Elucidating the Molecular Basis of the Interaction between the β2-integrin, αXβ2, and the low-affinity IgE Receptor, CD23
- Authors: Clarke, Stephen
- Date: 2019
- Subjects: CD23 antigen , Immune response Cellular immunity Molecular immunology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/37990 , 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.
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- Date Issued: 2019
Molecular and cellular analysis of the interaction between soluble CD23 and CD11/CD18 integrins
- Authors: Daniels, Brodie Belinda
- Date: 2010
- Subjects: CD23 antigen , Immune response -- Regulation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10303 , http://hdl.handle.net/10948/1217 , CD23 antigen , Immune response -- Regulation
- Description: The low affinity IgE receptor, CD23, is expressed by a wide variety of cells and cleaved from its original 45 kDa size to several smaller soluble CD23 proteins. Soluble CD23 function depends on the form of the protein and its interaction with various ligands. CD23 is believed to play an important role in regulating allergic responses and in inflammation, amongst others. β2 integrins are important in a variety of cell-adhesion reactions during immune-inflammatory mechanisms and the binding of their natural ligands generates outside-in cellular signalling, leading to cell activation. Although the binding of CD23 to β2 integrins contributes to this signalling in monocytes, the interaction site for CD23 is unknown. This study focused on the interaction of three soluble CD23 proteins with the β2 integrins CD11b/CD18 and CD11c/CD18. Differentiated HL60, THP1 and U937 monocytic cells were used to demonstrate the binding of three recombinant CD23 constructs (corresponding to 16, 25 and 33 kDa human soluble CD23) to upregulated CD11b/CD18 and CD11c/CD18. This binding was partially blocked by an antibody specific for the CD11b/CD18 αI domain, demonstrating that αI domains are involved in binding to CD23. Recombinant αI domain proteins of CD11b and CD11c were demonstrated to bind CD23 using ELISA and in surface plasmon resonance spectroscopy. The dissociation constants for CD23-CD11b/CD18 and CD23-CD11c/CD18 are comparable to other integrin ligands. This study has shown that CD23 interacts directly with the αI domains of β2 integrins and that the interaction surface likely spans the lectin domain as well as either the stalk and/or C-terminal tail of CD23. This study also looked at the effect that soluble CD23 proteins had on monocyte biology. It appears that iv sCD23 proteins have little effect on the phagocytic or chemotactic ability of monocytes, while an increase in oxidative burst was shown with the 16 kDa and 25 kDa CD23 proteins. Signalling pathways for the production of reactive oxygen species were investigated and it appears that the CD23 proteins signal mainly through the phosphoinositide-3 kinase pathway, although the mitogen activated protein kinase and Src kinase pathways may also play a role. These data suggest that sCD23 proteins induce outside-in signalling of β2 integrins and are able to change the activation state of CD11b/CD11c by stimulating oxidative burst. This needs to be further investigated by determining how the three sCD23 proteins are binding the CD11 proteins and investigating further leukocyte function and inflammatory responses by the cells.
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- Date Issued: 2010