Download The presence of monoglucosylated N196

Biochem. J. (2009) 421, 87–96 (Printed in Great Britain)
87
doi:10.1042/BJ20082170
The presence of monoglucosylated N196-glycan is important for the
structural stability of storage protein, arylphorin
Kyoung-Seok RYU*, Jie-Oh LEE†, Taek Hun KWON*, Han-Ho CHOI‡, Hong-Seog PARK‡, Soo Kyung HWANG§, Zee-Won LEE§,
Kyung-Bok LEE§, Young Hyun HAN*, Yun-Seok CHOI*, Young Ho JEON*, Chaejoon CHEONG*1 and Soohyun KIM§1
*Magnetic Resonance Team, Korea Basic Science Institute, Gwahangno 113, Daejeon 305-333, South Korea, †Department of Chemistry, Korea Advanced Institute of Science and
Technology, Gwahangno 335, Daejeon 305-701, South Korea, ‡Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Gwahangno 111,
Daejeon 305-806, South Korea, and §Division of Life Science, Korea Basic Science Institute, Gwahangno 113, Daejeon 305-333, South Korea
Although N-glycosylation has been known to increase the
stability of glycoproteins, it is difficult to assess the structural
importance of glycans in the stabilization of glycoproteins.
APA (Antheraea pernyi arylphorin) is an insect hexamerin that
has two N-glycosylations at Asn196 and Asn344 respectively.
The glycosylation of Asn344 is critical for the folding process;
however, glycosylation of Asn196 is not. Interestingly, the N196glycan (glycosylation of Asn196 ) remains in an immature form
(Glc1 Man9 GlcNAc2 ). The mutation of Asn196 to glutamine does
not change the ecdysone-binding activity relative to that of the
wild-type. In the present study, we determined the crystal structure
of APA, and all sugar moieties of the N196-glycan were clearly
observed in the electron-density map. Although the sugar moieties
of the glycan generally have high structural flexibility, most sugar
moieties of the N196-glycan were well organized in the deep
cleft of the subunit interface and mediated many inter- and intrasubunit hydrogen bonds. Analytical ultracentrifugation and
GdmCl (guanidinium chloride) unfolding experiments revealed
that the presence of the N196-glycan was important for stabilizing
the hexameric state and overall stability of APA respectively.
Our results could provide a structural basis for studying not only
other glycoproteins that carry an immature N-glycan, but also the
structural role of N-glycans that are located in the deep cleft of a
protein.
INTRODUCTION
the influence of this glycosylation on the overall Gibbs free
H2 O
) of the protein has not been evaluated. It has
energy (G unfold
been reported recently that the increased protein stabilization
by glycosylation originated from destabilization of the unfolded
state rather than stabilization of the folded state and this effect
is coupled with kinetic stabilization; the bulky polysaccharide
chains inhibit formation of a residual structure in the unfolded
glycoprotein [11]. Nevertheless, the analysis of glycosylation sites
in the protein structure databases still emphasizes the structural
role of glycosylation in the folded state, because 10 and 20 % of
the potential glycosylation sites were located in a deep cleft and
on the edges of grooves respectively [12].
The APA [Antheraea pernyi (Chinese oak silkworm)
arylphorin] is an insect hexamerin that consists of 688 amino
acids. APA belongs to a growing superfamily of proteins that
includes arthropod tyrosinase, arthropod haemocyanin, insect
hexamerin and dipteran arylphorin receptor [13]. It was reported
that at least two types of hexamerin exist in Lepidoptera: arylphorin and a methionine-rich storage protein [14]. Hexamerins
are synthesized in the fat body of a wide range of lepidopteran
and dipteran larvae, and they are highly accumulated in the
haemolymph. Hexamerins are storage proteins and are required
for somatic tissue metamorphosis, because they are reabsorbed
into the fat body and then metabolized as energy and amino acid
sources during insect maturation, a period during which there is
no feeding [15,16]. In addition to their role as a storage protein,
Glycosylation is an important cellular modification, and it is
related to many human diseases and developmental defects [1].
It introduces diversity into a biological system because of its
inherent structural heterogeneity, and thus plays critical roles
during a variety of cellular processes, such as protein folding,
protein–protein interaction, immune response and responses to
pathogens [2–4]. More than half of all eukaryotic proteins have
been reported to be glycosylated, and N-glycan is estimated to
be present in 90 % of these proteins [5]. The crystal structures
and NMR studies of glycoproteins indicate that glycans exist
primarily on the protein surface and most of the sugars are not
in direct contact with the protein, except for the two proximal
GlcNAc residues [6]. Many biochemical and genetic studies have
shown that N-glycosylation is important not only for folding,
but also for the stability of glycoproteins [3,7,8]. However, there
have been rare structural reports showing that the presence of the
glycan stabilizes the glycoprotein. NMR studies have shown that
the monofucosylation (O-glycosylation of Thr9 ) of the proteinase
inhibitor, Pars intercerebralis major peptide C, is critical for its
active structure [9]. A purely direct structural role of N-glycan has
only been proposed for quercetin 2,3-dioxygenase [10]. However,
the N-glycan of the dioxygenase is also located on the surface
of the protein, and only two proximal GlcNAc residues appear to
form the consistent intersubunit hydrogen bonds. Furthermore,
Key words: arylphorin, ecdysone, glycosylation, protein stability,
protein structure, X-ray crystallography.
Abbreviations used: APA, Antheraea pernyi arylphorin; BC, branched chain; ER, endoplasmic reticulum; GdmCl, guanidinium chloride; HEK, human
embryonic kidney; LC, longest chain; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; MFI, maximum fluorescence intensity; N,
native form; PIH, Panulirus interruptus haemocyanin; PNGase F, peptide N-glycosidase F; rAPA, recombinant APA; SASA, solvent-accessible surface
area; U, unfolded form.
1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
The crystal structure of Antheraea pernyi arylphorin has been deposited in the RCSB Protein Data Bank under code 3GWJ.
c The Authors Journal compilation c 2009 Biochemical Society
88
K.-S. Ryu and others
hexamerins appear to play other important roles during the
lifespan of insects. Hexamerins have been shown to be important
for maintaining high proportions of worker caste members in
termite societies, where a significant amount of two specific
hexamerins have been shown to suppress juvenile hormone-dependent worker differentiation into the soldier caste phenotype
[17]. It has also been suggested that hexamerins function as
ecdysteroid-carrier proteins in the haemolymph. This conclusion was based on the observation that spider (Eurypelma
californicum) haemocyanin and blowfly (Calliphora vicina)
calliphorin bind to the insect moulting hormones, ecdysone and
20-hydroxyecdysone [18]. 20-Hydroxyecdysone is the activated
form of ecdysone and has been shown to be critical for the
maturation of the arylphorin receptor in vivo and in vitro [19].
The structure of APA has attracted considerable interest,
because of the presence of monoglucosylated N-glycan and a
high content of aromatic amino acids (132 of 688 residues).
The presence of two glycosylation sites (Asn196 and Asn344 ) was
identified among four possible candidates (additional sites are
Asn57 and Asn623 ). The N344-glycan (glycosylation of Asn344 )
appears to be located on the protein surface, because it is easily
released from APA through treatment with PNGase F (peptide
N-glycosidase F) under non-denaturing conditions. However, the
N196-glycan (glycosylation of Asn196 ) remains in an immature
form, monoglucosylated oligosaccharide (Glc1 Man9 GlcNAc2 ),
and does not dissociate from the protein core after PNGase F
treatment [20]. During protein N-glycosylation, the precursor
oligosaccharide (Glc3 Man9 GlcNAc2 ) is pre-assembled on the
dolichol lipid by many ER (endoplasmic reticulum)-related
enzymes, and then is transferred to a specific asparagine residue
of a growing polypeptide chain by oligosaccharyltransferase.
