Structural and Molecular Basis of Carbohydrate-Protein Interaction Systems as Potential ...

13 Pages·2011·0.95 MB·English

1672 Current Pharmaceutical Design, 2011, 17, 1672-1684
Structural and Molecular Basis of Carbohydrate-Protein Interaction Systems as
Potential Therapeutic Targets
Yukiko Kamiya1,2, Maho Yagi-Utsumi1,2, Hirokazu Yagi2 and Koichi Kato1,2,3,4
1Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higa-
shiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan, 2Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1
Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan, 3The Glycoscience Institute, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku,
Tokyo 112-8610, Japan, 4GLYENCE Co, Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464-0858, Japan
Abstract: The sugar chains covalently modifying proteins and lipids are recognized by a variety of proteins, thereby mediating a broad
range of physiological and pathological events on cell surfaces as well as in cells. Hence, these carbohydrate–protein interaction systems
could be potential therapeutic targets for various diseases, including viral infections, autoimmune diseases and neurodegenerative disor-
ders. Cumulative crystallographic data of lectins complexed with their cognate carbohydrate ligands have elucidated the sugar recogni-
tion modes of these proteins, offering a structural basis for the design of drugs targeting carbohydrate–lectin interaction systems. In par-
ticular, structural and functional studies of animal L-type lectins, which possess a carbohydrate recognition domain with a structural re-
semblance to that of leguminous lectins such as concanavalin A, have demonstrated the molecular mechanisms underlying their distinct
roles in sorting and trafficking of glycoproteins in cells, exemplifying the structure-based engineering that manipulates the sugar-binding
properties of lectins. Furthermore, structural basis has been provided for the functional interplay between the L-type lectin ERGIC-53 and
the EF-hand Ca2+-binding protein MCFD2 in the intracellular transport of the coagulation factors V and VIII. This article also deals with
pathological carbohydrate–protein interactions involving ganglioside clusters on cell surfaces, particularly focusing on the interaction
between amyloid (cid:2) (A(cid:2)) and GM1 ganglioside. This interaction triggers conformational transition and consequent aggregation of A(cid:2), and
therefore, is considered to be a key step in Alzheimer’s disease. The recently reported structural information of the A(cid:2)–GM1 interaction
is presented, underscoring the significance of assemblages of glycoconjugates as therapeutic targets.
Keywords: carbohydrate–protein interaction, lectin, ganglioside, hemagglutinin, amyloid (cid:2), X-ray crystallography, NMR spectroscopy, fron-
tal affinity chromatography.
INTRODUCTION with a series of intracellular lectins operating as molecular chaper-
ones, cargo receptors and ubiquitin ligases [11-16]. Dysfunction of
Carbohydrates covalently modify proteins and lipids to produce
diverse glycoconjugates. More than half of proteins in nature are these systems may result in incurable diseases such as combined
estimated to be modified by sugar chains [1]. The carbohydrate factor V (FV) and factor VIII (FVIII) deficiency (F5F8D) [20, 21].
Furthermore, antibodies directed against sugar chains as
moieties affect not only the physical properties of proteins such as
solubility and thermostability, but also their biological properties self-antigens can cause autoimmune disorders. For instance, the
including serum half-life and functional protein–protein interactions production of autoantibodies reactive with GM1 ganglioside is seen
in Guillain-Barré syndrome [22, 23]. It has recently been reported
[2-5]. Hence, glycosylation is now considered to be one of the most
important factors in the design and development of biopharmaceu- that ganglioside clusters are involved in the interaction with pro-
ticals currently typified by antibody medicines [6-10]. teins associated with neurodegenerative disorders such as Alz-
heimer’s disease (AD) [24-27]. Thus, carbohydrate–protein interac-
The glycans of membrane glycoproteins and glycolipids cover
tion systems are crucial in physiological and pathological processes,
the entire surface of animal and plant cells, thereby mediating
and therefore, can be potential drug targets [28].
