(Wolf and Ploegh, 1995). However, the DM genes are different from the classical class II genes in several aspects. According to Kelly et al. (1991), the differences include low sequence homology between the two sets of genes, potentially more disulfide bridges in DM which would confer a more rigid structure and limited polymorphism (Sanderson et al., 1994; Pepteraux et al., 1996). It has been assumed that these properties may not allow tight binding of peptides in the groove of the DM molecule (Kelly et al., 1991).
1.4.2. The function of HLA-DM in the class II antigen presentation pathway
HLA-DM is not expressed on the cell surface (Sanderson et al., 1994), instead being retained within the endosomal system by the localization signal in the cytoplasmic tail of the ß-chain (which incorporates tyrosine 230, Lindstedt et al.,1995; Copier et al., 1996). Denzin et al. (1996) have reported that incubation of DR3-CLIP complexes with DM elicited release of CLIP in vitro. DM accumulates in the MIIC where peptide loading occurs (Sanderson et al., 1994) but is found throughout the endocytic pathway (Pierre et a l, 1996).
It has been shown that DM is expressed at a lower level than MHC class II molecules in .114 cells (Schafer et al., 1996). Subcellular fractionation studies of .114 cells have demonstrated that DM is present at one-fifth of the levels of HLA- DR in the MIIC and at one-twenty third of the amount of HLA-DR in the cell as a whole (Schafer et al., 1996). At endosomal pH, a sub-stoichiometric amount of DM (turnover number of 1 DM molecule per 3-12 DR molecules) efficiently mediated exchange of low affinity peptides, including CLIP, for antigenic peptides in an enzyme-like manner (Vogt et al., 1996).
Studies with mutant mice (C57BL/6, 129/SV) which fail to express H2-M have been performed in order to elucidate the function of DM in MHC class II antigen presentation (Martin et al., 1996; Miyazaki et al., 1996; Toum e et a l, 1997).
Splenocytes from these animals were unable to present peptides derived from protein antigens to class II-restricted T cells and positive selection of CD4+ T cells was reduced in these H2-M deficient mice (Martin et al., 1996; Tourne et al.,
1997). In these animals, the levels of MHC class II expression (I-Ab) on the B-cell surface were normal but I-Ah class II dimers were SDS-sensitive and they were found to be presenting CLIP predominantly (Martin et al., 1996; Miyazaki et al.,
1996). Additionally, in DR3 transfected T2 cells, which are DM deficient, proteolysis of Ii was less efficient and DR3-CLIP complexes were generated much more slowly than in wild-type cells (Riberdy et al., 1994). From these findings, it has been concluded that DM has several important functions, such as facilitating efficient proteolysis of Ii in the endocytic pathway, antigen presentation by MHC class II molecules and, ultimately, selective activation of CD4+ T cells by MHC class Il-mediated antigen presentation.
The requirement of HLA-DM or H2-M for effective MHC class II antigen presentation does not seem to be absolute however, since some MHC class II molecules, including DP4, DQ1, DR3 and I-Ab, require DM (or H2-M) for proper antigen presentation (Morris et al., 1994; Green et al., 1995; Sloan et al., 1995; van Ham et al., 1996; Miyazaki et al., 1996) while I-A^ does not (Stebbins et al.,
1996). However, for antigen presentation by I-Ad class II molecules, the requirement for H2-M seems to be less stringent than in the case of presentation mediated by I-Ab. Stebbins et al. (1995) have shown that the formation of I-Ad SDS-stable dimers and the dissociation of CLIP were not affected by the absence of DM in 9.5.3 cells, suggesting that I-Ad class II antigen presentation may be DM independent. However, Weenink et al. (1996) have demonstrated that I-Ad transfected T2 cells (T2.d), which were DM deficient, failed to form I-Ad SDS- stable dimers and to present peptides derived from intact proteins to specific T cell hybridomas. Furthermore, I-Ad molecules from these cells were predominantly
occupied with CLIP on the cell surface, indicating that DM (or H2-M) may be required for the I-Ad-mediated antigen presentation. After removal of CLIP by DM in the MIIC, the binding groove of the MHC class II molecules may be assumed to be temporarily empty. Stem et al. (1992) have reported that purified empty MHC class II molecules were not stable and tended to aggregate.
In summary, DM catalyzes dissociation of the CLIP from MHC class II molecules prior to peptide loading, a process which involves conformational changes in MHC class II molecules resulting from direct association of DM with the MHC class II molecules at lysosomal pH (Ullrich et a l , 1997). DM may stabilize empty MHC class II molecules in the MIIC by protecting them from aggregation until antigenic peptides are loaded (Denzin et al., 1996). In addition to facilitating CLIP removal, DM also catalyzes the release of other self-peptides from the MHC class II molecules (Kropshofer et al., 1996). This has led to the suggestion that DM has the potential to function as a “peptide editor” (van Ham et al., 1996) by selecting high stability MHC class II/peptide complexes before these complexes are presented to T cells, van Ham et al. (1996) showed that DM catalyses the release of peptides which do not have appropriate anchor residues and, hence, no optimal binding motif from HLA-DR3. In this way, DM facilitates selection of peptides that bind with high affinity to the MHC class II molecules for eventual presentation to the immune system from the pool of available peptides (van Ham et al., 1996).
1.5. Other molecules involved in MHC II antigen presentation
Since MHC class II a and ß subunits are synthesized and form heterodimers in the ER, additional proteins which are resident in this compartment may interact with the components of MHC class II antigen presentation system. Calnexin is a resident protein of the ER which stabilizes MHC class I molecules until antigenic