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Structure of the luminal domain of canine
calnexin 
The Ca2+ ion is shown in dark blue. The
transmembrane region follows the alpha-helix at the bottom of the structure.
Protein Data Bank structure ID:
1JHN.
N-linked glycans in the calnexin cycle
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Evolution of the calnexin family
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Calnexin is the prototype for a small group of ER-resident chaperone
proteins found throughout the eukaryotic kingdom. Calnexin
itself is the ancestor of the family and it is the only member found
in yeast. Calnexin and the calnexin homologue calreticulin
appear to be present in most organisms branching from the lineage
leading to vertebrates, including the slime mold Dictyostelium
discoideum, a very simple eukaroyote, the model invertebrates C
elegans and Drosophila melanogaster, and the chordate sea
squirts Ciona savignyi and Ciona intestinalis.
Both calnexin and calreticulin have undergone independent duplication
in different eukaryotic lineages; the plant Arabidopsis thaliana
has three calreticulins and two calnexins, for example. Humans
and most other mammals have four calnexin family proteins: calnexin,
calmegin, calreticulin and calreticulin 2.
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Calnexin and calreticulin
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Calnexin and calreticulin are components of the quality
control system that promotes correct folding of proteins that enter
the secretory pathway and targets misfolded proteins for degradation.
Lectins in other classes also participate in this system: some
L-type lectins recognize correctly folded
proteins and assist their exit from the ER and progression along the
secretory pathway, whereas some M-type lectins
recognize permanently misfolded proteins and assist their exit from
the ER into the cytoplasm for degradation. Recent evidence
suggests that some P-type lectins may play
similar roles, while some F-box lectins
recognize misfolded proteins that have been removed from the ER and
promote their proteasomal degradation.
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Both calnexin and calreticulin are resident in the ER; calnexin is
anchored to the ER membrane by a transmembrane domain, whereas
calreticulin possesses a C-terminal ER-retention signal to localize it
to the ER lumen. The structure of the luminal domain of calnexin
has been determined (left). The structure consists of a
membrane-proximal globular domain with a beta-sandwich fold similar to
that of the L-type lectins, and an
extended arm which projects away from the membrane and is formed by a antiparallel loop of polypeptide inserted into the globular domain,
towards its C-terminus. The arm consists of repeated polypeptide
segments, one of which is missing in calreticulin, producing a shorter
arm domain. The cytoplasmic region of calnexin is similar in
sequence to the extreme C-terminal region of calreticulin; the
transmembrane anchor is inserted between the globular domain and the
C-terminal region in calnexin only.
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The calnexin cycle for assisted protein folding
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The ligands for calnexin and calreticulin are glycoproteins bearing
Glc1Man9GlcNAc2 N-linked glycans (see
diagram, left).
The sugar binding site in calnexin family proteins is located in the
globular domain, while the polypeptide portion of glycoprotein ligands
may interact with the extended arm. Calnexin and calreticulin
bind different but overlapping sets of glycoproteins. The
extended arm also interacts with ERp57, a member of the protein
disulphide isomerase family. Whilst bound to calnexin or
calreticulin, glycoproteins are presented to ERp57 and other folding
factors, which assist in the attainment of the correct folded state.
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Removal of the final glucose residue from N-linked glycans is an
essential requirement before glycoproteins can exit the ER and
progress to the Golgi. The final glucose residue is removed by
ER glucosidase II, which may act on a glycoprotein whilst it is bound
to calnexin or calreticulin, triggering dissociation of the
glycoprotein from the chaperone, or when the glycoprotein dissociates
as part of equilibrium binding. Glycoproteins from which the
terminal glucose has been removed are checked for correct folding
before being allowed to exit the ER. The checking system
involves a membrane-bound glycosyltransferase enzyme, which recognizes
misfolded or partially unfolded proteins, probably through exposed
hydrophobic patches on the protein surface. Such proteins are
re-glucosylated, so that they once again become ligands for calnexin
and calreticulin. Cycles of deglucosylation and re-glucosylation
continue until the protein is correctly folded. Folded proteins
are not recognized by the glycosyltransferase and are able to proceed
to the Golgi. Proteins which become permanently misfolded are
removed from the ER and degraded in the cytoplasm by proteasomes, in a
process known as ER-associated degradation (ERAD, see
M-type lectins).
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Other calnexin family proteins in mammals
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Calmegin (calnexin-t) is a calnexin paralogue in mammals and some other
vertebrates. It is a testis-specific ER chaperone with regulated
expression, and in mice has been shown to be essential for male
fertility, probably by acting as a chaperone for sperm cell surface
receptors involved in sperm-egg interaction.
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Calreticulin 2 is a calreticulin paralogue in mammals and some
other vertebrates. In humans it is expressed in testis and is
suggested to work in conjunction with calmegin. In the rodent
lineage a further gene duplication has produced a calreticulin 3.
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