Polypeptide binding properties of the chaperone calreticulin.

Calreticulin is a highly conserved eukaryotic ubiquitious protein located mainly in the endoplasmic reticulum. Two major characteristics of calreticulin are its chaperone activity and its lectin properties, but its precise function in intracellular protein and peptide processing remains to be elucidated. We have investigated the interactions of human calreticulin with denatured ovalbumin, proteolytic digests of ovalbumin, and different available peptides by solid phase assays, size-exclusion chromatography, capillary electrophoresis, and MS. The results show that calreticulin interacts better with unfolded ovalbumin than with native ovalbumin, that calreticulin strongly binds components in proteolytic digests of denatured ovalbumin, and that calreticulin interacts strongly with certain synthetic peptides.     

Expanded binding specificity of the human histone chaperone NASP

NASP (nuclear autoantigenic sperm protein) has been reported to be an H1-specific histone chaperone. However, NASP shares a high degree of sequence similarity with the N1/N2 family of proteins, whose members are H3/H4-specific histone chaperones. To resolve this paradox, we have performed a detailed and quantitative analysis of the binding specificity of human NASP. Our results confirm that NASP can interact with histone H1 and that this interaction occurs with high affinity. In addition, multiple in vitro and in vivo experiments, including native gel electrophoresis, traditional and affinity chromatography assays and surface plasmon resonance, all indicate that NASP also forms distinct, high specificity complexes with histones H3 and H4. The interaction between NASP and histones H3 and H4 is functional as NASP is active in in vitro chromatin assembly assays using histone substrates depleted of H1.     

Dimerization and oligomerization of the chaperone calreticulin.

The chaperone calreticulin is a highly conserved eukaryotic protein mainly located in the endoplasmic reticulum. It contains a free cysteine SH group but does not form disulfide-bridged dimers under physiological conditions, indicating that the SH group may not be fully accessible in the native protein. Using PAGE, urea gradient gel electrophoresis, capillary electrophoresis and MS, we show that dimerization through the SH group can be induced by lowering the pH to 5-6, heating, or under conditions that favour partial unfolding such as urea concentrations above 2.6 m or SDS concentrations above 0.025%. Moreover, we show that calreticulin also has the ability to self-oligomerize through noncovalent interactions at urea concentrations above 2.6 m at pH below 4.6 or above pH 10, at temperatures above 40 degrees C, or in the presence of high concentrations of organic solvents (25%), conditions that favour partial unfolding or an intramolecular local conformational change that allows oligomerization, resulting in a heterogeneous mixture of oligomers consisting of up to 10 calreticulin monomers. The oligomeric calreticulin was very stable, but oligomerization was partially reversed by addition of 8 m urea or 1% SDS, and heat-induced oligomerization could be inhibited by 8 m urea or 1% SDS when present during heating. Comparison of the binding properties of monomeric and oligomeric calreticulin in solid-phase assays showed increased binding to peptides and denatured proteins when calreticulin was oligomerized. Thus, calreticulin shares the ability to self-oligomerize with other important chaperones such as GRP94 and HSP90, a property possibly associated with their chaperone activity.     

Delineation of the lectin site of the molecular chaperone calreticulin

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum that functions to promote protein folding as well as to retain misfolded proteins. Similar to its membrane-bound paralog calnexin (CNX), CRT is a lectin that preferentially interacts with glycoproteins bearing Glc1Man5-9GlcNAc2 oligosaccharides. Although the lectin site of CNX has been delineated through X-ray crystallographic and mutagenic studies, the corresponding site for CRT has not been as well characterized. To address this issue, we attempted to construct lectin-deficient CRT mutants, using the structure of CNX as a guide to identify potential oligosaccharide-binding residues. Mutation of 4 such CRT residues (Y109, K111, Y128, D317) completely abrogated oligosaccharide binding. In contrast, mutation of CRT residues M131 and D160, which correspond to important residues in the lectin site of CNX, had no effect on oligosaccharide binding. These findings suggest that the organization of the lectin site in CRT largely resembles that of CNX but is not identical. The deficiency in oligosaccharide binding by the mutants was not due to misfolding because they exhibited wild-type protease digestion patterns, were capable of binding the thiol oxidoreductase ERp57, and functioned just as efficiently as wild-type CRT in suppressing the aggregation of the nonglycosylated substrate citrate synthase. However, they were impaired in their ability to suppress the aggregation of the glycosylated substrate jack bean alpha-mannosidase. This provides the first direct demonstration of the importance of CRT's lectin site in suppressing the aggregation of nonnative glycoproteins.     

