MIP-1alpha: A Structure-Function Study

Ottersbach, Katrin (2001) MIP-1alpha: A Structure-Function Study. PhD thesis, University of Glasgow.

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Abstract

The observation that MIP-1alpha can inhibit the proliferation of transiently engrafting haemopoietic stem cells was first reported more than ten years ago. However, very little has since emerged about the molecular mechanism underlying stem cell inhibition. The work presented in this thesis therefore aimed at shedding some more light on the molecular mechanism and on how the structural properties of MIP-1alpha relate to its function as a stem cell inhibitor. Two properties of MIP-1alpha, which it shares with most other chemokines, were first of all considered, its ability to self-aggregate and its interaction with proteoglycans. It has already been demonstrated that the aggregation of murine MIP-1alpha is influenced by the pH and the ionic and hydrophobic strength of the buffer. In addition, three acidic residues in the carboxy terminal region of murine MIP-1alpha have been previously shown to be involved in self-association as their neutralisation generates aggregation-incompetent mutants. Since the aggregation of human MIP-1alpha has previously been found to be concentration-dependent, similar experiments were carried out for murine MIP-1alpha which established that its self-association is also controlled in a comparable way which allowed the isolation of differentially aggregated murine MIP-1alpha by progressive dilution. However, the forces that stabilise the oligomers appear to be stronger in human MIP-1alpha as compared to murine MIP-1alpha. In the recent determination of the crystal structure of murine MIP-1alpha, two calcium ions were found in association with the tetramer that were proposed to mediate the formation of higher order aggregates. However, experiments carried out as part of this thesis demonstrate that this is not the case since the removal of these ions by the addition of EDTA and EGTA has no influence on the aggregation process. Secondly, investigations were made into the way murine MIP-1alpha interacts with the glycosaminoglycan heparin. More specifically, experiments were aimed at establishing whether there is a link between the aggregation state of MIP-1alpha and its affinity for heparin since it is known for some chemokines, such as PF4, that the individual heparin binding sites in the monomers display positive cooperativity upon aggregation, resulting in a higher affinity of the tetramer for heparin. This may also have implications for a possible interaction of MIP-1alpha with proteoglycans in the bone marrow microenvironment. In a first approach, three aggregation-incompetent murine MIP-1alpha mutants, a monomer, dimer and a tetramer, were analysed for their binding to a heparin matrix. Surprisingly, the tetramer exhibited the lowest affinity for heparin, followed by the dimer and then the monomer with the highest affinity. One possible interpretation of this observation is that the heparin binding site becomes progressively occluded upon aggregation, thereby decreasing heparin binding in MIP-1alpha oligomers. Alternatively, the neutralisation of the negative charges during the generation of these aggregation mutants may have altered the strength with which they bind to heparin since glycosaminoglycan-protein interactions are predominantly determined by electrostatic forces. In order to resolve this question, differentially aggregated murine MlP-1? was prepared by progressive dilution (see above) and the different oligomers tested for their heparin binding affinity. No difference was observed in the strength with which the different aggregates bound to immobilised heparin which was also confirmed with stably cross-linked murine MIP-1alpha oligomers. This suggests, that the aggregation state of murine MIP-1alpha has no impact on its affinity for heparin which is instead controlled by its overall charge. Attention was then turned to the interaction of murine MIP-1alpha with the receptor on haemopoietic stem cells through which it mediates its inhibitory effect, as demonstrated in an in vitro assay, known as the CFU-A assay. It was first of all established that this inhibitory receptor is none of the four known murine MIP-1alpha receptors (CCR1, CCR3, CCR5 and D6) for the following reasons; (1) none of the other chemokines tested, which included other ligands for the four MIP-1alpha receptors as well as for all of the other known CC chemokine receptors, displayed any activity in the CFU-A assay, (2) the four known human MIP-1alpha variants, which show differential binding to murine CCR1, CCR5 and D6, have indistinguishable potencies as stem cell inhibitors, and (3) stem cells from CCR1-/-, CCR3-/-, CCR5-/- and D6-/- mice were still inhibited in their proliferation by MIP-1alpha, even when a chemokine analogue, which has the capacity to displace MIP-1alpha from all of its four receptors, was included in the assays. All of these data suggest that MIP-1alpha's inhibitory signal is conveyed by a novel, as yet uncharacterised receptor. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Gerry Graham
Keywords: Molecular biology
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-76259
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:13
Last Modified: 19 Nov 2019 16:13
URI: http://theses.gla.ac.uk/id/eprint/76259

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