Class I heavy chain (HC) must assemble with β-microglobulin (β2m) and acquire optimal peptide in order to be presented to cytotoxic T cells (CTLs). Calnexin is involved in the initial folding of class I HC and subsequent assembly with β2m. Incorporation of "empty" or suboptimally loaded class I molecules into the multimolecular loading complex is essential for them to acquire optimal peptides. The loading complex consists of several cofactors: TAP, tapasin, ERp57 and calreticulin. The precise role of calnexin and calreticulin in the regulated assembly and peptide loading and the significance of their physical interaction with other cofactors of the loading as well as preloading complex still remains unclear. Using mouse fibroblasts that lack calreticulin, I have studied the role of calreticulin in the assembly and loading of H2-K<sup>b</sup> and H2-D<sup>b</sup> expressed in these cells. MHC class I molecules in calreticulin-deficient cells are able to assemble with β2m normally, but their subsequent loading with optimal, stabilising peptides is defective despite their ability to interact with the TAP complex. The "empty" or suboptimally loaded class I molecules exit the ER rapidly. Reflecting the loading defect, presentation of endogenously processed antigens by class I molecules in calreticulin-deficient cells is impaired. I have used a human calnexin-deficient cell line CEM.NK<sup>R</sup> to study assembly of class I in the absence of calnexin. The results demonstrate that contrary to current understanding, calnexin has an important role in class I HC assembly with 32- microglobulin. The role of heavy chain glycosylation in class I biogenesis is still controversial. My findings suggest asparagine (N)-linked glycosylation of human class I heavy chain at position 86 is optimal and any deviations from "normal" glycosylation results in poor loading with peptides and some defect in the assembly with β2m. Despite affecting the loading function, glycosylation did not have significant effect on presentation of a high affinity binding epitope to HLA-A*0201 specific CTLs. Finally, I show that co-operation from all domains of calreticulin is essential in order to generate a fully functional calreticulin. Interestingly, proline-rich (P) -domain of calreticulin downregulated expression of a number of cellular proteins including MHC class I HC, despite restoring the cytosolic calcium levels in calreticulindeficient cells. The effect of P-domain on class I expression was at the level of transcription.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:249193 |
| Date | January 2002 |
| Creators | Adhikari, Raju |
| Contributors | Elliott, Timothy R. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:8d626bbd-4927-4a52-b892-461e968a1c96 |
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