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Calnexin, Calreticulin and the Folding of Glycoproteins

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Calnexin, Calreticulin and the Folding of Glycoproteins Protein folding in living cells is a complex, error-prone Calnexin, process. Numerous mechanisms are in place to ensure that newly synthesized proteins reach their folded calreticulin and functional form. One of the strategies is the covalent attachment of oligosaccharides to asparagine residues of nascent polypeptide chains. Such N-linked glyco- the folding of sylation occurs for the majority of soluble and mem- brane-bound proteins that are synthesized in the endoplasmic reticulum (ER). The addition of oligo- glycoproteins saccharides can be beneficial during maturation by making folding intermediates more soluble. In ad- dition, it allows the newly synthesized polypeptides to bind to calnexin and calreticulin, unique lectins pres- ent in the ER, which serve as molecular chaperones. Several reviews have been published on cabrexin and calreticulin in recent years that provided descriptions of the discovery and molecular characterization of these resident ER chaperoneslA. Here, we outline what Calnexin and calreticulin are molecular chaperones in the is currently known about calnexin, calreticulin and ac- endoplasmic reticulum (ER). They are lectins that interact with cessory enzymes in the process of glycoprotein folding. newly synthesized glycoproteins that have undergone partial Calnexin and calreticulin are lectins trimming of their core N-linked oligosaccharides. Together with the Calnexin is a nonglycosylated type I membrane protein (65 kDa, 573 amino acids) with its substrate- enzymes responsible for glucose removal and a glucosyltransferase binding domain in the lumen of the ER. It has an 89-residue cytoplasmic tail that is phosphorylated that re-glucosylates already-trimmed glycoproteins, they provide a and carries a C-terminal RKPRRE sequence that serves novel mechanism for promoting folding, oligometic assembly and as an ER-localization signalz. Calreticulin, the soluble homologue (46 kDa, 400 amino acids), has both a quality control in the ER. KDEL retrieval sequence and a highly negatively charged region responsible for Ca2+ binding and ER retention at its C-terminus4,5. The primary amino acid sequences of calreticulm and the lumenal domain of calnexln show extensive stretches of homology. by the oligosaccharyltransferase associated with the Although reported to be localized in several cell translocation machinery” (Fig. 1). They are attached compartments, calreticulin is clearly more abundant to the side chain of asparagine residues in the in the lumen of the ER, where it is one of the major consensus sequence Asn-X-Ser/Thr. Transfer of the proteins. A variety of functions have been ascribed to oligosaccharide generally occurs co-translationally as calreticulin, among them gene regulation, Ca2+ se- soon as the acceptor site enters the lumen of the ER. questration and RNA bindin&. It is now clear that, The original core glycans contain a string of three like calnexin, it is a molecular chaperone that binds glucose residues. Their removal begins on the grow- transiently to many glycoproteins in the ERGy. Both ing nascent chain. Glucosidase I removes the termi- seem to function as monomers, although they may nal oll,2-linked glucose, followed by elimination of be part of a larger dynamic network of proteins that the two remaining al,3-linked glucoses by glucosi- includes other ER chaperones and folding enzymes*. dase II (Fig. 1). Compared with other oligosaccharide- Initial experiments in tissue culture cells treated modification reactions in the secretory pathway, glu- with glycosylation inhibitors showed that calnexin cose trimming is exceptionally efficient; nearly all selectively associated with folding intermediates of N-linked chains are completely de-glucosylated. The glycoproteinsiO. Further studies using glucosidase Golgi complex of some cell types contains an endo- inhibitors demonstrated that calnexin bound to par- mannosidase that eliminates any residual glucose tially glucose-trimmed glycoproteins” . Subsequently, residues that may have been missed in the ER18. it was shown that calnexin1214 and calreticulin6,7 The monoglucosylated glycans that bind to calnexin The authors are in specifically associated with monoglucosylated glyco- and calreticulin arise in the ER as an intermediate dur- the Dept of proteins. Biochemical analysis using isolated oligo- ing the stepwise removal of glucoses. However, the same Cell Biology, saccharides has confirmed that they are a new type structure is also formed by re-glucosylation of fully Yale University of lectin and that they bind monoglucosylated core glucose-trimmed oligosaccharides. This occurs through School of glycans (Glc,Man,-,GlcNAc2), whereas they do not the action