cell–cell communication and viral infections. In cells, the carbohy-
drate moieties serve as tags for the quality control of glycoproteins To facilitate the design of drugs targeting carbohydrate recogni-
tion systems, deeper insights into the structural basis of carbohy-
in secretory and degradation pathways [11-16]. These carbohydrate
drate–lectin interactions are essential. However, it is difficult to
functions are generally expressed through interactions with carbo-
hydrate-recognition proteins collectively termed lectins. For exam- assess carbohydrate chains in structural analyses. One major reason
is that information concerning glycosylation is not directly coded in
ple, L-selectin, an animal lectin expressed on leucocytes, is in-
the genome. As a result, the glycan structures of glycoconjugates
volved in lymphocyte homing through its interaction with sialyl
6-sulfo Lewis X glycotope carried by CD34 and podocalyxin [17, are generally heterogeneous and the sequencing and production of
sugar chains cannot be performed conventionally. Hence, it is dif-
18]. Moreover, infections caused by bacteria and viruses are medi-
ficult to predict the glycoform of a protein, i.e. the sequence, link-
ated by interactions of their lectins with cognate sugar chains on the
surface of the host cells. For example, hemagglutinin (HA), a major age and profile of glycans. Moreover, determining the structure and
synthesis of sugar chains is unavoidably arduous because of their
envelope glycoprotein of influenza viruses, is a well-known viral
branched structures having a number of isomers. In addition, car-
lectin [19]. The binding of HA to sialylated glycans on host epithe-
lial cells is a critical initial step in their infection. The glycopro- bohydrate chains possess significant degrees of freedom in internal
motion, which hamper crystallization and interpretation of electron
tein-fates in cells, i.e. folding, translocation, and degradation, are
density. Structural information on sugar chains is thus extremely
determined through the interactions of their carbohydrate moieties
limited among the PDB-deposited data [29].
However, recent advances in glycobiology alongside rapid
*Address correspondence to this author at the Okazaki Institute for Integra-
technological progress have made it possible to realize preparations
tive Bioscience and Institute for Molecular Science, National Institutes of
and high-throughput analyses of sugar chains with complicated
Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787,
Japan; Tel: [phone]; Fax: [phone]; structures, which were previously considered to be impossible to
E-mail: [email protected] perform [30-34]. We have developed and applied our own system-
1381-6128/11 $58.00+.00 © 2011 Bentham Science Publishers
Carbohydrate–protein Interaction Systems as Therapeutic Targets Current Pharmaceutical Design, 2011, Vol. 17, No. 17 1673
atic methods for glycomics to address broader issues, including adopts (cid:2)+(cid:3) type topology and recognizes cognate oligosaccharides
glycosylation profiling and three-dimensional (3D) structural through the Ca2+ and the loop region [45-47] (Fig. 1E). To the best
analyses of sugar chains [35-39]. In this study, we illustrate several of our knowledge, no lectin has been reported to be composed of
examples of applications of structural analyses of carbohy- only (cid:2)-helices.
drate–protein interaction systems focusing on those potentially The sugar-binding modes of the lectins with a (cid:3)-sandwich
applicable to medicinal, diagnostic and clinical uses, and their re- structure are fundamentally classified into 2 groups [40, 48]. In one
lated practices. group, sugars are accommodated on a concave surface of a (cid:3)-sheet,
and in the other group, the sugar-binding site is constituted by the
BASIC STRUCTURES AND SUGAR-BINDING MODES OF
loops located at one end of the (cid:3)-barrel. The latter can be exempli-
LECTINS
fied by the crystal structures of influenza virus HA lectins com-
To better understand the biological functions of carbohydrates, plexed with their ligands (Fig. 2). HA is a homotrimer consisting of
it is essential to elucidate the sugar-binding modes of lectins as 3 structurally distinct regions, i.e. a globular head forming a
‘decoders of glycocodes’. Lectins are classified into more than 10 jelly-roll fold that serves as a CRD, a triple-stranded coiled-coil
families based on homology of amino acid sequences and/or 3D stalk and a globular foot composed of antiparallel (cid:3)-sheets [49, 50].