After the removal of the second glucose by glucosidase II, the
monoglucosylated glycoprotein enters the calnexin/calreticulin
cycle in the ER, which governs the folding process of glycoproteins. Finally, the removal of the last remaining Glc-a by
glucosidase II releases the glycoprotein from these chaperones
[4]. The properly folded glycoprotein then moves to the Golgi
apparatus, where the attached N-glycan undergoes further trimming and modification processes. Deglucosylation is generally
considered to be a major signal, indicating the completion of
protein folding [4], and thus monoglucosylated N-glycans are
rarely observed in mature glycoproteins. However, this glycan
has been detected in avian IgY [21], the egg jelly coat of starfish
[22] and human complement component C3 [23]. It has been
suggested that the hydrophobicity of large proteins is linked to
the presence of glucose-capped oligosaccharides [24].
In the present study, we characterized the specific roles of
two N-glycosylations at Asn196 and Asn344 with regard to protein
stability and folding. The structural roles of N196-glycan were
examined by determining the crystal structure of APA and by
employing various biophysical techniques.
MATERIALS AND METHODS
Protein purifications and identification of N-glycan
APA was purified from larval haemolymph by gel-permeation
and ion-exchange chromatography using a Superdex-200 column
(GE Healthcare) at pH 7.5 and a HiTrap-SP column (GE
Healthcare) at pH 5.0 respectively. Autumn APA was used for
further experiments, since the profile of N196-glycan was more
homogeneous than that of spring APA [16]. Expression vectors
containing either the FLAG-tagged wild-type rAPA (recombinant
APA) or mutant rAPA-N196Q were transiently transfected into
HEK (human embryonic kidney)-293a cells. After 4 days of
c The Authors Journal compilation c 2009 Biochemical Society
culture, medium was harvested, and the FLAG-tagged rAPA and
rAPA-N196Q were purified using an anti-FLAG M2–agarose
column (Sigma). To characterize the profiles of N-glycan, the
oligosaccharides released from denatured proteins were labelled
with 2-aminobenzamide, and then separated using normal-phase
HPLC as described previously [20]. The glycans were also
permethylated and analysed by MALDI–TOF (matrix-assisted
laser-desorption ionization–time-of-flight)-MS as described
previously [25].
Determining the binding constants of APA and rAPA-N196Q
for ecdysone
Ecdysone was purchased from Fluka, and the stock solution
(50 mM) was prepared in 100 % ethanol by directly weighing
the ecdysone powder. APA (0.2 μM) and rAPA-N196Q (0.1 μM)
were prepared in buffer (pH 7.5) containing 50 mM Tris/HCl
and 100 mM NaCl. The intrinsic fluorescence of both proteins
was measured using the PerkinElmer LS 50 spectrofluorimeter
at 25 ◦C. The excitation wavelength was fixed at 280 nm, and the
emission spectrum was recorded from 320 to 450 nm. The binding
constants were extracted by fitting the changes in maximum
intensity to a simple binding equation [18].
Crystallization and acquisition of diffraction data
A protein stock solution (10–12 mg/ml) was prepared in
buffer (10 mM Tris/HCl containing 100 mM NaCl, pH 7.5), and
crystallization conditions were screened using Crystal screen I
and II, and Index kits (Hampton Research). The crystals were
grown in a buffer [pH 4.6; 100 mM sodium acetate, 14–15 %
poly(ethylene glycol) 3350, 150 mM magnesium formate and
20 mM benzamidine chloride] using the hanging-drop method
at 20 ◦C. In addition, 30 % poly(ethylene glycol) 400 was added
to the crystal growth buffer as a cryoprotectant for freezing the
crystal, and the X-ray diffractions were performed under a cold
helium gas stream. Crystal screening was performed using the
synchrotron beamlines (4A and 6C) at the Pohang Accelerator
Laboratory. Diffraction data for the structure determination were
obtained using the synchrotron beamline at Spring-8, Japan, and
were processed using HKL-2000 package.
Crystal structure determination and analysis
The initial arylphorin model was generated by the MODELLER
program [26], using the structures of homologous haemocyanin
(PDB code 1HC1). Molecular replacement was performed using
the Molrep program, during which a reasonable solution was
obtained only when the trimeric structure of haemocyanin, and
not the monomeric structure, was used as an input model. The
model building was completed using the Coot program included
in the CCP4 package [27]. Structure refinement was performed
using the CNSsolve program [28]. A mild non-crystallographic
symmetry restraint was applied for each subunit, except for
the two GlcNAc sugar moieties of N334-glyan. The default
CNSsolve parameter of carbohydrate was corrected to maintain
the appropriate geometry of the N-acetyl group in the GlcNAc
residue. Figures showing the structure and the electron-density
map of arylphorin were generated by using the program Chimera
[29]. The cation–π interaction was analysed using the CaPTURE
program [30]. The areas of subunit interfaces and the SASAs
(solvent-accessible surface areas) of APA were calculated using
the NACCESS program (http://www.bioinf.manchester.ac.uk/
naccess/nacwelcome.html).
Monoglucosylated N-glycan enhances protein stability
Analytical ultracentrifugation
The oligomeric status of APA and rAPA-N196Q were analysed
by equilibrium sedimentation analysis at 20 ◦C. The experiments
were performed using an analytical ultracentrifuge (ProteomeLab
XL-A, Beckman Coulter) and An-60Ti analytical rotor. The
equilibrium sedimentation distributions of all proteins were
obtained after centrifugation for 48 h at 5000 rev./min. The
sedimentation coefficients of rAPA-N196Q were also obtained
from sedimentation velocity analysis. The SEDFIT program was
used for data analysis [31].
GdmCl (guanidinium chloride) equilibrium unfolding experiment
All three proteins (1 μM each of APA, rAPA and rAPAN196Q) were pre-equilibrated overnight in buffers (pH 7.5)
containing 50 mM Tris/HCl and an appropriate concentration of
guanidinium chloride (GdmCl) at 25 ◦C. However, the overnight
incubation seemed to be slightly insufficient for the reaction
to reach equilibrium for APA and rAPA, and thus a longer
incubation time (1 and 2 days) was used for the additional APA
unfolding experiments. The intrinsic fluorescence was measured
using the PerkinElmer LS 50 spectrofluorimeter at 25 ◦C with
280 nm excitation. The MFIs (maximum fluorescence intensities)
of emission spectra (320–450 nm) were used for the analysis of
Gunfold . The free energy (Gunfold , kcal · mol− 1 , where 1 kcal =
4.184 kJ) of all proteins was estimated from the ratio of the
unfolded form (U) to the native form (N) of the protein at
each concentration of GdmCl; Gunfold = −RT · ln[U/N]. The free
H2 O
energy of the native proteins (G unfold
, kcal · mol−1 ) was obtained
through linear extrapolation:
2
+ m · [GdmCl]
G unfold = G unfold
H O
where m is the number showing the dependence of the free energy
on the denaturant concentration. The unfolded fractions of rAPAN196Q were obtained after linear correction of the baselines that
were located before and after the denaturant-dependent transition
of MFI. However, a gradual pre-unfolding ahead of the main
unfolding transition was observed for both APA and rAPA, which
may have resulted from the presence of minor heterogeneity in
the N196-glycan [16]. The values of −RT · ln[U/N] were obtained
again by correction of the tentative population of the minor forms,
and the linearity of the plot of Gunfold against [GdmCl] was used
as an additional reference for determining the population of the
major transition.
