Role of cysteine amino acid residues in calnexin

Helen Coe • Jeannine D. Schneider •

Monika Dabrowska • Jody Groenendyk •

Joanna Jung • Marek Michalak

Received: 6 April 2011 / Accepted: 27 July 2011 / Published online: 13 August 2011

� Springer Science+Business Media, LLC. 2011

Abstract Calnexin is an endoplasmic reticulum protein

that has a role in folding newly synthesized glycoproteins.

In this study, we used site-specific mutagenesis to disrupt

cysteine and histidine amino acid residues in the N- and

P-domains of calnexin and determined whether these

mutations impact the structure and function of calnexin.

We identified that disruption of the N-domain cysteines

resulted in significant loss of the chaperone activity of

calnexin toward the glycosylated substrate, IgY, while

disruption of the P-domain cysteines only had a small

impact toward IgY. We observed that wild-type calnexin as

well as the P-domain double cysteine mutant contained an

intramolecular disulfide bond which is lost when the

N-domain cysteines are mutated. Mutation to the N-domain

histidine and N-domain cysteines resulted in increased

binding of ERp57. Mutations to the P-domain cysteines

further enhanced ERp57 binding to calnexin. Taken toge-

ther, these observations indicated that the cysteine residues

within calnexin were important for the structure and

function of calnexin.

Keywords Calnexin � Protein folding � Endoplasmic

reticulum � Glycoprotein � Cysteine � Histidine � Mutation �ERp57 � Structure � Function

Abbreviations

ER Endoplasmic reticulum

CNX Calnexin

MDH Malate dehydrogenase

Introduction

The endoplasmic reticulum (ER) is a large, membrane-

bound organelle that has many critical functions within the

cell, including protein synthesis and posttranslational mod-

ification, lipid synthesis, stress responses, Ca2? homeostasis

and a major role in the quality control cycle of newly syn-

thesized proteins [1]. To ensure proteins fold correctly, many

chaperone proteins such as calnexin and calreticulin and

foldases, such as ERp57, are found within the ER [1]. Spe-

cifically, calnexin and calreticulin bind newly synthesized

glycoproteins in the ER and support their proper folding [2],

while the oxidoreductase ERp57 catalyzes disulfide bond

formation in the newly synthesized protein [2].

Calnexin is a 90-kDa type I integral membrane protein

that has a structural and functional domain architecture

consisting of 4 major domains: the N-globular domain, the

P-arm domain, both located in the ER lumen, the trans-

membrane domain and the C-tail domain located in the

cytoplasm [3]. The N-globular and P-arm domains form a

folding module exposed in the lumen of ER [3]. The

N-globular domain contains the primary lectin binding site

and a Zn2? binding site [4], while the P-domain is a long

extended flexible arm domain that is involved in interaction

with ERp57 [3].

Recent site-specific mutagenesis studies with calreticu-

lin have identified H153 and Y302 in the N-domain and Y244

in the P-domain as being critical for calreticulin chaperone

H. Coe � M. Michalak

Departments of Pediatrics, School of Molecular and Systems

Medicine, University of Alberta, Edmonton,

AB T6G 2H7, Canada

H. Coe � J. D. Schneider � M. Dabrowska � J. Groenendyk �J. Jung � M. Michalak (&)

Department of Biochemistry, School of Molecular and Systems

Medicine, University of Alberta, Edmonton,

AB T6G 2H7, Canada

e-mail: Marek.Michalak@ualberta.ca

123

Mol Cell Biochem (2012) 359:271–281

DOI 10.1007/s11010-011-1021-0

function [5, 6]. Additionally, E239, E243, D241, Y302 and

Y244 in the P-domain of calreticulin play a role in binding

of ERp57 [5]. In calnexin, residues E351 and Y428 located in

the P-domain and N-domain, respectively, show decreased

chaperone activity for glycosylated substrates [7]. Calnexin

contains two highly conserved cysteine residues in the

N-domain, C161 and C195, similar to calreticulin, and two

cysteine residues in the P-domain, C361 and C367, that are

specific to calnexin and not found in the calreticulin

P-domain. These cysteine residues are conserved through-

out all species, and crystallographic analysis of calnexin

has predicted that they form putative disulfide bonds [8].

The contribution of these predicted disulfide bonds in both

the N-domain and P-domain to structure and/or function of

calnexin remains unknown; however, it is suggested that

disulfide bonds are important to maintain calnexin in the

proper conformation for ATP binding [9] and may affect

carbohydrate binding [8].

In this study, we examined the role that cysteine resi-

dues play in the chaperone function of calnexin. We

focused on the cysteine residues located within the

N-globular domain and in the P-domain of calnexin. We

found that mutations of C161, C195, C361, or C367 did not

significantly impact the trypsin accessible structure of

calnexin and had no impact on the chaperone function of

calnexin for non-glycosylated substrate, MDH. However,

the double mutation C161/195 efficiently prevented the

aggregation of the non-glycosylated substrate, MDH, better

than wild-type calnexin. Both double mutations C161/195

and C361/367 lost their ability to efficiently prevent thermal

aggregation of the glycosylated substrate, IgY, with the

C161/195 mutation completely losing any chaperone func-

tion. We examined the role of these mutations in calnexin

interaction with ERp57 and determined that mutations of

H202 and the N-domain cysteines enhanced ERp57 binding

by 2–2.5-fold, while the P-domain C361/367 mutation

resulted in greatly increased ERp57 binding, up to 5-fold.

This study demonstrates the importance of the cysteine

residues found in the N-globular domain and P-arm domain

toward the structure and function of calnexin.