structure [40, 41] (Fig. 1). To date, 16 kinds of HA subtypes have been identified in influenza
A major class of lectin folds is the jelly-roll topology, which is virus A isolated from animals, including birds and humans. Al-
described as a (cid:3)-sandwich with 12–14 strands in two sheets. This though these subtypes commonly share the location of the
topology is adopted by a number of leguminous lectins typified by sugar-binding loops and (cid:2)-helix of the CRDs, detailed modes of
concanavalin A (Con A) in jack bean seeds, animal lectins includ- carbohydrate–protein interactions are different in different
ing galectin and calnexin and viral lectins such as influenza virus HA–ligand complexes [51]. Fig. 2, as an example, illustrates the
HA [40, 41]. The plant lectin jacalin has a (cid:3)-prism fold in which 3 crystal structure of influenza virus A HA1 complexed with LSTc
(cid:3)-sheets (each of which contains a Greek key motif) encircle the (Sia(cid:2)2-6Gal(cid:3)1-4GlcNAc(cid:3)1-3Gal(cid:3)1-4Glc) [52]. In the CRD, the
threefold axis of symmetry [42] (Fig. 1B), while siglec, a Sia-Gal disaccharide is accommodated in the 2 loops at the mem-
sialic-acid-specific lectin, assumes an immunoglobulin fold [43] brane-distal tip interacting with galactose and sialic acid residues.
(Fig. 1D). A fucose-specific lectin from Aleuria aurantia exhibits a Such information provides the structural basis of selective recogni-
(cid:3)-propeller fold that possesses 5 fucose-binding sites between the tion of (cid:2)2-3 and (cid:2)2-6 sialyl linkages, which is a crucial factor in
propeller wings [44] (Fig. 1A). The carbohydrate recognition do- cross-species transfer of influenza viruses because the sialyl linkage
mains (CRDs) of mannan-binding lectins, selectins and the macro- type depends on the animal [19]. It has been reported that a mini-
phage mannose receptor belong to the C-type lectin family, which mum of 2 amino acid substitutions in the sugar-binding site of the
A B C
D E F
Fig. (1). Examples of 3D structures of lectins complexed with their cognate ligands
(A) Anguilla anguilla agglutinin complexed with fucose (PDB code: 1OFZ), (B) Jacalin complexed with Gal(cid:3)1-3GalNAc (PDB: 1M26), (C) Con A com-
plexed with Man(cid:2)1-2Man (PDB: 1I3H), (D) Siglec-1 complexed with NeuAc(cid:2)2-3Gal(cid:3)1-4Glc (PDB: 1QFO), (E) Mannan-binding protein complexed with
Man(cid:2)1-3Man (PDB: 1KWY), (F) Mannose-6-phosphate receptor complexed with Man-6-phosphate (PDB: 1M6P) The carbohydrate ligands are shown in
stick representation (red). The bound calcium ions and manganese ions are shown as spheres (yellow and orange, respectively).
Graphics were generated using PyMOL (http://www.pymol.org).
1674 Current Pharmaceutical Design, 2011, Vol. 17, No. 17 Kamiyaet al.
A Sugar-binding site B
Gal(cid:2)4 Glc(cid:2)5
GlcNAc(cid:2)3
Gal(cid:2)2
190-Helix
220-Loopp
Sia
113300-LLoop
Fig. (2). Crystal structures of the complex between HA and sialyl pentasaccharide (A) The overall structure of Human1918 HA1 with 3 subunits coloured
differently. One receptor-binding site is highlighted in red. The sugar ligand Sia(cid:2)2-6Gal(cid:3)1-4GlcNAc(cid:3)1-3Gal(cid:3)1-4Glc is shown in red. (B) Close-up view of
the sugar-binding site of Human1918 HA1. The key residues forming the sugar-binding site are shown in magenta and cyan. The residues involved in potential
hydrogen bonds between HA and the Sia(cid:2)2-6Gal moiety are highlighted in magenta.