RESULTS
Glycosylation of Asn344 is required for proper folding of APA,
but that of Asn196 is not
It has been well established that N-glycosylation plays a similar
role as protein chaperones, and thus its presence is important for
the proper folding of glycoproteins [3,7]. For example, it has been
shown that the activity of several ER-related chaperone proteins
is dependent on the presence of N-glycosylation in the substrate
protein [4,32]. APA has two occupied glycosylation sites, at Asn196
and Asn344 . We therefore tested the effect of glycan occupancy at
each glycosylation site on the expression of the APA protein
in mammalian cells. APA was expressed in an insoluble form in
Escherichia coli (results not shown). Substitution of glutamine
for the asparagine residue at the N-glycosylation sites prevented
attachment of the glycan in the ER. rAPA and two mutant
APA proteins (rAPA-N196Q and rAPA-N344Q) were expressed
in HEK-293a cells. The N196-glycan profile of rAPA was
determined to be very similar to that of purified APA, although
other minor forms were also present. We also confirmed that
89
rAPA-N196Q did not possess any glycan moiety at Gln196 (see
Supplementary Figure S1 at http://www.BiochemJ.org/bj/421/
bj4210087add.htm). The profile of the N344-glycans of APA
was different from that of rAPA and rAPA-N196Q. The N344glycan existed in a processed form, and thus a differential glycan processing in the Golgi apparatus of Antheraea pernyi and
HEK-293a cell is likely to be the cause of the different N344glycan profiles. On the other hand, the N196-glycan is in an
immature form, which suggests that further processing in the
ER and Golgi apparatus did not occur (see Supplementary Figure S1). Interestingly, rAPA-N196Q was expressed as a soluble
protein with a slight decrease in the amount of secreted protein;
however, rAPA-N344Q was not secreted into the culture medium,
but rather it accumulated inside the cell in an insoluble form (see
Supplementary Figure S2A at http://www.BiochemJ.org/bj/421/
bj4210087add.htm). Both rAPA and rAPA-N196Q seemed to be
properly folded. We have already found that the presence of
intermolecular disulfide bonds stabilizes the trimeric structure
of APA (results not shown), and the maintenance of the disulfide
bonds of rAPA and rAPA-N196Q was also confirmed by nonreducing SDS/PAGE analysis (Supplementary Figure S2B).
The glycosylation of Asn344 seems to be important for the
proper folding of rAPA; however, this is not the case for Asn196 .
The decreased amount of secreted rAPA-N196Q, compared with
rAPA, may have resulted from the loss of the N196-glycan. It
has been reported that glucosidase II can efficiently cleave the second glucose when an additional second glycan is present in the
protein chain [33]. The resulting monoglucosylated glycoprotein
enters the calnexin/calreticulin cycle in the ER, which governs
the folding process of glycoproteins [4]. The critical role of the
glycosylation of Asn344 during the folding process of APA would
be interesting, because the glycosylation motif of Asn344 is not
conserved in the arylphorin protein family (results not shown). It
is possible that another N-glycosylation could occur at a different
site on other arylphorins (e.g. Spodoptera litura arylphorin, Asn56
or Asn455 ), in addition to the common glycosylation site at Asn196
of APA.
The lack of glycosylation at Asn196 does not result in a stringent
structural change
We first investigated the secondary structures of APA, rAPA and
rAPA-N196Q using CD. The CD spectra of APA, rAPA
and rAPA-N196Q showed that all proteins were properly folded,
although the overall α-helical content of rAPA-N196Q seemed to
be slightly lower than that of APA or rAPA (results not shown). It
has been suggested that some hexamerins and haemocyanin bind
to ecdysteroid hormones and thus function as ecdysone-carrier
proteins [18]. We therefore evaluated the tertiary folds of APA
and rAPA-N196Q by comparing their ecdysone-binding affinities.
The protein–ecdysone dissociation constants (K d ) of both
proteins were determined using the intrinsic fluorescence of APA
and rAPA-N196Q. Their K d values were almost identical at
0.43 mM (Figure 1); thus, rAPA-N196Q exhibited identical
folding, at least in terms of ecdysone-binding and disulfide bond
formation. There were no clearly separate domains in the crystal
structure of APA (see below). The mutation of Asn196 to glutamine
did not seem to have a stringent effect on the overall structure of
APA and its effect would be localized in the interface of APA
hexamer. If the mutation makes a strong structural perturbation, it
is likely to propagate in an ecdysone-binding site of rAPA-N196Q,
and thus should affect the binding affinity.
Although the binding constant of APA to ecdysone was
obviously low, a high concentration of APA could facilitate the diffusion of ecdysone. We roughly estimated that APA accumulated
c The Authors Journal compilation c 2009 Biochemical Society
90
K.-S. Ryu and others
Table 1
Data collection and refinement statistics
Parameter
Figure 1
Binding of ecdysone to APA or rAPA-N196Q
The binding affinities of APA and rAPA-N196Q to ecdysone were estimated using the
intrinsic fluorescence of the proteins. Ecdysone was dissolved in absolute ethanol (Et-OH),
and fluorescence intensities of the proteins were measured at increasing ecdysone
concentrations. The fluorescence of either protein was not significantly changed by the addition
+
of pure ethanol only. APA (0.43 +
− 0.045 mM) and rAPA-N196Q (0.43 − 0.025 mM) have similar
binding affinities to ecdysone.
in the haemolymph up to a concentration of 0.5 mM at the last
stage of pupa (results not shown). Ecdysone is a non-polar steroid
hormone produced in the prothoracic gland, and its concentration
increases by more than 10 μM at the end of the final larval
stage [34]. Ecdysone has been known to mediate a variety of
developmental and physiological events in insects, including
hexamerin receptor maturation [19,34]. The insect moulting
process occurs within a short time, and thus the ecdysteroidcarrier activity of hexamerin may be required for synchronizing
the processes related to moulting.
Crystal structure of hexameric APA
We determined the crystal structure of APA (i) to study the influences of the N196-glycan on the structure of APA at the
molecular level, and (ii) to find out why the monoglucosylated
oligosaccharide of Asn196 survives the N-glycan trimming process
in the ER and Golgi apparatus. Fortunately, molecular replacement using the previously reported PIH [Panulirus interruptus
(California spiny lobster) haemocyanin] structure (PDB codes
1HC1) produced a reasonable solution, although the sequence
identity between the haemocyanin and APA was somewhat low
(∼ 28 %). The statistics of the crystal structure determined are
summarized in Table 1. The asymmetric unit of the APA crystal
contained one hexamer (Figure 2A). Although several studies
have predicted that arylphorins have a hexameric structure on
the basis of biochemical analysis [13,16], the crystal structure
and the analytical ultracentrifuge investigation (see below) clearly
confirm that the oligomeric state of APA is a hexamer.
The amino acid sequence alignment of APA and PIH (Figure 3)
and their structural comparison (see Supplementary Figure S3
at http://www.BiochemJ.org/bj/421/bj4210087add.htm) indicate
that arylphorin is likely to have evolved from haemocyanin,
although the population of aromatic amino acid is much higher for
arylphorin than for haemocyanin. The overall topology of APA
was very similar to that of the previously reported hexamerin
c The Authors Journal compilation c 2009 Biochemical Society
Data collection
Space group
a , b , c (Å)
α, β, γ (◦)
Resolution (Å)
R sym (%)
I /σ I
Completeness (%)
Redundancy
Refinement
Resolution limit (Å)
Number of reflections
R work /R free (%)
RMSD of bond length (Å)
RMSD of bond angle (◦)
Ramanchandran plot analysis
Most favoured region (%)
Allowed region (%)
Generously allowed region (%)
Disallowed region (%)
Value
P 21 2 1 2 1
121.196, 119.470, 319.878
90.0, 90.0, 90.0
50.0–2.42 (2.51–2.42)
14.1 (31.6)
20.0 (3.3)
95.3 (79.9)
5.9 (4.0)
50.0–2.42
168 198
18.2/23.0
0.0070
1.2367
92.4
7.1
0.5
0.0
structure of PIH [35], and only minor structural differences were
identified; APA has an additional β-sheet (residues 12–14 and
residues 580–582, β1), helix and one more β-strand (residues
176–182, α9, and residues 184–187, β2), and helix–turn–helix
(residues 670–679, α21a and α21b). The APA loop (residues
619–635, coloured red in Supplementary Figure S3B) has a very
high B-factor, but the corresponding loop in PIH is stabilized by
a disulfide bond.
