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Dissertation
Structural basis for autoantibody recognition of phosphatidylserine-[beta]2-glycoprotein I and apoptotic cells
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania and Hahnemann University
Aug 2001
DOI:
https://doi.org/10.17918/00003501
Abstract
The work described in this dissertation was undertaken to explore the nature of the autoantibody response in two antibody-mediated autoimmune diseases, systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS). Mutant versions of a murine anti-DNA and anti-phospholipid autoantibody, 3H9, were constructed and expressed as recombinant single chain Fv (scFv) in Escherichia coli. Purified scFv were tested for binding to phosphatidylserine and to phosphatidylserine as a complex with [beta]2-glycoprotein I ([beta]2GPI) in ELISA, revealing that the 3H9 heavy (H) chain V gene encodes specificity for these antigens. Moreover, it was determined that higher affinity for phosphatidylserine-[beta]2GPI could be achieved by the introduction of arginine residues into the CDR1, CDR2, and FWR3 of the 3H9 H chain at positions previously shown to be important for DNA binding. Flow cytometric analysis of structurally diverse variants of 3H9 established that the autoantibodies preferentially recognize Jurkat cells in advancing stages of apoptosis as defined by positive binding of annexin V and staining with propidium idodide. Confocal fluorescence microscopy revealed that the scFv initially bind to the cell surface at positions that overlap with the binding of annexin V and that the binding of the scFv and annexin V begin to segregate as the cell undergoes blebbing. The segregation of binding was found to culminate in the exclusive localization of the scFv to surface blebs on the apoptotic cells, with annexin V bound to regions between adjacent blebs. The results of these experiments suggest that, in SLE, B cells that express Ig receptors reactive against phosphatidylserine bind to apoptotic blebs and may have implications for the processing of nuclear antigens and the regulation of immune tolerance to self.
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Details
- Title
- Structural basis for autoantibody recognition of phosphatidylserine-[beta]2-glycoprotein I and apoptotic cells
- Creators
- Brian Anthony Cocca
- Contributors
- Marko Z. Radic (Advisor) - Drexel University, Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Awarding Institution
- Medical College of Pennsylvania and Hahnemann University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Medical College of Pennsylvania and Hahnemann University; Philadelphia, Pennsylvania
- Number of pages
- viii, 127 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Other Identifier
- 991021888798804721