title: Identification of an N-domain histidine essential for chaperone function in calreticulin.
abstract: 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.
authors.affiliation: Canadian Institutes of Health Research Membrane Protein Research Group and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.forename: Jody
authors.initials: J
authors.lastname: Groenendyk
authors.forename: Sylvia
authors.initials: S
authors.lastname: Papp
authors.forename: Monika
authors.initials: M
authors.lastname: Dabrowska
authors.forename: Barbara
authors.initials: B
authors.lastname: Knoblach
authors.forename: Cyril
authors.initials: C
authors.lastname: Kay
authors.forename: J M Robert
authors.initials: JM
authors.lastname: Parker
authors.forename: Michal
authors.initials: M
authors.lastname: Opas
authors.forename: Marek
authors.initials: M
authors.lastname: Michalak
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D037282:Calreticulin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D007136:Immunoglobulins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D037282:Calreticulin; C016512:IgY; D007136:Immunoglobulins; D018832:Molecular Chaperones; D006639:Histidine; D008291:Malate Dehydrogenase; D014357:Trypsin; D015032:Zinc; D001920:Bradykinin; D002118:Calcium