Calnexin Discriminates between Protein Conformational States and Functions as a Molecular Chaperone In Vitro

Ihara, Yoshito; Cohen-Doyle, Myrna F.; Saito, Yoshiro; Williams, David B.

doi:10.1016/s1097-2765(00)80335-4

Cited by 157 publications

(151 citation statements)

References 36 publications

(4 reference statements)

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“…42), to make them achieve their proper tertiary or quaternary structure (43). However, calnexin (44) and calreticulin (45) also have efficient oligosaccharideindependent chaperone activity in vitro.…”

Section: Fig 8 Thermal Stability Of Wild-type and Mutant Bmp-1fmentioning

confidence: 99%

Post-translational Modification of Bone Morphogenetic Protein-1 Is Required for Secretion and Stability of the Protein

Garrigue‐Antar

Hartigan

Kadler

2002

Journal of Biological Chemistry

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Bone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn 91 (prodomain), Asn 142 (metalloproteinase domain), Asn 332 and Asn 363 (CUB1 domain), Asn 599 (CUB3 domain), and Asn 726 in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn 726 presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule.Bone morphogenetic protein-1 (BMP-1), 1 also known as procollagen C-proteinase-1 (PCP-1) was first identified in osteogenic extracts of bone (1). BMP-1 is a smaller splice variant of mammalian tolloid, which is the vertebrate ortholog of tolloid, in Drosophila. BMP-1, mammalian tolloid, and two highly homologous relatives tolloid-like 1 and 2 constitute the tolloid clade of astacin-like metalloproteinases that have important functions in development and extracellular matrix formation (2). BMP-1 cleaves fibrillar procollagen type I, II, III (3, 4), and V (5-7), as well as type VII procollagen (8), prolysyl oxidase (9), probiglycan (10), and the ␥2 chain of prolaminin 5 (11). BMP-1 also cleaves chordin (2) therefore affecting dorsal-ventral patterning in vertebrates (12). Similarly, tolloid cleaves the chordin homologue short gastrulation during Drosophila embryo development (13). Bmp1 homozygous null mice are perinatal lethal, with defects in ventral body wall closure and collagen fibrillogenesis (14), which illustrates the importance of BMP-1 in tissue assembly and development.BMP-1 consists of a prodomain that is cleaved by a furin-like enzyme in the trans-Golgi network, 2 an astacin-like zinc metalloproteinase domain (15), one epidermal growth factor-like domain, and three CUB domains. In other proteins, CUB domains mediate protein-protein interactions (16).BMP-1 purified from mouse fibroblasts culture medium has been shown to be N-glycosylated (17). Sequence analysis reveals six potential N-glycosylation sites, one of which is located in the prodomain and five in the mature (active) molecule. However, no information is available on the structure and function of the glycosylation sites....

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Section: Fig 8 Thermal Stability Of Wild-type and Mutant Bmp-1fmentioning

confidence: 99%

Post-translational Modification of Bone Morphogenetic Protein-1 Is Required for Secretion and Stability of the Protein

Garrigue‐Antar

Hartigan

Kadler

2002

Journal of Biological Chemistry

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“…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.…”

mentioning

confidence: 99%

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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“…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).…”

mentioning

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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Calnexin Discriminates between Protein Conformational States and Functions as a Molecular Chaperone In Vitro

Cited by 157 publications

References 36 publications

Post-translational Modification of Bone Morphogenetic Protein-1 Is Required for Secretion and Stability of the Protein

Post-translational Modification of Bone Morphogenetic Protein-1 Is Required for Secretion and Stability of the Protein

NMR structure of the calreticulin P-domain

Calreticulin recognizes misfolded HLA-A2 heavy chains

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