From the Molecular Biophysics Unit, Indian
Institute of Science, Bangalore 560012, India, the
Department of
Organic Chemistry, Arrhenius laboratory, Stockholm University,
S-106 91 Stockholm, Sweden, and ** Institute of
Biochemistry, ETH-Zurich, Hoenggerberg, CH-8093, Switzerland
Received for publication, September 6, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Calreticulin is a molecular chaperone
found in the endoplasmic reticulum in eukaryotes, and its
interaction with N-glycosylated polypeptides is mediated by
the glycan Glc1Man7-9GlcNAc2 present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of
Glc1Man7-9GlcNAc2, determined by
the quantitative technique of isothermal titration calorimetry. This
method provides a direct estimate of the binding constants
(Kb) and changes in enthalpy of binding
( Calreticulin (CRT),1
along with calnexin, serves as a molecular chaperone in the
endoplasmic reticulum (ER) of eukaryotic cells. Although calreticulin
is a soluble, luminal protein, calnexin is a type I membrane protein
(1, 2). Segments of these proteins share amino acid identity ranging
from 42 to 78% (3). Calreticulin is a highly conserved ubiquitous
protein (Mr 46,000) and has been implicated in
Ca2+ storage and intracellular Ca2+ signaling
in the sarcoplasmic and endoplasmic reticula (4, 5). CRT has been
divided into three regions: the N-terminal, the C-terminal, and the
central P-domain, which consists of short sequence motifs repeated
three times in tandem. The N-terminal domain is highly conserved among
CRTs from different species and potentially mediates interactions
between CRT and the ER folding catalysts, protein disulfide
isomerase and ERp57 (6, 7). Recent studies show that the P-domain,
previously thought to be involved in oligosaccharide binding, interacts
directly with ERp57 (8-10). The C-domain is characterized by a high
content of acidic residues (4, 11), which is consistent with the
location of a low affinity (Kd = ~1-2
mM), high capacity (~25-50 mol) calcium-binding site
(12) and contains the ER retrieval sequence.
The ER plays an essential role in the folding and maturation of newly
synthesized proteins in the secretory pathway. ER quality control
operates at various levels; one of the most common modifications of
proteins translocated into the ER is the addition of
N-linked glycans (13). CRT has been found to bind to
partially trimmed, monoglucosylated glycans, and it has been shown that
the binding of calreticulin involves a direct oligosaccharide-protein
interaction specific for monoglucosylated oligosaccharides (14-17).
Recently the crystal structure of the luminal domain of calnexin has
been published (18), which shows a single carbohydrate-binding site in
its lectin domain. Given the sequence similarity between calnexin and
calreticulin, the carbohydrate-binding site in the latter should reside
in the corresponding homologous domain.
The binding of CRT is exquisitely specific for the monoglucosylated
N-linked oligosaccharides, which appear as transient
intermediates in vivo. The chaperone function of
calreticulin is assisted by two enzymes of contrasting catalytic
activities: UDP-glucose:glycoprotein glucosyltransferase and
glucosidase. The sequential action of glucosidase I and II removes two
glucose residues from
Glc3Man5-9GlcNAc2 from
N-linked glycoproteins in the ER (19), whereas
UDP-glucose:glycoprotein glucosyltransferase catalyzes the addition of
glucose on the nonreducing end mannose of the Substrate studies have identified the single-terminal glucose residue
as a critical determinant recognized by calreticulin because
oligosaccharides containing 0, 2, or 3 glucose residues fail to bind
(14, 23). Oligosaccharide binding is critical for the formation of
complexes between glycoproteins and calreticulin. If
N-linked glycosylation is blocked with tunicamycin or if
production of the Glc1Man9GlcNAc2
species is prevented by treatment with the glucosidase inhibitors
castenospermine or deoxynojirimycin, the binding of calreticulin to a
vast majority of proteins is inhibited (15, 24).
