N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin.

Rodan, Aylin R.; Simons, Jan Fredrik; Trombetta, E. Sergio; Helenius, Ari

doi:10.1002/j.1460-2075.1996.tb01084.x

Cited by 145 publications

(102 citation statements)

References 64 publications

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“…It is exceedingly unlikely that all of the examples of complexes between CNX or CRT with unglycosylated or nonglucose-trimmed proteins can be dismissed on the basis of their nonspecific inclusion in aggregates, trapping within detergent micelles, and insolubility of folding intermediates, as has frequently been claimed (1,(35)(36)(37). Indeed, in the present study, we have eliminated the possibilities of insolubility and aggregation through the use of high speed sedimentation and glycerol density gradient ultracentrifugation analyses.…”

Section: Calnexin Associates With Many Substrate Proteins When the Fomentioning

confidence: 76%

“…An increased affinity could result if CNX and CRT are oligomeric and capable of binding to multiple oligosaccharides on a glycoprotein substrate. Indeed there are several reports of enhanced CNX interactions when a glycoprotein is converted from a singly to a doubly glycosylated form (35,37,54). This could be due either to oligomeric CNX or to a bivalent CNX interaction that is induced by the precipitating anti-CNX antibody.…”

Section: Calnexin Associates With Many Substrate Proteins When the Fomentioning

confidence: 99%

“…It has been speculated that the lack of dissociation of CNX-substrate complexes after complete deglycosylation may be due to the trapping of the two species within the same detergent micelle (1,35) or that the substrate, being nonnative, might become insoluble upon dissociation (1,35,36). Similarly, it has been suggested that the association of CNX with nonglycosylated proteins in vivo may arise through nonspecific inclusion of CNX within misfolded protein aggregates (1,36,37).…”

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confidence: 99%

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The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo

Danilczyk¹,

Williams

2001

Journal of Biological Chemistry

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Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated (Glc 1 Man 7-9 GlcNAc 2 ) oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. We examined whether polypeptidebased interactions occur between calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. We show that if care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some calnexin-substrate complexes, but many others remain readily detectable. Furthermore, we demonstrate that calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. We conclude that both lectin-oligosaccharide and polypeptide-based interactions occur between calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates. Glycoprotein folding within the endoplasmic reticulum (ER)1 is facilitated in part by the membrane-bound chaperone calnexin (CNX) and its soluble homolog calreticulin (CRT) (1). These proteins are unique among molecular chaperones in that they utilize a lectin site as a means to associate with unfolded glycoproteins (2-4). The lectin site is specific for Glc 1 Man 7-9 GlcNAc 2 oligosaccharides, which exist transiently as intermediates during the processing of Asn-linked glycoproteins. It is a widely held view that the removal and re-addition of the terminal glucose residue of these oligosaccharides, catalyzed, respectively, by the ER enzymes glucosidase II and UDPglucose:glycoprotein glucosyltransferase, regulate cycles of CNX and CRT binding to glycoproteins (5, 6). In this model, CNX and CRT do not function as classical molecular chaperones that prevent aggregation by binding to hydrophobic polypeptide segments. Rather, they are thought to promote folding by recruiting other chaperones and folding enzymes, such as the thiol oxidoreductase ERp57 (7,8), to the glycoprotein substrate.The concept that CNX and CRT associate with glycoproteins solely through lectin-oligosaccharide interactions is based primarily on experiments wherein cultured cells were treated with tunicamycin to block Asn-linked oligosaccharide addition or with the glucosidase I and II inhibitors castanospermine or deoxynojirimycin to prevent the conversion of the Glc 3 Man 9 GlcNAc 2 precursor to the monoglucosylated Glc 1 Man 9 GlcNAc 2 species. Subsequent immunoprec...

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Section: Calnexin Associates With Many Substrate Proteins When the Fomentioning

confidence: 76%

Section: Calnexin Associates With Many Substrate Proteins When the Fomentioning

confidence: 99%

mentioning

confidence: 99%

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The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo

Danilczyk¹,

Williams

2001

Journal of Biological Chemistry

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“…The chain of glycan molecules is then biochemically modified within the ER and the Golgi apparatus to generate the diversified glycan moieties found in mature glycoproteins. ER ␣-glucosidases I and II are involved in the trimming of terminal glucose on core oligosaccharides, and the resulting monoglucosylated glycoproteins can bind to ER chaperones, the membrane-bound calnexin (CNX) and/or its soluble homologue calreticulin (CRT) (17,38,42). Removal of the last glucose from the glycans by glucosidase II releases the glycoprotein from CNX and/or CRT.…”

