Calnexin

Calnexin/CNX• Calnexin (CNX) is a 67kDa integral protein

of the endoplasmic reticulum (ER).• It consists of a large (50 kDa) N-terminal

calcium-binding lumenal domain, a single transmembrane helix and a short (90 residues), acidic cytoplasmic tail.

Function

• Calnexin is a chaperone, characterized by assisting protein folding and quality control, ensuring that only properly folded and assembled proteins proceed further along the secretory pathway.

• It specifically acts to retain unfolded or unassembled N-linked glycoproteins in the ER.

• N-linked glycosylation: Oligosaccharyltransferase recognizes the AA sequence NXS or NXT (with the S/T residue phosphorylated) and adds a 14-sugar backbone (2-N-acetylglucosamine, 9-branching mannose, and 3-glucose at the end) to the side-chain nitrogen of Asn.

• The asparagine-X-serine/threonine (NXS/T) motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation.

• These monoglucosylated oligosaccharides result from the trimming of two glucose residues by the sequential action of two glucosidases, I and II.

• Glucosidase II can also remove the third and last glucose residue.

• If the glycoprotein is not properly folded, an enzyme called UGGT (for UDP-glucose:glycoprotein glucosyltransferase) will add the glucose residue back onto the oligosaccharide thus regenerating the glycoprotein's ability to bind to calnexin.

• The improperly-folded glycoprotein chain thus loiters in the ER, risking the encounter with MNS1 (alpha-mannosidase), which eventually sentences the underperforming glycoprotein to degradation by removing its mannose residue.

• If the protein is correctly translated, the chance of it being correctly folded before it encounters MNS1 is high.

Cofactors

• ATP and calcium ions are cofactors involved in substrate binding for calnexin

Calreticulin (CTR or CALR):

• Calreticulin also known as calregulin, CRP55, CaBP3, calsequestrin-like protein, and endoplasmic reticulum resident protein 60 (ERp60) is a protein that in humans is encoded by the CALR gene.

• Calreticulin is a multifunctional protein that binds Ca2+ ions (a second messenger in signal transduction), rendering it inactive.

• Calreticulin is located in storage compartments associated with the endoplasmic reticulum.

• The term "Mobilferrin" is considered to be the same as calreticulin by some sources.

Function

• Calreticulin binds to misfolded proteins and prevents them from being exported from the endoplasmic reticulum to the Golgi apparatus.

• In normal cellular function, trimming of glucose residues off the core oligosaccharide added during N-linked glycosylation is a part of protein processing.

• If "overseer" enzymes note that residues are misfolded, proteins within the rER will re-add glucose residues so that other calreticulin/calnexin can bind to these proteins and prevent them from proceeding to the Golgi.

• This leads these aberrantly folded proteins down a path whereby they are targeted for degradation.

• Studies on transgenic mice reveal that Calreticulin and calnexin are also integral proteins in the production of MHC class I Proteins.

• As newly synthesized MHC class I α-chains enter the endoplasmic reticulum, calnexin binds on to them retaining them in a partly folded state.

• After the β2-microglobulin binds to the peptide-loading complex (PLC), calreticulin (along with ERp57 takes over the job of chaperoning the MHC class I protein while the tapasin links the complex to the transporter associated with antigen processing (TAP) complex.

• This association prepares the MHC class I for binding an antigen for presentation on the cell surface.

• Calnexin (CLN) and Calreticulin (CRT) are lectins. Calnexin is bound to luminal side of ER membrane and CRT is free from the membrane.

• As a nascent polypeptide chain enters the ER lumen, certain Asn residues are glycosylated through the addition of an oligosaccharide group consisting of Glc3Man9GlcNAc2.

• This happens even before the proteins are folded into proper shape..

• The outermost two glucoses of oligosaccharide group are rapidly removed through the action of glucosidases I and II to reveal the monoglucosylated species recognized by the lectins called calnexin/calreticulin

• In their ATP-bound state, calnexin and calreticulin bind to the monoglucosylated oligosaccharide (via their lectin sites) as well as to hydrophobic segments of the unfolded glycoprotein (via their polypeptide binding or chaperone sites).

• Glycoprotein dissociation involves not only the action of glucosidase II to remove the terminal glucose residue but also a change in affinity of the polypeptide binding site, possibly regulated by a shift from an ATP-bound to an ADP-bound or unbound state.

• After dissociation, if folding does not occur rapidly, the glycoprotein is glycosylated by another ER enzyme, UDP-glucose: glycoprotein glucosyltransferase.

• The said enzymes perform glycosylation only non-native protein conformers

• The glycoprotein can then re-bind in dual fashion to the ATP form of calnexin/calreticulin.

• In this model, both the glucosyl transferase and calnexin/calreticulin act as folding sensors.

Proposed mechanisms of action for calnexin and calreticulin.

David B. Williams J Cell Sci 2006;119:615-623

©2006 by The Company of Biologists Ltd

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