© 2015 John Wiley & Sons A/S.
Published by John Wiley & Sons Ltd
doi:10.1111/tra.12358
Review
N -Glycan-based ER Molecular Chaperone and
Protein Quality Control System: The Calnexin
Binding Cycle
Lydia Lamriben, Jill B. Graham, Benjamin M. Adams and Daniel N. Hebert∗
Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, Amherst,
MA 01003, USA
∗
Corresponding author: Daniel N. Hebert, [email protected]
Abstract
Helenius and colleagues proposed over 20-years ago a paradigm-
protein homeostasis, and its connection to disease states that are high-
shifting model for how chaperone binding in the endoplasmic reticulum
lighted in this review.
was mediated and controlled for a new type of molecular chaperone-
Keywords endoplasmic reticulum, molecular chaperones, N -glycans,
the carbohydrate-binding chaperones, calnexin and calreticulin. While
protein folding, quality control
the originally established basics for this lectin chaperone binding cycle
Received 25 November 2015, revised and accepted for publication 14
holds true today, there has been a number of important advances that
December 2015, uncorrected manuscript published online 17 December
have expanded our understanding of its mechanisms of action, role in
2015, published online 10 January 2016
Cells are challenged by the need to efficiently fold an expansive assortment of proteins that span three orders of magnitude in amino acid number under a variety of conditions.
Frequently, small single-domain proteins spontaneously
acquire their native fold with high efficiency immediately
upon translation (1). In contrast, larger complex proteins
fold more slowly aided by the intervention of cellular
factors such as molecular chaperones that are dedicated
to assisting and optimizing the folding process (2). How
molecular chaperones help with the maturation of proteins
is of important biological and medical interest, as there are
a growing number of diseases associated with protein folding defects involving difficult to fold multidomain proteins.
specifically recognize non-native structures for a large
range of client proteins; and (ii) how chaperones control
their binding cycle so substrates are transiently bound
then released. Protein folding involves sequestration
of hydrophobic residues in the core of the protein (or
membrane) and the exposure of hydrophilic side chains
to the aqueous environment. Classical chaperones from
the Hsp60, 70 and 90 families assist proteins by binding
to substrates possessing aqueous-exposed hydrophobic
domains that provide a signal indicating that the protein is
either a protein folding intermediate, misfolded or part of
an unassembled multimer (4). In this way, a chaperone can
specifically differentiate the folding stages for a large range
of proteins. The substrate binding affinities of Hsp60s,
70s and Hsp90s are controlled by adenine nucleotides.
The forward progression of the chaperone binding cycle
is mediated by the form of the bound adenine nucleotide
that alternates the conformation of the chaperone
Molecular chaperones are defined as proteins that assist
other proteins to reach their native structures through
transient interactions (3). There are two important concepts to understand for chaperones: (i) how chaperones
308 www.traffic.dk
The Calnexin Binding Cycle
from its high affinity bound state to the low affinity
released state.
An estimated one-third of the proteome is targeted to the
secretory pathway in mammalian cells and these proteins
fold in the endoplasmic reticulum (ER) (5). The ER of
metazoans contains Hsp70 (BiP/GRP78) and 90 (GRP94)
family members, but lack an Hsp60 component. The vast
majority of membrane and soluble proteins targeted for
the secretory pathway receive multiple N-linked carbohydrates as they are translated and translocated into the
ER (6,7). A molecular chaperone network of factors that
binds carbohydrates further assists the maturation of these
proteins (8–10).
N-linked glycans are originally transferred by oligosaccharyltransferase (OST) as a Glc3 Man9 GlcNAc2 appendage
preassembled on a dolichol phosphate that resides in
the ER membrane (11) (Figure 1). Once attached to the
protein, the glycan is rapidly restructured by ER resident glycosidases and a transferase to a glycoform that is
dependent upon the structure, age and the location of the
protein within the ER. The composition of glycans act as
protein fitness tags that signal the status and age of the
protein to which they are attached (8–10). Central to the
implementation of this glyco-code or signal is a specialized
carbohydrate-binding chaperone system that supports the
binding of the lectin chaperones, calnexin and calreticulin, specifically to monoglucosylated glycans. Therefore,
binding to the ER lectin chaperones is directly controlled
by the composition of the glycan. At first glance, the ER
lectin chaperone system provides a completely different
framework for assisting the maturation of proteins that
traverse the secretory pathway by binding the exposed
hydrophilic modification rather than misplaced exposed
hydrophobic side chains on a maturing polypeptide. In
this review, we will describe the discovery, mechanism
and role of this important ER lectin chaperone system in
cellular homeostasis and its association with disease states.
The Discovery of the Calnexin Binding Cycle
Understanding the mechanism of the lectin binding cycle
provided a paradigm shift in our thinking on how an alternative chaperone system could operate and assist in the cellular folding process. It also offered an explanation for the
Traffic 2016; 17: 308–326
A
B
C
Figure 1: Structure of N-linked glycan. N-linked glycans are transferred en bloc to asparagine residues in the
sequon Asn-X-Ser/Thr, where X denotes any amino acid except
for Pro. The precursor structure is composed of three glucoses (blue circles), nine mannoses (green circles) and two
N-acetylglucosamines (blue squares). Glycosidic bonds are
noted.
longstanding questions regarding the changes in composition of glycans as proteins progress through the secretory
pathway. Why build up an elaborate glycan structure in the
ER just to dismantle it and then build it up again in the
Golgi? This innovative carbohydrate-binding chaperone
model first proposed by Ari Helenius and his colleagues at
Yale University in 1994 was based on integrating a number
of seemingly disparate but important observations in the
literature with seminal results from his own lab (12,13).
Degen and Williams observed in 1991 that a protein of
88 kDa associated with major histocompatibility class I
heavy chain shortly after its translocation into the ER
(14). This heavy chain-associated protein was initially
termed p88 and it was insightfully proposed that it
might help retain class I in the ER until it associated
with β2-microglobulin and peptide, prior to its exit for
309
Lamriben et al.
peptide presentation at the cell surface. That same year,
Bergeron, Thomas and colleagues identified a number of
ER membrane phosphoproteins from canine rough-ER
derived microsomes (15). One of these proteins was
homologous to the soluble ER calcium-binding protein,
calreticulin, and was named calnexin. Calnexin and p88
were later demonstrated to be identical (16). Bergeron,
Thomas and colleagues later demonstrated that a number
of abundantly expressed glycoproteins (α1-antitrypsin,
α1-antichymotrypsin, transferrin, C3, apoB-100 and
α-fetoprotein) co-immunoprecipitated with calnexin in
HepG2 cells and the glycosylation inhibitor tunicamycin
abolished their interaction (17). Non-glycosylated albumin did not bind calnexin. These results lead to the
proposal that calnexin may play a role in the quality
control specifically of glycoproteins.
Earlier studies from Parodi and Cazzulo identified glucosylation activity initially in Trypanosoma cruzi, a parasitic
protozoan that assembles and transfers unglucosylated
Man9 GlcNAc2 glycans onto nascent chains in the ER (18).
Glucose was transferred onto the high mannose glycans
by using uridine disphosphate-glucose (UDP-Glc) as a
substrate. The transferring enzyme was later purified from
rat liver microsomes and named UDP-Glc: glycoprotein
glucosyltransferase (UGGT) (19). Purified UGGT was
found to modify denatured substrates more efficiently
than native substrates, providing a possible link between
the structure of the maturing protein and the glycan composition (20). The presence of a reglucosylation activity
that adds a single glucose to high mannose moieties in the
ER helped to provide an explanation for an earlier observation with a temperature-sensitive mutant of the vesicular
stomatitis virus, ts045, that encoded a mutant form of
the viral G protein (21). VSV G existed persistently in a
monoglucosylated state at the non-permissive temperature
but when the temperature was reduced to the permissive
temperature, the glucose was removed and the protein
exited the ER, once again associating the glycosylation
state of a protein with the structure of the polypeptide.
A major advance toward understanding the binding
properties of calnexin was discovered by Hammond et al.
who showed that binding to calnexin also required glucosidase trimming as interactions with calnexin were
blocked by pre-treatment with the glucosidase inhibitors
310
castanospermine or 1-deoxynojirimycin that supported
the accumulation of glycoproteins in tri-glucosylated
glycoforms (12). This indicated that not only were glycans
necessary for binding to calnexin but also these glycans
required some level of glucose trimming. Helenius and
colleagues reconciled these and earlier results into a central
model that described how the calnexin binding cycle is
controlled by the composition of the glycan (12,13,22). The
terminal glucose is initially trimmed in the ER by the membrane protein α-glucosidase I, followed by the trimming of
the second glucose by the soluble heterodimeric glycosidase, α-glucosidase II, to generate the monoglucosylated
substrate for calnexin. Binding to calnexin persists until
the final glucose is removed, once again by α-glucosidase
II. If the released substrate folds properly to its native state,
it exits the ER for the Golgi in COPII vesicles. However, if
it continues to display a non-native configuration, it is recognized by UGGT, which reglucosylates it to regenerate a
monoglucosylated protein that can rebind to calnexin until
it is deemed properly folded by UGGT. This model provides an explanation for: (i) how a carbohydrate-binding
chaperone cycle can be mediated; (ii) the persistent presence of a non-native protein in the monoglucosylated
state; and (iii) the important quality control role of the
reglucosylation activity of UGGT in the ER.
The calnexin binding cycle model was initially tested using
an in vitro translation system combined with rough-ER
derived microsomes that permitted the accumulation of
substrates in their tri-, di-, mono- and unglucosylated
states to demonstrate that calnexin bound specifically
to monoglucosylated proteins (23). Evidence was also
provided to support a central tenet of the model that reglucosylation in the ER could direct rebinding to calnexin.
The calnexin binding cycle was expanded to include the
soluble paralogue of calnexin, calreticulin, that also bound
monoglucosylated substrates (24). This novel method
of chaperone binding shifted the focus to the glycan in
directing the maturation, binding of chaperones and the
trafficking of glycoproteins in the early secretory pathway.
The ER Lectin Chaperone Network
The glucosidases
The deglucosylation events in the ER occur in a controlled
sequential manner which is initiated by α-glucosidase
Traffic 2016; 17: 308–326
The Calnexin Binding Cycle
I that cleaves the outer most α-1,2-linked glucose (11)
(Figures 1 and 3). α-Glucosidase I is a type II single-pass
transmembrane protein with a large globular luminal
portion that contains the catalytic domain and a short
N-terminal cytoplasmic tail (25). The crystal structure of
the soluble luminal domain of Saccharomyces cerevisiae
α-glucosidase I, Cwh41p, has been solved (26,27). Human
α-glucosidase I and Cwh41p share 24% sequence identity
overall and their catalytic domains share ∼45%, thus
Cwh41p may be used to model the human α-glucosidase
I catalytic activity and substrate binding properties. Two
acidic residues within the C-terminal domain of the
Cwh41p catalytic site confer the protein’s catalytic activity
and a number of conserved aromatic residues contribute
to the protein’s highly specific substrate binding properties.
α-Glucosidase I isolated from mouse fibroblasts was found
in a complex with Sec61 as observed in proteomics studies
of translocon-associated factors, which supports early
intervention by the enzyme in the protein folding pathway
(28). α-Glucosidase I null mutations in hamster cells, as
well as in S. cerevisiae, are tolerated (29,30). Once the
terminal glucose has been trimmed, the di-glucosylated
glycan becomes a substrate for α-glucosidase II, a soluble ER resident enzyme that specifically cleaves the
α-1,3-linked glucose moieties to generate deglucosylated
glycans (11). Human α-glucosidase II is comprised of
a large α-subunit (∼100 kDa) and a smaller β-subunit
(∼50 kDa) that associate non-covalently (31,32). The
α-subunit possesses catalytic activity and is retained
in the ER by its association with the β-subunit, which
contains an ER retention (KDEL) sequence (33). The
β-subunit is not required for the catalytic activity of the
enzyme; however, it appears necessary for its maturation,
solubility and stability. Additionally, the β-subunit contains a mannose 6-phosphate receptor homology (MRH)
domain, which has been proposed to bind the terminal
mannose on the trimmed C-branch of glycans as well
as the B-branch in order for the enzyme to efficiently
act on the A-branch glucoses (34,35). For translocating
glycoproteins emerging into the ER lumen, the first glycan
helps to recruit α-glucosidase II through binding to its
MRH domain, supporting the more efficient cleavage of
additional C-terminal N-glycans on the nascent chain
(36). The function of α-glucosidase II depends on the
presence as well as number of mannoses present on the
glycan. The enzymatic activity of α-glucosidase II on
Traffic 2016; 17: 308–326
the A-branch of the glycan is strongly influenced by the
presence of mannoses on the adjacent B- and C-branches.
In vitro data demonstrated a decrease in enzymatic activity
that correlated to a decrease in mannose content on the
targeted glycan (37). Like α-glucosidase I, α-glucosidase
II null mutations are tolerated in hamster and mouse
immortalized cell lines (38).
Calnexin and calreticulin
The type I membrane protein calnexin and its soluble paralogue calreticulin share a similar general organization
(Figure 2). Each is comprised of an N-terminal globular
domain, a central proline-rich domain and a C-terminal
domain. Human calnexin and calreticulin share 39% overall sequence identity (40).
The N-termini of the two proteins fold into globular domains consisting of anti-parallel β-sheets that
create a single carbohydrate-binding site (39). The
carbohydrate-binding site supports binding specifically
to monoglucosylated glycans (13). Isolated calreticulin
binds monoglucosylated glycans with micromolar affinity
(41). The crystal structure of calnexin shows that the
globular domain also contains a single bound calcium ion
that accounts for the high affinity low capacity binding
of calcium to the chaperones (39) (Figure 2). Calcium is
important for stabilizing the lectin chaperones and regulating their binding to substrates (42,43). ATP also appears
to modulate the conformation of the lectin chaperones, as
they become more resistant to proteases in its presence,
suggestive of them reaching more compact conformations (44,45). Recently, computational and mutagenesis
approaches have been used to identify residues in the globular domain of calreticulin that contribute to ATP binding
(45). ATP destabilized the substrate-calreticulin complex, indicative of it playing a role in regulating substrate
binding. High affinity calcium binding by calreticulin is
required for nucleotide binding and chaperone stabilization (45). Altogether the globular domain contains sites
for binding to carbohydrates located on the substrate, and
regulatory co-factors such as ATP and calcium.
The central Pro-rich or P-domain creates an arm that
extends away from the N-terminal lectin domain. The
P-domain of calnexin (140Å) is organized into a series of
sequential motifs, comprised of four consecutive copies of
311
Lamriben et al.
A
B
Figure 2: Structural and domain organization of calnexin and calreticulin. A) Linear cartoon representations of calnexin
and calreticulin. Both proteins possess a cleavable ER-targeting N-terminal signal sequence (green), a lectin domain (orange) and a
C-terminal domain (purple). Calnexin possesses a single-pass transmembrane domain (black) that couples calnexin to the ER membrane
and supports a cytoplasmic C-terminal domain. Calreticulin is soluble protein that is retained in the ER through its C-terminal KDEL
sequence. B) Surface and ribbon representations of the crystal structure of the calnexin luminal domain (PDB: 1JHN) (39). The globular
lectin domain (orange) forms a binding pocket to accommodate monoglucosylated clients and contains a calcium-binding site, with
a single Ca2+ ion shown in cyan. The P-domain (gray) protrudes from the globular domain into a hook-like shape. Cyclophilin B and
ERp57 bind at the tip of the P-domain.
312
Traffic 2016; 17: 308–326
The Calnexin Binding Cycle
motif 1 followed by four copies of motif 2 (46). The calreticulin P-domain is shorter than that of calnexin as it
involves two sets of three consecutive repeats rather than
four. Both P-domains are the site of interaction with two
folding factors: ERp57, a protein disulfide isomerase; and
cyclophilin B (CyB), a peptidyl-prolyl isomerase. Like PDI,
ERp57 consists of four thioredoxin domains of which the
two positioned at each termini possess the active CXXC
motifs, while the central thioredoxin folds lack enzymatic
activity (47). ERp57 contains a P-domain-binding region
that localizes it to the vicinity of lectin chaperone substrates
so it can assist disulfide bond formation and rearrangement
of lectin chaperone-associated substrates. The interaction
between the lectin chaperones and ERp57 is transient and
it occurs in the absence of a client substrate (48,49). The
region at the tip of the P-domain to which ERp57 binds
overlaps with that of CyB binding, making these interactions mutually exclusive (50).
Calnexin and calreticulin also appear to bind client
proteins in a glycan-independent manner (51). In vitro
studies show that they can inhibit the aggregation of
non-glycosylated substrates (52). The peptide-binding site
seems to be located in the vicinity of the glycan binding
site within the globular domain (53). While the affinities of calreticulin for glycosylated and non-glycosylated
substrates appear to be similar, the kinetics are distinct.
Binding to glycosylated clients is more rapid. Glycosylated and non-glycosylated clients support the formation
of open and closed conformations, respectively, where
the closed conformation involves the clamping down
of the P-domain on the substrate stabilizing substrate
interactions. The co-factor ERp57 also induces the closed
conformation of calreticulin as probed using FRET
measurements with purified components.
The C-terminal regions are divergent but they both contain ER retention signals. The membrane protein calnexin
possesses a single C-terminal transmembrane region and
a short cytoplasmic tail. Oriented in the cytoplasm is a
di-Arg sequence that aids in ER retention, along with a
number of phosphorylated residues that will be discussed
below. At its C-terminus, the soluble calreticulin has a
canonical KDEL sequence that supports ER retention and
retrieval. This region on calreticulin also contains a series of
acidic residues that support the low affinity (K d = 1–2 mM)
Traffic 2016; 17: 308–326
and high capacity (∼20 mol Ca2+ per mol protein) binding
of calcium (54,55). Calreticulin acquires its calcium buffering properties through the C-terminal domain. The tail of
calreticulin is intrinsically unstructured and only acquires
a rigid and folded structure when bound to calcium.
The glucosyltransferase protein folding sensor
Non-native unglucosylated proteins are reglucosylated by
UDP-glc: glycoprotein glucosyltransferase 1 (UGGT1) to
regenerate monoglucosylated side chains that can support rebinding to calnexin and calreticulin (13,56). Once
the glycoprotein is in a mono-glucosylated state, it also
becomes a substrate for α-glucosidase II, which competes
with UGGT1 with opposing effects (57) (Figure 3). This
positions UGGT1 to be a central gatekeeper in the ER that
decides whether glycoproteins traffic onto the Golgi and
beyond, or are retained in the ER for further assistance.
Human UGGT1 is a soluble (∼170 kDa) resident ER
enzyme. At its C-terminus, it possesses a catalytic glucosyltransferase domain. This domain contains a DxD motif
that is required for its catalytic activity (58,59). In an in
vitro study, purified rat UGGT1 required calcium to transfer glucose onto deglucosylated glycoprotein acceptors,
suggesting calcium dependence for the catalytic activity
of UGGT1 (19). Similar to α-glucosidase II, the catalytic
activity of UGGT1 is more effective on glycans with high
mannose content (20).
The N-terminal ∼80% of UGGT1 contains what is proposed to be the important protein folding sensor domain.
Bioinformatics analysis predicts that the N-terminal
region is comprised of three consecutive thioredoxin-like
domains (60). Thioredoxin folds are commonly associated
with protein disulfide isomerase family members that play
important roles in protein folding and quality control (47).
However, none of the thioredoxin-like domains of UGGT1
contain the catalytic CXXC motif that shuttles electrons in
disulfide formation, reduction or rearrangement reactions
(60). Instead, these thioredoxin-like domains appear to act
like the catalytically inactive domains found in many of the
protein disulfide isomerase family members including PDI
and ERp57. The three-dimensional structure of the third
thioredoxin-like domain of UGGT1 has been solved and it
involves a central β-sheet surrounded by a number of short
α-helices. It also possesses a distinct hydrophobic patch
313
Lamriben et al.
glucosyltransferase domain appears to be active when
swapped with the catalytic domain of UGGT1 or when
tested in vitro against synthetic substrates (62,63). Currently, no biological role has been assigned nor natural
substrates of UGGT2 have been identified.
Functional Roles of the Lectin Chaperone
Binding Cycle
The lectin chaperones are multifunctional factors playing
important roles in optimizing the efficiency of the protein folding program, minimizing deleterious side reactions such as aggregation, retaining immature or misfolded
cargo in the ER, and possibly targeting aberrant proteins
for degradation. Several experimental strategies have been
used to tease out the role of the lectin chaperone cycle
and their associated factors in the maturation and quality control of ER targeted client proteins. These approaches
include following the maturation of lectin chaperone substrates in the absence of their assistance and comparing it
to the natural pathway by using chemical inhibitors or cell
lines that lack the individual components, as well as studies
with purified components.
Figure 3: Processing of N-linked glycans and folding
pathway of secretory glycoproteins in the endoplasmic
reticulum. N-linked glycans, assembled by Alg gene protein
products (1), are transferred via the OST complex to proteins
translocated into the ER lumen (2). α-Glucosidases I and II
trim the first two glucoses (blue circles) from the glycan (3),
generating a monoglucosylated glycoform, which is a substrate
for calnexin (CNX) and calreticulin (CRT) (4). Trimming of the
remaining glucose by glucosidase II releases the substrate from
CNX/CRT (5). Glycoproteins that do not reach their native folded
state can be reglucosylated by UGGT1 (6), redirecting them
to CNX/CRT binding (7) or trimming by α-glucosidase II (8).
Glycoproteins that have reached their native state (star) (9) are
trimmed by mannosidases and directed for trafficking out of the
ER (10). Proteins that are terminally misfolded are trimmed by
mannosidases and directed for ERAD (11).
that might be involved in the recognition of folding intermediates or misfolded substrates. However, more studies
are required to test and confirm this proposed function.
UGGT1 also has a homologue UGGT2 that shares 55%
sequence identity with UGGT1 (61). The C-terminal
314
A main function of the lectin chaperone binding cycle is to
promote efficient folding and assembly of glycoprotein substrates to their native states. Inhibition of α-glucosidases I
and II with glucose analogues castanospermine (CST) or
N-butyl-1-deoxynojirimycn (DNJ) circumvents the lectin
chaperone binding cycle by accumulating substrates in
their tri-glucosylated state. This is commonly associated
with improper folding and reduced trafficking of glycoproteins from the ER as has been demonstrated for a number
of substrates including hemagglutinin, neuraminidase,
vesicular stomatitis G protein, class I histocompatibility
complex and tyrosinase (64–69). Validated targets of
calnexin and calreticulin assistance include endogenous
and pathogenic membrane and soluble glycoproteins.
The use of glucosidase inhibitors simultaneously bypasses
both lectin chaperones. To delineate the role of each
chaperone individually, knockout cell lines have been
used that are separately missing one of the chaperones or
co-factors. Calnexin deficiencies led to more error prone
folding of hemagglutinin in cnx−/− T lymphoblastoid
cells than observed in crt−/− mouse embryonic fibroblasts
Traffic 2016; 17: 308–326
The Calnexin Binding Cycle
(MEFs) (70). While hemagglutinin is known to interact
with both chaperones (71), these results are indicative of
hemagglutinin’s reliance on calnexin for its proper folding.
In contrast, studies in crt−/− cells have demonstrated the
requirement for calreticulin in the proper processing and
trafficking of MHC class I heavy chain and the bradykinin
receptor (72,73).
The interaction between calnexin or calreticulin with
substrates also promotes glycoprotein folding and degradation through its associated oxidoreductase, ERp57; or
the peptidyl-prolyl cis/trans isomerase, CyB (48,50,74).
As an oxidoreductase isolated ERp57 has been shown
to catalyze the oxidation, isomerization and reduction
of disulfide bonds (75). The catalytic activity of ERp57 is
enhanced when it is associated with calnexin or calreticulin
(76). The direct association of ERp57 with calnexin and
calreticulin has been suggested to be important for bringing ERp57 and its substrates together, thereby enhancing
local concentration of the isomerase for the catalysis of
disulfide bonds. ERp57 knockdown was found to specifically delay secretion of the glycosylated substrates (77).
Cellular ERp57 appears to oxidize HA post-translationally
but not co-translationally, as in the absence of ERp57 only
the post-translational oxidation processing of HA was
compromised (78). For further discussion of the role of
ERp57 in protein folding and quality control please see the
article by Ellgaard et al., in this issue (79).
Much less is known about the roles of PPIs in the ER.
CyB associates with the lectin chaperones through their
P-domain on a site that appears to overlap with the
ERp57 binding site (50). The immunosuppressive drug
cyclosporine A, an inhibitor of cyclophilins, has been used
to demonstrate that ER CyB helps prepare substrates for
ERAD, as it delayed the turnover of glycosylated ERAD
substrates (80). Isomerases associated with the lectin
chaperones can assist the processing and trafficking of
substrates associated with calnexin and calreticulin.
After release from lectin chaperones by α-glucosidase II
trimming, unglucosylated substrates that do not reach their
native state may be reglucosylated by UGGT1 (Figure 3).
The re-established monoglucosylated state directs rebinding to calnexin and calreticulin. Using a cell based reglucosylation assay that relies on the use of a mutant cell line
Traffic 2016; 17: 308–326
that transfers truncated carbohydrates to nascent chains,
UGGT1 was found to modify a wide spectrum of proteins
in live cells (81–83). The lysosomal protein prosaposin was
determined to be an obligate substrate of UGGT1, as in its
absence prosaposin poorly reached its proper locations but
instead accumulated in intracellular aggregates (82). By
supporting persistent chaperone binding, reglucosylation
promotes substrate solubility and prevents aggregation
(84). There is tremendous variability in the necessity of
reglucosylation to aid the maturation of secretory pathway
client proteins. Deletion of UGGT1 in mouse embryonic
fibroblast cells has shown that there are three general types
of glycoproteins (85). Class I proteins, such as VSV G, use
only one round of lectin chaperone binding, leading to
no alteration in folding rate or efficiency in the absence
of UGGT1. Class II proteins, such as BACE501, require
multiple rounds of binding, leading to early release from
the ER and a decrease in folding efficiency in uggt1−/−
cells. Class III proteins, such as hemagglutinin, exhibit
a decreased rate of release from calnexin when UGGT1
was deleted, possibly because UGGT1 itself is required
for their structural maturation or oligomerization. These
results suggest that the role of the lectin chaperone
binding cycle in glycoprotein folding is highly substrate
dependent.
Persistent calnexin and calreticulin binding, like classical
chaperones, act to slow the folding process (64,71,86).
Since chaperone binding is mediated by N-glycans, substrate binding occurs in a region-specific manner (71).
For larger multidomain proteins such as influenza hemagglutinin and neuraminidase, chaperone binding helps to
direct the molecular choreography of the cellular folding pathway (69,71,86). N-terminal glycans recruit early
co-translational calnexin binding (87), which can delay the
formation of intramolecular and intermolecular disulfide
bonds to create an efficient folding route (69,71,86). In the
absence of lectin chaperone binding, accelerated folding
and oligomerization can favor misfolding and aggregation. Trapping proteins on the chaperones delays folding,
oligomerization and trafficking (23,69,83). These results
suggest the possibility that the genetic code encodes an
additional layer of complexity that determines the positioning of N-linked glycans to help with the recruitment
of chaperones and the optimization of the cellular folding
process.
315
Lamriben et al.
An important role for the lectin chaperones is retaining intermediates, misfolded substrate or unassembled
multimers in the ER. ER retention of non-native clients
enhances proper glycoprotein maturation and prevents
non-functional and potentially harmful glycoproteins from
trafficking out of the ER (88). Glucosidase inhibition blocks
glycoprotein entry into the calnexin binding cycle and led
to an increased rate of RNAse secretion, as did the deletion of glycosylation sites (36). Glucosidase inhibition has
also been shown to increase export of non-native proteins from the ER in the case of MHC Class I molecules
(73). The cell line, MI8-5 CHO, which transfers glycans
with no glucoses to glycoproteins, has facilitated numerous
studies of the lectin chaperone binding cycle as glycoproteins can only enter the calnexin and calreticulin binding
cycle after glucosylation by UGGT1. Treatment with glucosidase inhibitors in this cell line traps substrates on the
lectin chaperones and delays glycoprotein secretion, as in
the case of α-1-antitrypsin and prosaposin (83). Calnexin
and/or calreticulin binding can also retain in the ER the
monomers of incompletely assembled oligomers, as is the
case of soybean agglutinin (89). UGGT1 can reglucosylate the folded monomers, directing them to re-associate
with lectin chaperones until complete assembly is achieved.
Collectively, these studies suggest that the ER lectin chaperones aid in retaining non-native glycoproteins in the
ER, thereby promoting correct folding and assembly prior
to ER exit.
While some eukaryotic cell lines can survive the knockout of calnexin, calreticulin, ERp57 or UGGT1, the deletion of these factors in mice causes severe phenotypes (90).
Calnexin knockout mice exhibited decreased survival rates
within 48 hr after birth, and survivors were smaller and displayed motor disorders such as unstable gait and truncial
ataxia, as well as a loss of myelinated nerve fibers (91). Calreticulin deletion in mice is embryonic lethal at 18 days due
to heart defects (92). ERp57 deletion in mice is embryonic
lethal at day 13.5, possibly due to increased STAT3 signaling (93). Finally, UGGT1 deletion is typically embryonic
lethal at day 13 in mice, although some mice survive to
birth (94). The number and diversity of glycoproteins that
rely on the lectin binding cycle for proper folding underscores its importance. Some of the obligate substrates of the
lectin chaperone network appear to be required for development and survival.
316
ER Organization of the Lectin Chaperone
Network
The function of the carbohydrate-binding chaperone network is in part controlled by the localization of its components within the cell. While the ER is comprised of a single
contiguous membrane and lumen, it is divided into a number of functional regions that help support the efficient progression of maturing proteins and a number of other cellular processes (95–97). At first glance, the ER is separated
into two morphologically distinct regions: the perinuclear
rough-ER decorated with ribosomes; and the smooth ER
that lacks ribosomes but extends throughout most mammalian cells to create a number of functional regions. The
lectin chaperones are dispersed throughout both distinct
areas (98,99). Calnexin and calreticulin have also been
found in locations outside the ER, suggestive of them playing roles beyond the confines of the ER boundaries (100).
Membrane-bound ribosomes directed to the ER by the
signal recognition particle (SRP) co-translocationally
translocate nascent chains into the ER through the proteinaceous channel comprised of Sec61. An assembly
line of translocon-associated factors awaits the emerging
nascent chain to create a privileged environment that
optimizes the efficient maturation of both membrane
and soluble client proteins (101). The spatial organization
of the factors is thought to temporally control access to
the nascent chain with two large multimeric complexes
that mediate N-glycosylation (OST) and signal sequence
cleavage (signal sequence peptidase complex) having early
access to maturing polypeptides. Calnexin and calreticulin
associate with both ribosome-arrested polypeptides, as well
as actively translating and translocating nascent chains
(86,87). This indicates that the membrane-integrated
α-glucosidase I that is a translocon-associated protein
(28), and soluble α-glucosidase II also have early access
to nascent chains. A single round of binding to the
lectin chaperones appears to occur during translation
as arrested chains were unable to be reglucosylated by
UGGT1 unless released from the translocon by puromycin
(81). Proteomics and morphological results indicated that
UGGT1 does not accumulate in the rough ER (98,99,102).
While the polysome-translocon complexes ensure spatial
separation of nascent chains to minimize unproductive
contacts that might lead to aggregation (103,104), binding
Traffic 2016; 17: 308–326
The Calnexin Binding Cycle
to the lectin chaperones provides an additional layer of
protection during this vulnerable stage of maturation
(64,65).
Maturing proteins progress from the rough to the smooth
ER, where they continue to fold and assemble (if applicable). In the smooth or transitional ER, clients are interrogated or subjected to quality control evaluations. UGGT1
plays a central role in the evaluation process and appears to
be located near ER exit sites as determined by immunolocalization as well as functional studies (102,105). Once a
protein is reglucosylated, it is proposed to move from the
calnexin-free vicinity of the ER exit sites toward a calnexin and calreticulin-rich region of the ER that required
∼20 min for the thermosensitive tsO45 mutant of VSV
G protein (105). Interestingly, while tsO45 VSV G is ER
retained at the non-permissive temperature, its mobility
was not retarded when compared to the mobility of the
protein at the permissive temperature (106). This may
be caused by weak and dynamic interactions between
the glycoprotein and the lectin chaperones (micromolar) (41). The properly folded, deglucosylated and released
substrate migrates to ER exit sites, is no longer recognized by UGGT1, and is therefore permitted to leave the
ER through COPII vesicles. Photobleaching studies with
calreticulin-GFP; however, call into question the presence
of a lectin chaperone free-zone as there was no region in the
ER to which calreticulin-GFP did not have access (107).
Terminally misfolded or irreparably folded glycoproteins
are efficiently reglucosylated by UGGT1 supporting persistent binding to the lectin chaperones (83). These clients
are eventually targeted for turnover by the ER-associated
protein degradation or ERAD process (108). Both calnexin
and calreticulin have been found to accumulate upon
proteasomal inhibition in a compartment with known
ERAD factors such as ER Man I/MAN1B1, EDEM1,
Derlin-1 and Os-9, as well as ERAD substrates (109,110).
This compartment has been called the ERQC (ER quality
control compartment) and has been proposed to be a
concentrated area where ERAD takes place (Figure 4).
As the ERQC lacks both UGGT1 and ERp57, it has been
proposed that persistent binding to the lectin chaperones
in the bulk ER may play a role in the delivery of aberrant
substrates to the ERQC for subsequent dislocation and
proteasomal degradation.
Traffic 2016; 17: 308–326
The ER directly contacts a number of organelles, including mitochondria (96) (Figure 4). Mitochondrial contact
regions are termed mitochondria-associated ER membranes or MAM. As the ER is the major calcium store in
the cell, it responds to metabolic needs through intracellular signals that can lead to rapid changes in free luminal
calcium levels (111). ER calcium channels including the
inositol 1,4,5-trisphosphate receptor and SERCA are
enriched in the MAM where they help to control calcium
exchange between the two organelles (112,113). The activity of SERCA is regulated by the lectin chaperones as they
both inhibit SERCA-mediated calcium uptake into the ER
(114,115). The SERCA activity appears to be modulated
by the recruitment of the lectin chaperone-associated
oxidoreductase ERp57 that reversibly controls the SERCA
redox state and activity (116). The involvement of the
lectin chaperones in SERCA regulation points to a role
for calnexin and calreticulin in controlling fundamental
cellular processes that extend beyond assisting protein
maturation and quality control.
The ER is a dynamic multifunctional organelle that
re-organizes itself in response to cellular stresses or the
needs of a particular cell type (117–120). Calnexin is differentially modified on its cytoplasmic-facing C-terminal
tail, altering its location within the ER in response to protein folding stress and calcium regulation. The C-terminus
of calnexin is phosphorylated at residues Ser534, Ser544
and Ser563 by casein kinase II (CKII) and the mitogen
activated protein kinase ERK1 (121). Phosphorylation
at these residues localizes calnexin to the rough ER and,
specifically, phosphorylation at Ser563 has been shown to
slow the maturation and secretion of α1-antitrypsin under
protein folding stress conditions (122). Four large-scale
profiling studies identified calnexin as being modified
by S-acylation; however, the effects of this modification
on dictating the location of calnexin within the ER are
controversial (123–126). One study shows that calnexin
associates with ribosome-translocon complexes through
palmitoylation at juxtamembrane residues Cys502 and
Cys503 and non-palmitoylated calnexin shows reduced
binding to its client proteins (127). Another study finds
that non-palmitoylated calnexin plays a more prominent
role in protein folding while palmitoylated calnexin is
implicated in calcium regulation localizing to the MAM
(128). Taken together, calnexin is subjected to multiple
317
Lamriben et al.
Figure 4: Cellular localization of the lectin chaperone network. In the rough ER (rER), which is characterized by the presence of
ribosomes on the cytoplasmic side of the membrane and translocation machinery, α-glucosidase I and II sequentially trim glucoses from
newly added N-linked glycans generating monoglucosylated glycoproteins. Calnexin and calreticulin associate with actively translating
and translocating proteins. As proteins continue to fold, they migrate to the smooth portion of the ER where they are interrogated by
UGGT1 in the peripheral ER at ER exit sites (ERES). If proteins are deemed terminally misfolded, they are targeted for ERAD. Calnexin,
calreticulin and misfolded proteins accumulate in the ERQC. The ER contacts the mitochondria at the mitochondria-associated membrane
(MAM). Calnexin in particular is shuttled between the rER and the MAM by post-translational modifications based on the current needs
of the cell. Calnexin is differentially phosphorylated (P – in yellow) on several residues within its C-terminal cytoplasmic tail, associating
it with translating ribosomes and enriching it in the rER. Additionally, calnexin has been shown to be palmitoylated (in blue).
forms of post-translational modification that accentuate
its many roles.
Calreticulin has also been found to act in locations outside the ER. While this proposal was originally met with
skepticism, there is accumulating cell biological and functional evidence for calreticulin acting in the cytoplasm,
the outer cell surface, and the extracellular matrix (129).
Cytoplasmic calreticulin has been shown to bind to the
cytoplasmic tail of a integrins to stabilize ligand binding
for calcium signaling and cell adhesion (130). Calreticulin
has also been found on the outer surface of a variety of cells
including melanocytes, fibroblasts, platelets, endothelial
and apoptotic cells. Cell surface calreticulin supports cell
spreading and migration (131,132). Calreticulin at the cell
surface in association with phosphatidylserine acts as an
318
‘eat me’ signal to direct the phagocytosis of apoptotic or
cancer cells by macrophages and neutrophils, as well as
fibroblasts (133,134). Interestingly, the cell surface roles of
calreticulin appear to contribute to its ability to accelerate
the rate of cutaneous wound healing when added topically
(135). Calnexin has also been found at the cell surface
for a number of cells (136). These studies point toward
an expanding influence of the lectin chaperones beyond
the ER.
Translational Implications Involving the Lectin
Chaperones
Calnexin and calreticulin bind and influence the maturation of a large number of proteins as they traffic through
Traffic 2016; 17: 308–326
The Calnexin Binding Cycle
the ER and many of these client proteins are associated with
disease states. Modulation of the lectin chaperone binding
cycle provides a potential avenue for therapeutic intervention for a variety of maladies. Modulation strategies can
involve either the derailment of the maturation of proteins
from pathogens, or the enhancement of essential endogenous activities that are associated with pathologies that
involve diminished protein levels due to aberrant or inefficient maturation.
Enveloped viruses usurp the mammalian secretory pathway for the production of their membrane glycoproteins
that serve essential functions in the viral life cycle. These
viral glycoproteins rely on the endogenous glycosylation
machinery and lectin chaperones to ensure their proper
maturation. Circumventing the lectin chaperone cycle
can lead to inefficient folding, and subsequent degradation of viral glycoproteins. Iminosugars that inhibit
glucosidases have been explored as possible anti-viral
agents for the treatment of enveloped viruses such as
influenza, HIV and Dengue virus (137,138). Deoxynojirimycin (NB-DNJ) derivatives (Miglustat or Zavesca)
and CST support the accumulation of glycoproteins in
their tri-glucosylated state thereby abolishing the influence
of the lectin chaperones on the maturation of the viral
glycoproteins. Both hemagglutinin and neuraminidase
fold inefficiently in the absence of calnexin and calreticulin assistance (64,69,71). Glucosidase inhibition has
been shown to diminish influenza virus A replication in
cell culture (139). The inhibition of glucosidase activity also caused structural alterations in Env or gp120,
and inhibited HIV replication in cells (140,141). Clinical trials were initiated using NB-DNJ on patients with
HIV but were halted due to side effects that included
gastrointestinal distress, and the discovery that cataracts
developed in rats upon NB-DNJ treatment (142). A DNJ
derivative (N-9-methoxynonyl-DNJ or UV-4) is also a
potent inhibitor of dengue virus infection in mice (138).
Disrupting the production of mature and infectious
virus by inhibiting the glycoprotein maturation process
provides a potential mechanism for the development
of broad-spectrum anti-viral agents directed toward
enveloped viruses.
Cells are relatively tolerant of glucosidase inhibition under
normal conditions. This includes treatment with chemical
Traffic 2016; 17: 308–326
inhibitors, as well as the development of stable cell lines
that lack either α-glucosidase I or II (30,38,143). In part,
this is likely explained by backup compensating chaperone
systems being in place; however, different outcomes can
occur if cells are stressed and this phenomenon can be
potentially exploited for therapeutic intervention. For
instance, CST analogues have been shown to possess
anti-tumor activity as iminosugar CST-derivatives inhibit
the proliferation of breast cancer cells by promoting both
cell cycle arrest and apoptosis (144). It is uncertain if this
anti-tumor property is related to the disruption of the
lectin chaperone binding cycle. Further investigation into
the influence of glucosidase inhibitors on cancer cells is
warranted.
There are a growing number of loss-of-functions diseases
associated with proteins that traverse the secretory pathway (108). For this class of disease, the malady is often
caused by a mutation in a secretory pathway client protein
that disrupts the proper maturation of the protein in the
ER resulting in its evaluation by the quality control process
as aberrant and its subsequent targeting for degradation
through the ER-associated degradation pathway. In some
cases this involves an overzealous evaluation decision that
supports the ER retention of a protein that possesses a slight
alteration but is functional. Overexpression of calnexin
has been shown to support the more efficient maturation
or correction of some proteins in live cells including the
pigment producing protein tyrosinase and the lysosomal protein glucocerebrosidase, proteins associated with
albinism and Gaucher disease, respectively (145,146). A
possible therapeutic strategy to extend cell culture findings
to the clinic has been developed by the Kelly lab, which
has used a thoughtful tactic of screening US Food and
Drug Administration-approved drugs for their ability to
augment the maturation of mutants associated with common lysosomal storage diseases in cell culture (147). They
discovered that the L-type calcium channel inhibitors, Diltiazem and Verapamil, partially corrected the trafficking
of two glucocerebrosidase mutations (N370S and L444P)
in fibroblast cells from Gaucher patients. These drugs,
approved for treating hypertension, increase the concentration of calcium in the ER by inhibiting ER ryanodine
calcium efflux channels. Enhanced glucocerebrosidase
maturation is proposed to be mediated by the higher calcium concentration supporting more effective assistance
319
Lamriben et al.
by the calcium-binding lectin chaperones calnexin and
calreticulin. As there is a number of calcium-binding
proteins in the ER, there is a number of activities expected
to be influenced by increased calcium levels.
The ER can be viewed as a protein production assembly line where products entering the ER are folded and
modified, before being packaged into COPII vesicles. In
recent years, there has been a great increase in the interest in the use of secretory pathway products as biologics or
biological compounds as therapeutic agents. These products include hormones, growth factors, enzymes, enzyme
inhibitors, vaccines and monoclonal antibodies (148). One
of the main hurdles for biologics is producing sufficient
quantities of active proteins. The creation of cell lines that
more efficiently produce properly folded secretory pathway client proteins could help to address this need. Cell
lines with activated UPR (ATF4 or Xbp1 [with ER01]
overexpression) boosted the production of antibodies in
CHO cells (149,150). Antibody secretion in CHO cells was
also enhanced by PDI overexpression (151). Enhancing
the involvement of calnexin and calreticulin might help
the production of N-glycosylated biologics by accelerating
their maturation within cells.
Myeloproliferative neoplasm is largely associated with
mutations in Janus kinase 2 (JAK2) that increase signaling
in the JAK/STAT pathway (152). Whole-exon sequencing
was used to identify mutations found in the approximate
one-third of patients that did not have a corresponding
JAK2 mutation. Two independent studies discovered
mutations in exon nine of calreticulin that were found in
the majority of the non-mutated JAK2 myeloproliferative
neoplasm patients tested (153,154). Interestingly, the most
predominant mutations identified (deletion of 52 base
pairs or insertion of 5 base pairs), which account for 85%
of the mutations identified, both cause a similar reading
frame shift that alters the C-terminus of calreticulin. Two
alterations generated by the mutated C-terminus are: (i) the
KDEL C-terminal ER retrieval/retention signal is absent;
and (ii) many of the negatively charge residues that create
the low affinity, high capacity calcium-binding property
of calreticulin are either missing or changed to positive
residues. These differences are expected to have a major
impact on the localization and the important calcium
signaling function of calreticulin. Further exploration is
320
required to understand how these mutations are linked to
chronic myeloid leukemia.
Closing Remarks
Major advances in our understanding of the protein maturation and quality control processes of the secretory pathway have occurred over the past two decades. Most notably,
N-glycans have emerged as key maturation and quality
control tags that signal the fate of the protein to which they
are attached (10). Many details of the glyco-code have been
solved and the players involved in encoding and decoding the protein fitness signals have been identified. Moreover, the glycan positioning on the maturating substrate
has been recognized to play an important role in directing the molecular choreography for the protein maturation
program of secretory pathway client proteins (71,86). Studies using additional substrates are now needed to elucidate
how this additional layer of the genetic code, that dictates
glycan positioning, helps to optimize the folding and maturation of glycoproteins.
The binding properties of the lectin and traditional molecular chaperones are seemingly the polar opposites-binding
to attached hydrophilic modifications or hydrophobic
patches on maturing or aberrant nascent chains, respectively. Bulky N-linked modifications are expected to be
aqueous-exposed regardless of the conformation of protein
so how can this modification be used to direct chaperone
binding and evaluate protein conformation? The initial
round of lectin binding, which is often co-translational,
is determined by the level and rate of glucose trimming.
α-Glucosidase II initiates binding as well as release by
trimming the second and third glucose residues on the
A-branch, respectively. Further studies are required to
determine the mechanism of α-glucosidase II trimming
and how its slow removal of the final glucose is regulated,
as this plays a significant part in controlling the initial
duration of lectin chaperone binding.
Though the lectin and traditional chaperone interactions
are differentially mediated, the protein determinants
that direct persistent chaperone binding or chaperone
rebinding seem to be similar. Chaperone rebinding is
directed by UGGT1, which appears to use a traditional
chaperone-like recognition process to interrogate nascent
Traffic 2016; 17: 308–326
The Calnexin Binding Cycle
chains before reglucosylation, as UGGT1 modifies proteins
with exposed hydrophobic residues (56). As UGGT1 is the
central gatekeeper that determines if a protein exits the ER
or is retained for further intervention, additional studies
are required to understand how UGGT1 is responsible for
evaluating the enormous number of proteins and conformations that exist in the ER lumen. Also of interest, is determining how misfolded proteins that are initially retained
are eventually relinquished and targeted for ERAD. Are
these proteins released after going through a certain number of reglucosylation-deglucosylation cycles or after a
particular duration of time? Does trimming of A-branch
mannoses by exo- or endomannosidase release a protein
from reglucosylation? Does the spatial organization of
the ER contribute to an iterative process for glycoprotein
maturation and quality control? If so, how is the spatial
organization maintained or shifted under stress conditions? While we have witnessed many advances in how
glycans direct the trafficking of nascent chains over the past
20 years, future studies will continue to provide further resolution to these important fundamental cellular processes
and how they can potentially be modulated for therapeutic
intervention.
Acknowledgments
This work was supported by the National Institutes of Health under award
number GM086874 (to D. N. H.); and a Chemistry-Biology Interface
Predoctoral training grant (T32 GM008515 to L. L.).
References
1. Radford SE. Protein folding: progress made and promises ahead.
Trends Biochem Sci 2000;25:611–618.
2. Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU. Molecular
chaperone functions in protein folding and proteostasis. Annu Rev
Biochem 2013;82:323–355.
3. Ellis RJ, Hemmingsen SM. Molecular chaperones: proteins essential
for the biogenesis of some macromolecular structures. Trends
Biochem Sci 1989;14:339–342.
4. Bukau B, Horwich AL. The hsp70 and hsp60 chaperone machines.
Cell 1998;92:351–366.
5. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS,
O’Shea EK. Global analysis of protein localization in budding yeast.
Nature 2003;425:686–691.
6. Apweiler R, Hermjakob H, Sharon N. On the frequency of protein
glycosylation, as deduced from analysis of the SWISS-PROT
database. Biochim Biophys Acta 1999;1473:4–8.
Traffic 2016; 17: 308–326
7. Zielinska DF, Gnad F, Wisniewski JR, Mann M. Precision mapping of
an in vivo N-glycoproteome reveals rigid topological and sequence
constraints. Cell 2010;141:897–907.
8. Helenius A, Aebi M. Intracellular functions of N-linked glycans.
Science 2001;291:2364–2369.
9. Hebert DN, Garman SC, Molinari M. The glycan code of the
endoplasmic reticulum: asparagine-linked carbohydrates as protein
maturation and quality-control tags. Trends Cell Biol
2005;15:364–370.
10. Hebert DN, Lamriben L, Powers ET, Kelly JW. The intrinsic and
extrinsic effects of N-linked glycans on glycoproteostasis. Nat Chem
Biol 2014;10:902–910.
11. Kornfeld R, Kornfeld S. Assembly of asparagine-linked
oligosaccharides. Annu Rev Biochem 1985;54:631–664.
12. Hammond C, Braakman I, Helenius A. Role of N-linked
oligosaccharides, glucose trimming and calnexin during glycoprotein
folding in the endoplasmic reticulum. Proc Natl Acad Sci USA
1994;91:913–917.
13. Helenius A. How N-linked oligosaccharides affect glycoprotein
folding in the endoplasmic reticulum. Mol Biol Cell
1994;5:253–265.
14. Degen E, Williams DB. Participation of a novel 88-kD protein in the
biogenesis of murine class I histocompatibility molecules. J Cell Biol
1991;112:1099–1115.
15. Wada I, Rindress D, Cameron PH, Ou WJ, Doherty JJ 2nd, Louvard D,
Bell AW, Dignard D, Thomas DY, Bergeron JJ. SSR alpha and
associated calnexin are major calcium binding proteins of the
endoplasmic reticulum membrane. J Biol Chem
1991;266:19599–19610.
16. Ahluwalia N, Bergeron JJ, Wada I, Degen E, Williams DB. The p88
molecular chaperone is identical to the endoplasmic reticulum
membrane protein, calnexin. J Biol Chem 1992;267:10914–10918.
17. Ou WJ, Cameron PH, Thomas DY, Bergeron JJM. Association of
folding intermediates of glycoproteins with calnexin during protein
maturation. Nature 1993;364:771–776.
18. Parodi AJ, Cazzulo JJ. Protein glycosylation in Trypanosoma cruzi. II.
Partial characterization of protein-bound oligosaccharides labeled
“in vivo”. J Biol Chem 1982;257:7641–7645.
19. Trombetta SE, Parodi AJ. Purification to apparent homogeneity and
partial characterization of rat liver UDP-glucose:glycoprotein
glucosyltransferase. J Biol Chem 1992;267:9236–9240.
20. Sousa MC, Ferrero-Garcia MA, Parodi AJ. Recognition of the
oligosaccharide and protein moieties of glycoproteins by the
UDP-Glc:glycoprotein glucosyltransferase. Biochemistry
1992;31:97–105.
21. Suh P, Bergmann JE, Gabel CA. Selective retention of
monoglycosylated high mannose oligosaccarides by a class of mutant
vesicular stomatitis virus G proteins. J Cell Biol 1989;108:811–819.
22. Hammond C, Helenius A. A chaperone with a sweet tooth. Curr Biol
1993;3:884–885.
23. Hebert DN, Foellmer B, Helenius A. Glucose trimming and
reglucosylation determine glycoprotein association with calnexin in
the endoplasmic reticulum. Cell 1995;81:425–433.
321
Lamriben et al.
24. Peterson JR, Ora A, Nguyen Van P, Helenius A. Transient, lectin-like
association of calreticulin with folding intermediates of cellular and
viral glycoproteins. Mol Biol Cell 1995;6:1173–1184.
25. Shailubhai K, Pukazhenti BS, Saxena ES, Vrama GM, Vijay IK.
Guocosidase I, a transmemebrane endoplasmic reticular glycoprotein
with a luminal catalytic domain. J Biol Chem
1991;266:16587–16593.
26. Barker MK, Rose DR. Specificity of processing alpha-glucosidase I is
guided by the substrate conformation: crystallographic and in silico
studies. J Biol Chem 2013;288:13563–13574.
27. Barker MK, Wilkinson BL, Faridmoayer A, Scaman CH, Fairbanks AJ,
Rose DR. Production and crystallization of processing
alpha-glucosidase I: Pichia pastoris expression and a two-step
purification toward structural determination. Protein Expr Purif
2011;79:96–101.
28. Dejgaard K, Theberge JF, Heath-Engel H, Chevet E, Tremblay ML,
Thomas DY. Organization of the Sec61 translocon, studied by high
resolution native electrophoresis. J Proteome Res
2010;9:1763–1771.
29. Esmon B, Esmon PC, Scheckman R. Eraly steps in processing of yeast
glycoproteins. J Biol Chem 1984;259:10322–10327.
30. Ray MK, Yang J, Sundaram S, Stanley P. A novel glycosylation
phenotype expressed by Lec23, Chinese hamster ovary mutant
deficient in a-glucosidase I. J Biol Chem 1991;266:
22818–22825.
31. Trombetta ES, Simons JF, Helenius A. Endoplasmic reticulum
glucosidase II is composed of a catalytic subunit, conserved from
yeast to mammals, and a tightly bound non-catalytic
HDEL-containing subunit. J Biol Chem 1996;271:27509–27516.
32. Stigliano ID, Caramelo JJ, Labriola CA, Parodi AJ, D’Alessio C.
Glucosidase II beta subunit modulates N-glycan trimming in fission
yeasts and mammals. Mol Biol Cell 2009;20:3974–3984.
33. Pelletier MF, Marcil A, Sevigny G, Jakob CA, Tessier DC, Chevet E,
Menard R, Bergeron JJ, Thomas DY. The heterodimeric structure of
glucosidase II is required for its activity, solubility, and localization in
vivo. Glycobiology 2000;10:815–827.
34. Munro S. The MRH domain suggests a shared ancestry for the
mannose 6-phosphate receptors and other N-glycan-recognising
proteins. Curr Biol 2001;11:R499–R501.
35. Stigliano ID, Alculumbre SG, Labriola CA, Parodi AJ, D’Alessio C.
Glucosidase II and N-glycan mannose content regulate the half-lives
of monoglucosylated species in vivo. Mol Biol Cell
2011;22:1810–1823.
36. Deprez P, Gautschi M, Helenius A. More than one glycan is needed
for ER glucosidase II to allow entry of glycoproteins into the
calnexin/calreticulin cycle. Mol Cell 2005;19:183–195.
37. Bosis E, Nachliel E, Cohen T, Takeda Y, Ito Y, Bar-Nun S, Gutman M.
Endoplasmic reticulum glucosidase II is inhibited by its end products.
Biochemistry 2008;47:10970–10980.
38. Reitman ML, Trowbrideg IS, Kornfeld S. A lectin-resistant mouse
lymphoma cell line is deficient in glucosidase II, a glycoprotein
processing enzyme. J Biol Chem 1982;257:10357–10363.
322
39. Schrag JD, Bergeron JJ, Li Y, Borisova S, Hahn M, Thomas DY, Cygler
M. The structure of calnexin, an ER chaperone involved in quality
control of protein folding. Mol Cell 2001;8:633–644.
40. Vassilakos A, Michalak M, Lehrman MA, Williams DB.
Oligosaccharide binding characteristics of the molecular chaperone
calnexin and calreticulin. Biochemistry 1998;37:3480–3490.
41. Kapoor M, Srinivas H, Kandiah E, Gemma E, Ellgaard L, Oscarson S,
Helenius A, Surolia A. Interactions of substrate with calreticulin, an
endoplasmic reticulum chaperone. J Biol Chem
2003;278:6194–6200.
42. Li Z, Stafford WF, Bouvier M. The metal ion binding properties of
calreticulin modulate its conformational flexibility and thermal
stability. Biochemistry 2001;40:11193–11201.
43. Brockmeier A, Williams DB. Potent lectin-independent chaperone
function of calnexin under conditions prevalent within the lumen of
the endoplasmic reticulum. Biochemistry 2006;45:12906–12916.
44. Ou WJ, Bergeron JJ, Li Y, Kang CY, Thomas DY. Conformational
changes induced in the endoplasmic reticulum luminal domain of
calnexin by Mg-ATP and Ca2+ . J Biol Chem
1995;270:18051–18059.
45. Wijeyesakere SJ, Gagnon JK, Arora K, Brooks CL 3rd, Raghavan M.
Regulation of calreticulin-major histocompatibility complex (MHC)
class I interactions by ATP. Proc Natl Acad Sci USA
2015;112:E5608–E5617.
46. Ellgaard L, Riek R, Herrmann T, Guntert P, Braun D, Helenius A,
Wuthrich K. NMR structure of the calreticulin P-domain. Proc Natl
Acad Sci 2001;98:3133–3138.
47. Ellgaard L, Ruddock LW. The human protein disulphide isomerase
family: substrate interactions and functional properties. EMBO Rep
2005;6:28–32.
48. Frickel EM, Riek R, Jelesarov I, Helenius A, Wuthrich K, Ellgaard L.
TROSY-NMR reveals interaction between ERp57 and the tip of the
calreticulin P-domain. Proc Natl Acad Sci USA 2002;99:1954–1959.
49. Kozlov G, Maattanen P, Schrag JD, Pollock S, Cygler M, Nagar B,
Thomas DY, Gehring K. Crystal structure of the bb’ domains of the
protein disulfide isomerase ERp57. Structure 2006;14:1331–1339.
50. Kozlov G, Bastos-Aristizabal S, Maattanen P, Rosenauer A, Zheng F,
Killikelly A, Trempe JF, Thomas DY, Gehring K. Structural basis of
cyclophilin B binding by the calnexin/calreticulin P-domain. J Biol
Chem 2010;285:35551–35557.
51. Danilczyk UG, Williams DB. The lectin chaperone calnexin utilizes
polypeptide-based interactions to associate with many of its
substrates in vivo. J Biol Chem 2001;276:25532–25540.
52. Saito Y, Ihara Y, Leach MR, Cohen-Doyle MF, Williams DB.
Calreticulin functions in vitro as a molecular chaperone for both
glycosylated and non-glycosylated proteins. EMBO J
1999;18:6718–6729.
53. Wijeyesakere SJ, Rizvi SM, Raghavan M. Glycan-dependent and
-independent interactions contribute to cellular substrate recruitment
by calreticulin. J Biol Chem 2013;288:35104–35116.
54. Baksh S, Michalak M. Expression of calreticulin in Escherichia coli
and identification of its Ca2+ binding domains. J Biol Chem
1991;266:21458–21465.
Traffic 2016; 17: 308–326
The Calnexin Binding Cycle
55. Conte IL, Keith N, Gutierrez-Gonzalez C, Parodi AJ, Caramelo JJ. The
interplay between calcium and the in vitro lectin and chaperone
activities of calreticulin. Biochemistry 2007;46:4671–4680.
56. Sousa M, Parodi AJ. The molecular basis for the recognition of
misfolded glycoproteins by the UDP-Glc: glycoprotein
glucosyltransferase. EMBO J 1995;14:4196–4203.
57. Caramelo JJ, Parodi AJ. Getting in and out from calnexin/calreticulin
cycles. J Biol Chem 2008;283:10221–10225.
58. Tessier DC, Dignard D, Zapun A, Radominska-Pandya A, Parodi AJ,
Bergeron JJ, Thomas DY. Cloning and characterization of mammalian
UDP-glucose glycoprotein: glucosyltransferase and the development
of a specific substrate for this enzyme. Glycobiology
2000;10:403–412.
59. Breton C, Snajdrova L, Jeanneau C, Koca J, Imberty A. Structures and
mechanisms of glycosyltransferases. Glycobiology
2006;16:29R–37R.
60. Zhu T, Satoh T, Kato K. Structural insight into substrate recognition
by the endoplasmic reticulum folding-sensor enzyme: crystal
structure of third thioredoxin-like domain of
UDP-glucose:glycoprotein glucosyltransferase. Sci Rep 2014;4:7322.
61. Arnold SM, Fessler LI, Fessler JH, Kaufman RJ. Two homologues
encoding human UDP-glucose:glycoprotein glucosyltransferase differ
in mRNA expression and enzymatic activity. Biochemistry
2000;39:2149–2163.
62. Arnold SM, Kaufman RJ. The noncatalytic portion of human
UDP-glucose: glycoprotein glucosyltransferase I confers UDP-glucose
binding and transferase function to the catalytic domain. J Biol Chem
2003;278:43320–43328.
63. Takeda Y, Seko A, Hachisu M, Daikoku S, Izumi M, Koizumi A,
Fujikawa K, Kajihara Y, Ito Y. Both isoforms of human
UDP-glucose:glycoprotein glucosyltransferase are enzymatically
active. Glycobiology 2014;24:344–350.
64. Hebert DN, Foellmer B, Helenius A. Calnexin and calreticulin promote
folding, delay oligomerization and suppress degradation of influenza
hemagglutinin in microsomes. EMBO J 1996;15:2961–2968.
65. Vassilakos A, Cohen-Doyle MF, Peterson PA, Jackson MR, Williams
DB. The molecular chaperone calnexin facilitates folding and
assembly of class I histocompatibility molecules. EMBO J
1996;15:1495–1506.