Substrate specificity of the SecB chaperone

The bacterial chaperone SecB assists translocation of proteins across the inner membrane. The mechanism by which it differentiates between secretory and cytosolic proteins is poorly understood. To identify its binding motif, we screened 2688 peptides covering sequences of 23 proteins for SecB binding. The motif is approximately 9 residues long and is enriched in aromatic and basic residues, whereas acidic residues are disfavored. Its identification allows the prediction of binding regions within protein sequences with up to 87% accuracy. SecB-binding regions occur statistically every 20-30 residues. The occurrence and affinity of binding regions are similar in SecB-dependent and -independent secretory proteins and in cytosolic proteins, and SecB lacks specificity toward signal sequences. SecB cannot thus differentiate between secretory and non-secretory proteins via its binding specificity. This conclusion is supported by the finding that SecB binds denatured luciferase, thereby allowing subsequent refolding by the DnaK system. SecB may rather be a general chaperone whose involvement in translocation is mediated by interactions of SecB and signal sequences of SecB-bound preproteins with the translocation apparatus.     

Interactions of substrate with calreticulin,an endoplasmic reticulum chaperone

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 (Delta H(b) degrees ) 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 x 10(4) m(-1) at 279 K is obtained when glucose occurs in alpha-1,3 linkage to Man alpha Me as in Glc alpha 1-3Man alpha Me. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha 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.     

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.     

Gentamicin binds to the lectin site of calreticulin and inhibits its chaperone activity

Recently, it became clear that aminoglycoside antibiotics affect protein-protein interactions involving protein disulfide isomerase as well as protein synthesis in the endoplasmic reticulum. In this study, we used affinity column chromatography to screen gentamicin-binding proteins in microsomes derived from bovine kidney in order to learn about the possible mechanisms of gentamicin-associated nephrotoxicity. One of the gentamicin-binding proteins was identified as calreticulin (CRT) by N-terminal amino acid sequence analysis. Interestingly, gentamicin inhibited the chaperone and oxidative refolding activities of CRT when N-glycosylated substrates such as alpha1-antitrypsin and alpha-mannosidase were used as substrates, but it did not inhibit the chaperone activity of CRT when unglycosylated citrate synthase was used. Moreover, CRT suppressed the aggregation of deglycosylated and denatured alpha-mannosidase, but gentamicin did not inhibit its chaperone activity. Experiments with domain mutants suggest that the lectin site of CRT is the main target for gentamicin binding and that binding of gentamicin to this site inhibits the chaperone activity of CRT.     

The interplay between calcium and the in vitro lectin and chaperone activities of calreticulin

The ER resident protein calreticulin fulfills at least two important roles. It works as a chaperone preventing Golgi exit of non-native protein species and enhancing protein folding efficiency in either N-glycan-dependent, lectin chaperone, or classical chaperone, N-glycan-independent, modes and is one of the main calcium buffers in the cell. This last feature is independent from the lectin chaperone properties of the protein as this last activity is also observed in a CRT fragment lacking calcium buffer capacity. Here we study the interplay between calcium and the lectin and chaperone activities of CRT. The affinity of CRT for monoglucosylated glycans measured in solution by equilibrium dialysis and fluorescence anisotropy was not affected by the absence of calcium. Binding of CRT to monoglucosylated neoglycoproteins displaying either native or molten globule-like conformations was also independent of calcium concentration. Moreover, calcium and monoglucosylated glycans stabilized the CRT structure in an apparent additive, independent manner when the protein was subjected to thermal or urea-induced denaturation. In addition, the ability of CRT to decrease the level of aggregation of a chemically denatured monoglucosylated and nonglycosylated protein was also independent of calcium level.     

The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.     

Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin

Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.     

