As shown in Figure 1 C, most protein interactions with the two mutants were either lost or severely diminished after the min incubation in contrast to wild-type Crt in K41 or K42 cells, which exhibited more stable association with newly synthesized proteins. It has been shown previously that ER chaperones can be detected in large, stable complexes with other components of the ER folding machinery Tatu and Helenius, ; Meunier et al. Presumably, Crt would not interact with components of this folding machinery through lectin-based interactions. Thus, it was important to determine whether the newly synthesized proteins coisolated with Crt, and shown to exhibit labile interactions with the lectin-deficient mutants, were indeed substrates that interact transiently with the chaperone during folding or whether they were stably associated components of the folding machinery.
To address this issue, cells expressing wild-type or lectin-deficient Crt were subjected to pulse-chase radiolabeling, and Crt-associated proteins were isolated at each time point. Because the efficiency of recovery of different Crt-associated proteins varied somewhat from experiment to experiment, we compared the patterns of associated proteins for wild-type Crt and the DA mutant in one experiment Figure 2 , top and for wild-type Crt and the YA mutant in a second experiment Figure 2 , bottom.
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In both experiments, Crt-deficient K42 cells were included as a control for the specificity of the immunoisolation, and, as expected, no proteins were recovered Figure 2 , K42 lanes. For all four cell lines, although the intensities of several Crt-associated proteins seemed unaltered during the chase, the majority of complexes dissociated extensively and at various rates over the min chase period.
This is consistent with previous studies that documented transient association of both Cnx and Crt during the folding of diverse glycoprotein substrates David et al. Furthermore, both the patterns of associated proteins and their dissociation kinetics seemed to be remarkably similar when wild-type Crt and the DA mutant were compared Figure 2 , top and when wild-type Crt and the YA mutant were compared Figure 2 , bottom.
These findings suggest that the majority of newly synthesized proteins associated with wild-type and lectin-deficient Crt are indeed substrates and that their dissociation from the mutant chaperones occurs at near normal rates despite the absence of any lectin—oligosaccharide interaction. Crt deficiency is associated with a three- to fourfold reduction in expression of class I molecules at the cell surface, a consequence of defective intracellular loading of class I molecules with stabilizing peptides Gao et al.
In K42 cells, both K b and D b were expressed at about one third the level of that observed in K41 cells. Normal expression of both molecules was restored upon transfection of K42 cells with wild-type Crt Figure 3 , K42 Wt. To determine whether the lectin site of Crt is important for restoring normal expression of class I molecules, the surface expression of K b and D b were examined in K42 cells transfected with the YA and DA lectin-deficient mutants.
Both mutant proteins restored the surface expression of K b and D b to the levels observed in cells expressing wild-type Crt. This was remarkable given previous studies suggesting that lectin—oligosaccharide interactions are the principal means whereby Crt associates with assembling class I molecules Harris et al.
A K42 cells stably expressing wild-type Crt or lectin-deficient mutants were incubated with mAbs Y-3 or B Cells were then incubated with phycoerythrin-conjugated goat anti-mouse IgG and analyzed by flow cytometry. K41 and K42 cells transfected with empty vector were also subjected to this analysis. As a negative control, cells were stained with secondary antibody alone. Error bars represent the SE values from three independent experiments. We next examined whether lectin-deficient Crt is capable of promoting peptide loading onto class I molecules.
Class I molecules that do not acquire stabilizing peptide during their biogenesis are capable of reaching the cell surface but rapidly disassemble into constituent subunits that are not reactive with conformation-sensitive mAb. These transient, peptide-receptive class I molecules can be stabilized by the addition of exogenous peptides to the culture medium, resulting in a substantial upregulation of surface expression Townsend et al.
In contrast, only a modest 1. This reflects the smaller population of peptide-receptive class I molecules that are produced in cells possessing Crt. Remarkably, when K42 cells expressing either the lectin-deficient YA and DA Crt mutant were incubated with peptide ligands, the extent of surface K b and D b upregulation was approximately twofold, similar to that observed in cells expressing wild-type Crt. These findings indicate that although the YA and DA Crt mutants lack the ability to bind monoglucosylated oligosaccharide, they retain the ability to promote the loading of peptides onto assembling class I molecules.
Peptide loading of class I molecules is promoted by wild-type and lectin-deficient Crt. Results are shown as the -fold increase in expression mean fluorescence value relative to cells incubated without peptide. We subsequently confirmed these results using an antigen presentation assay in which a specific class I-binding peptide is produced in the cytosol and delivered to class I molecules in the ER through the normal antigen processing machinery.