Materials and methods

Chemicals and reagents

Trypsin, malate dehydrogenase (MDH), ATP-Mg2?,

CaCl2, ZnCl2, and Dulbecco’s modified Eagle’s medium

were from Sigma. Fetal bovine serum was from Invitrogen.

SDS–PAGE reagents and molecular weight markers were

from Bio-Rad. Ni2?-nitrilotriacetic acid-agarose beads

(Ni–NTA) were from Qiagen. CM5 sensor chips, amine

coupling kit and the BIA evaluation analysis program were

from BIAcore Inc. (GE). All chemicals were of the highest

grade available.

Cloning, site-directed mutagenesis, and purification

of proteins

For E. coli expression of wild-type soluble calnexin with a

histidine tag, the wild-type soluble N- and P- domain of

calnexin was cloned into the restriction sites Nco1 and

Xba1 of pBAD/glIII to generate pBad-HisCNX, as

described previously [7]. Site-specific mutagenesis on

pBad-HisCNX was carried out using protocols from

QuickChange Site-Directed Mutagenesis Kit (Stratagene).

In brief, megaprimers were designed and mutants were

generated using Pfu DNA polymerase and a Gene Amp

PCR system 9700 Thermal Cycler. The following mutants

were created: CNX-H202A, CNX-H219A, CNX-C161A,

CNX-C195A, CNX-C161/195A, CNX-C361A, CNX-C367A

and CNX-C361/367A. Alanine was chosen in part due to

non-reactive conditions of this amino acid. Proteins were

expressed in Top10F’ E. coli, and the His-tagged proteins

were purified by one-step Ni2?-nitrilotriacetic acid-agarose

affinity chromatography under native conditions [5, 7].

Protein concentration was determined spectrophotometri-

cally (Bio-Rad protein assay) or using a Beckman System

6300 amino acid analyzer [5, 7]. Human ERp57 was

expressed in BL21 E. coli and purified as described pre-

viously [10, 11].

For expression in eukaryotic cells, cDNA encoding the

full-length calnexin was isolated from mouse brain library

using the Gateway Cloning Technology (Invitrogen) [12].

The following DNA primers were used for the amplifica-

tion of full-length calnexin: 50-GGG GAC AAG TTT

GTA CAA AAA AGC AGG CTT CAC CAT GGA AGG

GAA GTG GTT ACT-30 (forward) and 50-GGG GAC

CAC TTT GTA CAA GAA AGC TGG GTC TCA CTC

TCT TCG TGG CTT TCT0-30 (reverse). The forward pri-

mer contained an attB1 recombination site (bold), Kozak

site for expression in mammalian cells (italics) and caln-

exin gene-specific nucleotides (underlined). The reverse

primer contained attB2 recombination site (bold) and the

calnexin gene-specific nucleotides (underlined). First, to

generate entry clone vectors, a BP Clonase Reaction was

carried out as recommended by the manufacturer. Briefly,

the recombination reaction was carried out using BP

Clonase Enzyme Mix to insert full-length calnexin and the

ubiquitous promoter EF1a (cellular polypeptide chain

elongation factor 1 alpha) into the lentiviral destination

vector 2K7bsd (containing a blasticidine resistance gene for

cell selection). The resulting expression vector (p2K7-

CNX) was used as a template for site-specific mutagenesis.

The following mutants were created: CNX-H202A, CNX-

H219A, CNX-C161A, CNX-C195A, CNX-C161/195A, CNX-

272 Mol Cell Biochem (2012) 359:271–281

123

C361A, CNX-C367A, and CNX-C361/367A. Cells were

selected with 10 lg/ml of blasticidine (Gibco).

Western blot analysis and immunostaining

For Western blot analysis, cells were solubilized in RIPA

buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA,

1 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium

deoxycholate) containing protease inhibitors (0.5 mM

phenylmethylsufonyl fluoride (PMSF), 0.5 mM benzami-

dine, 0.05 lg/ml Na-tosyl-Lys-chloromethylketone (TLCK),

0.05 lg/ml 4-amidoinophenyl-methanesulfonyl fluoride

hydrochloride monohydrate (APMSF), 0.05 lg/ml (2S,3S)-

3-(N-f3dcarbamoyl)oxirane-2-carboxylic acid (E-64),

0.025 lg/ml leupeptin and 0.01 lg/ml pepstatin) [13]. Thirty

lg of protein lysate was separated by SDS–PAGE (10%

acrylamide), transferred to nitrocellulose membrane and

probed with rabbit-anti-calnexin antibodies (StressGen) at a

dilution of 1:1000 and rabbit-anti-tubulin antibodies (Stress-

Gen) at a dilution of 1:1000 [14]. Where indicated, protein

lysate was in 3X non-reducing buffer (30% glycerol, 6% SDS,

195 mM Tris, pH 8.0 and 0.01% bromophenol blue) or 3X

reducing buffer (30% glycerol, 6% SDS, 195 mM Tris, pH

8.0, 15% b-mercaptoethanol and 0.01% bromophenol blue).

Blots were developed using a chemiluminescent system [15].

