Related Experiment Videos
An unstable beta 2-microglobulin: major histocompatibility complex class I heavy chain intermediate dissociates from
1Department of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Insights
Proper assembly of MHC class I molecules requires heavy chain (HC), beta 2-microglobulin (beta 2m), and peptide in the endoplasmic reticulum. Calnexin binding to HC appears to be regulated by beta 2m association, influencing peptide loading.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- MHC class I molecule surface expression depends on the proper assembly of heavy chain (HC), beta 2-microglobulin (beta 2m), and peptide within the endoplasmic reticulum (ER).
- Newly synthesized HC initially associate with calnexin, an ER-resident chaperone, which plays a role in folding and assembly.
- Deficiencies in peptide transport (T2 cells) or beta 2m (FO-1 cells) provide insights into the assembly pathway and the role of chaperones.
Purpose of the Study:
- To investigate the precursor-product relationship of heavy chain pools during MHC class I assembly.
- To determine the role of beta 2m and peptide binding in the dissociation of HC from calnexin.
- To elucidate the stepwise assembly process of MHC class I molecules, including the function of calnexin.
Main Methods:
- Co-immunoprecipitation using HC10 and W6/32 antibodies to distinguish between beta 2m-unassociated and beta 2m-associated HC.
- In vitro peptide loading assays on cell lysates to assess the stability of HC complexes.
- Analysis of calnexin association with HC in peptide transporter-deficient (T2) and beta 2m-deficient (FO-1) cells, and beta 2m-transfected FO-1 cells.
Main Results:
- Calnexin co-immunoprecipitated with beta 2m-unassociated HC in T2 cells, but not with beta 2m-associated HC.
- In vitro addition of peptides stabilized pre-existing beta 2m-associated HC complexes but did not affect calnexin-associated HC lacking beta 2m.
- Dissociation of HC from calnexin was prolonged in beta 2m-deficient cells and rapid in beta 2m-transfected cells, suggesting beta 2m binding controls release.
- HC dissociation from calnexin was independent of peptide binding, occurring at similar rates in T2 and wild-type T1 cells.
Conclusions:
- Calnexin may mediate the dimerization of MHC class I HC with beta 2m during the initial stages of assembly.
- The dissociation of HC from calnexin, likely triggered by beta 2m binding, precedes or is independent of peptide binding.
- Unstable beta 2m+:HC:pep- complexes dissociate from calnexin and subsequently bind peptide to complete MHC class I assembly.
Abstract:
Proper assembly of the class I heavy chain (HC), beta 2-microglobulin (beta 2m), and peptide must occur in the endoplasmic reticulum (ER) in order for MHC class I molecules to be expressed on the cell surface. Newly synthesized class I HC bind calnexin, an ER resident chaperone. These calnexin-associated class I HC appeared to lack the stable association with beta 2m in peptide transporter-deficient T2 cells since beta 2m-unassociated class I HC-specific HC10 antibody, but not beta 2m-associated class I HC-specific W6/32 antibody, coimmunoprecipitated calnexin. To determine the precursor-product relationship of the pool of HC that bind peptide, class I-restricted peptides were added to lysates of T2 cells in vitro. These peptides stabilized preexisting beta 2m-associated HC complexes (beta 2m+:HC:pep-), but had no significant effect on the preexisting pool of calnexin-associated HC that lack beta 2m. Release of HC from calnexin appeared to be controlled by the binding of beta 2m, since beta 2m-deficient FO-1 cells showed a prolonged association of class I HC with calnexin, while beta 2m-transfected FO-1 cells displayed a more rapid dissociation of class I HC from calnexin. Consistent with this result, the dissociation of class I HC from calnexin did not appear to be dependent on peptide binding since the dissociation rates were similar in peptide transporter-deficient T2 cells and in wild-type T1 cells. From these observations, we speculate that in the stepwise assembly of class I molecules, calnexin may mediate dimerization of class I HC with beta 2m, and that the unstable beta 2m+:HC:pep- complexes, after dissociation from calnexin, subsequently bind peptide to complete the assembly.