Interactions of Substrate with Calreticulin, an Endoplasmic Reticulum Chaperone

Abstract

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc 1 Man 7-9 GlcNAc 2 present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc 1 Man 7-9 GlcNAc 2 , determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K b ) and changes in enthalpy of binding (⌬H b°) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 ؋ 10 4 M ؊1 at 279 K is obtained when glucose occurs in ␣-1,3 linkage to Man␣Me as in Glc␣1-3Man␣Me. The binding constant increases by 25-fold from di-to trisaccharide and doubles from tri-to tetrasaccharide, demonstrating that the entire Glc␣1-3Man␣1-2Man␣1-2Man␣Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed. Calreticulin (CRT),1 along with calnexin, serves as a molecular chaperone in the endoplasmic reticulum (ER) of eukaryotic cells. Although calreticulin is a soluble, luminal protein, calnexin is a type I membrane protein (1, 2). Segments of these proteins share amino acid identity ranging from 42 to 78% (3). Calreticulin is a highly conserved ubiquitous protein (M r 46,000) and has been implicated in Ca 2ϩ storage and intracellular Ca 2ϩ signaling in the sarcoplasmic and endoplasmic reticula (4, 5). CRT has been divided into three regions: the N-terminal, the C-terminal, and the central P-domain, which consists of short sequence motifs repeated three times in tandem. The N-terminal domain is highly conserved among CRTs from different species and potentially mediates interactions between CRT and the ER folding catalysts, protein disulfide isomerase and ERp57 (6, 7). Recent studies show that the P-domain, previously thought to be involved in oligosaccharide binding, interacts directly with ERp57 (8 -10). The C-domain is characterized by a high content of acidic residues (4, 11), which is consistent with the location of a low affinity (K d ϭ ϳ1-2 mM), high capacity (ϳ25-50 mol) calcium-binding site (12) and contains the ER retrieval sequence.The ER plays an essential role in the folding and maturation of newly synthesized proteins in the secretory pathway. ER quality control operates at various levels; one of the most comm...

Structural Basis of Carbohydrate Recognition by Calreticulin

“…The affinity of 0.7 M for Glc 1 Man 3 is very close to the reported value for intact CRT (supplemental Fig. 4) (29).…”

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

“…2D), Crt-GFP shows an unchanged mobile fraction but a reduced D eff . Thus, interactions between Crt and its substrates are dynamic, consistent with the weak affinity of Crt (K d ϭ 2 M) (25). Similar dynamic interactions are likely to occur with maturing polypeptides and Crt-GFP, because the introduction of this population further reduces D eff .…”

“…For the chaperone probe, we selected Crt for several reasons: the availability of a knockout cell line (19), chemical inhibitors of its function (20), well characterized biochemical properties (21)(22)(23)(24)(25)(26), and structural information (24,(27)(28)(29). Based on this information, we generated a GFP-tagged Crt (Crt-GFP) (Fig.…”

Cell Surface Expression of α1D-Adrenergic Receptors Is Controlled by Heterodimerization with α1B-Adrenergic Receptors

“…Some GPCRs contain ER retention motifs that must be removed or masked before they can be exported, including the CB1 N terminus (68) and GABA B -R1 C terminus (34). Other GPCRs may be targeted by ER retention proteins such as HSP79 analogue BiP/GRP78 (69) or the lectin-like proteins calnexin and calreticulin (70,71), preventing surface expression. We show here that ␣ 1B -ARs promote ␣ 1D -AR surface expression, presumably by direct heterodimerization.…”