title: Calreticulin promotes folding of functional human leukocyte antigen class I molecules in vitro.
abstract: The assembly of MHC class I molecules with beta(2)-microglobulin and peptides is assisted by the housekeeping chaperones calnexin, calreticulin, and Erp57 and the dedicated accessory protein, tapasin. Tapasin and calreticulin are essential for efficient MHC class I assembly, but their precise action during class I assembly remains to be elucidated. Previous in vitro studies have demonstrated that the lectin calreticulin interacts with monoglucosylated MHC class I heavy chains, whatever their state of assembly with light chains and peptide, and inhibits their aggregation above physiological temperature. We used a soluble single chain HLA-A2/beta(2)-microglobulin molecule, A2SC, to study the effect of calreticulin on the peptide binding capacity of HLA class I molecules. Calreticulin inhibited the formation of A2SC aggregates both when co-expressed in insect cells and during incubations at elevated temperature. Calreticulin dramatically enhanced acquisition of peptide binding capacity when added to denatured A2SC molecules during refolding at 4 degrees C. However, it had no effect on the rapid loss of A2SC peptide binding capacity at physiological temperature. We conclude that calreticulin promotes the folding of HLA class I molecules to a state in which, at low temperature, they spontaneously acquire peptide binding capacity. However, it does not induce or maintain a peptide-receptive state of the class I-binding site, which is likely to be promoted by one or several other components of the class I loading complexes. By being amenable to complementation with additional proteins, the described system should be useful for identification of these components.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale Unité 580, Université René Descartes Paris V, 75015 Paris, France.
authors.forename: Slobodan
authors.initials: S
authors.lastname: Culina
authors.forename: Grégoire
authors.initials: G
authors.lastname: Lauvau
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gubler
authors.forename: Peter M
authors.initials: PM
authors.lastname: van Endert
mesh_terms: D000818:Animals; D017920:Antiporters / Q000235:genetics / Q000502:physiology; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D037281:Calnexin / Q000235:genetics / Q000502:physiology; D037282:Calreticulin / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D002460:Cell Line; D004355:Drug Stability; D015870:Gene Expression; D015789:HLA-A2 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000502:physiology; D026901:Membrane Transport Proteins; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding; D011993:Recombinant Fusion Proteins; D018411:Spodoptera / Q000378:metabolism; D014162:Transfection; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D017920:Antiporters; D037282:Calreticulin; D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; C107762:tapasin; D037281:Calnexin