For immunostaining, cells were fixed with 3.7% parafor-

maldehyde in PBS for 20 min at room temperature and

washed 3 times with PBS [14]. Cells were permeabilized in a

buffer containing 0.1% Triton X-100, 100 mM PIPES, pH

6.9, 1 mM EGTA, and 4% w/v polyethylene glycol 8,000, for

2 min and washed 3 times with PBS [16]. Primary antibody

rabbit-anti-calnexin (StressGen) was used at a dilution of

1:100 in PBS, and the secondary antibody was rabbit conju-

gated-FITC (fluorescein isothiocynate) (Invitrogen) at a

dilution of 1:200 in PBS. Coverslips were co-stained with

Alexa Fluor 546-Concanavalin A at a dilution of 1:1000

(Sigma) [12]. The coverslips were visualized using spinning

disk confocal microscopy (WaveFX from Quorum Technol-

ogies, Guelph, Canada) with an Olympus IX-81 inverted

stand (Olympus). Images were acquired using a 60X objective

(N.A. 1.42) with an EMCCD camera (Hamamatsu, Japan).

The fluorescent dyes, FITC and Alexa Fluor 546, were excited

using a 491 and a 561 nm laser line, respectively (Spectral

Applied Research). Z-slices (0.25 lM) were acquired using

Volocity Software (Improvision) through the cells using a

piezo z-stage (Applied Scientific Instrumentation).

Aggregation assays

Protein aggregation analysis of non-glycosylated substrate

was carried out using 2 lM of denatured MDH re-sus-

pended in 50 mM sodium phosphate buffer, pH 7.5 and

0.2 lM of wild-type or mutant calnexin re-suspended in

10 mM Tris pH 7.0 and 1 mM EDTA. Buffer containing

50 mM sodium phosphate, pH 7.5, was incubated at 45�C

followed by the addition of both denatured MDH and

0.2 lM of wild-type or mutant calnexin, followed by

measurement for light scattering [6, 7, 17]. Protein aggre-

gation analysis of glycosylated substrate was carried out

using IgY isolated from chicken egg yolk using the EGG-

stract IgY purification method (Promega) [6]. After dena-

turing, 0.25 lM of IgY was re-suspended in a buffer con-

taining 10 mM Tris–HCl, pH 7.0, 150 mM NaCl and

5 mM CaCl2. Buffer containing 10 mM Tris–HCl, pH 7.0,

150 mM NaCl and 5 mM CaCl2 was incubated to 45�C

followed by the addition of both 0.2 lM of IgY and

0.125 lM of wild-type or mutant calnexin [6]. Light

scattering was measured at an excitation wavelength of

320 nm and an emission wavelength of 360 nm for 1 h

using a spectrofluorometer system C43/2000 (PTI) equip-

ped with a temperature-controlled cell holder [6, 17]. Both

MDH and IgY aggregation assays were performed 3 times

each with wild-type or mutant calnexin and averages were

plotted [5].

Proteolytic digestions

For trypsin digestion, 10 lg of purified, recombinant wild-

type or calnexin mutant protein was re-suspended in a

buffer containing 10 mM Tris–HCl, pH 7.0, 1 mM MgCl2,

and 100 mM KCl. Protein samples were incubated with

trypsin at 1:100 (trypsin:protein w/w) in the presence or

absence of 2 mM CaCl2, 1 mM ZnCl2? or 1 mM ATP-

Mg2?. Samples of digested proteins were taken at 0, 0.5, 2,

5, 10, and 20 min, separated by SDS–PAGE (12.5%

acrylamide) and stained with Coomassie Blue [5].

Surface plasmon resonance

A CM5 sensor chip was activated, coupled and blocked

using an Amine Coupling Kit from BIAcore, as described

previously [5, 7]. In brief, two lanes of a CM5 sensor chip

were activated using a 1:1 mixture of 1-ethyl-3-(3-dime-

thyl-aminopropyl) carbodiimide HCl (EDC) and

N-hydroxysuccinimide (NHS) for 7 min at a flow rate of

5 ll/min. One lM of purified wild-type calnexin or caln-

exin mutant protein was re-suspended in 10 mM sodium

acetate at pH 4.0 and coupled to one lane (second lane used

as control) at a flow rate of 5 ll/min until approximately

1,000–3,000 RU (resonance units) of protein were cova-

lently coupled. Both the ligand-bound lane and the control

lane were blocked using 1 M ethanolamine-HCl at pH 8.5

for 7 min at a flow rate of 5 ll/min. To monitor homoge-

nous, binary interactions, kinetic analysis experiments

were performed at 21�C in running buffer containing

20 mM Tris–HCl, pH 7.0, 135 mM KCl, 2 mM CaCl2,

Mol Cell Biochem (2012) 359:271–281 273

123

0.05% Tween-20 and protease inhibitors. His-tagged

human ERp57 was used as the analyte in a concentration

series curve from 5,000 to 5 nM. Kinetic analysis was

performed using a 1:1 Langmuir binding with drifting

baseline as a model (BIAanalysis software) to generate

equilibrium and rate constants. The maximum relative

response was calculated based on the predicted Rmax and

normalized to wild-type control.

Results

Expression of calnexin mutants in calnexin-deficient

cells

Site-specific mutagenesis of calnexin was carried out in

order to determine whether any of these mutations affected

the localization, structure, and function of calnexin. All

targeted amino acid residues were mutated to the neutral,

small, non-polar alanine residue because it does not alter

the main-chain conformation of the protein, nor does it

impose extreme electrostatic or steric effects [18]. In the

N-terminal domain, we created C161A, C195A, C161/195A

and H202A mutants. The H202 residue is conserved between

calnexin and calreticulin (H153), and in calreticulin,

mutation of H153 impacts chaperone function [6] (Fig. 1).

H219 is also important to investigate as it may have a role in

the chaperone function of calnexin through the coordina-

tion of divalent cations [6] (Fig. 1). The cysteine residues

(C361, C367, and C361/367) in the P-arm domain were

mutated (Fig. 1) as they were unique for calnexin and not

found in calreticulin.

