Calreticulin functions in vitro as a molecular chaperone for both glycosylated and non-glycosylated proteins

Saito, Yoshiro

doi:10.1093/emboj/18.23.6718

Cited by 243 publications

(242 citation statements)

References 35 publications

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“…Recombinant uniformly 13 C, 15 N-labeled CRT(189-288) was prepared as described previously (20). For the structure determination we used a 3 mM protein solution in 95% H 2 O͞5% D 2 O (vol͞vol) and a 1.5 mM solution in 100% D 2 O, which contained 10 mM CaCl 2 at pH 6.3.…”

Section: Methodsmentioning

confidence: 99%

“…For the collection of conformational constraints the following three experiments were recorded with a nuclear Overhauser enhancement (NOE) spectroscopy (NOESY) mixing time m ϭ 60 ms at T ϭ 20°C: (i) threedimensional (3D) combined 15 NOE cross peak assignments were obtained by using the module CANDID (T.H., P.G., and K.W., unpublished work) in the program DYANA (24). CANDID͞DYANA performs automated assignment and distance calibration of NOE intensities, removal of meaningless distance constraints, structure calculation with torsion angle dynamics, and automatic NOE upper distance limit violation analysis.…”

Section: Methodsmentioning

confidence: 99%

“…CNX and CRT also associate with a thiol oxidoreductase, ERp57, which interacts with cysteines in the substrate glycoproteins (12,13). In addition to their well-characterized association with glycan moieties, both CNX and CRT have been shown to bind to unfolded unglycosylated polypeptides and prevent their aggregation in vitro (14,15). It remains to be seen whether this type of interaction observed in vitro has a counterpart in the ER of living cells.…”

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confidence: 99%

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NMR structure of the calreticulin P-domain

Ellgaard¹,

Riek²,

Herrmann³

et al. 2001

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

179

124

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The NMR structure of the rat calreticulin P-domain, comprising residues 189 -288, CRT(189 -288), shows a hairpin fold that involves the entire polypeptide chain, has the two chain ends in close spatial proximity, and does not fold back on itself. This globally extended structure is stabilized by three antiparallel ␤-sheets, with the ␤-strands comprising the residues 189 -192 and 276 -279, 206 -209 and 262-265, and 223-226 and 248 -251, respectively. The hairpin loop of residues 227-247 and the two connecting regions between the ␤-sheets contain a hydrophobic cluster, where each of the three clusters includes two highly conserved tryptophyl residues, one from each strand of the hairpin. The three ␤-sheets and the three hydrophobic clusters form a repeating pattern of interactions across the hairpin that reflects the periodicity of the amino acid sequence, which consists of three 17-residue repeats followed by three 14-residue repeats. Within the global hairpin fold there are two well-ordered subdomains comprising the residues 219 -258, and 189 -209 and 262-284, respectively. These are separated by a poorly ordered linker region, so that the relative orientation of the two subdomains cannot be precisely described. The structure type observed for CRT(189 -288) provides an additional basis for functional studies of the abundant endoplasmic reticulum chaperone calreticulin.T he two homologous calcium-binding proteins calnexin (CNX) and calreticulin (CRT) serve as molecular chaperones in the endoplasmic reticulum (ER) of eukaryotic cells. They transiently bind to the majority of nascent and newly synthesized glycoproteins in the cell (1-5). The association promotes efficient folding and oligomeric assembly, and helps to retain the glycoproteins in the ER while they are still incompletely folded (5, 6). CNX and CRT are lectins that associate with the partially trimmed, monoglucosylated N-linked oligosaccharide moieties attached to the substrate proteins (5, 7-9). They cooperate in the ER with several independently acting enzymes that are involved in oligosaccharide trimming-i.e., glucosidases I and II and mannosidase I-and with the UDP-glucose:glycoprotein glucosyltransferase, which functions as a folding sensor (10-12). In this cooperative action, CNX and CRT have primarily a binding function, and the enzymes regulate the on-off cycle. CNX and CRT also associate with a thiol oxidoreductase, ERp57, which interacts with cysteines in the substrate glycoproteins (12, 13). In addition to their well-characterized association with glycan moieties, both CNX and CRT have been shown to bind to unfolded unglycosylated polypeptides and prevent their aggregation in vitro (14,15). It remains to be seen whether this type of interaction observed in vitro has a counterpart in the ER of living cells.In this study we used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of the central ''P-domain'' of CRT, comprising residues 189-288, CRT(189-288). The P-domain is thought to be involved in substrate ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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NMR structure of the calreticulin P-domain

Ellgaard¹,

Riek²,

Herrmann³

et al. 2001

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

179

124

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“…Calnexin is readily detected in association with free class I heavy chains in b 2 m-negative cell lines, while calreticulin is detected in association with heavy chain-b 2 m dimers in the early stages of assembly. This difference may reflect differences in glycan-independent chaperone -protein interactions, recently described for both calnexin and calreticulin (2,3). Alternatively, it may be because calnexin is a membrane protein while calreticulin is soluble (D. Williams, personal communication).…”

Section: Accessory Molecules In Mhc Class I Assemblymentioning

confidence: 99%

Intracellular Surveillance: Controlling the Assembly of MHC Class I‐Peptide Complexes

Cresswell

2000

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MHC class I molecules bind with high affinity to peptides in the endoplasmic reticulum and display them on the cell surface. Here they are screened by CD8-positive Tlymphocytes for the presence of foreign, pathogenderived peptides within the mass of self-peptides expressed. MHC class I assembly is a complicated process involving a number of accessory molecules, including specialized components as well as common chaperones. Our understanding of the mechanisms involved, while quite advanced, is far from complete.Key words: Calnexin, calreticulin, chaperone, ERp57, HLA, MHC, TAP, tapasin Received 13 December 1999, revised and accepted for publication 28 December 1999The expression of major histocompatibility complex (MHC) class I molecules on the surface of a cell is the end-point of a complicated process of assembly and transport which is initiated in the endoplasmic reticulum (ER). Class I molecules are effectively trimeric, consisting of a transmembrane glycoprotein, which is the product of the MHC-linked gene, a small protein called b 2 microglobulin (b 2 m), and a short peptide of 8 -10 amino acids. The peptide binds in a groove formed by two homologous polymorphic domains of the class I molecule. Binding is sequence-dependent and the specificity is mediated by interactions of the side chains of two or more amino acids of the peptide, the so-called anchor residues, with complementary 'pockets' in the binding groove. Additional binding energy is provided by interactions of the groove with the amino and carboxy-termini, and by hydrogen bonding interactions with the peptide backbone. In the absence of associated peptide, class I-b 2 m dimers are unstable in vitro and are not efficiently transported out of the ER in vivo.

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“…non-glucosylated proteins (Ihara, Cohen-Doyle et al 1999;Saito, Ihara et al 1999). As the newly synthesized MHC-I HC enters the ER via the translocon it is subjected to N-linked glycosylation of the sequon Asn-X-Ser/Thr (Bause, Muller et al 1983).…”

Section: Glycosylation and The Calnexin/calreticulin Cyclementioning

confidence: 99%