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The components were detected by fluorography and their migration compared to standard oligosaccharides. The observation that Glc Man GlcNAc was bound by immobilized calreticulin prompted us to determine the specificity of this interaction with a series of purified oligosaccharides, which differed in the number of glucose residues and the size of their polymannose component.

In the assay system employed, the 14 C-labeled oligosaccharides were individually chromatographed on calreticulin-Sepharose along with 3 H-labeled Glc Man GlcNAc to serve as reference for their position of emergence from the column Fig. From these analyses it became evident that binding of Man GlcNAc to calreticulin occurred only in its monoglucosylated state and, moreover, that effective although somewhat diminished interaction occurred with oligosaccharides in which the polymannose portion had been truncated Fig.

Assay of the binding capacity of several monoglucosylated polymannose oligosaccharides to a column of immobilized calreticulin. The scale refers only to the 14 C radioactivity; the 3 H-labeled Glc Man GlcNAc standard was present at approximately 3-fold greater radioactivity than the 14 C-labeled components. Assessment of the oligosaccharide binding specificity of calreticulin. Purified 14 C-labeled oligosaccharides and glycopeptides were chromatographed on calreticulin-Sepharose as in Fig.

Application of 14 C-radiolabeled glycopeptides from partially processed thyroid glycoproteins onto a calreticulin-Sepharose column resulted in a selective binding of Glc Man GlcNAc -containing peptides to the immobilized matrix, as revealed by thin layer chromatographic examination of the oligosaccharides released by endo H digestion of the bound and unbound fractions data not shown.

The immobilized calreticulin column also proved to be highly effective in removing Glc Man GlcNAc from complicated mixtures of polymannose intermediates even with the additional presence of the tri- and diglucosylated Man GlcNAc components Fig. Thin layer chromatography indicated that the unbound material was specifically freed from the Glc Man GlcNAc oligosaccharide, which was recovered in the bound fraction Fig. The minor saccharide component migrating ahead of the Glc Man GlcNAc in the bound fractions was identified as Glc Man GlcNAc, which has been reported to occur as an N -linked processing intermediate Selective retention of Glc Man GlcNAc on a calreticulin-Sepharose column from mixtures of oligosaccharide processing intermediates.

After desalting equal aliquots of the bound BD and unbound UN peaks as well as a portion of the initial mixture IN were chromatographed on a silica-coated plate in Solvent System B for 20 h, and the components were detected by fluorography G M to M , right panel.

Calreticulin | Marek Michalak | Springer

A mixture of Glc Man GlcNAc and Man GlcNAc was similarly chromatographed on an immobilized calreticulin column, and the initial, bound, and unbound oligosaccharides were examined by thin layer chromatography G M to M , right panel. The abbreviations are the same as in Fig. The effectiveness of the calreticulin-Sepharose in resolving metabolic intermediates was further made apparent when the cytosolic heptasaccharide fraction from HepG2 cells, which has been reported to consist of Glc Man GlcNAc and Man GlcNAc 27 , was loaded onto the column Fig. Thin layer chromatography after endomannosidase digestion did indeed reveal that the unabsorbed material consisted primarily of Man GlcNAc, which was resistant to the action of this enzyme, while the bound oligosaccharide represented Glc Man GlcNAc, which was converted to Man GlcNAc through the release of Glc Man Fig.

Separation of cytosolic heptasaccharide components from metabolically radiolabeled HepG2 cells by calreticulin-Sepharose chromatography. The desalted unbound UN and bound BD oligosaccharide fractions as well as several monoglucosylated standards were then treated with endomannosidase and subsequently chromatographed on cellulose-coated plates for h middle and right panels.

The components were detected by fluorography and their migration compared to standards. It is apparent from the present investigation that calreticulin cofractionates with endomannosidase during affinity chromatography of Triton-solubilized Golgi proteins on a Glc-Man-Affi-Gel column. This was surprising, particularly in view of the fact that only two polypeptide components in approximately equal amounts were retained by this matrix from a complex mixture of components, and alerted us to the possibility that calreticulin has a lectin-like binding capacity.

