- Title
- An investigation of IgE regulation by recombinant soluble IgE receptors and co-receptors in human cell culture models
- Creator
- Bowles, Sandra Lyn
- Subject
- Immunoglobulin E
- Subject
- Allergy
- Date Issued
- 2010
- Date
- 2010
- Type
- Thesis
- Type
- Doctoral
- Type
- PhD
- Identifier
- vital:10321
- Identifier
- http://hdl.handle.net/10948/1231
- Identifier
- Immunoglobulin E
- Identifier
- Allergy
- Description
- Type I hypersensitivities are mediated by the IgE antibody. The effector functions and synthesis of IgE result from interactions with a network of proteins that include a high affinity (FcRIα) and a low affinity (CD23, FcRII) Fc receptor in conjunction with the B lymphocyte receptor, CD21. CD23 is a multifunctional type II transmembrane protein that binds its known ligands through its ectodomain either as a membrane-bound or soluble receptor generated in vivo by specific proteolytic cleavages. IgE production is primarily regulated by interactions between IgE, CD23 and CD21. Despite its importance for development of strategies to limit hypersensitivity, precise information about the molecular interactions remains limited. During this study, I engineered, expressed and purified from bacteria three soluble human CD23 fragments that are normally formed in vivo and shed from the cell surface (1) derCD23, amino acids 156-298 (2) sCD23, amino acids 150-321 and (3) the entire ectodomain, exCD23, amino acids 48-321 to examine the comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to these fragments. Gel filtration HPLC revealed that derCD23 and sCD23 were monomeric whereas exCD23 assembled as a heterogeneous mixture that included trimers and monomers. At the concentrations utilized, CD23 fragments sCD23 and exCD23 bound CD21 with similar affinity, whereas interaction between derCD23 and CD21 was minimal when analyzed by surface plasmon resonance (SPR) spectroscopy. These findings suggest that penultimate “tail” amino acids between 298 and 321 stabilize CD21 attachment, although it cannot be ruled out, the region between Met 150 and Ser 156 may also play a role in binding CD21 SCR 1-2. In contrast, there is a progressive increment in the affinity of soluble fragments (exCD23>sCD23>derCD23) for IgE, upon increasing length of the proximal CD23 “stalk” domain. These findings highlight the differences in both the structural basis and affinity of the three physiological fragments of human CD23 for the ligands CD21 and IgE and underscore the complexity of CD23-mediated regulatory networks. It was found that B-cells only make up ~5% of the PBMC population, and that these cells were able to be activated, via STAT-6 phosphorylation, to enter class switch recombination (CSR) by the addition of switch factors (IL-4 and anti-CD40). Titration experiments dictated that 25 ng/mL of CD23 was the most efficient concentration to up-regulate IgE synthesis in PBMCs; furthermore, soluble CD23 proteins were incubated with PBMCs in the presence and absence of CD21 SCR 1-2 to investigate the effect that these recombinant proteins have on IgE synthesis. Results showed that the influence of recombinant proteins (both CD23 and CD21) on IgE synthesis was slight. It was shown that while derCD23 had no significant effect, monomeric sCD23 down-regulated, and the mixture of monomeric and oligomeric exCD23 up-regulated IgE synthesis. On addition of CD21 SCR 1-2 to the cells switched and treated with soluble CD23, it was found that in both cases for sCD23 and exCD23, IgE synthesis was increased, while for derCD23, there was no noticeable difference in IgE synthesis. This confirmed previous data showing the lack of binding between derCD23 and CD21 SCR 1-2. The exact binding site for CD21 on the CD23 molecule is unknown, and incompletely represented in the NMR and crystal structures. It is thought that CD21 binds to the C-terminal tail section, not present in derCD23. It is therefore likely that only a negative-feedback mechanism operates with derCD23 to regulate IgE synthesis. Further investigation of the binding of CD23 fragments to SCR 5-8 of CD21 and the effect of this on IgE synthesis may lead to a potential therapeutic role for derCD23 in the treatment of allergic disease. Data accumulated in this study suggests that investigating the modulation of oligomeric state and thus the activity of soluble CD23 fragments may be important in the construction of new regulators of IgE synthesis.
- Format
- 201 leaves
- Format
- Publisher
- Nelson Mandela Metropolitan University
- Publisher
- Faculty of Science
- Language
- English
- Rights
- Nelson Mandela Metropolitan University
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NMMU School of BioMolecular and Chemical Sciences
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