Poirier, Ghislaine; (1992) Cellular and structural requirements for peptide binding to MHC class II molecules. Doctoral thesis (Ph.D.), University College London.

Text Cellular_and_structural_requir.pdf Download (11MB)

Abstract

We have been interested in the final stage of antigen processing and presentation by MHC class II molecules. More precisely, the main aim of this project was to examine the cellular requirements for the binding of exogenous peptides to the mouse B lymphoma cell line, A20. In order to detect directly specific peptide/MHC class II complexes on A20 we have immunoselected a low class II expressor variant from A20. This cell line, called UV, was characterized biochemically and functionally. MHC Class II expression of UV is reduced to 5% of the level of the original cell line, due to a transcriptional deficiency, and is unable to present either antigen or peptide to specific T cell hybridoma. Moreover it has a decreased level of membrane MHC class I molecules. Using several negative control cell lines including UV, we have set up a two-step fluorescent peptide binding assay to A20, consisting in a first incubation with a biotinylated peptide and a second incubation in presence of a fluoresceinated derivative of streptavidin. This assay was validated in several ways. Using this assay, we have analysed the effect of various pharmacological and physical agents on peptide binding. Our results show that most of the peptide binding does not require either newly synthesised or recycling MHC class II molecules. Moreover, it still occurs in the absence of metabolic energy. We also showed that peptide binding depends on the membrane fluidity. Unexpectedly, peptide/MHC class II complexes have a remarkably low half-life as compared to that of soluble complexes. A cellular model for the binding of exogenous peptides to A20 is discussed. The secondary aim of this thesis was to examine the molecular characteristics of MHC class II molecules in relation to peptide binding, using a pan el of mutant MHC class II transfectants and the fluorescent peptide binding assay. The results of the analysis of mutations affecting the peptide binding site are consistent with the structural model of class II. We also showed the necessity of a close packing of the transmembrane domains of class II α and β chains for efficient peptide binding.

Download activity - last month

Export as CSVJSONXML

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as XMLCSVJSON

Archive Staff Only

View Item