Calnexin-deficient (cnx-/-) mouse embryonic fibroblast

were lentivirally transduced with wild-type calnexin or

calnexin mutants and stable cell lines were generated [12,

14]. Western blot analysis showed that all the cell lines

expressed recombinant protein (Fig. 2a), confirmed by

immunostaining for calnexin (Fig. 2b). Figure 2b shows

that, morphologically, all mutant cells lines appeared to

have intact ER by counterstaining with Concanavalin A, an

ER marker. Additionally, immunostaining demonstrated

that all calnexin mutants localized to an ER-like network

(Fig. 2b). Therefore, mutation of the calnexin residues

C161, C195, H202, H219, C361, and C367 to alanine had no

impact on expression or ER localization.

Structural analysis of calnexin mutants

We used limited proteolysis analysis to determine whether

the histidine and the cysteine residues of calnexin mutated

Fig. 1 Model of calnexin with

mutated residues are

highlighted. a A linear model of

truncated soluble dog calnexin

including the N-globular

domain (N) and P-domain

(P) with residues mutated

indicated. Amino acid

boundaries of domains is

shown. b The crystal structure

of truncated soluble calnexin.

c The crystal structure of the

N-domain of calnexin with

residues mutated in this study is

labeled. d The crystal structure

of the P-domain of calnexin

with cysteine residues mutated

in this study is labeled

274 Mol Cell Biochem (2012) 359:271–281

123

in this study contributed significantly to the structural sta-

bility of the protein [5–7]. For structural analysis, the ER

luminal domain (N ? P-domain) of calnexin and calnexin

mutants were expressed in E. coli and purified (Fig. 3),

followed by limited proteolysis analysis on soluble, wild-

type calnexin and calnexin mutants (H202A, H219A, C161A,

C195A, C161/195A, C361A, C367A, and C361/367A) in the

absence or presence of Ca2?, Zn2?, and ATP (Fig. 4).

Congruent with previous studies [5–7], we determined

that calnexin was partially resistant to trypsin digestion as a

fraction of full-length calnexin at the 20 min time point

was present (Fig. 4a). There was a slight increase in the

amount of protein resistant to trypsin digestion at the 5 min

time point in the C161/195 calnexin double mutant as com-

pared to the wild-type calnexin (Fig. 4a). There was no

observed impact on trypsin digestion with mutation of

H202A or C361/367 (Fig. 4a) and H219A, C161A, C195A,

C361A or C367A (data not shown).

Previous studies show that ATP binds to calnexin,

inducing a conformational change [9]. Mutation of residues

H202 and H219 in the globular domain of calnexin demon-

strated increased protection against trypsin digestion in the

presence of ATP (Fig. 4b), suggesting that the binding of

ATP to calnexin may be altered with these mutations,

tubulin

CNX

wt

cnx-/-

H202

H219

C195

C361

C367

C 161/

195

C361/

367

C161

ConA CNX MergeConA CNX Merge

H 219

wt

cnx

H 202

C161

C195

C367

C361

C361/367

C161/195

A

B

Fig. 2 Expression of calnexin mutants in calnexin-deficient mouse

embryonic fibroblasts is of normal quantity and location. a Western

blot analysis of protein lysates for wild-type (wt) and mutant calnexin

(CNX) using an anti-calnexin antibody and an antibody for tubulin

(loading control), isolated from calnexin-deficient cell lines stably

expressing calnexin mutants as described under ‘‘Materials and

methods’’. Mutant calnexin is labeled by residue number. b In the

second panel, normal immunolocalization is shown for calnexin and

calnexin mutants (CNX-labeled with FITC (fluorescein isothiocya-

nate)) in calnexin-deficient cell lines. In the first panel, Concanavalin

A (ConA) covalently linked to Alexa Fluor 546 is shown. The thirdpanel merges the first two panels. Both calnexin and mutated calnexin

localized to an ER-like network. Scale bar, 17 lm

Mol Cell Biochem (2012) 359:271–281 275

123

resulting in a different proteolysis pattern when compared

with wild-type calnexin. Additionally, we observed

increased protection against trypsin digestion with calnexin

containing the C367 mutation in the globular domain, sug-

gesting this residue may impact ATP binding (Fig. 4b). In

the presence of ATP, there was no observable change in the

trypsin digestion pattern in the Cys161 mutant (Fig. 4b), or

in C195, and the double mutants, C161/195 and C361/367 (data

not shown).

Calnexin contains a Ca2? binding site within the

N-globular domain, with binding altering protein confor-

mation [3, 8, 9]. In the presence of Ca2?, we observed

substantial differences in trypsin digestion patterns of wild-

type calnexin when compared with wild-type calnexin in

the absence of Ca2?, with loss of protection of full-length

protein upon the addition of Ca2? (Fig. 4a, c). This dem-

onstrated a role for Ca2? in the tertiary structure of caln-

exin. Interestingly, addition of Ca2? to the C161/195

mutation exhibited protection from limited trypsin prote-

olysis when compared with wild-type calnexin with Ca2?,

with full-length protein present at the 20 min time point

(Fig. 4c). The C161/195 residues are located in the globular

N-domain and may play a role in Ca2? binding, or orien-

tation of the Ca2? ion. No significant differences in trypsin

digestion patterns in the presence of Ca2? were observed in

H202, H219, C367, C361/367 (Fig. 4c), C161, C195, and C361

(data not shown) when compared with wild-type calnexin.