This was the case at all but the lowest levels of fusion protein expression Figure 5 , GFP gates 1—4 , confirming previous results obtained with various T cell-based assays Gao et al. Thus, using methods that detect both general and antigen-specific peptide loading, it is apparent that Crt does not require a functional lectin site to promote efficient peptide loading of class I molecules. Wild-type and lectin-deficient Crt enhance the presentation of a specific ovalbumin peptide by H-2K b molecules. After 24 h, cells were analyzed by flow cytometry by using mAb 25D1.
Two separate experiments are shown and include controls lacking plasmid or in which an isotype-matched primary antibody replaced mAb 25D1. Presumably, the ability of lectin-deficient Crt mutants to promote peptide loading of class I molecules should reflect their inclusion within the PLC. To test this, the composition of PLCs from the various cell lines was determined by immunoisolation from digitonin lysates of unlabeled or radiolabeled cells with anti-tapasin antiserum and detection of components by immunoblotting or by fluorography, respectively.
Thus, an intact lectin site is not required for recruitment of Crt to the PLC. Association of lectin-deficient Crt with members of the peptide loading complex. A The indicated cell lines were lysed in digitonin lysis buffer and then subjected to immunoisolation with either anti-tapasin antiserum lanes 1 or preimmune serum lanes 2. Note that the high background in the Crt panel is due to residual Ig heavy chain from the anti-tapasin antiserum 50 kDa , which migrates just below Crt 60 kDa. B The indicated cells were radiolabeled with [ 35 S]Met for 30 min, incubated on ice for 10 min with 20 mM N -ethylmaleimide in PBS, lysed in digitonin lysis buffer, and then subjected to immunoisolation with anti-tapasin antiserum.
The reason for the variability is unclear, but it probably reflects the relative fragility of the PLC lacking Crt compared with that in which Crt is incorporated either as a native protein or a lectin-deficient mutant. Class I molecules that fail to acquire a stabilizing peptide are subject to ER quality control, and they are exported slowly along the secretory pathway Townsend et al.
Crt seems to play a role in this quality control process because class I molecules that fail to acquire stabilizing peptides in Crt-deficient cells are exported rapidly from the ER Gao et al. This transport behavior is depicted in Figure 7 in which ER-to-Golgi transport rates of K b and D b molecules were determined by monitoring the kinetics at which their Asn-linked oligosaccharides are processed to complex forms that are resistant to digestion with endoglycosidase H endo H.
Both wild-type and lectin-deficient Crt retard ER-to-Golgi transport of assembling class I molecules. A The indicated cell lines were radiolabeled for 10 min with [ 35 S]Met and chased with unlabeled Met for various times. Cells were then lysed and H-2K b and D b molecules were immunoisolated sequentially first with anti-8 antiserum followed by a combination of mAbs S and B The mobilities of endo H-sensitive s and -resistant r heavy chains are indicated. These appear variably between experiments; the slower band corresponds to D b molecules with all three oligosaccharides processed to complex forms whereas the faster band corresponds to molecules that possess 1 immature and 2 complex oligosaccharides.
Both represent mature, Golgi-processed molecules and they are combined when calculating the percentage of endo H-resistant heavy chains. Asterisks denote nonspecific bands. The endo H-resistant heavy chain was then calculated as a percentage of the total heavy chain signal at each time point.
Error bars represent the SE values of three independent experiments for K b and two independent experiments for D b. To determine whether Crt's lectin function is required to influence the transport behavior of class I molecules, the ER-to-Golgi transport kinetics of K b and D b molecules were measured in K42 cells expressing the YA and DA lectin-deficient Crt mutants. Consistent with the recruitment of these mutants to the PLC, they were just as effective as wild-type Crt in normalizing the export kinetics of both K b and D b molecules. For the YA mutant, the corresponding half time for D b was similar to wild-type Crt at 90 min, whereas that for K b was somewhat slower than wild-type Crt at 40 min Figure 7 B.
The class I peptide loading complex is a complex structure that is stabilized by multiple interactions between its constituent components. At the center of the complex is tapasin, which interacts with both peptide-deficient class I molecules and the polytopic TAP peptide transporter, bridging the two.
Tapasin is also linked to the thiol oxidoreductase ERp57 through a stable disulfide linkage, and this tapasin—ERp57 subcomplex has been shown to promote the loading of peptides into the class I binding groove and their subsequent editing to favor peptides of higher affinity or slower off-rate Wearsch and Cresswell, How Crt functions to promote peptide loading onto class I molecules is unclear.
From the data in Figure 6 , it is apparent that it stabilizes some components of the peptide loading complex, such as ERp57, which is directly involved in peptide loading and editing Wearsch and Cresswell, Finally, given the more rapid export of peptide-receptive class I molecules out of the ER in cells lacking Crt, it is possible that Crt, through its C-terminal KDEL retrieval motif, contributes to quality control of class I molecules by recycling the PLC between the Golgi and ER until optimal peptides have been loaded Hsu et al.
