Identification of an N-domain Histidine Essential for Chaperone Function in Calreticulin

Abstract

Calreticulin is an endoplasmic reticulum (ER) luminalmutant 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.The endoplasmic reticulum (ER) 1 plays an essential role in a variety of cellular processes, including Ca 2ϩ homeostasis, protein and lipid synthesis, and post-translational modification and folding of membrane-associated and secreted proteins (1). The ER ensures that only correctly folded proteins proceed through the secretory pathway and directs misfolded proteins to ER-associated degradation (2, 3). The lumen of the ER is a dynamic environment that contains numerous molecular chaperones and Ca 2ϩ -binding proteins that are designed for these tasks. Molecular chaperones are proteins that bind to misfolded/unfolded proteins in a transient manner to assist in their folding.Calreticulin is a Ca 2ϩ -binding chaperone that resides in the lumen of the ER and is involved in modulation of Ca 2ϩ homeostasis and in the folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle" (4 -7). Calreticulin and calnexin are both ER lectins, which bind transiently to virtually all newly synthesized glycoproteins (5-7). Chaperone-assisted protein folding has been studied extensively using Escherichia coli GroEL heat shock proteins, which are cytoplasmic (8). Numerous studies have been carried out on ER-associated chaperones (2-7); yet, the molecular features of calreticulin that confer its chaperone function have not yet been determined (7).Three distinct structural domains have been identified in calreticulin: the amino-terminal, globular N-domain; the central P-domain; and the carboxyl-terminal C-domain (7). NMR (9), modeling (10), and biochemical studies (11) indicate that the globular N-domain and the "extended arm" P-domain of calreticulin may form a functional protein-folding unit (10). This region of calreticulin contains a Zn 2ϩ binding site and one disulfide bond, and it may also bind ATP (12)(13)(14). When calreticulin binds Zn 2ϩ , it undergoes dramatic conformational changes (15). Chemical modification of calreticulin has revealed that four histidines located in the N-domain of the protein (His 25 , His 82 , His 128 , and His 153 ) are involved in the Zn 2ϩ binding (12). The Zn 2ϩ -dependent conformational change in calreticulin affects its ability to bind to unfolded protein/ glycoprotein substrates in vitro (16), suggesting that conformational changes in calreticulin may modify its chaperone function. The role of the Zn 2ϩ binding histidine residues in calreticulin function is not known.Calreticulin deficiency is embryonic lethal, and cells derived from calreticulin knockout embryos have impaired Ca 2ϩ homeostasis and compromised protein folding and quality control (11,17). The availability of calret...

Identification by Mutational Analysis of Amino Acid Residues Essential in the Chaperone Function of Calreticulin

“…For expression in eukaryotic cells, the rab-bit calreticulin gene was amplified and cloned into pcDNA3.1/Zeo (12). For detection of the recombinant protein, a hemagglutinin epitope (HA) tag was engineered to the C terminus of calreticulin to generate pcDNA-CRT-HA with the C-terminal ER retrieval KDEL amino acid sequence left intact (11,12).…”

“…Plasmids and Site-directed Mutagenesis-For Escherichia coli expression of calreticulin, the wild type full-length rabbit calreticulin gene was amplified and cloned into pBAD/glIII to generate pBAD-CRT as previously described (12). For expression in eukaryotic cells, the rab-bit calreticulin gene was amplified and cloned into pcDNA3.1/Zeo (12).…”

“…For example, in calreticulin-deficient cells, folding of the bradykinin receptor is altered, and this impairs its ability to initiate InsP 3 -dependent Ca 2ϩ release (11). The InsP 3 -dependent Ca 2ϩ release is, however, fully restored in calreticulin-deficient cells that express recombinant calreticulin (11,12). Therefore, in this study, we used restoration of bradykinin-dependent Ca 2ϩ release from the ER to compare the function of several site-specific calreticulin mutants with that of wild-type calreticulin.…”

“…The N-domain of calreticulin contains a carbohydrate binding site (13) and one disulfide bond (14). In a recent study, site-specific mutation of histidine residues in calreticulin revealed that His 153 , which is located in the N-domain of the protein, is essential for its chaperone function (12). The "extended arm" structure of the P-domain is involved in the association of calreticulin with ERp57, a thiol-disulfide oxidoreductase that is a close homolog of protein-disulfide isomerase (15)(16)(17).…”

“…NMR (8), structural modeling (9,10), and biochemical studies (11,12) have indicated that, together, the globular N-domain and the "extended arm" P-domain of calreticulin form a functional protein-folding unit (9). The N-domain of calreticulin contains a carbohydrate binding site (13) and one disulfide bond (14).…”