Calnexin binds Zn2? within its N-globular domain [4],

and the importance of the co-factor Zn2? in the function of

calnexin has been previously demonstrated [4]. We

examined whether mutations within the N-globular domain

or P-arm domain of calnexin resulted in conformational

changes in the presence of Zn2?. The addition of Zn2?

resulted in greater trypsin accessibility toward wild-type

calnexin protein (compare CNX, Fig. 4a, d). This was not

surprising as previous studies with calreticulin have shown

that in the presence of 100 lM Zn2?, large hydrophobic

patches of the protein are exposed, suggesting that a similar

event may be occurring in calnexin, in that the binding of

Zn2? may result in the exposure of large hydrophobic

patches in calnexin [4, 19]. Mutation of N-globular domain

residues, H202, H219, and C161/195 resulted in increased

protection of calnexin to trypsin digestion in the presence

of Zn2? when compared with wild type, with the presence

of full-length calnexin as late as 10 min (Fig. 4d). As well,

we observed increased protection to trypsin digestion with

the mutation of residues found in the P-arm domain (C361

and C367) (Fig. 4d). Most strikingly, there was increased

protection to trypsin digestions in the double C361/367

mutant in the presence of Zn2?, with a strong, persistent

presence of full-length calnexin up to 20 min (Fig. 4d),

suggesting that these residues may have a role in the

coordination of Zn2?. This was of interest as ERp57

binding has been mapped to the P-arm domain of calnexin

[20] and may be a Zn2?-dependent interaction [4].

To determine whether disulfide bridges are formed

within calnexin, we isolated protein lysates from cnx-/-

cells expressing wild-type calnexin or the mutants C161/195

and C361/367A, and analyzed the lysate by SDS–PAGE

under reducing and non-reducing conditions, followed by

Western blot analysis with calnexin antibodies. As expec-

ted, under non-reducing conditions, we distinguished a

shift in mobility in wild-type calnexin as compared to

reducing conditions, indicating the presence of disulfide

bridge(s) (Fig. 5). We also saw a shift in the mobility under

non-reducing versus reducing conditions for cells

expressing the P-domain C361/367 mutant (Fig. 5), sug-

gesting the continued presence of a disulfide bond. How-

ever, in cells expressing the N-domain C161/195 mutant, the

mobility shift under non-reducing versus reducing condi-

tions was lost (Fig. 5). This qualitative approach suggested

that calnexin contains a disulfide bond forming between the

N-globular C161-C195 residues that affected protein

mobility. When the P-domain C361/367 mutant was ana-

lyzed, the mobility shift was not lost and remained similar

to the wild type mobility shift, suggesting that the

P-domain C361-C367 did not form a disulfide bridge or that

conformational changes under reducing and non-reducing

conditions were too small to be detected using SDS–

PAGE. This indicates that the disulfide bond in calnexin

must be forming between C161-C195 and not C361-C367.

Cysteine mutants lost their ability to prevent

aggregation of IgY but not MDH

In order to investigate the roles of residues H202, H219,

C161, C195, C361, and C367 in calnexin chaperone activity,

we measured their ability to prevent thermal aggregation of

two substrates, MDH, a non-glycosylated substrate and

IgY, a glycosylated substrate. Soluble, wild-type calnexin

Fig. 3 SDS–PAGE of calnexin and calnexin mutants expressed in E.coli and purified. Wild-type (wt) soluble calnexin and soluble

calnexin mutants indicated by mutant residue were expressed in E.

coli and purified. Proteins were separated by SDS–PAGE (10%

acrylamide) followed by staining with Coomassie Blue as described

under ‘‘Materials and methods’’

276 Mol Cell Biochem (2012) 359:271–281

123

was able to prevent thermal aggregation of the MDH

substrate (Fig. 6). Mutation of residues H202, H219, C161,

C195, C361, C367 (data not shown), and C361/367 in calnexin

did not significantly change the thermal aggregation of

MDH as compared to wild-type calnexin (Fig. 6).

However, the double mutant, C161/195 in the N-globular

domain of calnexin, demonstrated enhanced prevention of

aggregation of the MDH substrate when compared with

wild-type protein (Fig. 6). This suggested that the double

mutation to the residues C161 and C195 positively impacted

A B D

C

Fig. 4 Trypsin digestion patterns of calnexin and calnexin mutants.

Calnexin and calnexin mutants indicated by mutant residue were

expressed in E. coli, purified, and incubated with trypsin at 1:100

(trypsin/protein; w/w) at 37�C. Aliquots were taken at the time points

indicated, and the proteins were separated by SDS–PAGE and stained

with Coomassie Blue. Proteins were treated with trypsin in the

presence of 0.1 mM EGTA (no Ca2?) (a), 1 mM Mg-ATP (b), 1 mM

Ca2? (c) or 0.1 mM Zn2? (d) as described under ‘‘Materials and

methods’’. The arrows indicate the location of calnexin. CNX,

calnexin. Location of protein ladder is indicated to the left of each

panel

Mol Cell Biochem (2012) 359:271–281 277

123

the folding ability of calnexin toward non-glycosylated

substrate.

Next, we examined the effect of calnexin mutations on

the glycosylated substrate, IgY. Figure 7 shows that both

double cysteine mutations, C161/195 and C361/367, were less

efficient in preventing thermal aggregation of the IgY

substrate as compared to wild-type calnexin, while IgY

thermal aggregation with the H202 mutation was not sig-

nificantly different than wild-type calnexin (Fig. 7). Spe-

cifically, mutations to the N-globular domain cysteines

(C161/195) actually resulted in enhanced aggregation of IgY,

higher than IgY alone, losing any ability to prevent

aggregation of IgY. This suggested that mutation of these

cysteine residues significantly impacted folding of the gly-

cosylated substrate. The P-arm domain cysteines (C361/367)

were not able to completely prevent aggregation of IgY

(approximately 50%) as compared to wild-type calnexin.

