Identification of an N-domain histidine essential for chaperone function in calreticulin.

Calreticulin is an endoplasmic reticulum (ER) luminal Ca(2+)-binding chaperone involved in folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle." We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt(-/-) cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders.