Role of Calnexin and Calreticulin in protein folding in the ER

Special to the ER is the cotranslational addition of N-glycans to client proteins. Glc3Man9GlcNAc2-core glycans are transferred onto nascent chains at the side-chains of asparagine residues within the context NXS/T (where X stands for any residue except proline) As soon as an N-glycan is added to a folding protein, Glucosidase I will remove the terminal glucose moiety. Next, Glucosidase II removes the second glucose moiety and, eventually, the third glucose

After removal of the first two glucoses from their N-glycans, ER client proteins become substrate for the chaperones calnexin (CNX) and calreticulin (CRT). These two chaperones are lectins, i.e. proteins that can associate with carbohydrate structures. The lectin chaperones, CNX and CRT, bind monoglucosylated N-glycans. CNX and CRT are homologous proteins with similar structure and function, except that CNX is membrane-anchored, whereas CRT is a lumenal protein The two lectin chaperones have no equivalents in other cellular compartments and their chaperoning mechanism is unique to the ER. The primary role of lectin chaperones is to keep folding intermediates in a folding competent state. The lectin chaperones also contribute to oxidative folding, since both proteins form a tandem with the oxidoreductase ERp57.

After removal of the last glucose from the N-glycan, UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as folding sensor. UGGT probes the folded state of the substrate by interacting with both the glycan structure and peptide backbone of the folding substrate. Minor local deviations from the native state already determine that the ER substrate fails according to the strict UGGT standards. If so, UGGT reglucosylates the N-glycan, thereby allowing the incompletely folded substrate to (re-)associate with CNX and/or CRT for an additional folding cycle. As a consequence, the substrate cannot leave the ER yet. The cycles of deglucosylation and reglucosylation continue until the substrate is correctly folded or targeted for degradation.

Whereas removal of glucose moieties on N-glycans are used as signal for correct folding and exit to the Golgi, removal of the terminal mannose of the middle branch of N-glycans by a1,2-mannosidase provides a signal for folding failure. Mannose trimming of ER client glycans render them prone to degradation, since mannosidase inhibitor kifunensin delays degradation of misfolded ER substrates. Mannose trimmed N-glycans (Man8) in turn are poor substrates for reglucosylation by UGGT. They are substrates for the Man8-specific lectin EDEM instead. EDEM targets misfolded proteins for dislocation out of the ER, a process referred to as ER-associated degradation (ERAD). Dislocated misfolded proteins are degraded in the cytosol by the proteasome.

Currently, we are investigating the role of CNX and CRT in the folding process of the model proteins LDL receptor, HIV Envelope, Influenza HA and CFTR.

• Braakman I A novel lectin in the secretory pathway. An elegant mechanism for glycoprotein elimination. EMBO Rep. 2001;2:666-8. pdf
• Ermonval M, Duvet S, Zonneveld D, Cacan R, Buttin G & Braakman I Truncated N-glycans affect protein folding in the ER of CHO-derived mutant cell lines without preventing calnexin binding. Glycobiology 2000;10:77-87. pdf
• Zhang JX, Braakman I, Matlack KE & Helenius A Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. Mol Biol Cell. 1997;8:1943-54. pdf
• Chen W, Helenius J, Braakman I & Helenius A Cotranslational folding and calnexin binding during glycoprotein synthesis. Proc Natl Acad Sci U S A 1995;92:6229-33. pdf
• Hammond C, Braakman I & Helenius A Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci U S A 1994;91:913-7. pdf