This suggested that the C361/367 residues were moderately

influential toward the chaperone function of calnexin. In

summary, we determined that the N-globular domain

cysteine residues C161 and C195 were important for the

chaperone activity of calnexin toward both glycosylated

and non-glycosylated substrates.

Interaction of calnexin alanine mutants with ERp57

Cross-linking studies demonstrate a functional association

of calnexin with ERp57 that brings ERp57 into close

proximity to newly synthesized proteins to carry out

isomerization of disulfide bonds [10, 21]. Genetic screens,

NMR studies and mutagenesis analysis show that the

C-domain of ERp57 interacts with the P-domain of caln-

exin [20]. We used BIAcore Surface Plasmon Resonance

techniques to investigate whether the residues H202,

C161/195, and C361/367 had any impact on the interaction of

calnexin with ERp57. Consistent with previous observa-

tions [7], an increase in mass was seen for wild-type

calnexin and ERp57, demonstrating an interaction between

the two proteins (Fig. 8). Both the binding constants

(association tendency, KA and dissociation tendency, KD)

of ERp57 to wild-type and calnexin mutants were within

the normal parameters, not only confirming that the system

was functional, but also indicating affinity of ERp57 for

calnexin (Table 1). The rate constants (Ka, Kd) of ERp57

for wild-type calnexin and the calnexin mutants were also

within normal parameters; however, differences in the rate

of association and disassociation of ERp57 to calnexin and

calnexin mutants were observed (Table 1). When com-

paring the rates of complex formation (Ka), the C361/367

P-domain mutant had a slower complex formation than

wild-type, H202 and C161/195 calnexin mutants (Table 1).

When observing the dissociation rate, all three calnexin

Fig. 5 Calnexin under non-reducing and reducing conditions.

Western blot analysis of calnexin and calnexin double cysteine

mutants expressed in calnexin-deficient (cnx-/-) cells under reducing

or non-reducing conditions as described under ‘‘Materials and

methods’’. The location of reduced and non-reduced calnexin is

indicated by the arrows. WT, wild-type

Fig. 6 Effect of soluble wild-type calnexin and calnexin mutants on

the thermal aggregation of the non-glycosylated substrate, malate

dehydrogenase (MDH). Wild-type, C161/195, and C361/367 calnexin

mutants were incubated with heat-treated MDH and thermal aggre-

gation was monitored over time at 360 nm as described under

‘‘Materials and methods’’. All graphs were normalized to soluble

wild-type calnexin. Experiments were performed 3 times with the

average plotted

Fig. 7 Effect of soluble calnexin and calnexin mutants H202, C161/195,

and C361/367 on the thermal aggregation of the glycosylated substrate,

IgY. Wild-type, H202, C161/195, and C361/367 calnexin mutants were

incubated with IgY and thermal aggregation was monitored over time

at 360 nm as described under ‘‘Materials and methods’’. All graphs

were normalized to soluble wild-type calnexin. Experiments were

performed 3 times with the average plotted

278 Mol Cell Biochem (2012) 359:271–281

123

mutants formed a less stable complex with ERp57 when

compared with wild-type, with a faster dissociation rate

(the C361/367 mutant having the least stable complex)

(Table 1). The H202 and C161/195 N-globular domain

mutants bound 2–2.5-fold more ERp57 than wild-type

calnexin, while the C361/367 P-domain mutant bound

approximately 5-fold more ERp57 when compared with

wild-type (Fig. 8). The observed increase in bound ERp57

to the N-domain mutants may result from an indirect

influence on the P-domain that could potentially expose

novel residues involved in ERp57 binding. This phenom-

enon has been observed previously with calreticulin, where

N-domain mutations result in increased ERp57 binding [5,

7]. Interestingly, the C361/367 mutation resulted in a 5-fold

increase in binding of ERp57 as compared to wild-type

calnexin (Fig. 8), suggesting mutation of the P-domain

cysteines may have induced conformational exposure of

nascent amino acid residues involved in ERp57 binding.

Discussion

In this study, we carried out functional and structural anal-

ysis of N- and P-domain mutants of calnexin. We focused

on the cysteine residues in the N-domain (C161, C195) which

are predicted to form a disulfide bridge [8, 9] and the

P-domain cysteine residues (C361, C367) that are unique to

calnexin [8, 9]. We examined the role of histidine residues

in the N-globular domain of calnexin, particularly H202 as

the corresponding histidine in calreticulin (H153) is critical

to the chaperone function of calreticulin [6]. Consistent

with earlier studies, we found that wild-type, soluble caln-

exin prevented aggregation of both glycosylated substrate

(IgY) and to a lesser extent, non-glycosylated substrate

(MDH) [7]. Therefore, calnexin may bind both glycosylated

and non-glycosylated substrates, but shows specificity for

glycosylated substrate [7]. Both the C361/367 and the C161/195

residues were important for folding of glycosylated sub-

strates, as we observed an increase in the thermal aggre-

gation of IgY when these residues were mutated. The

N-globular domain cysteines are conserved within calreti-

culin (C88 and C120), and mutation to these residues results

in partial disruption of chaperone activity of calreticulin [5].

