Further support for this model is provided by kinetic stability of pMHCII complexes in the presence of DM and the absence of an exchange peptide.[52, 57, 47] In consideration of the correlation between two-peptide intermediates and ‘open’ conformers, the observed DM-associated increase
in inter-peptide FRET has been interpreted as evidence that DM recognizes the ‘open’ MHCII resulting from the interaction with the two peptides. An important step in defining the two-peptide/MHCII intermediate and refining the exchange mechanism in general will be mapping the location where the exchange peptide interacts with the pre-bound peptide/MHCII complex. Exchange peptides with different chemistry need to be recognized, so one possibility is that the competitor peptide interacts with a distinct (presumably less Selleck Pexidartinib polymorphic) site present across MHCII alleles. Analysing the ‘peptide exchangeability’ of MHCII molecules carrying ad hoc mutations in the absence or presence of DM might be an approach to address these questions. Interestingly, the possibility GSK-3 activation that the two-peptide/MHCII intermediate and the push-off
mechanism occur both in the absence of DM at neutral pH and in the presence of DM at acid pH broadens the possibilities for loading MHCII molecules efficiently under different conditions. Consequently, the question arises as to whether a similar breadth of binding conditions also takes place in vivo and whether it might regulate alternative loading or recycling pathways of class II MHC molecules. The extensive
CYTH4 polymorphism characterizing MHCII molecules affects the stabilities of class II heterodimers and plays a role in determining the extent to which DM exerts its function. In vitro experiments have shown allele-dependent association of DM with empty class II. Studies performed in transfected cells have identified the allele-specific requirement of DM during class II-restricted antigen presentation, however different groups reached contradictory conclusions.[61-64] It is likely that the complementation assays adopted in those works to investigate DM activity could be affected by additional experimental variables, such as abnormal expression levels and functional contributions by recipient cell lines, impairing our ability to evaluate the significance of these observations. To rectify these technique-related inconsistencies, mutant mice were generated expressing known ratios of different MHC class II alleles and Ii chain via homologous recombination in embryonic stem cells. Experiments conducted in these animals showed clear evidence for distinctive isotype-specific modes of peptide capture and dependence on DM.[65, 66] These studies led to an investigation of the possibility that human MHCII molecules also feature a diversified DM and/or Ii requirement for appropriate trafficking and antigen presentation.