Thus, because of the importance of the calreticulin-glycoprotein
interaction in various biological processes, the study of calreticulin-oligosaccharide interactions at the molecular level has
become imperative. The oligosaccharide binding studies on calreticulin
and calnexin have been limited because of the nonavailability of
N-linked sugar
Glc1Man9GlcNAc2 in sufficient
amounts from natural sources as well as the difficulty in synthesizing
such large oligosaccharide structures. Here, we report the interaction
of di-, tri-, and tetrasaccharide with calreticulin using the
quantitative technique of isothermal titration calorimetry. We report
the stoichiometry, binding constant, and various thermodynamic
parameters for these interactions. Calreticulin-sugar binding is
enthalpically driven, the binding enthalpies of tetrasaccharide being
larger than those of the trisaccharide and the enthalpies of
trisaccharide binding being larger than those observed for
disaccharide. These thermodynamic data thus define in quantitative
terms the extended combining site of calreticulin for the glucosylated
arm of the oligosaccharide. Moreover, these data are consistent with
the model of the complexes of calreticulin with these sugars.
Materials--
Media components were obtained from
Hi-media (Delhi, India). GST-agarose, reduced glutathione,
isopropyl-1-thio- Synthesis of Oligosaccharides--
All three of the
oligosaccharides have been synthesized previously (25-27). However, in
our approach the glucose moiety was introduced last, using
thioglycoside donors in both tetrasaccharide and trisaccharide
synthesis. This is the route followed by Matta and colleagues
(25) in tetrasaccharide synthesis, whereas Cherif et
al. (27) used a 2 + 2 block approach. The overall yield over the
three glycosylations was 24% (66, 76, and 48%) as compared with those
of Matta and colleagues (25) 19% (78, 64, and 39%) and
Monneret and colleagues (27) 11% (49, 48, and 47%). In the trisaccharide synthesis, this approach (1 + 2) has been employed earlier by Cherif and Monneret (26) using a glucose
trichloroacetimidate donor advantageously. Our attempt, using a glucose
thioglycoside donor, gave comparable results (19% as compared with a
22% overall yield). Carbohydrate concentrations were determined by the
phenol-sulfuric acid method of Dubois et al. (28) using
mannose as a standard. The details of the oligosaccharide synthesis
used are shown in Scheme 1 and in the
Supplemental Material (available on line at http://www.jbc.org).
Expression and Purification of Recombinant Calreticulin--
The
GST-CRT full-length fusion protein was purified essentially as
described by Peterson and Helenius (29). Briefly, the construct was
transformed into Escherichia coli DH5 Protein Estimation--
The E280 of
calreticulin was estimated as 1.94 using the method of Gill and von
Hippel (30). For a 1-mg ml Isothermal Titration Calorimetry--
ITC experiments were
performed using a VP-ITC calorimeter from Microcal Inc.
(Northampton, MA) as described previously by Wiseman et al.
(31). For titrations involving tri- and tetrasaccharide, typically 90 µM protein (in 10 mM MOPS, 5 mM CaCl2, 150 mM NaCl, pH 7.4) in a
sample cell, volume 1.4181 ml, was titrated with 10× excess sugar
(present in same buffer) from the 300-µl stirrer syringe. For the
titration of disaccharide, for example, 100 µM protein
was used, and the sugar (1.3 mM) was added as 30-40
injections of 6 µl into the sample cell. The titrations were
performed while samples were being stirred at 400 rpm at the required
temperatures. An interval of 3 min between each injection was given for
the base line to stabilize. The heat-of-dilution values of the
sugar were subtracted from the titration data. The data so obtained were fitted via the nonlinear least-squares minimization method to
determine binding stoichiometry (n), the binding constant
(Kb), and the change in enthalpy
( Modeling Studies--
Because the crystal structure of
calreticulin has not been determined, the substrate specificity of this
protein to various sugars was investigated by computer modeling using
the MODELLER suite of programs (33, 34). The crystal structure of
calnexin (Ref. 17, PDB code 1JHN) provided the template needed for modeling the structure of calreticulin. The modeled structure of
calreticulin was then subjected to simulated annealing and positional
refinement using the CNS suite of programs (35, 36). All of the
oligosaccharide structures used to examine the binding mode to
calreticulin were obtained from SWEET dB (37). The mode of binding of
the following oligosaccharides was examined: Glc/Glc
The sugar-binding site in the modeled calreticulin was deduced from
analogy to the crystal structure of calnexin. The monosaccharide was
docked into the binding site using the docking package SYBYL (SYBYL®
6.7.1 Tripos Inc., St. Louis, MO). The docking geometry was improved by
optimizing the overlap between the functional description of the
binding site and the ligand, generating multiple solutions. The least