Similar mutation to the N-globular domain cysteines of

calnexin, C161/195, resulted in considerable loss of chaper-

one activity, as demonstrated by significantly enhanced

thermal aggregation of IgY. Structural studies on calnexin

have modeled a putative carbohydrate binding site in the

N-globular domain of calnexin with the third mannose

residue of the oligosaccharide wrapping around the C161

and C195 residues [8]. The involvement of the C161/195

residues in the carbohydrate binding site [8] was further

demonstrated by mutation of the C161 and C195 residues,

resulting in enhanced folding of the non-glycosylated sub-

strate, MDH, but complete loss of folding toward the gly-

cosylated substrate IgY, suggesting these two cysteines may

be forming an important part of the carbohydrate binding

Fig. 8 Maximum interaction of soluble wild-type calnexin and H202,

C161/195, and Cys361/367 calnexin mutants with recombinant ERp57 by

Surface Plasmon Resonance. Purified soluble calnexin and calnexin

mutants were covalently bound to a sensor chip and ERp57 injected

over the chip with mass changes recorded as described under

‘‘Materials and methods’’. The bar graphs are the Rmax values of

wild-type calnexin and calnexin mutants normalized for molecular

weight and valence. Rmax was plotted in arbitrary units as compared to

wild-type, taking into account the following parameters; the predicted

Rmax, the molecular weight of both ligand and analyte and the valence

of these two molecules. The wild-type calnexin: ERp57 binding was

arbitrarily set to ‘‘1’’, and the obtained Rmax values of the calnexin

mutants were normalized to this

Table 1 Kinetic analysis of surface plasmon resonance of recombinant ERp57 binding to soluble wild-type calnexin and calnexin mutants

Mutant Ka (1/Ms) Kd (1/s) KA (1/M) KD (M)

CNX-wt 1.98e4 1.03e-2 1.93e6 5.19e-7

CNX-H202 1.05e4 3.24e-3 3.25e6 3.07e-7

CNX-C161/195 2.2e4 4.35e-3 5.06e6 1.97e-7

CNX-C361/367 1.93e3 4.54e-4 4.36e6 2.3e-7

Calnexin and calnexin mutants were covalently bound to the sensor chip with recombinant ERp57 injected at different concentrations over the

chip, with binding measured. Kinetic analysis was generated based on a 1:1 Langmuir binding with drifting baseline model. The residues

indicated were mutated to alanine residues

CNX calnexin

Mol Cell Biochem (2012) 359:271–281 279

123

site and have a role in the folding of glycosylated substrate.

Interestingly, we observed that the H202 residue had no role

in the chaperone function of calnexin for either glycosylated

(Fig. 7) or non-glycosylated (data not shown) substrate.

Although this was surprising due to the fact that the con-

served histidine residue in calreticulin (H153) is critical for

chaperone function [5], this suggested that calnexin and

calreticulin may have divergent chaperone functions that

may contribute to the specificity of their substrates. The

H219 mutation did not impact thermal aggregation of gly-

cosylated or non-glycosylated substrates (data not shown),

suggesting that histidine residues in the N-globular domain

of calnexin may not play a role in chaperone function in

calnexin.

The P-domain of calnexin is a flexible arm domain that

contains 4 copies of two unique proline-rich motifs

arranged in a 1111–2222 pattern [3, 8]. The four copies of

motif 1 and motif 2 extend away from the lectin domain

and fold back on one another, forming a large hairpin [8].

Interestingly, mutations to the C361/367 in the P-arm

domain did not impact the thermal aggregation of the

non-glycosylated substrate MDH but partially enhanced

the thermal aggregation of the glycosylated substrate IgY.

We observed that the C361/367 mutation in the P-arm

domain were important in folding of the glycosylated

substrate IgY, although to lesser extent than the N-glob-

ular domain cysteines. These cysteines are not found in

calreticulin [8] and therefore, may play a novel structural

or functional role in calnexin.

The P-arm domain is the site of ERp57 binding to

calnexin, specifically through residues located at the tip,

including residues Y343 and D344 in the 4th repeat of motif

1 and G349 and E352 located in the 4th repeat of motif 2

[20]. Mutation of the P-domain C361/367 located in the 4th

repeat of motif 2 resulted in an increased interaction with

ERp57. We observed there was slower association and

faster dissociation with ERp57 in the C361/367 mutant, but

with higher overall binding, suggesting that mutation of the

C361/367 resulted in local conformational changes that

potentially enhanced novel binding sites while disrupting

nearby established ERp57 interaction residues. This result

also provided further evidence for the model of ERp57

binding to calnexin to specifically target substrates rather

than binding to calnexin to form disulfide bonds in caln-

exin. Interestingly, we determined that the N-globular

domain H202 and C161/195 were involved in ERp57 binding

and when mutated, interaction with ERp57 was enhanced,

potentially by the exposure of nascent ERp57 binding sites.

Although ERp57 interaction with calnexin is not mediated

through its N-domain, previous studies have shown that

mutations to the N-domain of calnexin [7] and calreticulin

[5] may result in enhanced ERp57 binding, suggesting that

modifications to the carbohydrate binding site results in

conformational changes and functional properties affecting

ERp57 binding.

In conclusion, we carried out site-specific mutagenesis to

generate mutations to cysteines (C161, C195, C161/195, C361,

C367, and C361/367) and histidines (H202 and H219). Our study

has shown the cysteine residues in the N- and P-domain of

calnexin were involved in the function of the protein. We

identified the N-globular cysteines C161 and C195, near the

carbohydrate binding pocket, as having a major role in the

chaperone activity of glycosylated proteins, while the

P-domain cysteines C361 and C367 as having a minor role in

the chaperone activity toward glycosylated proteins. We

determined that C161/195 and H202 enhanced ERp57 binding,

with C361/367, located near residues involved in ERp57

binding, significantly increasing the interaction with ERp57,

potentially by minor conformational changes occurring, with

exposure of nascent ERp57 binding sites.