In comparison with PIH, the structure of APA has two distinguishing features. One is the presence of intermolecular disulfide
bonds between Cys73 and Cys649 , which form a circular structure
within each top (A-B-C) and bottom (D-E-F) trimer unit. The
other is the presence of a monoglucosylated oligosaccharide at
Asn196 (Figures 2A and 2B). However, the presence of disulfide
bonds does not seem to be critical for stabilizing the structure of
the arylphorin protein family, because sequence alignment of the
insect hexamerin proteins shows that these cysteine residues of
APA (Cys73 and Cys649 ) are not conserved even in the arylphorin
protein family (results not shown). Analysis of the interface
areas between the subunits of the APA hexamer revealed that
the interface area between subunit pairs A-D, B-F and C-E was
largest. The overall contact surface areas between subunit B and
subunit A, C, D and F were 1115.3 (19.8), 1078.3 (17.9), 860.5
(381.4), and 2817.7 (544.7) Å2 (1 Å = 0.1 nm) respectively, where
the values in parentheses are the area that is mediated by the
N196-glycan. The N196-sugar moieties of subunit A are mainly
in contact with subunit D, and those of subunit D are also in major
contact with subunit A (Figure 2C). It is likely that the dimeric
pair of subunits A and D (similarly, subunits B and F, and subunits
C and E) is the core interaction responsible for the formation of
the hexameric arylphorin, and the presence of the disulfide bonds
of APA that make the circular connection of each top or bottom
trimer seems to be incidental. On the other hand, the glycosylation
motif of Asn196 is clearly conserved only in the arylphorin protein
family (Figure 4). The residue corresponding to Asn196 of APA
is not present in very or moderately methionine-rich hexamerin
proteins.
The N196-glycan of APA is located in the deep cleft of the
subunit interface (Figure 2A), and the presence of all sugar
moieties was identified in the annealed omit electron-density map
(Supplementary Figure S4 at http://www.BiochemJ.org/bj/421/
Monoglucosylated N-glycan enhances protein stability
Figure 2
91
Crystal structure of APA
(A) The crystal asymmetric unit of APA consisted of six monomers (A, blue; B, yellow; C, aquamarine; D, green; E, cyan; F, brown). The different subunits are indicated using a chain code (.A, .B, .C,
.D, .E and .F). All 12 sugar moieties of monoglucosylated N196-glycan (S-N196 ) in each subunit are shown in the crystal structure; however, only two proximal GlcNAc residues of N344-glycan
(S-N344 ) were barely detected. The N196-glycan from subunit A are located in the deep cleft that was formed by subunits A, D and E. Two cysteine residues (C649.E and C73.D) forming intersubunit
disulfide bonds are indicated using a yellow colour (inset). (B) A different view of the top trimer unit of the APA hexamer (subunits D, E and F) shows that each top and bottom trimer unit were
stabilized by the intersubunit disulfide bonds. (C) The orientation and colours for each subunit were the same as in (A). The density of the blue colour represents the variation of the crystal B -factor
for subunit A, where a strong colour indicates a high B -factor. Cys649 of subunit E (C649.E) forms intermolecular disulfide bonds with Cys73 of subunit-D (C73.D) (marked with yellow spheres).
The LC of subunit A had a lower B -factor in comparison with the BC1 and BC2, and not only had multiple intrasubunit interactions, but also participated in many intersubunit interactions with
subunit D (marked with a star). The BC1 of subunit E was spatially close to subunit A, but the BC2s of all subunits were positioned outside the protein core. The regions that were located within
5 Å of the N196-glycan were decorated using the same colours as the contacting sugars. (D) Schematic illustration of the monoglucosylated N196-glycan and the N344-glycan. Sugar chains of the
N196-glycan were divided further into LC (GlcNAc-1 to Glc-a), BC1 (Man-A and Man-D2) and BC2 (Man-B and Man-D3).
bj4210087add.htm). The longest chain of the N196-glycan (LC
in Figure 2C, GlcNAc-1 to Glc-a) is well defined in the electrondensity map in comparison with the first (BC1 in Figure 2C,
Man-A to Man-D2) and second (BC2 in Figure 2C, Man-B
to Man-D3) branched sugar chains, and thus the B-factors of LC
are much lower than that of BC1 and BC2 (see Supplementary Figure S5 at http://www.BiochemJ.org/bj/421/bj4210087add.htm).
The sugar moieties of LC form more than 20 direct or watermediated hydrogen bonds with adjacent amino acids, which are
located in the same or different subunits (Figure 5). Especially,
the sugar moieties (Man-4 to Glc-a) of subunit A form hydrogen
bonds with Tyr43 , Lys47 , Phe75 , Leu76 , Lys78 , Val118 , His119 and
Tyr182 of subunit D, and the same sugar moieties of subunit D
form the reciprocal hydrogen bonds with the same residues of
subunit A. The LC region seems to be important for enhancing
the interaction between subunits A and D, subunits B and F, and
subunits C and E, with regard to the hydrogen-bond network.
Therefore this sugar chain could additionally stabilize the overall
trimer–trimer interaction (see below) by enhancing the interaction
between each top and bottom dimer (Figures 2B and 2C).
Moreover, many direct or water-mediated intrasugar hydrogen
bonds (∼ 20) were identified in the LC, and not in the BC1
and BC2 regions. This means that the sugar moieties of the LC
are well placed to seize adjacent water molecules. The BC1 of
subunit A is positioned close to Leu528 , Glu529 , Cys649 and Phe650
of subunit E and Lys392 of subunit D, in which the Man-A moiety
c The Authors Journal compilation c 2009 Biochemical Society
92
Figure 3
K.-S. Ryu and others
Secondary structures and sequence alignments of arylphorin and haemocyanin
Structure-based sequence alignments were performed using the Chimera program (http://www.cgl.ucsf.edu/chimera/). The open and grey-coloured boxes represent the β-sheet and α-helix
respectively. Asn196 and Asn344 of APA are indicated with an asterisk (*), and Cys73 and Cys649 are indicated with a plus sign (+), respectively. Two intramolecular disulfide bonds (Cys483 and Cys502 ;
Cys562 and Cys609 ) were observed in the structure of PIH (underlined), where the disulfide bond between Cys562 and Cys609 seems to stabilize the loop structure of PIH (see Supplementary Figure
S3 at http://www.BiochemJ.org/bj/421/bj4210087add.htm).
Figure 4
Sequence alignment of hexamerins in the Asn196 region of APA
The identical and conserved residues in the hexamerin protein family are indicated with an asterisk
(*) and a plus sign (+) respectively. The N-glycosylation motif is indicated with a box. ApAry,
arylphorin from Antheraea pernyi (Chinese oak silkmoth); HcAry, arylphorin from Hyalophora
cecropia (cecropia moth); BmSp2, sex-specific storage-protein 2 precursor arylphorin from
Bombyx mori (domestic silkworm); MsAry, arylphorin subunit alpha precursor from Manduca
sexta (tobacco hornworm); SlAry, arylphorin subunit from Spodoptera litura (common cutworm);
HcVmr, very methionine-rich storage protein; HcMmr, moderate methionine-rich storage
protein.
of subunit A (O3 atom) consistently forms hydrogen bonds with
the Cys649 of subunit E (carbonyl O atom). The Man-D2 moiety
of subunit A (O4 and O6 atoms) is close to the side chains of
Glu529 of subunit E and Lys392 of subunit D (within 3.5–3.9 Å),
and thus it might form a transient hydrogen bond due to its
dynamic characteristics in comparison with the sugar moieties
of the LC. The structural role of BC1 might be important in
the other arylphorins that do not contain intermolecular disulfide
bonds, because the hydrogen-bond formation between subunit A
and subunit E, which is mediated by the sugar moieties of BC1,
can mimic the same effect of intersubunit disulfide bonds. The
c The Authors Journal compilation c 2009 Biochemical Society
sugar moieties of BC2 from all subunits are positioned outside
the protein core.