HAs of human viruses lead to switching of their binding the (cid:3)-sandwich. In this canonical site, an aspartate residue in loop
specificities from avian to human receptors [53, 54]. Several ani- A, an asparagine residue in loop C and an arginine residue in loop
mals, including quails, chickens, cats and dogs, express both (cid:2)2-3- B form a hydrogen bond network with O-6 and O-4 of the mannose
and (cid:2)2-6-linked sialic acids and probably serve as ‘mixing vessels’, residue. This sugar-binding mode is widely conserved among the
through which avian–human reassortant viruses might be generated leguminous lectins.
following co-infections [55-58]. Crystallographic data also indicate Maackia amurensis leucoagglutinin (MAL) is a leguminous
that the (cid:2)-helix in the sugar-binding pocket is in contact with the lectin with a unique specificity for sialyl oligosaccharides contain-
inner structures of sialyl receptors such as Gal-4 and Glc-5 residues ing NeuAc(cid:2)2-3Gal(cid:3)1-4GlcNAc/Glc(cid:3) [63, 64]. The crystal structure
of LSTc, offering a structural basis for designing a ligand with im- of this lectin with sialyllactose shows that the 4 loops constitute a
proved affinity for a specific HA (Fig. 2B). sugar-binding cleft [65]. The central galactose residue occupies the
The sugar-binding affinity of lectins is generally lower, some- canonical site interacting with loops A and C but not with loop B.
times with binding constants in the millimolar range, and can be Instead, another loop, which is not involved in mannose binding in
avidly enhanced through multivalent interactions. HA possesses 3 Con A, participates in galactose binding (Fig. 3C). These structural
carbohydrate-binding sites in the trimeric form, which are further data indicate that leguminous lectins sharing the same fold exhibit
clustered on the viral surfaces. Hence, clustering of sugar or sugar sugar-binding specificities through a variation of structures and
mimetics is a straightforward strategy for designing ligands with combinations of loops at their conserved binding sites.
improved affinity. Synthetic glycopolymers, such as polyacryla-
mide or polyglutamic acid carrying multivalent sialyl carbohy- STRUCTURAL BASIS OF SUGAR RECOGNITION BY
drates, have been shown to inhibit influenza virus infection effec- L-TYPE LECTINS
tively [59-61]. The CRDs homologous with leguminous lectins are shared
Con A is another representative lectin with a jelly-roll fold. The among certain kinds of animal lectins. Intriguing examples include
crystal structure of its complex with Man(cid:2)1-2Man-OMe reveals intracellular lectins; 53-kDa membrane protein of the ER–Golgi
that the sugar-binding site is located on the concave (cid:3)-sheet, in intermediate compartment (ERGIC-53), vesicular integral protein
which 4 loops (referred to as A, B, C and D) are involved in the of 36 kDa (VIP36), and VIP36-like (VIPL), which are related type
interaction with ligands [62]. Although Con A forms a tetramer 1 membrane proteins of the mammalian early secretory pathway
with 4 equivalent active sites, distinct sugar-binding modes have [66-68] (Fig. 4). Because of the homology of their luminal CRD to
been observed in 1 crystal structure, in which the that of leguminous lectins, these lectins are classified as L-type
Man(cid:2)1-2Man-OMe disaccharide exhibited unambiguous electron lectins [66-71]. ERGIC-53 forms a disulphide-linked hexameric
density in 2 of the 4 subunits. Despite such multiplicity of interac- structure composed of 6 identical subunits [72]; in each of these,
tion modes, the nonreducing terminal mannose in one subunit (Fig. the CRD is connected to a coiled-coil domain, followed by a trans-
3A) and the reducing terminal mannose in another subunit (Fig. 3B) membrane region, along with a cytosolic region that contains both
were accommodated in the same manner at corresponding sites of endoplasmic reticulum (ER) exit and retrieval motifs [73, 74].
Carbohydrate–protein Interaction Systems as Therapeutic Targets Current Pharmaceutical Design, 2011, Vol. 17, No. 17 1675
Man
Man Gal
Asn14 AAsn1144 Asp137
Asp208 Asp208 Asp87
LLoooopp(cid:2)AA
Loop(cid:2)D
Loop(cid:2)C
Loop(cid:2)B
Fig. (3). Crystal structures of Con A complexed with Man(cid:2)1-2Man-OMe (A and B) and MAL complexed with NeuAc(cid:2)2-3Gal(cid:3)1-4GlcNAc (C) The overall
structures of lectins are presented at the bottom. Close-up views of the sugar-binding sites are shown at the top of the figure. The key amino acid residues and
sugars forming the hydrogen bonds are drawn in stick representation. The hydrogen bonds are shown as dashed-line sticks.
