Interaction of the chaperone calreticulin with proteins and peptides of different structural classes.

The interaction of calreticulin with native and denatured forms and polypeptides in proteolytic digests of proteins representing structural classes of all-alpha-helix (hemoglobin, serum albumin), all-beta-sheet (IgG) and alpha-helix + beta-sheets (lysozyme, ovalbumin) was investigated. The binding of calreticulin to denatured proteins was found to depend on conformation and structural class of the protein. No interaction was observed with the native proteins, whereas binding was seen for the denatured proteins, the order of interaction being lysozyme = IgG > ovalbumin >> hemoglobin = serum albumin. Moreover, the interaction between calreticulin and the heat-denatured proteins depended on the temperature and time used for denaturation and the degree of proteolytic fragmentation. Calreticulin bound well to peptides in proteolytic digests from protease K or chymotrypsin treatment of lysozyme, IgG and ovalbumin but weakly or not at all to peptides in proteolytic digests of hemoglobin and serum albumin. Synthetic peptides from lysozyme and ovalbumin confirmed binding to hydrophobic peptides from these proteins. These results show that calreticulin has the ability to interact with denatured and fragmented forms of proteins with a preference for beta-strand structure and hydrophobicity.

Interaction of C1q with the receptor calreticulin requires a conformational change in C1q.

The interaction between C1q and the chaperone calreticulin was studied under various conditions. When both proteins were present in equal amounts in solution, no interaction could be demonstrated. However, C1q immobilized on a hydrophobic surface, exposed to heat-treatment or bound to immunoglobulins (Igs) showed a strong, rapid and specific binding of calreticulin. The interaction appeared to be a two-step process, and the initial phase of interaction was sensitive to high concentrations of salt but not to a physiological salt concentration. The following strong binding was insensitive to salt and extremes of pH but sensitive to strongly denaturing agents (urea and guanidine). The sensitivity to salt during the initial phase of interaction was practically identical to that observed when calreticulin was bound to type V collagen. Binding between C1q and calreticulin could be inhibited by serum amyloid P component and by proteinase K-digested ovalbumin, and the binding of calreticulin to proteinase K-digested ovalbumin was shown to be inhibited by C1q. The data indicate that C1q binds stably to the peptide-binding site of calreticulin and that the initial binding of calreticulin to C1q involves the collagen-like domain of the C1q molecule. In conclusion, our results suggest calreticulin as a potential receptor for an altered conformation of C1q as occurs during binding to Igs. Thus, the chaperone and protein-scavenging function of calreticulin may extend from the endoplasmic reticulum to the topologically equivalent cell surface, where it may contribute to the elimination of immune complexes and apoptotic cells.