energy solution was taken as the best conformation of the glucose to
interact with the protein. This solution was then subjected to
positional refinement using CNS. Retaining the position and orientation
of the glucose ring with respect to the protein, the disaccharide was
superposed on the monosaccharide using the program ALIGN. The protein
and sugar coordinates were further examined using INSIGHT II. All
possible conformations relating the two sugar moieties were explored at
intervals of 10°. The best conformation was chosen as the one making
the maximum number of hydrogen bonds and minimum short contacts with
the protein. Following a similar procedure, the trisaccharide was
superposed on the best conformer of the disaccharide. Retaining the
torsion angles along the We studied the oligosaccharide binding properties of calreticulin
by isothermal titration calorimetry as it provides a direct estimate of
the binding constants (Kb) and changes in
enthalpy of binding ( Thermodynamics of Calreticulin-Sugar Interactions--
The results
of a representative titration of the disaccharide, trisaccharide, and
tetrasaccharide to calreticulin at 293 K are shown in Fig.
1. Each of these titrations exhibits a
monotonic decrease in the exothermic heat of binding with each
successive injection until saturation is achieved. A nonlinear
least-squares fit of the ITC data to the identical site model is also
shown. The close fit of the data to the identical site model shows that calreticulin has only one type of site for its complementary sugar ligand. The thermodynamic parameters,
The thermodynamics of the binding reaction presented in Table I
shows that the binding reaction is entropically driven for Glc Calreticulin Modeling Studies--
A model of calreticulin based
upon the structure of calnexin is shown in Fig.
3B. The modeling studies
provided a rationale for the sugar specificity of calreticulin. The
binding site residues in calreticulin for glucose corresponding to
those in calnexin are Tyr-109, Lys-111, Tyr-128, Met-131, Asp-135,
Gly-124, and Asp-317 (Fig. 4 and Ref.
18). The monosaccharide, i.e. Glc The Glc1Man9GlcNAc2 glycan
intermediate formed after cleavage of two glucose residues by
The recently solved crystal structure of calnexin shows that its
sugar-binding domain has a striking similarity with that of a "legume
lectin" fold (40-42). Thus, a wealth of data on the molecular
features of carbohydrate recognition by legume lectins helps us to
explain in molecular terms the mode of interaction of the saccharides
to calreticulin (43-45). In CRT, as in legume lectins, the concave and
convex sheets are made of six and seven strands, respectively, whereas
the roof region consists of only two strands as compared with five in
legume lectins (Fig. 3B). An important finding that comes
from the modeling studies is that the presence of a helix behind the
convex Elucidation of the stoichiometry of interaction of calreticulin with
its substrate has been a matter of speculations and debate. The ITC
data presented here establish unambiguously that calreticulin-sugar interactions have a stoichiometry of 1 (Fig. 1). Indeed, the recent structural solution of calnexin also reports one site for glucose binding in this molecule, although the low affinity of this ligand and
the consequent low occupancy of the chaperone with glucose cannot be
ignored at this moment. The extremely low affinity of glucose for
calreticulin is not surprising (Kb ~ 60 M The affinity of glucose per se or methyl Hb°) as well as the stoichiometry of the
reaction. Unlike past speculations, these studies demonstrate
unambiguously that calreticulin has only one site per molecule for
binding its complementary glucosylated ligands. Although the
binding of glucose by itself is not detectable, a binding constant of
4.19 × 104 M
1 at 279 K is
obtained when glucose occurs in
-1,3 linkage to Man
Me as in
Glc
1-3Man
Me. The binding constant increases by 25-fold from di-
to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glc
1-3Man
1-2Man
1-2Man
Me
structure of the oligosaccharide is recognized by calreticulin. The
thermodynamic parameters thus obtained were supported by modeling
studies, which showed that increased number of hydrogen bonds and van
der Waals interactions occur as the size of the oligosaccharide is
increased. Also, several novel findings about the recognition of
saccharide ligands by calreticulin vis á vis legume
lectins, which have the same fold as this chaperone, are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3 arm of
Man5-9GlcNAc2 chains of unfolded glycoproteins, thereby acting as a folding sensor (20, 21). Repeated
cycles of reglucosylation by the glucosyltransferase lead to prolonged
association of calreticulin with the unfolded protein until its
appropriate folding occurs. Should this process fail, the protein is
retrotranslocated to the cytosol and subsequently degraded by the
proteasome (22).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, MOPS, and
SDS-PAGE reagents were obtained from Sigma.