ER ERGIC/cis-Golgi
ppHH(cid:3)77.44 ppHH(cid:3)55.88(cid:2)66.66
VIP36
COP I
Malfolded glycoprotein
ERGIC-53
Folded glycoprotein
COP II
MCFD2
VIPL
Mannose
N-Acetylglucosamine
Fig. (4). Schematic drawing of the functional interplay among the L-type lectins in sorting and trafficking of the cargo glycoproteins in cells. (adapted from
reference [15] with modification).
1676 Current Pharmaceutical Design, 2011, Vol. 17, No. 17 Kamiyaet al.
Thus, this lectin cycles between the ER and the ER-Golgi interme- FAC data demonstrated that VIPL and VIP36 specifically in-
diate compartment (ERGIC) [73]. On the other hand, VIP36 and teract with the high-mannose-type oligosaccharides retaining the
VIPL have a stalk region instead of a coiled-coil domain [67, 75]. trimannosyl branch, i.e. Man(cid:2)1-2Man(cid:2)1-2Man, in the D1 branch
VIP36 cycles between the Golgi complex and the ER [68], while without terminal glucosylation, while ERGIC-53 has broader speci-
VIPL resides in the ER [67]. Hence, these 3 lectins are considered ficity and lower affinity in binding to oligosaccharides containing 6
to play distinct roles in the secretory pathway, notwithstanding the or more mannose residues, irrespective of the presence or absence
structural similarities of their CRDs. It is well known that mutations of the terminal glucose residue [78, 79] (Fig. 5). The terminal glu-
in the gene encoding ERGIC-53 can cause F5F8D, which is an cose residue on the D1 branch is removed and reattached during the
autosomal recessive disorder characterized by coordinated reduc- chaperone-assisted folding process of nascent glycoproteins in the
tion in the plasma levels of FV and FVIII [21]. This disorder is also ER[81]. Hence, the different specificities of L-type lectins for glu-
associated with mutations in another gene, i.e. multiple coagulation cosylated oligosaccharides suggest their distinct roles in sorting and
factor deficiency 2 (MCFD2) [20, 76], which encodes a soluble trafficking, coupled with quality control of the cargo glycoproteins.
luminal protein with 2 EF-hand Ca2+-binding motifs. It has been
Satoh et al. determined the crystal structures of VIP36-CRD
demonstrated that these 2 causative gene products of F5F8D form a complexed with mannose, Man(cid:2)1-2Man and Man(cid:2)1-2Man(cid:2)1-
complex responsible for the intracellular transport of FV and FVIII 3Man(cid:3)1-4GlcNAc(cid:3)1-4GlcNAc [71]. As observed in the crystal
[20, 77]. In this transport system, the sugar-binding activity of ER-
structures of Con A in complex with mannose-containing sugars
GIC-53 is considered important for capturing the cargos that are [62], all the crystal structures exhibited interaction of a mannose
heavily glycosylated proteins. To better understand the molecular residue with the aspartate residue in loop B and the asparagine resi-
mechanisms underlying the biological functions of the L-type lect-
due in loop C, both of which are conserved in the pocket near the
ins, it is crucial to elucidate the structural basis of carbohydrate Ca2+-binding site. On the basis of these crystallographic data, along
recognition by these lectins. with the knowledge of its specificity for the D1 trimmanosyl
The first step is to define the sugar-binding specificities of the branch, they proposed a 3D structure model of VIP36-CRD bound
homologous L-type lectins; however, this is difficult to predict to Man GlcNAc (Fig. 6A). The model indicates that in addition to
8 2
solely on the basis of the crystal structures of unliganded CRDs. the key residues located in the canonical site, Ser96 and Asp261 are
For comprehensive and quantitative characterization of the in contact with Man-D1, providing no space for accommodation of
sugar-binding properties of these lectins, we performed frontal af- the additional glucose residue apparently due to steric hindrance by
finity chromatography (FAC) using the pyridylaminated sugar li- Glu98 (Fig. 6B). While VIPL conserve these residues and therefore
brary for comprehensive characterization of the sugar-binding show similar profiles of sugar-binding specificities, the carbohy-
properties of ERGIC-53, VIPL and VIP36 [78, 79]. In this tech- drate-binding pocket of ERGIC-53 is significantly shallower be-
nique, individual sugars are applied onto a column on which the cause it lacks the protrusive Asp261 side chain (Fig. 6C). This led
lectin is immobilized. On this column, sugar chains with higher to the hypothesis that removal of the structural constraint caused by
affinity for the lectin are expected to be eluted later. Based on the the Asp261 side chain may enable conformational rearrangement of
retardation of the elution of the sugar chains in comparison with the D1 outer branch, avoiding the steric crush of the glucose residue
that of a control ligand that has not interacted with the lectin, the in VIP36-CRD (Fig. 6D). Indeed, our FAC data demonstrated that
binding constants for the lectin can be computed. This method en- mutated VIP36-CRD, in which asparatate was substituted with
ables a quantitative characterization of carbohydrate–protein inter- glycine (VIP36-D261G), could bind mono-glucosylated
actions with lower affinities (K: ~104 M(cid:2)1) [80]. high-mannose-type oligosaccharides [78].
