of this unique autoimmune dermatosis.