However Crt functions to promote peptide loading, a substantial body of evidence has accumulated suggesting that its interaction with class I H chains and with the PLC is mediated largely through its ability to bind monoglucosylated oligosaccharides. For example, treatment of cells with a glucosidase inhibitor to prevent the formation of monoglucosylated oligosaccharides results in a reduced but not eliminated Crt-class I interaction and a corresponding reduction of class I in the PLC Sadasivan et al. Reduced Crt association was also observed with a class I molecule mutated to remove one of its glycans at residue 86 Harris et al.
In the absence of the lectin—oligosaccharide interaction, Crt failed to bind to any of these species, suggesting a lack of ability to discriminate between class I conformational states through polypeptide-based interactions Wearsch et al. On peptide loading, UGGT no longer acts on the native conformer and deglucosylation and Crt dissociation can occur Elliott and Williams, In this context, it is remarkable that our Crt mutants, demonstrably lacking lectin function both in vitro and in cells, are fully capable of substituting for wild type Crt in class I biogenesis.
These mutants were incorporated into the PLC and they supported the assembly of other components into the PLC as well. This contrasts with previous studies using a glucosidase inhibitor to block the lectin component of the class I—Crt interaction wherein reduced incorporation of class I into the PLC was observed Sadasivan et al.
Furthermore, we found that in the absence of lectin function the mutants complemented the peptide loading defect observed in Crt-deficient cells just as efficiently as wild-type Crt. They also were as effective as wild-type Crt in ER quality control, restoring normal ER-to-Golgi export kinetics to assembling class I molecules. How can these seemingly disparate results be rationalized?
First, regarding the different phenotypes observed in class I biogenesis when glucosidase inhibitors are used versus lectin-deficient Crt, it is important to note that inhibitors such as castanospermine are not selective for preventing Crt interactions with class I oligosaccharides. Castanospermine also affects Cnx interactions that occur at the earliest stages of H chain folding and, furthermore, alters the oligosaccharide structure of all nascent Asn-linked glycoproteins including class I H chains with unknown effects on their folding.
Cnx is also involved in the assembly of the PLC before class I molecules are incorporated Diedrich et al. Thus, the use of lectin-deficient Crt mutants offers a much more selective approach to assess the importance of the lectin component of the Crt—H chain interaction in class I biogenesis. Second, concerning the reported importance of monoglucosylated oligosaccharides in recruiting Crt or Cnx to class I molecules, there are several contrary studies indicating that Crt or Cnx are indeed capable of associating with class I molecules through polypeptide-based interactions.
For example, immunoisolated complexes of Cnx and mouse or human class I H chains could be completely deglycosylated by endo H treatment without dissociation of the complex Ware et al. In addition, treatment of cells with glucosidase inhibitors did not prevent interactions of Cnx with certain human or mouse class I molecules Danilczyk and Williams, We have also shown that lectin-deficient Cnx is fully competent to associate with free class I H chains and prevent their premature degradation when coexpressed in Drosophila cells Leach and Williams, Coupled with demonstrations that Cnx or Crt can selectively bind nonnative and unglycosylated protein conformers and suppress their aggregation under physiological conditions Saito et al.
Why have such contacts been difficult to observe in the previous studies on Crt and class I molecules described above? In the experiments testing for Crt—substrate interactions by coimmunoisolation, we have shown that polypeptide-mediated interactions are quite labile, differ in strength between different substrates, and can be missed if the immunoisolations are not performed rapidly and under mild conditions Danilczyk and Williams, Indeed, previous investigations on Crt—class I interactions showed that they were strongly reduced but not eliminated after treatment with glucosidase inhibitors or if H chain glycans were removed Sadasivan et al.
In the in vitro interactions between recombinant Crt and various assembly states of class I, it is possible that relevant polypeptide interaction sites were not accessible when the substrates were immobilized on beads or on plastic in the binding assays Wearsch et al. The information about patients' clinicopathological characteristics was obtained from medical records. Informed consent was waived because this was a historical cohort study. Immunostained tissue sections were reviewed and scored independently by two authors A. We used a previously described scoring method 23 with minor modification.
Briefly, the staining intensity of tumor cells was scored as: i absent or weak, 1 point; ii moderate, 2 points; and iii strong, 3 points. Each intensity was calculated by multiplying the intensity score by the percentage of positive tumor cells and then summing the values to obtain the final IHC score. After sample deparaffinization, rehydration, and antigen retrieval by heating in citrate buffer 10 mM, pH 6.
Independent prognostic factors for overall survival were analyzed with Cox's proportional hazard regression model in a stepwise manner. A flow chart of our study is shown in Figure S1. A protein spot with the expression 4. Identification of calreticulin. Matched peptides are shown in bold red. MW, molecular weight.