The N344-glycan is located on the surface of APA, and
two proximal GlcNAc moieties were barely detected in the
annealed omit map (see Supplementary Figures S4 and S5).
The two proximal GlcNAc moieties on the N344-glycan have a
much higher B-factor compared with the corresponding GlcNAc
moieties of N196-glycan, and their values vary significantly with
the subunit origins. The B-factor of GlcNAc-2 of N344-glycan,
which originates from subunit D, is even higher than that of the
last sugar moiety of N196-glycan (Man-D3), making the exact
model building of the GlcNAc-2 moiety located in subunit D very
difficult.
The monoglucosylated oligosaccharide of APA can survive the
extensive trimming process that occurs in the ER and Golgi
apparatus, because the location of N196-glycan in the deep cleft of
the subunit interface and the presence of many N196-glycan that
mediate the intra- and inter-subunit hydrogen bonds are likely to
restrict the access of various glycosidases. Interestingly, the ribbon
presentation of the APA monomer indicates that the N196-glycancontacting residues are mainly part of short α-helical structures
(Figure 5), and thus the N196-glycan could also stabilize these
local α-helices.
N196-glycan enhances interaction between top and bottom
subunits
We compared the oligomeric states of rAPA-N196Q with
that of rAPA by analytical ultracentrifugation (Figure 6). The
Monoglucosylated N-glycan enhances protein stability
Figure 5
93
Stereo view of N196-glycan-mediated intra- and inter-subunit hydrogen bonds
Subunits A, D and E are coloured blue, green and cyan respectively, and the observed water molecules are indicated using purple-coloured balls. The density of ribbon colour represents the
variation of the crystal B -factor, where a strong colour indicates a higher B -factor. The N196-glycan of subunit A is displayed as a ball-and-stick model. The residues located within 5 Å of the sugar
moieties of N196-glycan are displayed as a stick model. The hydrogen bonds satisfying the precise and moderate criteria were indicated using cyan- and orange-coloured lines respectively. The
residues that participate in the N196-glycan-mediated hydrogen bonds are listed in the bottom panel. The subunit origination of residues was indicated using a chain code (.A, .B and .E). The names
of the atoms that participate in the hydrogen bonds are shown in parentheses (blue, direct hydrogen bond; red, water-mediated hydrogen bond).
sedimentation equilibrium analysis clearly showed that rAPA
mainly exists as hexamer. However, the equilibrium sedimentation
profile of rAPA-N196Q could not be fitted to any single
component models, which indicates that rAPA-N196Q undergoes
a reversible hexamer–trimer equilibrium, although the hexameric
state is still predominant. The hexamer–trimer equilibrium of
rAPA-N196Q is likely to be observed, simply due to the presence
of intermolecular disulfide bonds that maintains the trimeric state
(see Supplementary Figure S2B). The hexameric state of rAPA is
more stable than that of rAPA-N196Q (hexamer compared with
hexamer–trimer equilibrium). The crystal structure shows that the
N196-glycan mediated not only intrasubunit hydrogen bonds, but
also many intersubunit hydrogen bonds for each pair of subunits:
A and D, B and F, and C and E. The loss of N196-glycan is likely
to decrease the overall trimer–trimer interaction and thus shift the
equilibrium of APA from the hexameric state to the hexamer–
trimer equilibrium.
The presence of N196-glycan is also important for the overall
protein stability
The overall stabilities of APA and rAPA-N196Q were evaluated
with a GdmCl equilibrium-unfolding experiment at 25 ◦C. The
denaturation midpoint of GdmCl unfolding (Cm ) of rAPA-N196Q
was much lower than that of APA. Interestingly, a gradually
unfolding ahead of the main transition was observed for APA, but
not for rAPA-N196Q (Figure 7). Although it is difficult to explain
the molecular basis of this gradual pre-unfolding, it is likely to
result from the presence of less populated, more processed N196glycan in the purified APA. The presence of minor heterogeneity
in the N196-glycan (20–30 %) has already been characterized
and its population also varies with the cultural season of A. pernyi
larva [16]. N196-glycans that have a reduced length in sugar
moieties are likely less capable of forming intra- and inter-subunit
hydrogen bonds.
H2 O
) at a
The Gibbs free energy of the unfolded state (G unfold
zero concentration of GdmCl was obtained following the simple assumption that the Gunfold is linearly dependent on the
H2 O
concentration of denaturant [36]. Using this method, (G unfold
)
−1
of rAPA-N196Q was determined to be 14.8 +
0.6
kcal
·
mol
.
−
H2 O
value for APA was roughly estimated to be 23–
The G unfold
24 kcal · mol− 1 for the two tentative populations of the gradual
pre-transition (22.9 and 24.0 kcal · mol− 1 for 20 and 25 %
H2 O
respectively), where the linearity values (R2 ) of the plot of G unfold
against [GdmCl] were 0.997 and 0.996 respectively (Figure 7).
H2 O
The G unfold
of APA was 18.2 and 28.1 kcal · mol− 1 when the
populations of the gradual pre-transition were assumed to be 15
and 30 % respectively, where the linearity values were 0.989 and
H2 O
0.994. Although it is difficult to determine the exact G unfold
value
of APA, probably due to the presence of minor heterogeneity
in the N196-glycan, it is clear that the presence of N196-glycan
stabilizes the overall stability of APA by at least ∼ 10 kcal · mol− 1 .
The overall GdmCl-unfolding profile of rAPA was similar to that
of APA (results not shown). We cannot completely rule out the
possibility that decreased stability of rAPA-N196Q could result
from a structural perturbation by the mutation of Asn196 into
c The Authors Journal compilation c 2009 Biochemical Society
94
K.-S. Ryu and others
Figure 7
Figure 6
Differential oligomeric states of APA and rAPA-N196Q
The oligomeric states of rAPA (A) and rAPA-N196Q (B) were resolved using an equilibrium
sedimentation analysis at 20 ◦C. Experimental data are shown with small open circles and the
fitting results are shown as lines. The rAPA exists as an almost pure hexamer, because the fit
residuals of the hexamer and hexamer–trimer equilibrium models were almost identical. However,
a hexamer–trimer equilibrium was observed for rAPA-N196Q. tri, trimer; hexa, hexamer; octa,
octamer.
glutamine. However, the side chain of Asn196 is exposed to the
protein surface and there are many hydrogen bonds mediated by
N196-glycan (Figure 5), where even GlcNAc-1 moiety forms six
hydrogen bonds with the protein core. Therefore the results from
the GdmCl-unfolding experiments of APA and rAPA-N196Q are
likely to show that the presence of N196-glycan is important not
only for stabilizing the hexameric state, but also for increasing the
overall stability of APA. The presence of N196-glycan-mediated
intra- and inter-subunit hydrogen bonds seems to be the primary
reason for the decreased stability of rAPA-N196Q in comparison
with APA and rAPA.
