- Title
- Elucidating the Molecular Basis of the Interaction between the β2-integrin, αXβ2, and the low-affinity IgE Receptor, CD23
- Creator
- Clarke, Stephen
- Subject
- CD23 antigen
- Subject
- Immune response Cellular immunity Molecular immunology
- Date Issued
- 2019
- Date
- 2019
- Type
- Thesis
- Type
- Doctoral
- Type
- PhD
- Identifier
- http://hdl.handle.net/10948/37990
- Identifier
- vital:34277
- Description
- The low affinity IgE receptor, CD23, is involved in a myriad of immune reactions. It is not only a receptor for IgE, but also functions in the regulation of IgE synthesis, isotype switching in B cells, and induction of the inflammatory response. These effector functions of CD23 arise through its interaction with another leukocyte-specific cell surface receptor – the β2 integrin subfamily. However, this interaction has not been fully described. It has been shown that CD23 is capable of interacting with the β3 and β5 integrin β-subunit of integrins via a basic RKC motif in a metal cation-independent fashion. The currently proposed mechanism for the interaction between CD23 and the integrin superfamily was applied to the β2 integrin subfamily. In this study the interaction was probed for whether or not the RKC motif governs the interaction as well. This was done by performing bioinformatics docking predictions between the CD23 and αXβ2 integrin proteins. This revealed that in the absence of cations, the RKC motif is involved in interaction with the integrin αI domain. However, since physiologically integrin activity has been shown to be regulated by metal cations, docking predictions were also performed in the presence of such cations. This showed the interaction to involve novel acidic motifs within the CD23 protein, GEF and LDL. This same pattern of interaction was seen in docking predictions between CD23 and the β2- and β3I-like domains. To further investigate, recombinant proteins of sCD23 and the αXI domain were produced using E. coli expression systems. The DNA sequence was mutated to produce mutant versions of the CD23 RKC and GEF motifs as well as a high-affinity locked αXI domain. These proteins were used in subsequent SPR spectroscopy analysis of the binding affinity between immobilised integrin and CD23 analyte. It was shown that the mutation within the RKC motif reduced the binding affinity under cation-independence, especially when the Arg172 residue was substituted. However binding was not completely lost. This result was supported by synthetic peptides containing the same RKC motif and substitutions. These showed complete loss in binding in the double RKΔAA substitution, suggesting the involvement of other residues in the RKC-dependent interaction. In contrast, under cation dependence, the RKC motif substitutions showed no effect on binding affinity, while the GEF motif substitution exhibited near complete loss in binding. This same effect on binding was validated by U937 cell-based ELISA using live cells. This showed decreased capture of differentiated U937 cells, expressing the αXβ2 integrin, by immobilised recombinant sCD23 protein. In this study it was noted that, 2 contrary to the SPR analysis, metal cations allowed for a higher titre of cells to be captured in comparison to the cation-free binding.
- Format
- x, 175 leaves
- Format
- Publisher
- Nelson Mandela University
- Publisher
- Faculty of Science
- Language
- English
- Rights
- Nelson Mandela University
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NMU School of BioMolecular and Chemical Sciences
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | SOURCE1 | Stephen Clarke.pdf | 14 MB | Adobe Acrobat PDF | View Details Download |