Eukaryotic Expression Of Autoantigen Dsg3 In Pemphigus Vulgaris And Application On The Clinical Detection

ObjectivePemphigus vulgaris (PV) is a potentially fatal blistering disease of the skin and mucous membranes characterized by the loss of intercellular adhesion (acantholysis) of keratinocytes due to binding of autoantibodies to the cell surface. Autoreactive IgG found in patients with human PV binds the cell surface of keratinocytes in stratified squamous epithetlia, and causes loss of cell-cell adhesion with resultant blister formation. The target molecule for autoantibodies in human PV has been identified as desmoglein (Dsg) 3, a transmembrane desmosomal component that belongs to the cadherin supergene family. The deduced amino acid sequences of Dsg showed marked homology with members of Ca²⁺-dependent cell adhesion molecules, cadherins, especially with desmogleinâ… (Dsgl), which is a transmembrane glycoprotein of desmosome complex and also the autoantigen for pemphigus foliaceus (PF)。Like classic cadherins, PVA has five extracellular subdomnains of approximately equal size, termed EC1 (amino terminus) through EC5 (carboxy terminus of the extracellular portion). The three-dimensional structure of the NH2-terminal EC domain (EC1) of N-cadherin determined by x-ray crystallography revealed an important element of dimerization, called the strand dimer. Indeed, several findings in the literature provide evidence that the rest of the cadherin EC domain has a function in adhesion beyond serving as a simple spacer region. Although some adhesion blocking antibodies have been found to bind to EC1, many adhesion blocking antibodies and an adhesion activating antibody have been found to recognize other EC domains, including EC5 and EC3. In addition, naturally occurring missense mutations in EC2 and EC3 domains of E-cadherin have been found in several human tumors, and mutations in one Ca²⁺-binding site of E-cadherin between EC domains abolish adhesive function.Furthermore, a biophysical study measuring direct molecular forces between cadherin ectodomains found evidence for multiple adhesive interactions, with maximal adhesive force developing when the ectodomains domains overlap entirely. Although none of these studies identified specific binding sites, they do suggest that five extracellular subdomnains EC domains other than EC1 play important roles in the homophilic binding interactions.The recombinant protein of human Dsg3, which includes the entire extracellular domain of human Dsg3, has become obtainable with the use of a baculovirus expression system that express most, if not all, of the conformational epitopes of the native antigen recognized by human PV sera However, mammalian-expression system is more superior than baculovirus-expression system in protein processing, and recombinant protein in mammalian-expression system is more coincidence with natural protein structure of Dsg3.In this study, we obtained recombinant of the entire coding region of Dsg3 in mammalian-expression system. We focused on immunoreactivity of PV sero against recombinant protein Dsg3 and appear to be recognized by a subset of sera from PV patients. In addition, we attempted to utilize recombinant Dsg3 to represent the entire extracellular domain of Dsg3 and develop specific ELISA for the detection od Abs directed against these Ags. We examined the utility of the ELISA in the diagnosis of PV.Materials and MethodsCase and capital Materials1. Patient of Pemphigus vulgaris: The entire 40 patient were from Liaoning Province, 22 of whom were men, the rest 18 women, with the age from 14 to 60, an average age 38±14. They were collected from dermatological institutions, The First Affiliated Hospital of China Medical University. All the patient were given clinic, histopathology and indirect immunofluorescence staining. Healthy control sera were obtained from 40 normal individuals.2. Plasmid,strain and cell: Plasmid pcDNA3.1^TM/myc-His (-) B were provided by Pro. Zhang Zhaoshan from academy of military medical sciences in Beijing. Strain TG1, Hela cell were provided by pathogenic microorganism department of China Medical University.3. Capital Materials: Ni-NTA purification system, transfection kit, Lipofectamine^TM 2000 and G418 (Geneticin) were bought from Company Invitrogen of America. QIAprep Spin Miniprep Kit was bought from Qiagen, Trypsin. RPMI1640 medium were bought from Company Qiagen of America. Restriction enzyme, alkali phosphatase and T4 DNA joining enzyme, RNA easy Mini kit. Plasmid purification Miniprep kit and etc were bought respectively from Takara Bio-engineering Company and Company Promega.4. Experimental instruments: CO₂ incubator, superclean bench, low temperature refrigeration, ultraviolet spectrophotometer, automatic running gel imaging system, GP electrophoresis apparatus,-80℃deep freeze refrigerator, microscope.Methods1. The extraction of RNA template and the obtaining of cDNA.2. The amplification,clone,recombination and identification of Dsg3 ECl-EC5 cDNA frag.3. Stable transfection of Dsg3 into Hela cell line mediate by Lipofectamine^TM 2000. 