66. Cannon KS, Hebert DN, Helenius A. Glycan dependent and
independent association of Vesicular Stomatitis Virus G Protein with
Calnexin. J Biol Chem 1996;271:14280–14284.
67. Branza-Nichita N, Petrescu AJ, Dwek RA, Wormald MR, Platt FM,
Petrescu SM. Tyrosinase folding and copper loading in vivo: a crucial
role for calnexin and alpha-glucosidase II. Biochem Biophys Res
Commun 1999;261:720–725.
68. Wang N, Daniels R, Hebert DN. The cotranslational maturation of the
type I membrane glycoprotein tyrosinase: the heat shock protein 70
system hands off to the lectin-based chaperone system. Mol Biol Cell
2005;16:3740–3752.
69. Wang N, Glidden EJ, Murphy SR, Pearse BR, Hebert DN. The
cotranslational maturation program for the type II membrane
Traffic 2016; 17: 308–326
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
glycoprotein influenza neuraminidase. J Biol Chem
2008;283:33826–33837.
Molinari M, Eriksson KK, Calanca V, Galli C, Cresswell P, Michalak
M, Helenius A. Contrasting functions of calreticulin and calnexin in
glycoprotein folding and ER quality control. Mol Cell
2004;13:125–135.
Hebert DN, Zhang JX, Chen W, Foellmer B, Helenius A. The number
and location of glycans on influenza hemagglutinin determine
folding and association with calnexin and calreticulin. J Cell Biol
1997;139:613–623.
Nakamura K, Zuppini A, Arnaudeau S, Lynch J, Ahsan I, Krause R,
Papp S, De Smedt H, Parys JB, Muller-Esterl W, Lew DP, Krause K-H,
Demaurex N, Opas M, Michalak M. Functional specialization of
calreticulin domains. J Cell Biol 2001;154:961–972.
Gao B, Adhikari R, Howarth M, Nakamura K, Gold MC, Hill AB, Knee
R, Michalak M, Elliott T. Assembly and antigen-presenting function
of MHC class I molecules in cells lacking the ER chaperone
calreticulin. Immunity 2002;16:99–109.
Jessop CE, Tavender TJ, Watkins RH, Chambers JE, Bulleid NJ.
Substrate specificity of the oxidoreductase ERp57 is determined
primarily by its interaction with calnexin and calreticulin. J Biol Chem
2009;284:2194–2202.
Frickel EM, Frei P, Bouvier M, Stafford WF, Helenius A, Glockshuber
R, Ellgaard L. ERp57 is a multifunctional thiol-disulfide
oxidoreductase. J Biol Chem 2004;279:18277–18287.
Zapun A, Darby NJ, Tessier DC, Michalak M, Bergeron JJ, Thomas DY.
Enhanced catalysis of ribonuclease B folding by the interaction of
calnexin or calreticulin with ERp57. J Biol Chem
1998;273:6009–6012.
Rutkevich LA, Cohen-Doyle MF, Brockmeier U, Williams DB.
Functional relationship between protein disulfide isomerase family
members during the oxidative folding of human secretory proteins.
Mol Biol Cell 2010;21:3093–3105.
Solda T, Garbi N, Hammerling GJ, Molinari M. Consequences of
ERp57 deletion on oxidative folding of obligate and facultative
clients of the calnexin cycle. J Biol Chem 2006;281:6219–6226.
Ellgaard L, McCaul N, Chatsisvili A, Braakman I. Co- and
post-translation protein folding in the ER. Traffic in press.
Bernasconi R, Solda T, Galli C, Pertel T, Luban J, Molinari M.
Cyclosporine A-sensitive, cyclophilin B-dependent endoplasmic
reticulum-associated degradation. PLoS One 2010:5(9).
Pearse BR, Gabriel L, Wang N, Hebert DN. A cell-based
reglucosylation assay demonstrates the role of GT1 in the quality
control of a maturing glycoprotein. J Cell Biol 2008;181:309–320.
Pearse BR, Tamura T, Sunryd JC, Grabowski GA, Kaufman RJ, Hebert
DN. The role of UDP-Glc:glycoprotein glucosyltransferase 1 in the
maturation of an obligate substrate prosaposin. J Cell Biol
2010;189:829–841.
Tannous A, Patel N, Tamura T, Hebert DN. Reglucosylation by
UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein
secretion but not degradation. Mol Biol Cell 2015;26:390–405.
Ferris SP, Jaber NS, Molinari M, Arvan P, Kaufman RJ.
UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes
323
Lamriben et al.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
324
substrate solubility in the endoplasmic reticulum. Mol Biol Cell
2013;24:2597–2608.
Solda T, Galli C, Kaufman RJ, Molinari M. Substrate-specific
requirements for UGT1-dependent release from calnexin. Mol Cell
2007;27:238–249.
Daniels R, Kurowski B, Johnson AE, Hebert DN. N-linked glycans
direct the cotranslational folding pathway of influenza
hemagglutinin. Mol Cell 2003;11:79–90.
Chen W, Helenius J, Braakman I, Helenius A. Cotranslational folding
and calnexin binding of influenza hemagglutinin in the endoplasmic
reticulum. Proc Natl Acad Sci USA 1995;92:6229–6233.
Ellgaard L, Helenius A. Quality control in the endoplasmic reticulum.
Nat Rev Mol Cell Biol 2003;4:181–191.
Keith N, Parodi AJ, Caramelo JJ. Glycoprotein tertiary and quaternary
structures are monitored by the same quality control mechanism.
J Biol Chem 2005;280:18138–18141.
Williams DB. Beyond lectins: the calnexin/calreticulin chaperone
system of the endoplasmic reticulum. J Cell Sci 2006;119:615–623.
Denzel A, Molinari M, Trigueros C, Martin JE, Velmurgan S, Brown S,
Stamp G, Owen MJ. Early postnatal death and motor disorders in
mice congenitally deficient in calnexin expression. Mol Cell Biol
2002;22:7398–7404.
Mesaeli N, Nakamura K, Zvaritch E, Dickie P, Dziak E, Krause K-H,
Opas M, MacLennan DH, Michalak M. Calreticulin is essential for
cardiac development. J Cell Biol 1999;144:857–868.
Coe H, Jung J, Groenendyk J, Prins D, Michalak M. ERp57 modulates
STAT3 signaling from the lumen of the endoplasmic reticulum. J Biol
Chem 2010;285:6725–6738.
Molinari M, Galli C, Vanoni O, Arnold SM, Kaufman RJ. Persistent
glycoprotein misfolding activates the glucosidase II/UGT1-driven
calnexin cycle to delay aggregation and loss of folding competence.
Mol Cell 2005;20:503–512.
Voeltz GK, Rolls MM, Rapoport TA. Structural organization of the
endoplasmic reticulum. EMBO Rep 2002;3:944–950.
Levine T, Rabouille C. Endoplasmic reticulum: one continuous
network compartmentalized by extrinsic cues. Curr Opin Cell Biol
2005;17:362–368.
Lynes EM, Simmen T. Urban planning of the endoplasmic reticulum
(ER): how diverse mechanisms segregate the many functions of the
ER. Biochim Biophys Acta 2011;1813:1893–1905.
Gilchrist A, Au CE, Hiding J, Bell AW, Fernandez-Rodriguez J,
Lesimple S, Nagaya H, Roy L, Gosline SJ, Hallett M, Paiement J,
Kearney RE, Nilsson T, Bergeron JJ. Quantitative proteomics analysis
of the secretory pathway. Cell 2006;127:1265–1281.
Smirle J, Au CE, Jain M, Dejgaard K, Nilsson T, Bergeron J. Cell
biology of the endoplasmic reticulum and the Golgi apparatus
through proteomics. Cold Spring Harb Perspect Biol
2013;5:a015073.
Wang WA, Groenendyk J, Michalak M. Calreticulin signaling in
health and disease. Int J Biochem Cell Biol 2012;44:842–846.
Schnell DJ, Hebert DN. Protein translocons: multifunctional mediators
of protein translocation across membranes. Cell 2003;112:491–505.
102. Zuber C, Fan JY, Guhl B, Parodi A, Fessler JH, Parker C, Roth J.
Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase
indicates involvement of pre-Golgi intermediates in protein quality
control. Proc Natl Acad Sci USA 2001;98:10710–10715.
103. Chen W, Helenius A. Role of ribosome and translocon complex
during folding of influenza hemagglutinin in the endoplasmic
reticulum of living cells. Mol Biol Cell 2000;11:765–772.
104. Brandt F, Etchells SA, Ortiz JO, Elcock AH, Hartl FU, Baumeister W.
The native 3D organization of bacterial polysomes. Cell
2009;136:261–271.
105. Cannon KS, Helenius A. Trimming and readdition of glucose to
N-linked oligosaccharides determines calnexin association of a
substrate glycoprotein in living cells. J Biol Chem
1999;274:7537–7544.
106. Nehls S, Snapp EL, Cole NB, Zaal KJ, Kenworthy AK, Roberts TH,
Ellenberg J, Presley JF, Siggia E, Lippincott-Schwartz J. Dynamics and
retention of misfolded proteins in native ER membranes. Nat Cell
Biol 2000;2:288–295.
107. Snapp EL, Sharma A, Lippincott-Schwartz J, Hegde RS. Monitoring
chaperone engagement of substrates in the endoplasmic reticulum of
live cells. Proc Natl Acad Sci USA 2006;103:6536–6541.
108. Hebert DN, Molinari M. In and out of the ER: protein folding, quality
control, degradation, and related human diseases. Physiol Rev
2007;87:1377–1408.
109. Frenkel Z, Gregory W, Kornfeld S, Lederkremer GZ. Endoplasmic
reticulum-associated degradation of mammalian glycoproteins
involves sugar chain trimming to Man6-5GlcNAc2. J Biol Chem
2003;278:34119–34124.
110. Benyair R, Ogen-Shtern N, Lederkremer GZ. Glycan regulation of
ER-associated degradation through compartmentalization. Semin
Cell Dev Biol 2015;41:99–109.
111. Clapham DE. Calcium signaling. Cell 2007;131:1047–1058.
112. Goetz JG, Genty H, St-Pierre P, Dang T, Joshi B, Sauve R, Vogl W,
Nabi IR. Reversible interactions between smooth domains of the
endoplasmic reticulum and mitochondria are regulated by
physiological cytosolic Ca2+ levels. J Cell Sci 2007;120:3553–3564.