a
(cid:2)(cid:3)(cid:4)
4.0
VIP36
VIPL
3.(cid:6)0(cid:3)(cid:4) ERGIC-53
1
(cid:3)M
)(cid:2) 22.(cid:5)(cid:5)00(cid:3)(cid:4)(cid:4)
44
0
1
(cid:2)
x
(
a1.(cid:7)0(cid:3)(cid:4)
K
0.(cid:4)0(cid:3)(cid:4)
(cid:10) (cid:10) (cid:12) (cid:13) (cid:14) (cid:12) (cid:13) (cid:15) (cid:14) (cid:12) (cid:13) (cid:16) (cid:17) (cid:20) (cid:17) (cid:2) (cid:6) (cid:5)
(cid:9) (cid:9) (cid:11) (cid:11) (cid:11) (cid:11) (cid:11) (cid:9) (cid:15) (cid:15) (cid:15) (cid:9) (cid:9) (cid:18)(cid:19) (cid:9) (cid:9) (cid:9) (cid:9)
(cid:16)
(cid:8)(cid:8) (cid:9)(cid:9) (cid:9)(cid:9) (cid:9)(cid:9) (cid:9)(cid:9) (cid:9)(cid:9) (cid:8)(cid:8) (cid:9)(cid:9) (cid:9)(cid:9) (cid:9)(cid:9) (cid:8)(cid:8)
(cid:8) (cid:8) (cid:9)
Mannose
N-Acetylglucosamine
Glucose
Fig. (5).K values of high-mannose-type oligosaccharides for VIP36, VIPL and ERGIC-53 (adapted from reference [122]).
a
Carbohydrate–protein Interaction Systems as Therapeutic Targets Current Pharmaceutical Design, 2011, Vol. 17, No. 17 1677
A B
Asp261
Asp261
Leu262
Ser96 Gly260
Glu168
Tyr164
Asp131 Asn166 Thr169 Glu98
His190
C D
G(cid:2)y260 Leu261 Gly261
Gly259
Ser96
C
Phe154 D1 4
Asp112211
Asn156
Arg98
His190
Fig. (6). 3D structural models of sugar-binding sites of VIP36 (A and B), ERGIC-53 (C) and VIP36-D261G (D) (adapted from reference [122] with modifica-
tions)
The models of VIP36 complexed with oligosaccharides are based on the crystal structures as previously described in reference [78].
ManGlcNAc-Asn (A) and GlcManGlcNAc-Asn (B and D) are shown in a stick representation in which the glucose residue and Man(cid:2)1-2Man(cid:2)1-2Man
8 2 8 2
residues are coloured magenta and green, respectively, while the rest of the ligand molecule is coloured purple.