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confidence: 99%

Antiviral Effects of an Iminosugar Derivative on Flavivirus Infections

Lee

Liao

et al. 2002

J Virol

204

164

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Endoplasmic reticulum (ER) ␣-glucosidase inhibitors, which block the trimming step of N-linked glycosylation, have been shown to eliminate the production of several ER-budding viruses. Here we investigated the effects of one such inhibitor, N-nonyl-deoxynojirimycin (NN-DNJ), a 9-carbon alkyl iminosugar derivative, on infection by Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2). In the presence of NN-DNJ, JEV and DEN-2 infections were suppressed in a dose-dependent manner. This inhibitory effect appeared to influence DEN-2 infection more than JEV infection, since lower concentrations of NN-DNJ substantially blocked DEN-2 replication. Secretion of the flaviviral glycoproteins E and NS1 was greatly reduced, and levels of DEN-2 viral RNA replication measured by fluorogenic reverse transcription-PCR were also decreased, by NN-DNJ. Notably, the viral glycoproteins, prM, E, and NS1 were found to associate transiently with the ER chaperone calnexin, and this interaction was affected by NN-DNJ, suggesting a potential role of calnexin in the folding of flaviviral glycoproteins. Additionally, in a mouse model of lethal challenge by JEV infection, oral delivery of NN-DNJ reduced the mortality rate. These findings show that NN-DNJ has an antiviral effect on flavivirus infection, likely through interference with virus replication at the posttranslational modification level, occurring mainly in the ER.The Flaviviridae family includes the three genera Hepacivirus (e.g., Hepatitis C virus), Flavivirus, and Pestivirus (e.g., Bovine viral diarrhea virus [BVDV]). The genus Flavivirus comprises more than 70 viruses, many of which are potent human pathogens that can cause severe encephalitic, hemorrhagic, hepatic, or febrile illnesses (35). Of particular importance for public health are the mosquito-borne flaviviruses, such as Yellow fever virus (41, 48), Japanese encephalitis virus (JEV) (45), West Nile virus (25), and dengue viruses (DENs) (16). Severe forms of dengue-related diseases, such as dengue hemorrhagic fever and dengue shock syndrome, have recently become the most serious vector-borne viral diseases in humans. It is estimated that more than 50 million people suffer DEN infection annually, with approximately 2.5 billion people living in at-risk areas (16,34). Even though these flaviviruses have a major clinical impact, there is still no vaccine for DENs, nor are there any specific antiviral therapeutics available for treatment of infections with JEV or DENs.Flaviviral virions are composed of a lipid bilayer with two or more envelope proteins surrounding a nucleocapsid, which consists of a single-stranded positive-sense genome RNA associated with multiple copies of capsid proteins. After entering a host cell, flaviviral RNA first translates into a long polyprotein, which is cleaved by cellular and viral proteases into individual structural and nonstructural proteins. RNA replication begins with the synthesis of complementary negative strands, which are then used as templates for reproduction of positiv...

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“…It was originally believed that recognition and binding of monoglucosylated oligosaccharides present on glycoproteins were necessary and sufficient for calnexin and calreticulin to promote folding of the glycoproteins (see, for example, ref. 6). However, more recent experiments have indicated that both calnexin and calreticulin directly discriminate between protein conformational states, and that both proteins can function in vitro as chaperones for glycosylated, as well as nonglycosylated, substrates (7,8).…”

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confidence: 99%

Calreticulin recognizes misfolded HLA-A2 heavy chains

Mancino

Rizvi

Lapinski

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Our studies investigated functional interactions between calreticulin, an endoplasmic reticulum chaperone, and major histocompatibility complex (MHC) class I molecules. Using in vitro thermal aggregation assays, we established that calreticulin can inhibit heat-induced aggregation of soluble, peptide-deficient HLA-A2 purified from supernatants of insect cells. The presence of HLA-A2-specific peptides also inhibits heat-induced aggregation. Inhibition of heat-induced aggregation of peptide-deficient HLA-A2 by calreticulin correlates with a rescue of the HLA-A2 heavy chain from precipitation, by forming high-molecular-weight complexes with calreticulin. Complex formation between HLA-A2 heavy chains and calreticulin occurs at 50°C but not 37°C, suggesting polypeptide-based interactions between the HLA-A2 heavy chain and calreticulin. Once complexes are formed, the addition of peptide is not sufficient to trigger efficient assembly of heavy chain͞␤2m͞peptide complexes. Using a fluorescent peptidebased binding assay, we show that calreticulin does not enhance peptide binding by HLA-A2 at 37°C. We also show that calreticulin itself is converted to oligomeric species on exposure to 37°C or higher temperatures, and that oligomeric forms of calreticulin are active in inhibiting thermal aggregation of peptide-deficient HLA-A2. Taken together, these results suggest that calreticulin functions in the recognition of misfolded MHC class I heavy chains in the endoplasmic reticulum. However, in the absence of other endoplasmic reticulum components, calreticulin by itself does not enhance the assembly of misfolded MHC class I heavy chains with ␤2m and peptides.

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N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin.

Cited by 145 publications

References 64 publications

The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo

The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo

Antiviral Effects of an Iminosugar Derivative on Flavivirus Infections

Calreticulin recognizes misfolded HLA-A2 heavy chains

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