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Scheme 1.
Synthesis of the di-, tri-, and
tetrasaccharides. i, NIS/AgOTf,
CH2Cl2; ii, NaOMe, MeOH;
iii, 8, DMTST, Et2O; iv,
H2, Pd/C; v, DMTST,
CH2Cl2; vi, aqueous AcOH
70%; vii, a, 8, Br2,
CH2Cl2, and b,
Bu4NBr.
cells. An overnight
plateau phase culture was used to inoculate fresh Luria broth
containing 50 µg/ml ampicillin, 2 mM calcium chloride at a dilution of 1:100. Cells were grown at 30 °C to an
A600 of 0.4-0.6 and were induced by 0.2 mM isopropyl-1-thio-
-D-galactopyranoside followed by incubation for an additional 6 h. The cells were
harvested and resuspended in a 1:10 culture volume of lysis buffer (500 mM Tris-HCl, 100 mM NaCl, pH 7.5) and sonicated
on ice using a Branson tip sonicator. Lysates thus obtained were
loaded onto GST-agarose (equilibrated with 2 bed volumes of lysis
buffer). The specifically bound protein was eluted by 5 mM
reduced glutathione in Tris buffer. The eluate was concentrated using a
10-kDa Amicon concentrator and dialyzed against a 10 mM
MOPS, 5 mM CaCl2, 150 mM NaCl, pH
7.5, buffer.
1 GST-CRT solution,
A280 was determined as 1.06.
Hb°) using Origin software (Microcal) as
described previously (31, 32). The experimental conditions ensured that
the c value ranged between 2 and 130, where
c = Kb × Mt (0) and Mt (0) is the
initial macromolecule concentration for all of the titrations.
Me, Glc
1-3Man
Me, Glc
1-3Man
1-2Man
Me,
Glc
1-3Man
1-2Man
1-2Man
Me, Man
1-3Man
Me.
-1,3 linkage of the disaccharide, the
dihedral angle of the 1-2 linkage was allowed to vary from 0 to 360°
in intervals of 10°. A similar procedure was followed for building the tetrasaccharide. The conformation along the
-1,2 linkage between
the third and fourth mannose sugars was varied through 0 to 360° at
intervals of 10°. The best conformer at every stage was subjected to
positional refinement using CNS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Hb°) as well as the
stoichiometry of the reaction (n). Additionally, the results
obtained were rationalized on the model of calreticulin built by its
homology with the recently solved structure of calnexin (18).
Gb°,
Hb°, and
Sb at
279 and 293 K, determined from the titration calorimetry measurements are presented in Table I. The values of
Kb increase almost by a factor of 2 from tri- to
tetrasaccharide. Also, there is a greater than 25-fold increase in the
binding constant from di- to trisaccharide. These findings demonstrate
that calreticulin recognizes all four of the residues on the
glucosylated branch of the
Glc1Man9GlcNAc2 oligosaccharide.
The disaccharide Man
1-2Man
Me and Man
1-3Man
Me did not show
any binding to the protein, thus demonstrating the importance of
the terminal glucose residue for the interaction. These studies are
qualitatively consistent with those of Williams and colleagues (38),
who tested a variety of mono-, di-, and oligosaccharides for their
ability to inhibit the binding of
[3H]Glc1Man9GlcNAc2
to GST-CRT immobilized on glutathione-agarose. The smallest
inhibitory compounds were the disaccharides, Glc
1-3Man and
Glc
1-3Glc. Also, the trisaccharide, Glc
1-3Man
1-2Man, and the tetrasaccharide, Glc
1-3Man
1-2Man
1-2Man, were ~100-
and ~500-fold, respectively, more potent inhibitors than the
disaccharides.