Another important aspect of understanding the carbohydrate VIP36 cannot be ascribed to a single titratable group. Interestingly,
binding of L-type lectins is to elucidate the molecular mechanisms VIP36 and VIPL, which share 70% amino acid identity in the
underlying the catching and releasing of glycosylated cargos during CRDs but exhibit distinct pH dependence of sugar binding [78, 79],
their intracellular translocation. Progressive acidification occurs share all titratable groups in and around the sugar-binding site, ex-
from the ER through the Golgi complex to the trans-Golgi network cept for an extra lysine residue (Lys166) near the putative
because of H+ pumping by v-ATPase: the typical pH values of the sugar-binding site of VIPL-CRD [78]. This prompted us to design a
ER, cis-Golgi network and trans-Golgi network have been reported mutated VIP36-CRD in which a lysine residue is inserted into the
to be 7.2, 6.4 and 5.4, respectively [82]. It has therefore been sup- corresponding position, i.e. between Glu168 and Thr169
posed that the subcellular pH difference is a crucial factor affecting (VIP36-168K169). Our FAC analysis demonstrated that this mutant
the interactions of L-type lectins with cognate ligands. Indeed, bio- retained the sugar-binding selectivity of the wild type, but no longer
chemical and NMR data indicate that ERGIC-53 and VIPL effec- exhibited bell-shaped pH dependence; rather, it showed a steady
tively bind the carbohydrate ligands under neutral conditions but increase in affinity for oligosaccharides with increasing pH, very
not at lower pH, whereas VIP36 exhibits a bell-shaped binding much like VIPL (Fig. 7). These studies exemplify the struc-
dependence on pH with a lower pH optimum (pH 6.5) [15, 78, 79, ture-based engineering of lectins demonstrating that the
83] (Fig. 7). The pH dependence of sugar binding of L-type lectins sugar-binding properties of lectins can be switched by a single
and their subcellular localization, together with the organellar pH, amino acid substitution or insertion.
suggests that ERGIC-53 binds glycosylated cargos in the ER and Cumulative evidence indicates that intracellular lectins cooper-
releases them upon arrival in lower-pH environments on the secre-
ate with other proteins in the interaction with their client glycopro-
tory pathway, while VIP36 captures glycoproteins in the cis-Golgi teins [11, 12, 84, 85]. ERGIC-53 forms a complex with MCFD2,
network and recycles to the ER, where it releases the cargos be- the product of the MCFD2 gene, in a Ca2+-dependent manner,
cause of higher pH, thereby contributing to post-ER quality control
thereby functioning as a cargo receptor of FV and FVIII in their
of malfolded glycoproteins [11, 15] (Fig. 4). early secretory pathway (vide supra) [20, 77, 86]. ERGIC-53 also
The attenuation of sugar-binding affinities of ERGIC-53 at interacts with ERp44, a member of the protein disulphide isomerase
lower pH can be attributed to ionization of His178 (in human ER- family, thereby contributing to the hexameric structure formation of
GIC-53) [83], which destabilizes the coordination of neighbouring immunoglobulin M [87, 88]. Recently, we determined the 3D
Ca2+ and consequently disrupts the structural integrity of the structure of the complex of MCFD2 with the CRD of ERGIC-53 by
sugar-binding loop [69, 70]. The bell-shaped pH dependence of X-ray crystallographic analysis in conjunction with NMR and bio-
1678 Current Pharmaceutical Design, 2011, Vol. 17, No. 17 Kamiyaet al.
3.0
VIPL
VVIIPP3366
2.5 VIP36(cid:3)168K169
1 22.00
(cid:3)(cid:3)M
)(cid:2)
4
0
1 1.5
x(cid:2)
(
a
K
1.0
00.55
0.0
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
pH
Fig. (7). Effects of pH on affinities of VIPL, VIP36 and VIP36-168K169 for pyridilaminated ManGlcNAc (adapted from reference [122]).
