Calreticulin recognizes misfolded HLA-A2 heavy chains

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°C but not 37°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 chain͞␤2m͞peptide complexes. Using a fluorescent peptidebased binding assay, we show that calreticulin does not enhance peptide binding by HLA-A2 at 37°C. We also show that calreticulin itself is converted to oligomeric species on exposure to 37°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 ␤2m and peptides.

Calreticulin Promotes Folding of Functional Human Leukocyte Antigen Class I Molecules in Vitro

“…However, these experiments must be interpreted with caution because we cannot rule out that deglycosylation destabilizes class I molecules in a fashion that cannot be reversed by CRT. Moreover, we confirmed that CRT inhibits class I aggregation, both in a direct and indirect fashion (26). CRT directly inhibited precipitation of refolded empty A2SC molecules at 45°C.…”

“…Wearsch et al (11) showed that monoglucosylation of soluble HLA-B8 is both necessary and sufficient to induce class I/CRT interaction but did not study the effect of CRT on peptide assembly with class I (11). The latter issue was investigated by Mancino et al (26) who found that CRT prevented HLA-A2 aggregation at 45-50°C, whereas it had no effect in a peptide exchange assay at physiological temperature.…”

Calreticulin Is a Thermostable Protein with Distinct Structural Responses to Different Divalent Cation Environments

“…3), coupled with exposure of hydrophobic residues (Fig. 7) at temperatures approaching the T Trans values is likely responsible for the occurrence of calreticulin oligomerization upon heating and cooling (40,41). As such, the findings presented here provide further structural evidence for the notion that the cellular requirements for calreticulin may extend beyond its classical lectin-based functions.…”

Major Histocompatibility Complex Class I Molecules Expressed with Monoglucosylated N-Linked Glycans Bind Calreticulin Independently of Their Assembly Status

“…The interaction with calnexin can persist in the presence of castanospermine or in glucosidase-deficient cells for some, but not all, murine and human class I alleles (31,48). In addition, calreticulin can suppress the aggregation at 45-50°C of insect cell-expressed HLA-A2, which is glycosylated but with an unknown and likely processed structure (49). In that study, however, no interaction was observed at physiological temperatures.…”

“…In that study, however, no interaction was observed at physiological temperatures. To date, aggregation inhibition assays are the only in vitro techniques successfully used to demonstrate the interaction of calnexin or calreticulin with substrates that are either non-glycosylated or glycosylated with the incorrect structure (37,47,49). The published assays were performed at 45°C, which has been shown to be the T m for the thermal denaturation of calreticulin (50).…”