Acknowledgments This work was supported by a grant to M.M.

from the Canadian Institutes of Health Research (MOP-53050). H.C.

and J.J. are supported by a studentship from the Alberta Innovates-

Health Solutions.

Conflict of interest These authors have no conflicting interest.

References

1. Hebert DN, Molinari M (2007) In and out of the ER: protein

folding, quality control, degradation, and related human diseases.

Physiol Rev 87:1377–1408

2. Williams DB (2006) Beyond lectins: the calnexin/calreticulin

chaperone system of the endoplasmic reticulum. J Cell Sci

119:615–623

3. Jung J, Coe H, Opas M, Michalak M (2006) Calnexin: an

endoplasmic reticulum integral membrane chaperone. Calcium

Binding Proteins 1:67–71

4. Leach MR, Cohen-Doyle MF, Thomas DY, Williams DB (2002)

Localization of the lectin, ERp57 binding, and polypeptide

binding sites of calnexin and calreticulin. J Biol Chem 277:

29686–29697

5. Martin V, Groenendyk J, Steiner SS, Guo L, Dabrowska M,

Parker JM, Muller-Esterl W, Opas M, Michalak M (2006) Iden-

tification by mutational analysis of amino acid residues essential

in the chaperone function of calreticulin. J Biol Chem 281:

2338–2346

6. Guo L, Groenendyk J, Papp S, Dabrowska M, Knoblach B, Kay

C, Parker JMR, Opas M, Michalak M (2003) Identification of an

N-domain histidine essential for chaperone function in calreti-

culin. J Biol Chem 278:50645–50653

7. Groenendyk J, Dabrowska M, Michalak M (2010) Mutational

analysis of calnexin. Biochim Biophys Acta 1808:1435–1440

8. Schrag JD, Bergeron JJM, Li Y, Borisova S, Hahn M, Thomas DY,

Cygler M (2001) The structure of calnexin, an ER chaperone

involved in quality control of protein folding. Mol Cell 8:633–644

9. Ou WJ, Bergeron JJ, Li Y, Kang CY, Thomas DY (1995) Con-

formational changes induced in the endoplasmic reticulum

luminal domain of calnexin by Mg-ATP and Ca2?. J Biol Chem

270:18051–18059

280 Mol Cell Biochem (2012) 359:271–281

123

10. Zapun A, Darby NJ, Tessier DC, Michalak M, Bergeron JJ,

Thomas DY (1998) Enhanced catalysis of ribonuclease B folding

by the interaction of calnexin or calreticulin with ERp57. J Biol

Chem 273:6009–6012

11. Corbett EF, Oikawa K, Francois P, Tessier DC, Kay C, Bergeron

JJ, Thomas DY, Krause KH, Michalak M (1999) Ca2? regulation

of interactions between endoplasmic reticulum chaperones. J Biol

Chem 274:6203–6211

12. Coe H, Jung J, Groenendyk J, Prins D, Michalak M (2009)

ERp57 modulates STAT3 signalling from the lumen of the

endoplasmic reticulum. J Biol Chem 285:6725–6738

13. Milner RE, Baksh S, Shemanko C, Carpenter MR, Smillie L,

Vance JE, Opas M, Michalak M (1991) Calreticulin, and not

calsequestrin, is the major calcium binding protein of smooth

muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

J Biol Chem 266:7155–7165

14. Coe H, Bedard K, Groenendyk J, Jung J, Michalak M (2008)

Endoplasmic reticulum stress in the absence of calnexin. Cell

Stress Chaperones 13:497–507

15. Nakamura K, Bossy-Wetzel E, Burns K, Fadel M, Lozyk M,

Goping IS, Opas M, Bleackley RC, Green DR, Michalak M

(2000) Changes in endoplasmic reticulum luminal environment

affect cell sensitivity to apoptosis. J Cell Biol 150:731–740

16. Balakier H, Dziak E, Sojecki A, Librach C, Michalak M, Opas M

(2002) Calcium-binding proteins and calcium-release channels in

human maturing oocytes, pronuclear zygotes and early preim-

plantation embryos. Hum Reprod 17:2938–2947

17. Saito Y, Ihara Y, Leach MR, Cohen-Doyle MF, Williams DB

(1999) Calreticulin functions in vitro as a molecular chaperone

for both glycosylated and non-glycosylated proteins. EMBO J

18:6718–6729

18. Lefevre F, Remy MH, Masson JM (1997) Alanine-stretch scan-

ning mutagenesis: a simple and efficient method to probe protein

structure and function. Nucleic Acids Res 25:447–448

19. Corbett EF, Michalak KM, Oikawa K, Johnson S, Campbell ID,

Eggleton P, Kay C, Michalak M (2000) The conformation of

calreticulin is influenced by the endoplasmic reticulum lumenal

environment. J Biol Chem 275:27177–27185

20. Pollock S, Kozlov G, Pelletier MF, Trempe JF, Jansen G, Sitni-

kov D, Bergeron JJ, Gehring K, Ekiel I, Thomas DY (2004)

Specific interaction of ERp57 and calnexin determined by NMR

spectroscopy and an ER two-hybrid system. EMBO J 23:

1020–1029

21. Oliver JD, Roderick HL, Llewellyn DH, High S (1999) ERp57

functions as a subunit of specific complexes formed with the ER

lectins calreticulin and calnexin. Mol Biol Cell 10:2573–2582

Mol Cell Biochem (2012) 359:271–281 281

123