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Fig. 1.
Isothermal calorimetric titration of
calreticulin with different sugars. Raw data obtained after 6-µl
injections of 1 mM sugar solution into 100 µM
calreticulin in 10 mM MOPS buffer containing 5 mM CaCl2 and 150 mM NaCl at 293 K
(top). Nonlinear least-squares fit ( ) of the heat
released as a function of the added ligand (
) for the titration
(bottom) is also shown. The data were fitted to a
single-site model to obtain n,
Hb°, and Kb,
respectively, for: disaccharide, 0.94 (±0.04), 2.2 (±0.15) kcal/mol,
and 2.4 × 104 (±0.5) M
1
(A); trisaccharide, 0.89 (± 0.002), 6.9 (± 0.031)
kcal/mol, and 6.1 × 104 (± 0.42)
M
1 (B); and tetrasaccharide, 0.99 (±0.006), 10.44 (±0.088) kcal/mol, and 1.09 × 106
(±0.05) M
1 (C). The values in
parentheses are the errors associated with each fitted parameter for a
given experiment.
Thermodynamic quantities for the binding of sugars to calreticulin
1-3Man
Me and enthalpically driven for the
tetrasaccharide, whereas for the trisaccharide,
Glc
1-3Man
1-2Man
Me, the switch between these two modes occur.
The changes in enthalpy observed upon the binding of tetrasaccharide
are the highest. Also from the data in Table I, it appears that the
changes in enthalpy and entropy upon binding are compensatory (Fig.
2). Similar enthalpy-entropy compensation
has been observed earlier for many protein-ligand interactions
(39).
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Fig. 2.
Enthalpy-entropy compensation plot of
Hb° as a function of
T
Sb for the binding of
di (
)-, tri (
)-, and tetrasaccharide (
) to calreticulin.
The plot shows a linear relationship with a slope of 1.25.
Me, was
hydrogen-bonded to the side chains of Tyr-109, Tyr-128, Asp-135, and
Asp-317. The best conformer of the disaccharide interacted with the
protein through six hydrogen bonds made by the second sugar moiety
along with the five hydrogen bonds contributed by the first sugar. The
addition of the third sugar to the disaccharide introduced three
more hydrogen bonds with the protein. The fourth sugar of the
tetrasaccharide contributed three additional hydrogen bonds to the
interactions with the protein. Likewise, the nonpolar interactions also
increased with the size of the oligosaccharide chain. The
contacts with the residues of the protein made by the four sugars are
listed in Table II.
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Fig. 3.
A, the sequence alignment of calnexin
and calreticulin. The structure of calreticulin was obtained by
homology modeling using the crystal structure of calnexin (PDB code
1JHN). The figure shows the sequence alignment of those regions of
calreticulin that were modeled against calnexin (CNX).
Residues 21-349 of calreticulin share similarity with residues 70-458
of calnexin. SSHM, secondary
structure of the homology modeled
structure and E- -strand, H-helix, L-loop. This figure was made using
CHROMA. B, modeled structure of calreticulin using calnexin
as the template. The structure contains six
-strands in the concave
sheet (red), seven
-strands in the convex sheet
(green), and two additional
-strands forming the roof
sheet (blue). The
-helix, which shields the hydrophobic
regions of the convex
-sheet, thus preventing the oligomerization of
monomers, is shown in yellow. As the structure of the
P-domain was not of importance to the present study, it is not shown in
the model.
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Fig. 4.
A, LIGPLOT representation of the
tetrasaccharide interactions in the modeled
calreticulin-tetrasaccharide complex. Hydrogen bonds within a distance
of 2.5 to 3.35 Å are denoted by dotted lines. Hydrophobic
interactions within 3.9 Å are included. B, view of the
sugar-binding site of calreticulin along with the tetrasaccharide. The
residues that may be involved in hydrogen bond interactions with the
sugar are shown as sticks. C, a Conolly
surface representation of the sugar-binding site of calreticulin with
the tetrasaccharide shown as sticks.