4. Ues SDS-PAGE identify PCR product.5. Purification of recombinant protein and identification of Western-blot.6. Clinical application of recombinant protein.Results1. Identification of recombinant plasmid pcDNA3.1(-)-Dsg 3Using RT-PCR amplification epithelial cell RNA by specific primer, it has been proved that PCR product sequencing is consistent with Dsg3 EC1-EC5 sequence of Gene Bank. Insert PCR product into expression vector pcDNA3.1^TM/myc-His(-)B, and then turn it into E. coli TG1, picking cloned bacterium grown on LB/Amp medium then extract plasmid. Carrying out enzyme cutting by homologous restriction enzyme, respectively cutting out the clone of target gene frag and carrying agen frag, that is the masculine clone named pcDNA3.1^TM/myc-His(-)B-Dsg3.2. The SDS-PAGE identifing of recombination protein pcDNA3.1^TM/myc-His(-)B-Dsg3 expressed product in Hela cellIn the Hela cell of recombinant plasmid pcDNA3.1^TM/ myc-His (-) B-Dsg3 stable transfection, pcDNA3.1^TM/myc-His (-) B-Dsg3 gene expressed fusion protein with the molecular mass of 66kDa or so. Hela cell without plasmid transfection and Hela cells transfection by plasmid pcDNA3.1TM/myc-His (-) B did not express the protein above.3. Identification product of pcDNA3.1^TM/myc-His (-) B-Dsg3 expressedWestern-blot displayed recombination gene (pcDNA3.1TM/myc-His (-) B-Dsg3) expressing fusion protein band can be identified by autoantibody IgG in sera of Pemphigus vulgaris patient.4. Detect Clinical sampleWe examined the reactivity of sera from 40 patients with PV, and 40 normal sera as control sera. On the Dsg3 ELISA, 38 of 40 seras (95%) from patients exceed the cutoff value. In contrast, none of 40 normal sera (0%) exceeded the cutoff value. None of 40 normal control sera were positive against Dsg3.DiscussionPatients with PV have the circulating autoantibodies against the cell surface of keratinocytes. The major target for autoantibodies in human PV is Dsg3. Characterization of the autoantibodies in human PV has been advanced by generation of the recombinant extracellular domains of human Dsg3 by baculovirus expression. The aim of this study was to cloned the entired coding region of Dsg3 by the mammalian expression system, and to develop a novel recombinant protein in mammalian to represent the entire extracellular domain of Dsg3, which will be a valuable tool to characterize the autoantibodies in Human PV.Isolation of cDNA clones encoding the pemphigus antigens is one of the major advancements in the investigations of pemphigus and has led to a greater understanding of the pathophysiology of those disease. In the study we aimed to produce a recombinant protein of PVA, which can absorb pathogenic autoantibodies from PV patients' sera. The previously produced bacterial fusion proteins for PVA represented only the sequential epitopes, and failed to absorb out pathogenic antibodies from PV sera. Later, Amagai constructed a chimeric molecule of the entire extracellular domain of PVA tailed with the constant region of human IgG1, termed PV Ig, and produced it in COS7 cells and in Sf9 insect cells by baculovirus expression system. In the study, we constructed recombinant fusion protein by mammalian expression system. Compared with baculovirus expression system, mammalian expression system is more superior for modify and processing of recombinant protein. Therefore, we attempted to produce PVA recombinant proteins in mammalian expression system, which can mediated posttranslational modification like glycosylation, proteolytic processing, and protein folding. Compared with previous recombinant Dsg3 protein, the recombinant proteins produced in mammalian cells are more facilitated in purification of recombinant Dgs3 protein and much more likely to contain more natural epitopes. The recombinant pemphigus Ags, which possess the native conformation, have enabled us to develop ELISAs as sensitive and specific diagnostic tools for pemphigus. ELISA studies have shown that sera from patients with mucosal-dominant PV react mainly against Dsg3. Conventional immunofiuorescence testing using a variety of animal and human tissue as substrate is universally used to diagnose and follow the disease activity of patients with pemphigus. Routine immunofiuorescence testing, however, is unable to distinguish between Abs directed against Dsg3 and Dsgl and also Abs against other nonrelevant Ags present in the tissue substrate. Use of the Dsg3 ELISAs allowed for the precise qualitation of Ab against the specific target Ags in PV. The diagnosis of PV can made utilizing these ELISAs by the following criteria: positive reactivity against Dsg3 regardless of reactivity against Dsgl indicateds PV. This information is of critical importance in determining the appropriate prognosis for these patients. Although immunoblot assay using various Ag sources has been helpful for this purpose, these ELISAs are superior because the ELISA can detect Abs against conformation dependent epitopes on the Dsg3 molecules.In summary, we have developed ELISAs using the recombinant pemphigus Ags. which when used together provide a sensitive, specific tool for the evaluation of patients with PV. This diagnostic tool will be useful not only for clinical application but also for the more precise characterization of the Ab response in patients with PV and for understanding fundamental immunopathologic methanism of PV.ConclusionDsg3 EC1-EC5 contained the epitopes relevant to PV antibodies, which play an important role in the pathogenesis of pemphigus vulgaris.