The presence of N196-glycan decreases the hydrophobic SASA of
APA
APA has a high proportion of aromatic residues compared with
PIH (19.2 and 11.2 % respectively), and thus its overall proportion
of hydrophobic residues is also higher than that of PIH (47.7 and
39.7 % respectively). Although the increased number of aromatic
c The Authors Journal compilation c 2009 Biochemical Society
GdmCl unfolding of APA and rAPA-N196Q
The fluorescence intensity maxima of rAPA-N196Q were obtained with 280 nm excitation after
overnight incubation. However, two additional incubation times (1 and 2 days) were used to test
whether complete equilibrium was reached during the GdmCl-unfolding of APA. Fractions of the
unfolded form (U) of APA and rAPA-N196Q were obtained from the baseline correction of
the measured intrinsic fluorescence. A gradual unfolding before the main transition was observed
for APA, but not for rAPA-N196Q. This probably resulted from the presence of a minor population
of more processed N196-glycans (20–30 %) [16,20]. The unfolded fraction at the main transition
was recalculated after subtraction of the population from the minor transition. Two representative
populations of the minor transition (a, 20 %; b, 25 %) were used to show the influence of the
H2 O
). Inset, The G unfold of all prominor transition on the overall stability of APA (G unfold
teins was estimated from the ratio of the unfolded form (U) to the native form (N) of the protein:
H2 O
, kcal · mol−1 ) was
G unfold = −RT × ln[U/N]. The free energy of the native proteins (G unfold
H2 O
+ m · [GdmCl]. APA (22.9 +
obtained through linear extrapolation; G unfold = G unfold
− 0.5
−1
was more stable than rAPA-N196Q
and 24.0 +
− 0.7 kcal · mol for a and b respectively)
H2 O
−1
(14.8 +
− 0.6 kcal · mol ). The errors of G unfold were obtained from the errors of the linear
fitting.
residues for APA compared with PIH increases the number of the
cation–π interactions between positively charged and aromatic
residues (137 in APA compared with 78 in PIH) [30,37], the
higher population of hydrophobic residues possibly decreases
the overall stability via increasing the hydrophobic SASA. The
hydrophobic SASAs of APA (without glycans) and PIH were
76752 and 73278 Å2 respectively, and the presence of glycans in
APA reduced the hydrophobic SASA from 76752 to 73738 Å2 ,
and thus probably decreased the overall unfavourable exposure of
hydrophobic surfaces to the aqueous phase.
It is also possible that the N196-glycan additionally stabilizes
the structure of APA by decreasing the Cp (heat capacity of
unfolding) of APA. Recently, chemical glycosylation studies have
shown that increasing the degree of glycosylation by lactose or
dextran linearly enhances the thermal stability of proteins by
decreasing the Cp , where the effect is intensified by increasing
the size of the modified glycan, and by increasing the protein
melting temperature [38]. The decreased Cp supposedly result
from the association of the exposed non-polar residues of the
unfolded protein with the glycan moieties [39].
DISCUSSION
Although the crystal structure of APA and the biophysical
results indicate that the N196-glycan plays an important role in
Monoglucosylated N-glycan enhances protein stability
stabilizing the APA structure, more detailed studies to determine
its structural roles must be conducted. The inner region of APA is
much more hydrophobic than that of the hexamerin homologue
(PIH), and thus the Cp of APA would be higher [40,41].
The hydrophobic SASAs of unfolded and folded APA (without
glycans) were 347 399.4 and 76 752.2 Å2 respectively, and those
of the unfolded and folded PIH were 310 556.4 and 73 277.9 Å2
respectively. Monoglucosylated N-glycan (Glc1 Man9 GlcNAc2 ),
which corresponds to the N196-glycan of APA, seems to be
present mainly in the arylphorin protein family and this is related
to the high population of hydrophobic residues since members
contain many aromatic residues. Therefore characterizing more
H2 O
H2 O
and Sunfold
),
detailed thermodynamic parameters (Cp , Hunfold
H2 O
in addition to G unfold , for both APA and rAPA-N196Q is still
required for achieving a better understanding of the complete
mechanism by which N196-glycan stabilizes the structure of APA.
It has been reported that the presence of glycans stabilize
RNase A and B (∼ 1.2 kcal · mol− 1 ) [42], and α 1 -antitrypsin
(2.1 kcal · mol− 1 ) [43]. Moreover, the increased Gunfold of αchymotrypsin even after multiple dextran chemical modifications
is also less than 10 kcal · mol− 1 [38]. However, the glycosylation
of Asn196 seems to be critical for enhancing the stability of APA,
because its stabilizing effect is very large. This may be due to the
location of N196-glycan (the deep cleft of subunit interface) and
also due to the presence of N196-glycan mediated intra- and intersubunit hydrogen bonds. The backbone of the glycan is known
to be very flexible, and thus it is implausible that a structurally
pre-assembled N196-gycan forms hydrogen bonds with a protein
partner. Paradoxically, this high flexibility could provide a
sufficient degree of freedom for glycan moieties to adapt to its
surrounding protein environment, and to form hydrogen bonds
via their many hydroxy groups, although this is a somewhat
unfavourable process in terms of entropy. The effect of the N196glycan on the overall stability may originate from its location in
APA. It would be interesting to study the effect of glycosylation
sites on the overall stability of the glycoprotein (surface compared
with the deep cleft) and to see whether the glycan could adapt to
the deep cleft of the subunit interface and generate additional
hydrogen bonds.
The crystal structure of APA also explains why the N196-glycan
can survive the intensive trimming processes that occur in the ER
and Golgi apparatus. The remaining Glc-a residue of the mature
APA suggests that at least a dimer formation (subunits A and D,
or B and F, or C and E pairs) occurs before the completion of
folding. The largest interface area was observed for these dimer
pairs, and each pair was additionally stabilized by reciprocal
intersubunit hydrogen bonds that involved the sugar moieties
of Asn196 , including Glc-a. Therefore the accessibility of Glc-a
to glucosidase II, which exists in N196-glycan, may be greatly
reduced. Monoglucosylated N-glycans that exist in other proteins,
such as avian IgY, the egg jelly coat of starfish and human
complement component C3, probably face a similar environment
as the monoglucosylated N-glycan of APA. The complete hexamer and interdisulfide bridge formation of APA would also be
cleaved during the calnexin/calreticulin cycle, because it includes
ERp57 (ER protein 57), a thiol-disulfide oxidoreductase [4]. The
chaperone-like activity of N-glycosylation may also be dependent
on the location of N-glycosylation in the partially folded protein,
which could influence the accessibility of the calnexin/calreticulin
chaperon complex to the monoglucosylated N-glycan.
The arylphorin protein family, which appears to have been
evolved from haemocyanin, has an exceptionally high proportion
of aromatic amino acids. These proteins may have acquired
glycosylation during evolution to prevent hydrophobic aggregation during the folding process and/or to enhance their stability.
95
It is expected that the high stability of APA contributes to its
long half-life during the insect lifespan, even in the absence of
protease activity in the haemolymph, because (i) the insect larva
seems to face a hostile environment involving large deviations in
temperature, and (ii) APA should be accumulated and remained
in large amounts before being used for insect maturation. Finally,
ecdysone activates the maturation of the arylphorin receptor,
and the receptor incorporates APA into the larval fat body for
utilization as energy and amino acid sources. These factors could
constitute a reason to generate a unique trafficking pathway for
arylphorin in the class Insecta, in which arylphorin is secreted
from the fat body to the haemolymph and is then reabsorbed into
the fat body.
AUTHOR CONTRIBUTION
Kyoung-Seok Ryu mainly performed the structural determination, biophysical studies and
the writing of the paper. Jie-Oh Lee helped with the data collection and the crystal symmetry determination. Taek Hun Kwon, Young Hyan Han, Yun-Seok Choi and Young
Ho Jeon assisted with the X-ray data analysis and the biochemical experiments. Han-Ho
Choi and Hong-Seog Park purified the natural APA protein. Soo Kyung Hwang, Zee-Won
Lee and Kyung-Bok Lee conducted the purification and characterization of recombinant
arylphorin proteins from HEK-293a cells and the analysis of glycosylation. Chaejoon
Cheong and Soohyun Kim outlined experimental direction, performed data analysis and
writing of the paper.