of a lumenal enzyme, the UDP-Glc:glyco- Medicine, bind glycans with two or three or no glucose resi- protein glucosyltransferase1y*20 (Fig. 1). This enzyme 333 Cedar Street, duesi5,16. Both the affinity for the oligosaccharides counteracts the function of glucosidase II by adding New Haven, and the number of binding sites are unknown. glucose residues back to the oligosaccharides, thus re- CT 06520-8002, generating monoglucosylated glycans2i. The existence USA. Generating the oligosaccharide ligands of de- and re-glucosylation reactions introduces the possi- E-mail: N-linked glycans are added to growing polypeptide bility of a cycle that involves association with, and dis- ari.helenius@ chains as 14resIdue oIigosaccharides (Glc,Mar+GlcNAc,) sociation from, calnexin and calreticulin (see below). yale.edu trends in CELL BIOLOGY (Vol. 7) May 1997 Copyright 0 1997 Elsevier Science Ltd. All rights reserved. 0962-8924/97/$17.00 193 PII: 50962-8924(97)01032-5 Glucosyl- transferase E Glucosidase II -Asn- -Asn- -Asn- T-Asn- A Glucose 0 Mannose 0 N-acetylglucosamine FIGURE 1 N-glycosylation and glucose trimming in the endoplasmic reticulum (ER). The core oligosaccharide (Glc,Man,GlcNAc,) is synthesized in the ER membrane and transferred to the consensus N-glycosylation sites (Am-X-Ser/lhr) on nascent polypeptide chains. Immediately after transfer, the glucose residues are removed by the sequential action of glucosidases I and II. Clucosidase I removes the terminal al-24inked glucose, whereas glucosidase II removes the two remaining al-3-linked residues17. Fully glucose-trimmed high-mannose glycans can be re-glucosylated by UDP-glucose:glycoprotein glucosyltransferase if present on incompletely folded or assembled proteinslg. The enzymes that generate the monoglucosylated A model for transient glycoprotein binding to glycoproteins have been known for many years, but calnexin and calreticulin their genes have been cloned only recently (Table 1). Binding of glycoproteins to calnexin and calreticu- Glucosidase I is a type-11 membrane glycoprotein lin is a transient step during early maturation. Depend- (92 kDa). Glucosidase II, by contrast, is soluble and ing on the distribution of glycans in the polypeptide does not share any primary sequence homology with chain, associationcan begin with the growing nascent glucosidase I. In mammalian cells, it is a heterodimer chain30. It continues posttranslationally for variable in which the cx subunit (104 kDa) contains the active periods of time-ranging from a few minutes to hours. site, and the B subunit (58 kDa) carries retrieval and With permanently misfolded proteins, or unassembled retention sequences for ER localization. subunits of oligomers, the association usually con- UDP-Glc:glycoprotein glucosyltransferase is a sol- tinues until the protein is degraded9J*J2J3,31-33.Final uble enzyme of 170 kDa. It and glucosidase II are the releasefrom calnexin and calreticulin occurs when only soluble enzymes involved in the processing of the polypeptides have reached a fully folded confor- N-linked carbohydrates in the secretory pathway. Its mation or when they have undergone oligomeric C-terminal 500 amino acids share homology between assembly (for reviews, seeRefs 2, 34 and 35). different species and with other known glucosyl- According to one model (Fig. 2a; Ref. 36), glyco- transferases, suggesting that it probably contains the protein folding intermediates are retained in the ER catalytic domain. by a cycle of glucose removal and addition coupled The glucosyltransferase only recognizes non-native to chaperone binding and release.Glucosidase II per- glycoproteins as substratesz2J3. Among the proteins forms a dual role: it is needed to uncover the mono- capable of distinguishing native from non-native poly- glucosylated glycans for initial binding and afterwards peptides, it is the only one known to modify its sub- to de-glucosylate them completely, thus releasing strates covalently. As long as glycoproteins are incom- the glycoproteins from the chaperones.The glucosyl- pletely folded or misfolded, they are re-glucosylated transferase allows reassociation by re-glucosylating by the transferase and continue to be bound to cal- unbound non-native proteins. The glycoprotein nexin and calreticulin. This is illustrated by the ts045 exits the cycle when it has reached its native form vesicular stomatitis virus (VSV) G protein, which has and becomes ‘invisible’ to the glucosyltransferase. a thermoreversible folding defect. At the nonpermiss- Consistent with the need for glucose removal for ive temperature, the G protein undergoes continu- substrate release, inhibition of glucosidaseII activ- ous de- and re-glucosylation in the ER of infected ity inhibits release of substrates from calnexin and cells2* and it remains calnexin associatedz4. The ma- calreticulin9,13,28,37*38.It
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