HLA-DM is present in one-fifth the amount of HLA-DR in the class II peptide-loading compartment where it associates with leupeptin-induced peptide (LIP)-HLA-DR complexes.
HLA-DM has been shown in vitro to catalyze the release of invariant chain (Ii) derived peptides from the peptide-binding groove of class II molecules, thereby facilitating the binding of antigenic peptides. Previous studies showed that at steady state, the majority of DM resides in the class II peptide-loading compartment (IIPLC) where Ii dissociates from class II molecules and antigenic peptides are bound. Here we characterize the expression of DM in vivo in subcellular fractions containing the IIPLC. Using quantitative immunoblotting, we show that in the cell as a whole, class II molecules are expressed in 23-fold molar excess of DM. However, DM is concentrated in the IIPLC, where it is present in a considerably higher concentration relative to the class II molecules, in a molar ratio of 5DR:1 DM. This molar ratio of DM to DR in the IIPLC in vivo is consistent with the catalytic function proposed for DM from studies in vitro. We also provide both biochemical and genetic evidence that DM associates with complexes which contain Ii fragments and class II molecules in the IIPLC. Such complexes are only observed in leupeptin-treated cells in which Ii fails to be completely degraded and complexes containing the leupeptin-induced fragment of Ii (LIP) and class II molecules accumulate in the IIPLC. This observation is consistent with LIP-class II complexes being a substrate for DM in vivo and suggests that interactions of DM and LIP-class II are extremely transient under normal conditions.
Schafer
P H
PH
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
Green
J M
JM
Malapati
S
S
Gu
L
L
Pierce
S K
SK
D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism*; D001402:B-Lymphocytes / Q000502:physiology*; D002451:Cell Compartmentation; D006681:HLA-D Antigens / Q000378:metabolism*; D006684:HLA-DR Antigens / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000378:metabolism*; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000276:immunology* / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding
D000944:Antigens, Differentiation, B-Lymphocyte; C416189:H2-M antigens; D006681:HLA-D Antigens; C084245:HLA-DM antigens; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D007976:Leupeptins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; C045084:invariant chain; C032854:leupeptin