Hydrogen bonding interactions made by calreticulin residues with sugar
moieties
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucosidase I and II from the precursor N-glycan
Glc3Man9GlcNAc2 in the ER mediates
the interaction of glycoproteins with calreticulin and calnexin. These
two lectin chaperones promote the correct folding of their
glycoprotein substrates.
-sheet perhaps shields the hydrophobic regions of the
sheet, thus preventing the oligomerization through interactions between
the "convex" sheets of the monomers. This in turn explains why CRT,
in contrast to the legume lectins, exists as a monomer.
1),2
as the modeling studies do not show any aromatic residue within the
striking distance for stacking with the monosaccharide. The importance
of the stacking interactions between the hydrophobic
-face of the
pyranose ring of the monosaccharide and the lectins is well
known (46). For example, the studies of Sharma and Surolia (47)
explicitly noted that the failure of Phaseolus vulgaris lectin to bind to monosaccharides is related to the absence of such a
stacking interaction. Indeed, the introduction of an aromatic residue
in a P. vulgaris mutant that can stack with the
monosaccharide makes up for this deficiency of the native protein (48).
The inability of manno-oligosaccharides by themselves to bind shows that glucose at the nonreducing end is necessary for saccharide substrates to bind to calreticulin. An essential requirement of glucose
in
-1,3 linkage to mannose is supported by modeling studies showing
that the equatorially oriented hydroxyl group at C-2 of glucose
in the saccharide substrates is making a favorable contribution to the
binding process by hydrogen bonding with Asp-317 and Tyr-109 of
calreticulin (Table II). The lack of such an interaction between oligosaccharides devoid of
-1,3-linked nonreducing end glucose, i.e. as in Man
1-3Man, thus explains the obligatory
requirement of glucose in
-1,3 linkage for binding to CRT.
-glucopyranoside
to calreticulin is very poor. However, its extension by mannose in
-1,3 linkage increases the binding constant in a striking manner.
The extension of this structure by mannose in
-1,2 linkage, as in
Gluc
1-3Man
1-2Man
Me and
Gluc
1-3Man
1-2Man
1-2Man
Me, enhances the binding further
by 25- and 50-fold, respectively, indicating that the combining site of
the chaperone consists of a number of subsites each of which can
accommodate a hexapyranosyl residue. As the
G for any
of the saccharides equals the sum of the free energies from each
subsite filled, the contribution of
G from each subsite
of calreticulin is obtained by comparing a pair of saccharides. These
values together with the corresponding subsite occupied by the
constituent sugar units of the saccharides are shown in Table
III. An increase in
G as
the successive units of mannose are added supports our suggestion that
subsites A-D are contiguous, favorable subsites. Of these
subsites, B, C, and D are the strong binding subsites, with subsite C
being the strongest, whereas subsite A is a weakly interacting subsite.
These interpretations are supported by favorable changes in enthalpies
as the size of the oligosaccharides is increased. Thus, subsites C and
D contribute
5.0 and
3.14 kcal/mol, respectively, to the enthalpy
of binding.
Subsite model for calreticulin
Gb°,
Hb°,
and
S values for pairs of saccharides that differ by a
single sugar residue. It is assumed that an additional sugar residue
(italicized) is bound at the subsite listed above.
