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Calreticulin recognizes misfolded HLA-A2 heavy chains
Laura Mancino1, Syed Monem Rizvi, Philip Edward Lapinski
1Department of Microbiology and Immunology and Immunology Graduate Program, University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA.
Insights
Calreticulin, an endoplasmic reticulum chaperone, prevents heat-induced aggregation of major histocompatibility complex (MHC) class I heavy chains. Oligomeric calreticulin binds misfolded MHC I, but doesn't enhance peptide assembly alone.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Calreticulin is an endoplasmic reticulum (ER) chaperone protein.
- Major histocompatibility complex (MHC) class I molecules are crucial for immune recognition.
- Understanding their interactions is key to cellular immunity.
Purpose of the Study:
- To investigate the functional interactions between calreticulin and MHC class I molecules.
- To determine calreticulin's role in the folding and assembly of MHC class I.
Main Methods:
- In vitro thermal aggregation assays were used to assess protein stability.
- Fluorescent peptide-based binding assays evaluated peptide binding efficiency.
- Analysis of high-molecular-weight complex formation between proteins.
Main Results:
- Calreticulin inhibits heat-induced aggregation of peptide-deficient HLA-A2 heavy chains.
- Complex formation occurs at elevated temperatures (50°C), indicating polypeptide interactions.
- Oligomeric calreticulin is active in preventing MHC I aggregation.
- Calreticulin does not enhance peptide binding to HLA-A2 at physiological temperatures.
- Calreticulin alone does not promote the assembly of MHC I with beta2m and peptides.
Conclusions:
- Calreticulin recognizes and stabilizes misfolded MHC class I heavy chains in the ER.
- Its function is primarily in preventing aggregation rather than directly facilitating peptide loading.
- Calreticulin's role in MHC I quality control is context-dependent, requiring other ER components for full assembly.
Abstract:
Our studies investigated functional interactions between calreticulin, an endoplasmic reticulum chaperone, and major histocompatibility complex (MHC) class I molecules. Using in vitro thermal aggregation assays, we established that calreticulin can inhibit heat-induced aggregation of soluble, peptide-deficient HLA-A2 purified from supernatants of insect cells. The presence of HLA-A2-specific peptides also inhibits heat-induced aggregation. Inhibition of heat-induced aggregation of peptide-deficient HLA-A2 by calreticulin correlates with a rescue of the HLA-A2 heavy chain from precipitation, by forming high-molecular-weight complexes with calreticulin. Complex formation between HLA-A2 heavy chains and calreticulin occurs at 50 degrees C but not 37 degrees C, suggesting polypeptide-based interactions between the HLA-A2 heavy chain and calreticulin. Once complexes are formed, the addition of peptide is not sufficient to trigger efficient assembly of heavy chain/beta2m/peptide complexes. Using a fluorescent peptide-based binding assay, we show that calreticulin does not enhance peptide binding by HLA-A2 at 37 degrees C. We also show that calreticulin itself is converted to oligomeric species on exposure to 37 degrees C or higher temperatures, and that oligomeric forms of calreticulin are active in inhibiting thermal aggregation of peptide-deficient HLA-A2. Taken together, these results suggest that calreticulin functions in the recognition of misfolded MHC class I heavy chains in the endoplasmic reticulum. However, in the absence of other endoplasmic reticulum components, calreticulin by itself does not enhance the assembly of misfolded MHC class I heavy chains with beta2m and peptides.