ACKNOWLEDGEMENTS
We thank S. M. Lee (Pusan National University, Busan, South Korea) and O. K. Cho
(Korea Basic Science Institute) for the generous supply of insect haemolymph and mutant
generation respectively. We also thank S. J. Park and N. Y. Oh (both at the Korea Basic
Science Institute) for glycan analysis.
FUNDING
This study was supported by a Korea Basic Science Institute (KBSI) grant [grant number
T27010] to S. K.
REFERENCES
1 Dennis, J. W., Granovsky, M. and Warren, C. E. (1999) Protein glycosylation in
development and disease. BioEssays 21, 412–421
2 Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct.
Glycobiology 3, 97–130
3 Mitra, N., Sinha, S., Ramya, T. N. and Surolia, A. (2006) N-linked oligosaccharides as
outfitters for glycoprotein folding, form and function. Trends Biochem. Sci. 31, 156–163
4 Helenius, A. and Aebi, M. (2004) Roles of N-linked glycans in the endoplasmic reticulum.
Annu. Rev. Biochem. 73, 1019–1049
5 Apweiler, R., Hermjakob, H. and Sharon, N. (1999) On the frequency of protein
glycosylation, as deduced from analysis of the SWISS-PROT database.
Biochim. Biophys. Acta 1473, 4–8
6 Wormald, M. R., Petrescu, A. J., Pao, Y. L., Glithero, A., Elliott, T. and Dwek, R. A. (2002)
Conformational studies of oligosaccharides and glycopeptides: complementarity of NMR,
X-ray crystallography, and molecular modelling. Chem. Rev. 102, 371–386
7 Mitra, N., Sharon, N. and Surolia, A. (2003) Role of N-linked glycan in the unfolding
pathway of Erythrina corallodendron lectin. Biochemistry 42, 12208–12216
8 Buck, T. M., Eledge, J. and Skach, W. R. (2004) Evidence for stabilization of aquaporin-2
folding mutants by N-linked glycosylation in endoplasmic reticulum. Am. J. Physiol.
Cell Physiol. 287, C1292–C1299
9 Mer, G., Hietter, H. and Lefevre, J. F. (1996) Stabilization of proteins by glycosylation
examined by NMR analysis of a fucosylated proteinase inhibitor. Nat. Struct. Biol. 3,
45–53
10 Fusetti, F., Schroter, K. H., Steiner, R. A., van Noort, P. I., Pijning, T., Rozeboom, H. J., Kalk,
K. H., Egmond, M. R. and Dijkstra, B. W. (2002) Crystal structure of the copper-containing
quercetin 2,3-dioxygenase from Aspergillus japonicus . Structure 10, 259–268
11 Shental-Bechor, D. and Levy, Y. (2008) Effect of glycosylation on protein folding: a close
look at thermodynamic stabilization. Proc. Natl. Acad. Sci. U.S.A. 105, 8256–8261
12 Petrescu, A. J., Milac, A. L., Petrescu, S. M., Dwek, R. A. and Wormald, M. R. (2004)
Statistical analysis of the protein environment of N-glycosylation sites: implications for
occupancy, structure, and folding. Glycobiology 14, 103–114
c The Authors Journal compilation c 2009 Biochemical Society
96
K.-S. Ryu and others
13 Burmester, T. and Scheller, K. (1996) Common origin of arthropod tyrosinase, arthropod
hemocyanin, insect hexamerin, and dipteran arylphorin receptor. J. Mol. Evol. 42,
713–728
14 Telfer, W. H. and Kunkel, J. G. (1991) The function and evolution of insect storage
hexamers. Annu. Rev. Entomol. 36, 205–228
15 Telfer, W. H. and Pan, M. L. (2003) Storage hexamer utilization in Manduca sexta .
J. Insect Sci. 3, 26
16 Jung, H. I., Kim, Y. H. and Kim, S. (2005) Structural basis for the presence of a
monoglucosylated oligosaccharide in mature glycoproteins. Biochem. Biophys.
Res. Commun. 331, 100–106
17 Zhou, X., Oi, F. M. and Scharf, M. E. (2006) Social exploitation of hexamerin: RNAi
reveals a major caste-regulatory factor in termites. Proc. Natl. Acad. Sci. U.S.A.
103, 4499–4504
18 Jaenicke, E., Foll, R. and Decker, H. (1999) Spider hemocyanin binds ecdysone and
20-OH-ecdysone. J. Biol. Chem. 274, 34267–34271
19 Burmester, T. and Scheller, K. (1997) Developmentally controlled cleavage of the
Calliphora arylphorin receptor and posttranslational action of the steroid hormone
20-hydroxyecdysone. Eur. J. Biochem. 247, 695–702
20 Kim, S., Hwang, S. K., Dwek, R. A., Rudd, P. M., Ahn, Y. H., Kim, E. H., Cheong, C., Kim,
S. I., Park, N. S. and Lee, S. M. (2003) Structural determination of the N-glycans of a
lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in
the storage protein. Glycobiology 13, 147–157
21 Ohta, M., Hamako, J., Yamamoto, S., Hatta, H., Kim, M., Yamamoto, T., Oka, S.,
Mizuochi, T. and Matsuura, F. (1991) Structures of asparagine-linked oligosaccharides
from hen egg-yolk antibody (IgY): occurrence of unusual glucosylated oligo-mannose
type oligosaccharides in a mature glycoprotein. Glycoconjugate J. 8, 400–413
22 Endo, T., Hoshi, M., Endo, S., Arata, Y. and Kobata, A. (1987) Structures of the sugar
chains of a major glycoprotein present in the egg jelly coat of a starfish, Asterias
amurensis . Arch. Biochem. Biophys. 252, 105–112
23 Crispin, M. D., Ritchie, G. E., Critchley, A. J., Morgan, B. P., Wilson, I. A., Dwek, R. A.,
Sim, R. B. and Rudd, P. M. (2004) Monoglucosylated glycans in the secreted human
complement component C3: implications for protein biosynthesis and structure.
FEBS Lett. 566, 270–274
24 Khalaila, I., Peter-Katalinic, J., Tsang, C., Radcliffe, C. M., Aflalo, E. D., Harvey, D. J.,
Dwek, R. A., Rudd, P. M. and Sagi, A. (2004) Structural characterization of the N-glycan
moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax
quadricarinatus . Glycobiology 14, 767–774
25 Kang, P., Mechref, Y., Klouckova, I. and Novotny, M. V. (2005) Solid-phase
permethylation of glycans for mass spectrometric analysis. Rapid Commun.
Mass Spectrom. 19, 3421–3428
26 Marti-Renom, M. A., Stuart, A. C., Fiser, A., Sanchez, R., Melo, F. and Sali, A. (2000)
Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys.
Biomol. Struct. 29, 291–325
27 Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for
protein crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760–763
Received 3 November 2008/3 April 2009; accepted 9 April 2009
Published as BJ Immediate Publication 9 April 2009, doi:10.1042/BJ20082170
c The Authors Journal compilation c 2009 Biochemical Society
28 Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve,
R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S. et al. (1998) Crystallography &
NMR system: a new software suite for macromolecular structure determination. Acta
Crystallogr. Sect. D Biol. Crystallogr. 54, 905–921
29 Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng,
E. C. and Ferrin, T. E. (2004) UCSF Chimera: a visualization system for exploratory
research and analysis. J. Comput. Chem. 25, 1605–1612
30 Gallivan, J. P. and Dougherty, D. A. (1999) Cation–π interactions in structural biology.
Proc. Natl. Acad. Sci. U.S.A. 96, 9459–9464
31 Brown, P. H. and Schuck, P. (2006) Macromolecular size-and-shape distributions by
sedimentation velocity analytical ultracentrifugation. Biophys. J. 90, 4651–4661
32 Vandenbroeck, K., Martens, E. and Alloza, I. (2006) Multi-chaperone complexes
regulate the folding of interferon-γ in the endoplasmic reticulum. Cytokine 33,
264–273
33 Deprez, P., Gautschi, M. and Helenius, A. (2005) More than one glycan is needed for ER
glucosidase II to allow entry of glycoproteins into the calnexin/calreticulin cycle.