Studies carried out with immobilized calreticulin and an array of oligosaccharides derived from N -linked carbohydrate units revealed that protein-saccharide interactions were limited to monoglucosylated polymannose components. While the presence of a terminal mannose-linked glucose residue was clearly critical for the binding to take place, substantial interaction was retained after extensive trimming of the two unglucosylated chains of the polymannose unit.

On the other hand, the carbohydrate-peptide linkage region appeared to have no discernible influence on binding, as monoglucosylated oligosaccharides in N -glycosidic linkage or in their unconjugated state terminating in either N -acetylglucosamine or di- N -acetylchitobiose interacted with the calreticulin to the same extent. The selectivity of the immobilized calreticulin for monoglucosylated polymannose oligosaccharides or glycopeptides made it a highly effective tool for sorting out these components from complex mixtures of processing intermediates. While calreticulin and endomannosidase are believed to function quite differently, namely as molecular chaperone 1 , 36 , 37 and processing enzyme 13 , 14 , respectively, our study demonstrates some intriguing similarities that merit comment.

The inability of calreticulin to bind tri- and diglucosylated oligosaccharides was mirrored by the previously reported low in vitro reactivity of endomannosidase with such saccharide species. Also relevant was the finding that monoglucosylated oligosaccharides with extensively truncated mannose chains could still effectively interact with both the chaperone and the enzyme 14 , particularly since this property stands in pronounced contrast to the specificity of glucosidase II, which is known to require the untrimmed mannose branches for interaction with its substrate Although calreticulin is generally believed to be primarily situated in the ER 32 , our finding of this protein in the Golgi is consistent with reports indicating its presence at the cell surface 39 , 40 and other subcellular compartments 41 , 42 , Indeed, it is apparent that calreticulin takes part in intracellular trafficking which accounts for this wide distribution 43 , 44 and distinguishes it from calnexin, the other lectin-like chaperone, which is a membrane-bound ER-resident protein The presence of molecular chaperones with affinity for proteins with monoglucosylated N -linked oligosaccharides has provided the basis for a model 1 that accounts for their preferential association with glycoproteins at an early stage of processing and explains the observed accelerated protein degradation due to impaired folding or oligomerization during a glucosidase blockade 4 , 5.

However, as this scheme also postulated that dissociation of glycoproteins from the chaperone is brought about by the action of glucosidase II, its relevance would be limited to the ER locale where this enzyme is situated If deglucosylation is required to dissociate the calreticulin-glycoprotein complexes in a more distal location, which would be either the Golgi itself or an ER-Golgi intermediate compartment, another mechanism is required.

This has prompted us to propose a tentative modified model Fig. This scheme would take into account the occurrence of calreticulin and endomannosidase in comparable amounts in this location and, more importantly, the fact that endomannosidase in marked contrast to glucosidase II has the capacity to interact with N -linked oligosaccharides in which the mannose chains have been trimmed. The latter characteristic is relevant, as glycoproteins that exit from the ER will have already undergone a substantial degree of processing though the action of ER-resident mannosidases 25 , Although the ER may be the primary site for protein folding and oligomerization to take place, a number of instances have already been described in which such quality controlling events take place in more distal compartments 47 , Schematic proposal for two distinct mechanisms of dissociating glycoproteins with monoglucosylated N -linked oligosaccharides from their interaction with calreticulin subsequent to folding of their polypeptide chain.

The highly specific lectin-like interaction of molecular chaperones like calreticulin and calnexin with the N -linked oligosaccharides of glycoproteins represents an intriguing example of the biological role of saccharide chains and in particular extends the function of the polymannose-linked glucose residues beyond that of their well known involvement in the process of cotranslational N -glycosylation 49 , Since it is quite likely, however, that the carbohydrate-protein interaction is only one manner in which the binding of chaperones to polypeptide intermediates is mediated 10 , 45 , 51 , definition of the mechanisms utilized by various cell types for their diverse secretory proteins will require extensive further investigation.

We thank Dr. Van for providing the antiserum against calreticulin and J. Kuduz for preparation of the immobilized calreticulin. The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. All sugars mentioned in the text are in the D-configuration.

Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum.

You'll be in good company. Journal of Lipid Research. Previous Section Next Section. Ligand Affinity Chromatography of Triton-solubilized Golgi Membranes Solubilized Golgi membranes 12 and calreticulin, isolated from rat liver by a slightly modified version of the procedure described by Van et al. Preparation of Radiolabeled Oligosaccharides and Glycopeptides Metabolically radiolabeled [ 14 C]Glc Man GlcNAc and [ 14 C]Man GlcNAc were prepared from thyroid glycoproteins after incubation of slices with [ 14 C]glucose as described previously 14 , 24 ; in this procedure the oligosaccharides released from Pronase-generated glycopeptides by endo H are purified by preparative thin layer chromatography.

Thin Layer Chromatographic Procedures Resolution of small oligosaccharides, including fragments from acetolysis treatment, was achieved on plastic sheets precoated with cellulose 0. Radioactivity Measurements Liquid scintillation counting was carried out with Ultrafluor with a Beckman LS instrument; double channel measurements were made when 14 C- and 3 H-labeled oligosaccharides were present together.

Presence of Calreticulin in the Rat Liver Golgi Endomannosidase Preparation Obtained by Glc-Man-Affi-Gel Chromatography An examination of amino acid sequences occurring in the electrophoretically resolved protein components 60 and 56 kDa of the ligand affinity-purified endomannosidase preparation Fig. Figure 1: Immunochemical identification of calreticulin in the ligand affinity chromatographically purified rat liver endomannosidase preparation and the Golgi membranes from which it was obtained. Figure 2: Immunochemical detection of calreticulin in the eluted fractions from a Glc-Man-Affi-Gel column.

Immobilized Calreticulin Resolves Glc Man GlcNAc from Unglucosylated Man GlcNAc In view of our previously demonstrated understanding that endomannosidase has a high degree of specificity for monoglucosylated polymannose oligosaccharides, which is responsible for its retention on Glc-Man-Affi-Gel 12 , we explored the possibility that the binding of calreticulin to this matrix has a similar basis. Evaluation of the Binding Specificity of Calreticulin The observation that Glc Man GlcNAc was bound by immobilized calreticulin prompted us to determine the specificity of this interaction with a series of purified oligosaccharides, which differed in the number of glucose residues and the size of their polymannose component.

Figure 4: Assay of the binding capacity of several monoglucosylated polymannose oligosaccharides to a column of immobilized calreticulin. Figure 5: Assessment of the oligosaccharide binding specificity of calreticulin. Immobilized Calreticulin Can Effectively Separate Free and N-linked Monoglucosylated Polymannose Oligosaccharides from Mixtures of Processing Intermediates Application of 14 C-radiolabeled glycopeptides from partially processed thyroid glycoproteins onto a calreticulin-Sepharose column resulted in a selective binding of Glc Man GlcNAc -containing peptides to the immobilized matrix, as revealed by thin layer chromatographic examination of the oligosaccharides released by endo H digestion of the bound and unbound fractions data not shown.

Figure 6: Selective retention of Glc Man GlcNAc on a calreticulin-Sepharose column from mixtures of oligosaccharide processing intermediates. Figure 7: Separation of cytosolic heptasaccharide components from metabolically radiolabeled HepG2 cells by calreticulin-Sepharose chromatography. Figure 8: Schematic proposal for two distinct mechanisms of dissociating glycoproteins with monoglucosylated N -linked oligosaccharides from their interaction with calreticulin subsequent to folding of their polypeptide chain.

Previous Section. Helenius A. CrossRef Google Scholar. CrossRef Medline Google Scholar. Fiedler K. Moore S. Kearse K. Medline Google Scholar. Lodish H. Cell Biol. Sasak V. Rabouille C. Hebert D. Ware F. Peterson J.


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Cell 6 , — Hiraizumi S. Lubas W. Leelavathi D. Acta , — Tulsiani D. Maddy A. Depierre J. Google Scholar. The authors thank Servier for the figures that were produced using Servier Medical Art www. Feng et al. However, although several mechanisms of CRT exposure on the surface of cells undergoing immunogenic apoptosis have been described 11 , which mechanisms of CRT secretion are used by macrophages to enhance PrCP are currently unknown and many more interesting and challenging findings are expected.

