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.
- Full Text:
- Date Issued: 2019
- 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.
- Full Text:
- Date Issued: 2019
Liver steatosis and insulin-resistance : reversal by Sutherlandia frutescens
- Authors: Clarke, Stephen
- Date: 2014
- Subjects: Insulin resistance , Diabetes -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10348 , http://hdl.handle.net/10948/d1020788
- Description: Type 2 diabetes mellitus (T2DM) is rapidly emerging as one of the greatest global health issues of the 21st century. Insulin-resistance is a condition associated with T2DM and in the cell it is defined as the inadequate strength of insulin signalling from the insulin receptor downstream to the final substrates of insulin action involved in multiple metabolic, gene expression, and mitogenic aspects of cellular function. To investigate the potential mechanisms involved in the development of insulin-resistance, two in vitro liver cell models were established using palmitate or a combination of insulin and fructose as inducers. The development of insulin-resistance was determined via the capacity of the hepatocytes to maintain normal glucose metabolism functionality by measuring hepatic gluconeogenesis and glycogenolysis. It was established that the treatments induced the development of insulinresistance after 24 hours chronic exposure. Previous studies have investigated the potential of Sutherlandia frutescens extracts as therapeutic agents for insulin-resistance. The aim of this study was thus to investigate the ability of a hot aqueous extract of S. frutescens to reverse the insulin-resistant state, via measuring gluconeogenesis and glycogenolysis, the associated changes in cellular physiology (lipid accumulation, oxidative stress, and acetyl- CoA levels), and changes in mRNA expression. The results showed that S. frutescens had a significant effect on reversing the insulin-resistant state in both models of insulin-resistance. Furthermore, S. frutescens was capable of reducing lipid accumulation in the form of triacylglycerol in the high insulin/fructose model, while this was unaffected in the palmitate model. However, S. frutescens did reduce the accumulation of diacylglycerol in the palmitate model. Oxidative stress, seen to be associated with the insulin-resistant state, was successfully treated using the extract, as indicated by a reduction in reactive oxygen species. However no change was seen in the nitric oxide levels, in either model. Interestingly, although S. frutescens had no effect on the level of acetyl-CoA in the insulin/fructose model, it was found to increase this in the palmitate model. It is suggested that this may be due to increased β-oxidation and metabolic activity induced by the extract. The analysis of mRNA expression gave some insight into possible mechanisms by which insulin-resistance develops, although the results were inconclusive due to high variability in samples and the possibility of the RNA being compromised. Future studies will address this issue. The results of this study reflect different proposed clinical causes of insulin-resistance through the responses seen in the two cell models. These indicate that liver steatosis and insulin-resistance are induced by high palmitate as well as high insulin and fructose levels, and reversed by S. frutescens. Therefore the potential of S. frutescens to be used as a therapeutic agent in the treatment of insulin-resistance is indicated by this study.
- Full Text:
- Date Issued: 2014
- Authors: Clarke, Stephen
- Date: 2014
- Subjects: Insulin resistance , Diabetes -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10348 , http://hdl.handle.net/10948/d1020788
- Description: Type 2 diabetes mellitus (T2DM) is rapidly emerging as one of the greatest global health issues of the 21st century. Insulin-resistance is a condition associated with T2DM and in the cell it is defined as the inadequate strength of insulin signalling from the insulin receptor downstream to the final substrates of insulin action involved in multiple metabolic, gene expression, and mitogenic aspects of cellular function. To investigate the potential mechanisms involved in the development of insulin-resistance, two in vitro liver cell models were established using palmitate or a combination of insulin and fructose as inducers. The development of insulin-resistance was determined via the capacity of the hepatocytes to maintain normal glucose metabolism functionality by measuring hepatic gluconeogenesis and glycogenolysis. It was established that the treatments induced the development of insulinresistance after 24 hours chronic exposure. Previous studies have investigated the potential of Sutherlandia frutescens extracts as therapeutic agents for insulin-resistance. The aim of this study was thus to investigate the ability of a hot aqueous extract of S. frutescens to reverse the insulin-resistant state, via measuring gluconeogenesis and glycogenolysis, the associated changes in cellular physiology (lipid accumulation, oxidative stress, and acetyl- CoA levels), and changes in mRNA expression. The results showed that S. frutescens had a significant effect on reversing the insulin-resistant state in both models of insulin-resistance. Furthermore, S. frutescens was capable of reducing lipid accumulation in the form of triacylglycerol in the high insulin/fructose model, while this was unaffected in the palmitate model. However, S. frutescens did reduce the accumulation of diacylglycerol in the palmitate model. Oxidative stress, seen to be associated with the insulin-resistant state, was successfully treated using the extract, as indicated by a reduction in reactive oxygen species. However no change was seen in the nitric oxide levels, in either model. Interestingly, although S. frutescens had no effect on the level of acetyl-CoA in the insulin/fructose model, it was found to increase this in the palmitate model. It is suggested that this may be due to increased β-oxidation and metabolic activity induced by the extract. The analysis of mRNA expression gave some insight into possible mechanisms by which insulin-resistance develops, although the results were inconclusive due to high variability in samples and the possibility of the RNA being compromised. Future studies will address this issue. The results of this study reflect different proposed clinical causes of insulin-resistance through the responses seen in the two cell models. These indicate that liver steatosis and insulin-resistance are induced by high palmitate as well as high insulin and fructose levels, and reversed by S. frutescens. Therefore the potential of S. frutescens to be used as a therapeutic agent in the treatment of insulin-resistance is indicated by this study.
- Full Text:
- Date Issued: 2014
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