Mol. Cell 19, 183–195
34 Burmester, T., Antoniewski, C. and Lepesant, J. A. (1999) Ecdysone-regulation of
synthesis and processing of fat body protein 1, the larval serum protein receptor
of Drosophila melanogaster . Eur. J. Biochem. 262, 49–55
35 Volbeda, A. and Hol, W. G. (1989) Crystal structure of hexameric haemocyanin from
Panulirus interruptus refined at 3.2 Å resolution. J. Mol. Biol. 209, 249–279
36 Creighton, T. E. (1993) Stability of the folded conformation. In Proteins: Structures and
Molecular Properties, 2nd edn, pp. 287–291, W. H. Freeman, New York
37 Pack, S. P. and Yoo, Y. J. (2004) Protein thermostability: structure-based difference
of amino acid between thermophilic and mesophilic proteins. J. Biotechnol. 111,
269–277
38 Sola, R. J., Rodriguez-Martinez, J. A. and Griebenow, K. (2007) Modulation of protein
biophysical properties by chemical glycosylation: biochemical insights and biomedical
implications. Cell. Mol. Life Sci. 64, 2133–2152
39 van Teeffelen, A. M., Broersen, K. and de Jongh, H. H. (2005) Glucosylation of
β-lactoglobulin lowers the heat capacity change of unfolding; a unique way to affect
protein thermodynamics. Protein Sci. 14, 2187–2194
40 Makhatadze, G. I. and Privalov, P. L. (1990) Heat capacity of proteins. I. Partial molar heat
capacity of individual amino acid residues in aqueous solution: hydration effect. J. Mol.
Biol. 213, 375–384
41 Privalov, P. L. and Makhatadze, G. I. (1990) Heat capacity of proteins. II. Partial molar heat
capacity of the unfolded polypeptide chain of proteins: protein unfolding effects. J. Mol.
Biol. 213, 385–391
42 Joao, H. C. and Dwek, R. A. (1993) Effects of glycosylation on protein structure and
dynamics in ribonuclease B and some of its individual glycoforms. Eur. J. Biochem. 218,
239–244
43 Kwon, K. S. and Yu, M. H. (1997) Effect of glycosylation on the stability of α 1 -antitrypsin
toward urea denaturation and thermal deactivation. Biochim. Biophys. Acta 1335,
265–272
Biochem. J. (2009) 421, 87–96 (Printed in Great Britain)
doi:10.1042/BJ20082170
SUPPLEMENTARY ONLINE DATA
The presence of monoglucosylated N196-glycan is important for the
structural stability of storage protein, arylphorin
Kyoung-Seok RYU*, Jie-Oh LEE†, Taek Hun KWON*, Han-Ho CHOI‡, Hong-Seog PARK‡, Soo Kyung HWANG§, Zee-Won LEE§,
Kyung-Bok LEE§, Young Hyun HAN*, Yun-Seok CHOI*, Young Ho JEON*, Chaejoon CHEONG*1 and Soohyun KIM§1
*Magnetic Resonance Team, Korea Basic Science Institute, Gwahangno 113, Daejeon 305-333, South Korea, †Department of Chemistry, Korea Advanced Institute of Science and
Technology, Gwahangno 335, Daejeon 305-701, South Korea, ‡Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Gwahangno 111,
Daejeon 305-806, South Korea, and §Division of Life Science, Korea Basic Science Institute, Gwahangno 113, Daejeon 305-333, South Korea
Figure S2 Characterizations of the wild-type and mutant APA proteins that
were expressed in HEK-293a cells
Figure S1 Identification of N-glycans attached to Asn196 and Asn344 using
normal-phase HPLC and MALDI–TOF-MS
(A) Oligosaccharides released from denatured proteins were labelled with 2-aminobenzamide,
and then separated by normal-phase HPLC. APA, rAPA and rAPA-N196Q were expressed in
HEK-293a cells. The monoglucosylated N196-glycan is indicated with an asterisk (*). The
N196-glycan profile of rAPA was very similar to that of APA. However, the N344-glycan profile
of rAPA was different from that of APA, possibly due to the differential glycan processing
between the Golgi apparatus of insect and mammalian cells. GU, glucose units. (B) The same
glycans were permethylated and analysed by MALDI–TOF-MS to confirm the HPLC results. The
monoglucosylated glycan is indicated with an asterisk (*).
(A) Top panel: Western blot analysis of the total cells using anti-FLAG antibody confirmed that
wild-type rAPA and two mutant APA proteins (rAPA-N196Q and rAPA-N344Q) were expressed
in HEK-293a cells. Middle panel: the same Western blot analysis of the culture medium shows
that the rAPA and rAPA-N196Q proteins were secreted from the cell, but the rAPA-N344Q was
not. The amount of secreted rAPA-N196Q was less than that of rAPA. Bottom panel: Western
blot analysis with anti-actin antibodies was used to confirm that the same amount of cells was
used as in the top panel. (B) rAPA and rAPA-N196Q were purified using anti-FLAG affinity
column, and then the presence of intermolecular disulfide bonds in rAPA and rAPA-N196Q was
determined from non-reducing SDS-PAGE analysis [ETSH (ethanethiol), 2-mercaptoethanol],
which stabilizes the trimeric structure of APA. The monomeric, dimeric and trimeric bands are
indicated by the arrows.
1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
The crystal structure of Antheraea pernyi arylphorin has been deposited in the RCSB Protein Data Bank under code 3GWJ.
c The Authors Journal compilation c 2009 Biochemical Society
K.-S. Ryu and others
Figure S3
Structure comparison of arylphorin and haemocyanin
(A) The overall hexameric structure of APS (left-hand panel) was very similar to that of PIH (right-hand panel). The subunit origination of residues was indicated using a chain code. The cysteine
residues that form disulfide bonds are shown as yellow spheres. (B) Superimposed ribbon presentation of APA (brown) and PIH (cyan) monomers. The overall structures of both proteins were very
similar, except for some additional local regions. The additional secondary structures of APA are labelled. The APA loop (residues 619–635, coloured red) has a very high B -factor; the corresponding
loop in PIH was stabilized by a disulfide bond between Cys562 and Cys609 .
c The Authors Journal compilation c 2009 Biochemical Society
Monoglucosylated N-glycan enhances protein stability
Figure S4
Stereo view annealed omit map of Asn196 monoglucosylated oligosaccharide and N344-glycan
(A) All the sugar moieties of N196-glycan can be observed in the electron-density map. We clearly determined the positions of most sugar moieties from GlcNAc-1 to Man-B, and moderately for
those of Man-D2 and Man-D3, which were located in the terminus. (B) Only two proximal sugar moieties of N344-glycan were observed in the electron-density map.
Figure S5
B -factor analysis of N196- and N344-glycans
The B -factors of N196-glycans did not vary with their subunit origins, but those of N344-glycans
had diverse distributions according to their subunit origins. The position of the sugar moieties of
BC2 (Man-B and Man-D3) were outside of the protein core and thus had the highest B -factors.
On the other hand, the sugar moieties of LC (GlcNAc-1 to Glc-a) were involved in many inter- and
intra-subunit hydrogen bonds, and thus their B -factors were lower than those of BC1 (Man-A
and Man-D2) and BC2.
Received 3 November 2008/3 April 2009; accepted 9 April 2009
Published as BJ Immediate Publication 9 April 2009, doi:10.1042/BJ20082170
c The Authors Journal compilation c 2009 Biochemical Society