Flow cytometry analysis of pancreatic cell lines. In total, 77 patients from Yamaguchi University Hospital and 64 patients from Osaka University Hospital were analyzed; among them, 61 were excluded and 80 were eligible for the study. Flow chart of patient selection for this study. Calreticulin was mainly found in the cytoplasm of normal and cancerous tissues, and had higher expression in the acinus and lower in the islets and ducts of normal pancreatic tissues. Representative images of CD44v9 expression are shown in Figure S7 a. Variant isoform 9 of CD44 was found in the cytoplasm and membrane of normal and cancerous tissues; in normal pancreatic tissues, its expression level was the same in the acinus, islets, and ducts.
The intensity of CD44v9 expression in the membrane was scored as strong. Calreticulin CRT expression in resected pancreatic tumor samples. CRT levels were high in the acinus, moderate in the islets, and weak in the ducts of normal pancreatic tissue left upper panel. In cancerous tissues, CRT expression was categorized as absent middle upper panel , weak right upper panel , moderate left lower panel , and strong middle lower panel. Cox's regression analysis was used to assess the relationship between clinicopathological features and overall survival.
Stepwise backward elimination was used to select significant independent variables. Cox's proportional hazard analysis of overall survival in 80 patients with pancreatic cancer. Because the median value of the IHC score for CRT was and the best balancing point of sensitivity and specificity for recurrence prediction within a year was The relationship between clinical features and CRT levels is shown in Table 2.
In contrast, CD44v9 level was unrelated to the clinical outcome Fig. Relationship between calreticulin CRT expression and clinical features of pancreatic cancer patients. Bold values indicate significance. CD44v9, CD44 variant isoform 9. Interestingly, there were no differences in CRT scores and CRT expression sites between patients treated or not with preoperative chemoradiotherapy The cases with high levels of both CRT and CD44v9 were examined for intratumor localization of these proteins by immunochemistry.
Immunofluorescence staining of CRT green and CD44 variant isoform 9 CD44v9 red showing their partial colocalization in pancreatic cancer tissues left lower panel, merged image, arrows. Nuclei were stained with DAPI blue. No definitive reasons for the high CRT expression of acinar cells in clinical samples were found in the previous reports, or in our study. Because the ER is a place where protein is synthesized in cells, acinar cells have a large ER to facilitate the synthesis of many proteins; 35 therefore, abundant CRT may exist in normal acinar cells.
The function of each cell subset is unclear as the findings are controversial. Our results are not in agreement with previous data showing that CRT surface expression did not differ between cancer cells and CSCs in bladder tumors and glioblastoma, 19 which may be attributed to the difference in cancer types. Furthermore, Chao et al. There are two major discrepancies in CRT and CD44v9 expression between cell lines and clinical samples. However, we could not obtain these two findings in clinical samples. In clinical samples, most of the stained cells were cancer cells.
Thus our observations in vivo included cytoplasmic expression of CRT in cancer cells, which might have given rise to the discrepancy between the in vitro and in vivo results. There are two possible explanations for this discrepancy. Second, CRT surface expression could contribute to an aggressive phenotype of cancer cells not associated with their resistance to phagocytosis. However, CRT can be one of the few candidate therapeutic targets in cancer because its expression on CSLC surface may present an exceptional mechanism used by cancer cells to evade immune surveillance.
Immunotherapy combined with chemotherapy can be used to induce CRT expression on the surface of tumor cells. This difference is useful for immunological targeting and avoiding adverse effects. It is possible that immunotherapy targeting CRT inappropriately recognizes normal tissues expressing CRT at a much lower level. However, the level of recognition in such cases is not high, and the adverse effects of immunotherapies are low. Further investigations on CRT expression on CSLCs will lead to the development of novel therapeutic targets to prevent the progression of pancreatic cancer.
Shigefumi Yoshino received honoraria as a lecture fee from MSD corporation, outside the submitted work. The other authors have no conflicts of interest to declare. Representative images of CD44 variant isoform 9 CD44v9 expression in pancreatic tissues. Authors: Breanna S. Ireland 2 ,. Monika Niggemann 1 ,. David B. Williams 1 , 2.
Calreticulin - Paul Eggleton, Marek Michalak - Bok () | Bokus
Breanna S. Full text PDF Related articles. Abstract Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum ER whose folding-promoting functions are directed predominantly toward aspargine-linked glycoproteins. Citations 1 Recent citations: Oh Kwang Kwon et al. Related articles Based on techniques. References Vassilakos, A. EMBO J. Danilczyk, U. Gao, B.
Immunity 16 , 99— Parodi, A. Leach, M. In Calreticulin , 2nd ed. Eggleton, P. Jackson, M.
Delineation of the lectin site of the molecular chaperone calreticulin
Science , — Rajagopalan, S. Ou, W. Nature , —