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Title: The role of tapasin in MHC class 1 antigen presentation
Author: Williams, Anthony Peter
ISNI:       0000 0001 3568 7672
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2004
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MHC class I molecules are heterotrimeric complexes of a polymorphic MHC class I heavy chain, beta-2 microglobulin (β2 M) and a short peptide. These complexes present self and foreign peptides to host effector cells. This allows for monitoring of the internal milieu of cells through representation of the protein breakdown products (peptides) within MHC class I complexes at the cell surface. This thesis examines the role of the endoplasmic reticulum resident MHC class I chaperone tapasin in this process. The initial characterisation was undertaken at a genetic level, identifying polymorphic variants of human tapasin. A search for linkage with MHC class I and II genes was carried out, with linkage only identified for the nearest MHC class II loci. The limited genomic variation was further analysed in primate tapasin and 15 amino acid differences from human tapasin were identified. Further work was directed at developing a functional test for the role of tapasin in MHC class I presentation, so that such differences could be analysed. A model system was set up that examined the role of tapasin upon previously identified tapasin dependent and independent alleles (B*4402 and B*2705 respectively). A thermostability assay was developed that assessed the peptide cargo of BM402 and B*2705 within cells that were competent or deficient for tapasin. This work identified that tapasin was important for the optimal assembly of both B*4402 and B*2705. Tapasin was shown to improve both the rate and extent of optimal peptide acquisition. More detailed studies showed that the improvement in the thermostability of such complexes could occur independently of tapasin for B*2705, although to a lesser extent to that seen in the presence of tapasin. B*4402 was unable to load optimal peptides in the absence of tapasin, although it was shown that these B*4402 complexes were unlikely to be peptide empty as their thermostability fell further upon TAP blockade. When the loading of these alleles was assessed in the presence of a soluble tapasin construct that did not abridge the MHC class I complexes to TAP, peptide loading was achieved but the thermostability of such complexes was reduced when compared to full length tapasin. This suggested that the association of nascent MHC class I molecules with the TAP complex is necessary for maximal optimisation of the MHC class I complex peptide cargo. Finally, a single point mutation was identified that permitted tapasin independent loading of B*4402. This single change at position 116 was identical to that seen in the allele B*4405. B*4402 was shown to achieve a greater thermostability than B*4405 in the presence of tapasin. B*4405 was able to load optimal peptides in the absence of tapasin, with a further improvement in the presence of tapasin. Both B*2705 and B*4405 were less thermostable than B*4402 in the presence of tapasin. These experimental observations have led to a reassessment of the multiple functions of tapasin in facilitating peptide loading and a consideration of the molecular mechanisms that may permit such allele specific interactions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral