Blockade of Complement Activation in Bullous Pemphigoid by Using Recombinant CD55-CD46 Fusion Protein
Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by the presence of autoantibodies targeting components of the basement membrane zone (BMZ). The pathogenesis of BP is closely associated with complement activation, which is triggered by these autoantibodies. Complement activation leads to the formation of membrane attack complexes, causing tissue damage and blister formation. Complement-regulatory proteins (CRPs), such as CD35, CD46, CD55, and CD59, play a crucial role in controlling the complement system by regulating enzyme cascades, preventing the assembly of membrane attack complexes, and maintaining homeostasis. Among these, CD55 and CD46 are particularly important as they inhibit the production of C3 and C5 convertases and accelerate the degradation of existing convertases, thereby protecting cells from complement-mediated damage.
In this study, a novel recombinant fusion protein, designated as DM (CD55-CD46), was developed to enhance the inhibitory activity against complement activation in BP. The fusion protein was designed by combining the coding sequences of CD55 and CD46, which share similar structural features, including a signal peptide, four complement control protein-repeat domains, and a serine/threonine-rich domain. The fusion gene was constructed by joining the coding sequences of CD55 and CD46 in-frame, resulting in a chimeric protein with potentially greater complement regulatory activity.
The amino acid and nucleotide sequences of CD55 and CD46 were analyzed and codon-optimized using bioinformatics tools such as ClustalW and Vector NTI Viewer. The fusion gene was then ligated into the pET-28a expression vector using Nde I and Xho I restriction sites. The recombinant plasmid, pET-28a-DM, was confirmed through double-enzyme digestion and DNA sequencing, ensuring the correct insertion of the fusion gene. The plasmid was transformed into E. coli Top10 cells, and the expression of the DM fusion protein was induced using isopropyl-b-D thiogalactopyranoside (IPTG). The fusion protein was predominantly found in inclusion bodies, which were solubilized and purified using NI-NTA affinity chromatography. The purified protein had a molecular weight of 71,000, as determined by SDS-PAGE, and its identity was further confirmed by Western blot analysis using monoclonal antibodies against CD55 and CD46.
To evaluate the functional efficacy of the DM fusion protein, HaCaT cells and healthy skin sections were incubated with pathogenic IgG from BP patients and fresh serum from healthy volunteers containing complement components. C3b deposition, a marker of complement activation, was assessed using immunofluorescence staining. The results demonstrated significant C3b deposition in HaCaT cells and the BMZ of healthy skin sections incubated with pathogenic IgG. However, this deposition was markedly inhibited by treatment with 10 mg/mL of the DM fusion protein. The inhibitory effect of the DM protein was significantly higher than that of CD55 or CD46 alone, as evidenced by reduced C3b deposition and lower C3a levels in the supernatant of HaCaT cells, as measured by enzyme-linked immunosorbent assay (ELISA).
The findings of this study highlight the potential therapeutic application of the DM fusion protein in BP. By combining the complement regulatory functions of CD55 and CD46, the DM fusion protein exhibited enhanced inhibitory activity against complement activation, which is a key driver of BP pathogenesis. This approach represents a novel strategy for targeting the complement system in autoimmune blistering diseases. The successful construction and functional validation of the DM fusion protein provide a foundation for further preclinical and clinical studies to explore its therapeutic potential in BP and other complement-mediated diseases.
The study also underscores the importance of complement regulatory proteins in maintaining immune homeostasis and preventing tissue damage in autoimmune diseases. Dysregulation of CD55 and CD46 has been implicated in various autoimmune conditions, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and myasthenia gravis. The development of chimeric CRP molecules, such as the DM fusion protein, offers a promising avenue for enhancing complement regulation and mitigating the pathological effects of autoantibody-mediated complement activation.
In conclusion, the recombinant CD55-CD46 fusion protein, DM, demonstrated significant inhibitory activity against complement activation in BP. The fusion protein effectively reduced C3b deposition and C3a production, highlighting its potential as a therapeutic agent for BP. Further research is needed to evaluate the safety and efficacy of the DM fusion protein in clinical settings and to explore its applicability in other complement-mediated diseases. This study contributes to the growing body of evidence supporting the therapeutic targeting of the complement system in autoimmune diseases and provides a novel approach for the development of complement regulatory therapies.
doi.org/10.1097/CM9.0000000000001312
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