Peptide binding inhibits aggregation of soluble MHC class II in solution.
Affinity-purified major histocompatability complex (MHC) class II molecules are known to bind antigenic peptide in vitro. This peptide-bound MHC class II is known to undergo a change in structure upon stable binding of antigenic peptide. Previous results from our, and other laboratories, have suggested a relationship between MHC class II structure and peptide association that enables class II to enter into a stable conformation upon peptide binding. In this report we describe that stable binding of high-affinity antigenic peptide to MHC class II molecule results in transition of aggregated purified MHC class II proteins to a stable heterodimeric state. Such transition was demonstrated by using purified human HLA-DR2 class II molecule and high-affinity myelin basic protein (MBP) 83-102)Y83 peptide. Highly aggregated purified DR2 (high molecular weight; HMW) was first separated from heterodimer (low molecular weight: LMW) in the presence of 50-fold molar excess of MBP(83-102)Y83 peptide. We then show that the aggregated HMW preparation can be successfully converted into a stable dimer by further incubation with MBP(83-102)Y83 and changing various binding parameters such as pH, temperature, reducing agent, and peptide concentrations. Under optimized conditions, the highly aggregated inactive DR2 molecules can be completely loaded with the antigenic peptide. The transformed heterodimers with bound peptide prepared by this method are biologically active, as shown by their ability to induce the production of gamma-interferon by SS8T-transformed human T cells. These results suggest that in solution, MHC class II molecules may be aggregated in the absence of bound peptide. Such aggregated MHC class II molecules can be converted to stable and biologically active heterodimers in the presence of high-affinity antigenic peptide.
Arimilli
S
S
Corixa Corporation, Redwood City, California 94063, USA. arimilli@corixa.com
Astafieva
I
I
Mukku
P V
PV
Cardoso
C
C
Deshpande
S
S
Nag
B
B
D000595:Amino Acid Sequence; D002461:Cell Line, Transformed; D015802:HLA-DR2 Antigen / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D012995:Solubility; D013696:Temperature
D015802:HLA-DR2 Antigen; D004676:Myelin Basic Protein; D010446:Peptide Fragments; C113463:myelin basic protein 83-102