Analyses of carbohydrate binding property of lectin-chaperone calreticulin

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum. It is a lectin that promotes the folding of proteins carrying N-linked glycans. Recent investigations have revealed that glucosylated high-mannose-type glycans are employed as key elements in this process. Here, we performed quantitative analyses of the interaction of CRT with various disaccharides, including fluorine-substituted analogues using a quartz-crystal microbalance (QCM). These experiments revealed the weak affinity of 2- and 3-fluoroglucose derivatives. On the other hand, 6-fluoroglucose derivatives exhibited a significant affinity, indicating that the role of 6-position of OH is less significant for binding to CRT. We also characterized binding epitope of the Glcα1-3ManαMe to CRT by saturation transfer difference (STD) NMR spectroscopy. It is proposed that 2-, 3-, and 4-positions of Glc and 3-, 4-, and 6-positions of Man are in close contact with CRT binding pocket, while 6-position of Glc and 2-position of Man are not. These finding are in excellent agreement with our QCM experiment.     

Nerve growth factor induces the expression of chaperone protein calreticulin in human epithelial ovarian cells

Epithelial ovarian cancer is highly angiogenic and high expression of Nerve Growth Factor (NGF), a proangiogenic protein. Calreticulin is a multifunctional protein with anti-angiogenic properties and its translocation to the tumor cell membrane promotes recognition and engulfment by dendritic cells. The aim of this work was to evaluate calreticulin expression in human normal ovaries, benign and borderline tumors, and epithelial ovarian cancer samples and to evaluate whether NGF regulates calreticulin expression in human ovarian surface epithelium and in epithelial ovarian cancer cell lines. Calreticulin mRNA and protein levels were analyzed using RT-PCR, Western blot and immunohistochemistry in 67 human ovarian samples obtained from our Institution. Calreticulin expression induced by NGF stimulation in cell lines was evaluated using RT-PCR, Western blot and immunocytochemistry. We found a significant increase of calreticulin mRNA levels in epithelial ovarian cancer samples as compared to normal ovaries, benign tumors, and borderline tumors. Calreticulin protein levels, evaluated by Western blot, were also increased in epithelial ovarian cancer with respect to benign and borderline tumors. When HOSE and A2780 cell lines were stimulated with Nerve Growth Factor, we found an increase in calreticulin protein levels compared to controls. This effect was reverted by GW441756, a TRKA specific inhibitor. These results suggest that NGF regulates calreticulin protein levels in epithelial ovarian cells through TRKA receptor activation.     

Peptide specificity of protein prenyltransferases is determined mainly by reactivity rather than binding affinity

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of lipid groups from farnesyl diphosphate and geranylgeranyl diphosphate, respectively, to a cysteine near the C-terminus of protein substrates. FTase and GGTase I modify several important signaling and regulatory proteins with C-terminal CaaX sequences ("C" refers to the cysteine residue that becomes prenylated, "a" refers to any aliphatic amino acid, and "X" refers to any amino acid). In the CaaX paradigm, the C-terminal X-residue of the protein/peptide confers specificity for FTase or GGTase I. However, some proteins, such as K-Ras, RhoB, and TC21, are substrates for both FTase and GGTase I. Here we demonstrate that the C-terminal amino acid affects the binding affinity of K-Ras4B-derived hexapeptides (TKCVIX) to FTase and GGTase I modestly. In contrast, reactivity, as indicated by transient and steady-state kinetics, varies significantly and correlates with hydrophobicity, volume, and structure of the C-terminal amino acid. The reactivity of FTase decreases as the hydrophobicity of the C-terminal amino acid increases whereas the reactivity of GGTase I increases with the hydrophobicity of the X-group. Therefore, the hydrophobicity, as well as the structure of the X-group, determines whether peptides are specific for farnesylation, geranylgeranylation, or dual prenylation.     

Alternative chaperone machinery may compensate for calreticulin/calnexin deficiency in Caenorhabditis elegans

Proper folding and maintenance of the native structure are central to protein function and are assisted by a family of proteins called chaperones. Calreticulin and calnexin are ER resident chaperones well conserved from worm to human. Calreticulin/calnexin knock-out mice exhibit a severe phenotype, whereas in Caenorhabditis elegans, calreticulin [crt-1(jh101)]- and calnexin [cnx-1(nr2009)]-null mutant worms exhibit only a mild phenotype, suggesting the possible existence of alternative chaperone machinery that can compensate for the deficiency of calreticulin and/or calnexin. In order to rapidly identify the compensatory chaperone components involved in this process, we analyzed the proteome of crt-1(jh101) mutants and [crt-1(jh101);cnx-1(nr2009)] double mutants. When grown at 20 degrees C, we found that five proteins were up-regulated and two proteins were down-regulated in crt-1(jh101) mutants; nine proteins were up-regulated and five proteins were down-regulated in [crt-1(jh101);cnx-1(nr2009)] double mutants. In addition, elevation of the cultivation temperature to 25 degrees C, which is still permissive to growth but causes specific defects in mutants, led to the identification of several additional proteins. Interestingly, the consistent increment of heat shock protein-70 family members (hsp70) together with protein disulfide isomerase (PDI) at all the examined conditions suggests the possible compensatory function imparted by hsp70 and PDI family members in the absence of calreticulin and/or calnexin.     

