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Although our recent studies have provided the first evidence demonstrating the direct binding of Mycoplasma arthritidis-derived mitogen (MAM) to MHC class II molecules, it is not yet established how MAM interacts with these molecules. Herein, we demonstrate that MAM binds preferentially and with high affinity to HLA-DR molecules in a zinc-dependent manner. MAM's affinity (25 nM) for HLA-DR molecules is comparable to that of staphylococcal superantigens, and is slightly higher than that for murine MHC class II molecules expressed on the A20 B cell line (111 nM). The amino acid residues located between 14 - 31 and 76 - 90 of the MAM N-terminus play a critical role in MAM / HLA-DR interactions. Histidine at position 81 of the HLA-DR beta-chain, known to be critical for binding of zinc-coordinated superantigens, is not necessary for MAM / HLA-DR interactions. The HLA-DR residues involved in MAM binding are located in the proximal binding groove of the HLA-DR molecule, where the nature of the peptide of the binding groove plays an important role in MAM / HLA-DR interaction. This is the first detailed characterization of MAM's interactions with MHC class II molecules showing a mode of interaction with HLA-DR distinct from that of other superantigens.

Mycoplasma arthritidis-derived mitogen (MAM) is considered to be a member of the super-antigen family despite the fact that there is no evidence until now indicating its binding to MHC class II molecules. To demonstrate its direct binding and to determine the regions involved in MHC class II and TCR interactions, we generated a recombinant wild-type and two truncated forms of the MAM protein. Data obtained in the course of the present investigation show that MAM binds specifically and significantly to human MHC class II molecules. Evidence is also provided that MAM bears two distinct binding regions: one is located within its N terminus and interacts with MHC class II molecules, while the second region which is located in its C terminus mediates its recognition by the TCR. Association of the MHC class II-associated invariant chain peptide with the peptide binding groove on the cell surface completely abolished MAM binding and presentation. This inhibitory effect is restored by the expression of HLA-DM molecules, suggesting that the nature of the peptide within the binding groove and/or the stability of the MHC class II molecules on the cell surface may modulate MAM/MHC class II interactions.

Although our recent studies have provided the first evidence demonstrating the direct binding of Mycoplasma arthritidis-derived mitogen (MAM) to MHC class II molecules, it is not yet established how MAM interacts with these molecules. Herein, we demonstrate that MAM binds preferentially and with high affinity to HLA-DR molecules in a zinc-dependent manner. MAM's affinity (25 nM) for HLA-DR molecules is comparable to that of staphylococcal superantigens, and is slightly higher than that for murine MHC class II molecules expressed on the A20 B cell line (111 nM). The amino acid residues located between 14 - 31 and 76 - 90 of the MAM N-terminus play a critical role in MAM / HLA-DR interactions. Histidine at position 81 of the HLA-DR beta-chain, known to be critical for binding of zinc-coordinated superantigens, is not necessary for MAM / HLA-DR interactions. The HLA-DR residues involved in MAM binding are located in the proximal binding groove of the HLA-DR molecule, where the nature of the peptide of the binding groove plays an important role in MAM / HLA-DR interaction. This is the first detailed characterization of MAM's interactions with MHC class II molecules showing a mode of interaction with HLA-DR distinct from that of other superantigens.