Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM.
The nonclassical major histocompatibility complex class II molecule HLA-DM (DM) has recently been shown to play a central role in the class II-associated antigen presentation pathway: DM releases invariant chain-derived CLIP peptides (class II-associated invariant chain protein peptide) from HLA-DR (DR) molecules and thereby facilitates loading with antigenic peptides. Some observations have led to the suggestion that DM acts in a catalytic manner, but so far direct proof is missing. Here, we investigated in vitro the kinetics of exchange of endogenously bound CLIP for various peptides on DR1 and DR2a molecules: we found that in the presence of DM the peptide loading process follows Michaelis-Menten kinetics with turnover numbers of 3-12 DR molecules per minute per DM molecule, and with KM values of 500-1000 nM. In addition, surface plasmon resonance measurements showed that DM interacts efficiently with DR-CLIP complexes but only weakly with DR-peptide complexes isolated from DM-positive cells. Taken together, our data provide evidence that DM functions as an enzyme-like catalyst of peptide exchange and favors the generation of long-lived DR-peptide complexes that are no longer substrates for DM.
Vogt
A B
AB
Department of Molecular Immunology, German Cancer Research Center, Heidelberg, Germany.
Kropshofer
H
H
Moldenhauer
G
G
Hämmerling
G J
GJ
D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000378:metabolism*; D002461:Cell Line, Transformed; D006681:HLA-D Antigens / Q000378:metabolism*; D006684:HLA-DR Antigens / Q000378:metabolism*; D015798:HLA-DR1 Antigen / Q000378:metabolism; D015802:HLA-DR2 Antigen / Q000378:metabolism; D004854:Herpesvirus 4, Human; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding
D000944:Antigens, Differentiation, B-Lymphocyte; D000951:Antigens, Neoplasm; C416189:H2-M antigens; D006681:HLA-D Antigens; C084245:HLA-DM antigens; D006684:HLA-DR Antigens; D015798:HLA-DR1 Antigen; D015802:HLA-DR2 Antigen; D000949:Histocompatibility Antigens Class II; C045084:invariant chain