Lectin-deficient calreticulin retains full functionality as a chaperone for class I histocompatibility molecules

Calreticulin is a molecular chaperone of the endoplasmic reticulum that uses both a lectin site specific for Glc(1)Man(5-9)GlcNAc(2) oligosaccharides and a polypeptide binding site to interact with nascent glycoproteins. The latter mode of substrate recognition is controversial. To examine the relevance of polypeptide binding to protein folding in living cells, we prepared lectin-deficient mutants of calreticulin and examined their abilities to support the assembly and quality control of mouse class I histocompatibility molecules. In cells lacking calreticulin, class I molecules exhibit inefficient loading of peptide ligands, reduced cell surface expression and aberrantly rapid export from the endoplasmic reticulum. Remarkably, expression of calreticulin mutants that are completely devoid of lectin function fully complemented all of the class I biosynthetic defects. We conclude that calreticulin can use nonlectin-based modes of substrate interaction to effect its chaperone and quality control functions on class I molecules in living cells. Furthermore, pulse-chase coimmunoisolation experiments revealed that lectin-deficient calreticulin bound to a similar spectrum of client proteins as wild-type calreticulin and dissociated with similar kinetics, suggesting that lectin-independent interactions are commonplace in cells and that they seem to be regulated during client protein maturation.     

Abundant accumulation of the calcium-binding molecular chaperone calreticulin in specific floral tissues of Arabidopsis thaliana.

Calreticulin (CRT) is a calcium-binding protein in the endoplasmic reticulum (ER) with an established role as a molecular chaper-one. An additional function in signal transduction, specifically in calcium distribution, is suggested but not proven. We have analyzed the expression pattern of Arabidopsis thaliana CRTs for a comparison with these proposed roles. Three CRT genes were expressed, with identities of the encoded proteins ranging from 54 to 86%. Protein motifs with established functions found in CRTs of other species were conserved. CRT was found in all of the cells in low amounts, whereas three distinct floral tissues showed abundant expression: secreting nectaries, ovules early in development, and a set of subepidermal cells near the abaxial surface of the anther. Localization in the developing endosperm, which is characterized by high protein synthesis rates, can be reconciled with a specific chaperone function. Equally, nectar production and secretion, a developmental stage marked by abundant ER, may require abundant CRT to accommodate the traffic of secretory proteins through the ER. Localization of CRT in the anthers, which are degenerating at the time of maximum expression of CRT, cannot easily be reconciled with a chaperone function but may indicate a role for CRT in anther maturation or dehiscence.     

Thermodynamics of substrate binding to the chaperone SecB

The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and alpha-lactalbumin. SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded disulfide intermediates of alpha-lactalbumin, which have 40-60% of native secondary structure. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29, and -0.41 kcal mol(-1) K(-1), respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. There is no evidence for two separate types of binding sites for positively charged and hydrophobic ligands. Spectroscopic and proteolysis protection studies of the binding of SecB to poly-L-Lys show that binding of highly positively charged peptide ligands to negatively charged SecB leads to charge neutralization and subsequent aggregation of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft. SecB aggregation in the absence of substrate is prevented by electrostatic repulsion between negatively charged SecB tetramers.     

PDI family protein ERp29 forms 1:1 complex with lectin chaperone calreticulin

Lectin chaperone calreticulin is well known to interact with ERp57 which is one of PDI family proteins. The interaction of ERp57 with calreticulin is believed to assist disulfide bond formation of nascent glycoprotein in the ER. Various kinds of PDI family proteins are present in the ER, however, their precise roles have been unclear. In this study, interaction assay between PDI family proteins and calreticulin by SPR analysis was performed. Our analysis revealed for the first time formation of a 1:1 complex between ERp29 and calreticulin. The dissociation constant of interaction between ERp29 and calreticulin was shown to be almost identical to ERp57–calreticulin interaction. We speculate that the recognition site of ERp29 within calreticulin is different from that of ERp57.