113. Simmen T, Lynes EM, Gesson K, Thomas G. Oxidative protein folding
in the endoplasmic reticulum: tight links to the
mitochondria-associated membrane (MAM). Biochim Biophys Acta
2010;1798:1465–1473.
114. John LM, Lechleiter JD, Camacho P. Differential modulation of
SERCA2 isoforms by calreticulin. J Cell Biol 1998;142:963–973.
115. Roderick HL, Lechleiter JD, Camacho P. Cytosolic phosphorylation of
calnexin controls intracellular Ca(2+) oscillations via an interaction
with SERCA2b. J Cell Biol 2000;149:1235–1248.
116. Li Y, Camacho P. Ca2+−dependent redox modulation of SERCA 2b
by ERp57. J Cell Biol 2004;164:35–46.
117. Mehlmann LM, Terasaki M, Jaffe LA, Kline D. Reorganization of the
endoplasmic reticulum during meiotic maturation of the mouse
oocyte. Dev Biol 1995;170:607–615.
118. Kamhi-Nesher S, Shenkman M, Tolchinsky S, Vigodman Fromm S,
Ehrlich R, Lederkremer GZ. A novel quality control compartment
Traffic 2016; 17: 308–326
The Calnexin Binding Cycle
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
derived from the endoplasmic reticulum. Mol Biol Cell
2001;10:1711–1723.
van Anken E, Romijn EP, Maggioni C, Mezghrani A, Sitia R,
Braakman I, Heck AJ. Sequential waves of functionally related
proteins are expressed when B cells prepare for antibody secretion.
Immunity 2003;18:243–253.
Lieber CS. The discovery of the microsomal ethanol oxidizing system
and its physiologic and pathologic role. Drug Metab Rev
2004;36:511–529.
Chevet E, Wong HN, Gerber D, Cochet C, Fazel A, Cameron PH,
Gushue JN, Thomas DY, Bergeron JJ. Phosphorylation by CK2 and
MAPK enhances calnexin association with ribosomes. EMBO J
1999;18:3655–3666.
Cameron PH, Chevet E, Pluquet O, Thomas DY, Bergeron JJ. Calnexin
phosphorylation attenuates the release of partially misfolded
alpha1-antitrypsin to the secretory pathway. J Biol Chem
2009;284:34570–34579.
Martin BR, Cravatt BF. Large-scale profiling of protein palmitoylation
in mammalian cells. Nat Methods 2009;6:135–138.
Yount JS, Moltedo B, Yang YY, Charron G, Moran TM, Lopez CB,
Hang HC. Palmitoylome profiling reveals S-palmitoylation-dependent
antiviral activity of IFITM3. Nat Chem Biol 2010;6:610–614.
Yang W, Di Vizio D, Kirchner M, Steen H, Freeman MR. Proteome
scale characterization of human S-acylated proteins in lipid
raft-enriched and non-raft membranes. Mol Cell Proteomics
2010;9:54–70.
Merrick BA, Dhungana S, Williams JG, Aloor JJ, Peddada S, Tomer
KB, Fessler MB. Proteomic profiling of S-acylated macrophage
proteins identifies a role for palmitoylation in mitochondrial targeting
of phospholipid scramblase 3. Mol Cell Proteomics 2011;10:M110
006007.
Lakkaraju AK, Abrami L, Lemmin T, Blaskovic S, Kunz B, Kihara A,
Dal Peraro M, van der Goot FG. Palmitoylated calnexin is a key
component of the ribosome-translocon complex. EMBO J
2012;31:1823–1835.
Lynes EM, Raturi A, Shenkman M, Ortiz Sandoval C, Yap MC, Wu J,
Janowicz A, Myhill N, Benson MD, Campbell RE, Berthiaume LG,
Lederkremer GZ, Simmen T. Palmitoylation is the switch that assigns
calnexin to quality control or ER calcium signaling. J Cell Sci
2013;126:3893–3903.
Gold LI, Eggleton P, Sweetwyne MT, Van Duyn LB, Greives MR,
Naylor SM, Michalak M, Murphy-Ullrich JE. Calreticulin:
non-endoplasmic reticulum functions in physiology and disease.
FASEB J 2010;24:665–683.
Coppolino MG, Woodside MJ, Demaurex N, Grinstein S, St-Arnaud
R, Dedhar S. Calreticulin is essential for integrin-mediated calcium
signalling and cell adhesion. Nature 1997;386:843–847.
White TK, Zhu Q, Tanzer ML. Cell surface calreticulin is a putative
mannoside lectin which triggers mouse melanoma cell spreading. J
Biol Chem 1995;270:15926–15929.
Orr AW, Elzie CA, Kucik DF, Murphy-Ullrich JE. Thrombospondin
signaling through the calreticulin/LDL receptor-related protein
Traffic 2016; 17: 308–326
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
co-complex stimulates random and directed cell migration. J Cell Sci
2003;116:2917–2927.
Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A,
Murphy-Ullrich JE, Bratton DL, Oldenborg PA, Michalak M, Henson
PM. Cell-surface calreticulin initiates clearance of viable or apoptotic
cells through trans-activation of LRP on the phagocyte. Cell
2005;123:321–334.
Feng M, Chen JY, Weissman-Tsukamoto R, Volkmer JP, Ho PY,
McKenna KM, Cheshier S, Zhang M, Guo N, Gip P, Mitra SS,
Weissman IL. Macrophages eat cancer cells using their own
calreticulin as a guide: roles of TLR and Btk. Proc Natl Acad Sci USA
2015;112:2145–2150.
Nanney LB, Woodrell CD, Greives MR, Cardwell NL, Pollins AC,
Bancroft TA, Chesser A, Michalak M, Rahman M, Siebert JW, Gold LI.
Calreticulin enhances porcine wound repair by diverse biological
effects. Am J Pathol 2008;173:610–630.
Okazaki Y, Ohno H, Takase K, Ochiai T, Saito T. Cell surface
expression of calnexin, a molecular chaperone in the endoplasmic
reticulum. J Biol Chem 2000;275:35751–35758.
Dalziel M, Crispin M, Scanlan CN, Zitzmann N, Dwek RA. Emerging
principles for the therapeutic exploitation of glycosylation. Science
2014;343:1235681.
Perry ST, Buck MD, Plummer EM, Penmasta RA, Batra H, Stavale EJ,
Warfield KL, Dwek RA, Butters TD, Alonzi DS, Lada SM, King K, Klose
B, Ramstedt U, Shresta S. An iminosugar with potent inhibition of
dengue virus infection in vivo. Antiviral Res 2013;98:35–43.
Hussain S, Miller JL, Harvey DJ, Gu Y, Rosenthal PB, Zitzmann N,
McCauley JW. Strain-specific antiviral activity of iminosugars against
human influenza A viruses. J Antimicrob Chemother
2015;70:136–152.
Fischer PB, Collin M, Karlsson GB, James W, Butters TD, Davis SJ,
Gordon S, Dwek RA, Platt FM. The alpha-glucosidase inhibitor
N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry
at the level of post-CD4 binding. J Virol 1995;69:5791–5797.
Scanlan CN, Offer J, Zitzmann N, Dwek RA. Exploiting the defensive
sugars of HIV-1 for drug and vaccine design. Nature
2007;446:1038–1045.
Tierney M, Pottage J, Kessler H, Fischl M, Richman D, Merigan T,
Powderly W, Smith S, Karim A, Sherman J, et al. The tolerability and
pharmacokinetics of N-butyl-deoxynojirimycin in patients with
advanced HIV disease (ACTG 100). The AIDS Clinical Trials Group
(ACTG) of the National Institute of Allergy and Infectious Diseases. J
Acquir Immune Defic Syndr Hum Retrovirol 1995;10:549–553.
Butters TD, Dwek RA, Platt FM. Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses. Glycobiology 2005;15:43R–52R.
Allan G, Ouadid-Ahidouch H, Sanchez-Fernandez EM,
Risquez-Cuadro R, Fernandez JM, Ortiz-Mellet C, Ahidouch A. New
castanospermine glycoside analogues inhibit breast cancer cell
proliferation and induce apoptosis without affecting normal cells.
PLoS One 2013;8:e76411.
Toyofuku K, Wada I, Hirosaki K, Park JS, Hori Y, Jimbow K.
Promotion of tyrosinase folding in COS 7 cells by calnexin. J Biochem
(Tokyo) 1999;125:82–89.
325
Lamriben et al.
146. Ong DS, Mu TW, Palmer AE, Kelly JW. Endoplasmic reticulum Ca2+
increases enhance mutant glucocerebrosidase proteostasis. Nat
Chem Biol 2010;6:424–432.
147. Mu TW, Fowler DM, Kelly JW. Partial restoration of mutant enzyme
homeostasis in three distinct lysosomal storage disease cell lines by
altering calcium homeostasis. PLoS Biol 2008;6:e26.
148. Walsh G. Biopharmaceutical benchmarks--2003. Nat Biotechnol
2003;21:865–870.
149. Haredy AM, Nishizawa A, Honda K, Ohya T, Ohtake H, Omasa T.
Improved antibody production in Chinese hamster ovary cells by
ATF4 overexpression. Cytotechnology 2013;65:993–1002.
150. Cain K, Peters S, Hailu H, Sweeney B, Stephens P, Heads J, Sarkar K,
Ventom A, Page C, Dickson A. A CHO cell line engineered to express
XBP1 and ERO1-Lalpha has increased levels of transient protein
expression. Biotechnol Prog 2013;29:697–706.
151. Borth N, Mattanovich D, Kunert R, Katinger H. Effect of increased
expression of protein disulfide isomerase and heavy chain binding
326
protein on antibody secretion in a recombinant CHO cell line.
Biotechnol Prog 2005;21:106–111.
152. Vainchenker W, Delhommeau F, Constantinescu SN, Bernard OA.
New mutations and pathogenesis of myeloproliferative neoplasms.
Blood 2011;118:1723–1735.
153. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E,
Milosevic JD, Them NC, Berg T, Gisslinger B, Pietra D, Chen D,
Vladimer GI, Bagienski K, Milanesi C, Casetti IC, et al. Somatic
mutations of calreticulin in myeloproliferative neoplasms. N Engl J
Med 2013;369:2379–2390.
154. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC,
Avezov E, Li J, Kollmann K, Kent DG, Aziz A, Godfrey AL, Hinton J,
Martincorena I, Van Loo P, et al. Somatic CALR mutations in
myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med
2013;369:2391–2405.
Traffic 2016; 17: 308–326