The decrease in the enthalpy of binding of saccharides with the
decrease in their sizes implies more extensive hydrogen bonding and van der Waals interactions for the larger saccharides. This is also
borne out by modeling studies, which show a progressive increase in the
number of hydrogen bonds and van der Waals interactions from glucose to
the tetrasaccharide. As the maximum increment in H occurs
for subsite C, it is not surprising to find that the mannosyl residue
occupying this subsite is involved in a maximum number of hydrogen
bonds. Generally, the hydrogen bond between the charged atom and a
hydroxyl group is stronger than that involving a neutral atom (49). A
perusal of the model of calreticulin-sugar interaction indeed shows
many hydrogen bonds between sugar hydroxyls and the charged atoms of
CRT. This explains the greater affinities of CRT-oligosaccharide
interactions as compared with other lectin-sugar interactions. In
addition to hydrogen bonding, residues Arg-73, Tyr-109, Asp-125,
Met-131, Leu-318, and Tyr-319 make hydrophobic interactions with the
sugar. Interestingly, the phenyl ring of Trp-319 may possibly make an
OH-
interaction with the third sugar (mannose of
Glc
1-3Man
1-2Man
1-2Man
Me). The O-4 hydroxyl group of the
mannose moiety makes contacts with all of the carbon atoms of the
phenyl ring of Trp-319 within a distance ranging from 2.9 to 3.6 Å.
OH-
interactions have been observed only recently in protein
structures (50, 51). In the case of protein-sugar interactions,
there is only one recent example from our own laboratory (52).
Interestingly, calreticulin-saccharide interactions, unlike most other
protein-carbohydrate reactions, show that for the binding of
Glc1-3Man
Me and Glc
1-3Man
1-2Man
Me, the values of free energies are more negative than those of the enthalpies (44). This
indicates the favorable entropic effects for binding, especially for
Glc
1-3Man
Me. This favorable entropic contribution could reflect
the hydrophobic nature of the reaction. This is also borne out by
greater burial of the apolar surface as the size of the oligosaccharide increases and is more pronounced for the disaccharide. The burial of the hydrophobic surface area was calculated by
subtracting the accessible hydrophobic surface area (calreticulin and
sugar) from that of the complex. A value of 122 Å was obtained for the monosaccharide, and it increased to 220 Å for the disaccharide. For
the trisaccharide and tetrasaccharide, this value was 233 and 260 Å,
respectively. However, as the plot of enthalpy-entropy compensation has
a slope of 1.25, enthalpic factors, viz. hydrogen bonding
and van der Waals interactions, appear to dominate
calreticulin-saccharide interactions as is also attested to by modeling
studies. The observation of enthalpy-entropy compensation in
calreticulin-saccharide recognition can be explained in two ways (Fig.
2). In one, the release or uptake of water has been explained as the
primary source of this compensation (53). Another and perhaps more
likely explanation is that a progressively greater portion of the
hydrophobic surface gets buried with an increase in the size of
ligand. However, this favorable term is countered by the effect of the
restriction of the ligand in the combining site; the larger the ligand,
the tighter the binding (i.e. more number of interactions),
and consequently, the greater the extent of restriction of degrees of
freedom and the larger the change in the unfavorable entropy term with
increasing ligand size, as observed experimentally.
In conclusion, our calorimetric studies demonstrate
that calreticulin has a single binding site for its respective glycan moiety and provide evidence for the interaction of various
oligosaccharides with calreticulin, showing that the entire
Glc1-3Man
1-2Man
1-2Man structure of the oligosaccharide is
recognized by calreticulin. Our modeling studies demonstrate
that an increased number of hydrogen bonds and van der Waals
interactions occurs as the size of the oligosaccharide is increased,
which supports the observed thermodynamic data.
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ACKNOWLEDGEMENT |
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We thank Prof. Raghavan Varadarajan for the use of the VP-ITC calorimeter.
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FOOTNOTES |
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* This work was supported by grants from the Department of Biotechnology and Department of Science and Technology, Government of India (to A. S.), the Swiss National Science Foundation (to A. H.), and the European Union (Contract No. HPRN-CT-2000-00001 to S. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains protocols
for synthesis of oligosaccharides.
§ Joint first co-authors.
¶ Present address: Paul Scherrer Institute, Villigen CH-5232, Switzerland.
To whom correspondence should be addressed. Tel.:
91-80-3942714; Fax: 91-80-3600535; E-mail:
surolia@mbu.iisc.ernet.in.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc. M209132200
2 M. Kapoor and A. Surolia, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CRT, calreticulin; ER, endoplasmic reticulum; ITC, isothermal titration calorimetry; PDB, Protein Data Bank; CNS, crystallography NMR software; MOPS, 4-morpholinepropanesulfonic acid.
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