In this regard it has also been suggested the links between ER calcium depletion and detection of ER-resident proteins, including CRT, in the extracellular space Since CRT is involved in the quality control of glycoproteins in the ER by a lectin-like function, the authors asked whether during PrCP, the extracellular binding of CRT on neutrophils and other living cells may also involve sugar moieties.

These findings demonstrate that this CRT binding and specificity can be extended to other cell types than neutrophils and suggest that PrCP could be a general phenomenon that may occur during different biological processes such as inflammation resolution and anti-cancer surveillance Fig. Having identified the asialoglycan epitope involved in CRT binding, the authors then examined whether CRT-binding and PrCP could be modulated by neuraminidase treatment 1. They found that this treatment significantly promoted PrCP of different types of viable human cancer cells by both human and mouse macrophages.

In a complementary approach, Feng et al. To test the potential usefulness of these observations to clinical settings, the authors mined transcriptome databases of cancers and this revealed higher expression of genes promoting the removal of sialic acids such as neuraminidases correlated with an improved survival, while higher expression of the genes enhancing sialic acid expression such as sialyl transferases correlated with a worse outcome.

Also, during normal and pathological haematopoiesis the level of CRT or PHA-L binding correlated inversely with cellular half-life, with highest binding in blast cells and lowest in multipotent progenitors, leukemia stem cells and haematopoeitic stem cells. These studies indicate that regulation of asialoglycan levels on the cell surface and the subsequent PrCP due to the CRT mediated recognition could be important during homeostasis and inflammation resolution and as well as immunosurveilance.

This work by Feng et al. First, previous studies have suggested that CRT on the surface of apoptotic cells functions as a DAMP responsible for the immunogenicity of apoptotic cancerous cells 3 , 13 , This current work places CRT in a new context, i.

Whether modulation of the asialoglycan epitope by neuraminidase treatment also modulate the uptake of apoptotic cells, whether this will modulate their immunogenicity is a question that could also be relevant for tumor immunotherapy.

Bibliographic Information

In this regard, Feng et al. Future studies addressing the anti-tumor responses could be useful. This raises also the question whether other phagocytic cells, such dendritic cells, can secrete CRT to decorate their preys, and whether engulfment of viable cancer cells would lead to antigen presentation. Second, the current work suggesting the modulation of asialoglycoproteins on the surface of viable cells and the section of CRT from macrophages, begin to provide mechanistic insights.

Although not focus of this work, whether different types of tissue macrophages as well as their differentiation such as M1-like versus M2-like signatures might alter CRT secretion, or whether the PrCP is rather a feature of tumor associated macrophages remain to be seen. Finally, beyond tumor immunosurveillance, an intriguing observation is the interaction between viral neurominidases such as Influenza virus and the host cells. Indeed Influenza neurominidases have been shown to enhance phagocytosis of Influenza-infected cells through desialylation Future investigations will lead to a better understanding of the molecular mechanisms of the clearance of viable cells during cancer and viral infections with relevance to anti-tumor therapies as well as normal hematopoietic development.

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Additional information

Feng, M. Programmed cell removal by calerticulin in tissue homeostasis and cancer. Gardai, S. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell , — Galluzzi, L. Immunogenic cell death in cancer and infectious disease. Chao, M. Programmed cell removal: a new obstacle in the road to developing cancer.


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Cancer 12 , 58—67 Sierro, F. Suicidal emperipolesis: a process leading to cell-in-cell structures, T cell clearance and immune homeostasis. Krishna, S. Mechanisms and consequences of entosis. Cell Mol. Life Sci. Metayer, L. Anti-CD47 antibodies induce phagocytosis of live, malignant B cells by macrophages via the Fc domain, resulting in cell death by phagoptosis. Oncotarget 8 , — Lozupone, F. Cancer cell cannibalism: a primeval option to survive. Henson, P. Cell removal: efferocytosis. Cell Dev.


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