9 2
physical methods [89]. The crystallographic data indicate that crystallographic analyses of their complexes with the pentasaccha-
MCFD2 primarily binds the segment spanning the first and second ride released from GM1 [95, 97] (Figs. 9A and 9B). In these struc-
(cid:2) strands of ERGIC-53, which are remote from the sugar-binding tures, the outer branch of the carbohydrate chain is involved in the
site and not conserved in other L-type lectins (Fig. 8). The missense interaction. On the other hand, the conformations of the GM1 oli-
mutations of MCFD2 reported until date in F5F8D result in amino gosaccharide and its analogues have long been characterized by
acid substitutions at the binding interface of ERGIC-53 or the Ca2+ solution NMR methods [98-101]. Recently, we determined the 3D
coordination site. These data indicate that the mutations of MCFD2 structure of the pentasaccharide in lyso-GM aqueous micelles (mo-
hamper complex formation with ERGIC-53, rendering the intracel- lecular mass: ~60 kDa) based on NMR spectral data. Our molecular
lular transport of FV and FVIII less efficient and ultimately offering dynamics calculations in conjunction with the NOE-derived dis-
a structural basis for F5F8D. tance constraints as pseudoenergies converged to a well-defined
In comparison with the previously reported 3D structure of bouquet-like structure [102] (Figs. 9C and 9D). Comparison of the
MCFD2 [90] and ERGIC-53-CRD [70], our crystallographic data sugar structure with the sugars complexed with the cholera toxin
and polyoma virus indicate that the conformation of the carbohy-
also indicate that MCFD2 undergoes significant conformational
alteration upon binding to ERGIC-53, while MCFD2 binding has drate chains is altered upon binding to the proteins in a
little impact on the conformation of ERGIC-53-CRD [89]. These ligand-dependent manner (Figs. 10A and 10B).
observations suggest that the sugar-binding affinity and specificity Recently, growing evidence has indicated that gangliosides
of ERGIC-53 are not influenced by interaction with MCFD2. One interact with amyloid (cid:2) (A(cid:2)), which is a 40- or 42-amino-acid pep-
intriguing possibility is that MCFD2 is converted into its active tide cleaved from its precursor membrane protein by sequential
form upon complex formation with ERGIC-53 and is therefore able actions of (cid:2)- and (cid:3)-secretases, and promote the assembly of A(cid:2),
to capture polypeptide segments of FV and FVIII in cooperation which is considered to be a crucial step in AD [25, 26]. In 1995,
with ERGIC-53 interacting with their carbohydrate moieties. The Yanagisawa et al. reported a unique A(cid:2) species in cerebral cortices
ERGIC-53/MCFD2 complex has been proposed to be a therapeutic of AD patients, which is closely associated with GM1 ganglioside
target in anticoagulation [20, 91]. Our model suggests that the de- [103]. Subsequently, a series of in vitro studies have indicated that
sign of drugs targeting this system should take into account the GM1-bound A(cid:2) (GA(cid:2)) possesses an extremely high potential to
allosteric conformational change of MCFD2 upon complex forma- facilitate A(cid:2) assembly [104, 105], which prompted the hypothesis
tion with ERGIC-53. Thus, proteins cooperating with lectins could that GA(cid:2) acts as a seed for A(cid:2) fibrillogenesis in the brain [103,
also be drug targets in an attempt to modulate their interactions with 106, 107]. Hence, ganglioside–A(cid:2) interaction systems are potential
glycoproteins. therapeutic targets in AD treatment. To better understand the un-
derlying mechanisms of pathological functions of the gangliosides
GANGLIOSIDE CLUSTERS AS DRUG TARGETS in AD, it is highly desirable to obtain detailed structural information
The carbohydrate moieties on animal cell surfaces can also be of these interaction systems.
therapeutic targets because glycosphingolipids, including gangli- Ultra-high-field NMR data also showed that the sugar–lipid
osides, are known to play a variety of physiological and pathologi- interface is primarily responsible for binding to A(cid:2)(1–40), suggest-
cal roles as receptors for microbial toxins, mediators of cell adhe- ing that the inner part of the ganglioside clusters for accommodat-
sion and modulators of signal transduction [92, 93]. For instance, ing A(cid:2) molecules is more important than the outer carbohydrate
GM1 ganglioside acts as a target of cholera toxin [94], polyoma branches that provide microbial toxin- and virus-binding sites (Figs.
virus [95], growth-regulatory galectin-1 [96] and autoantibodies 9A and 9B) [102]. Because the structure of the inner part is shared
associated with Guillain-Barré syndrome [22]. The sugar-binding among gangliosides [108-110], A(cid:2)(1–40) would be expected to
modes of cholera toxin and polyoma virus have been visualized by interact with various ganglioside micelles. A(cid:2)(1–40) has been
Carbohydrate–protein Interaction Systems as Therapeutic Targets Current Pharmaceutical Design, 2011, Vol. 17, No. 17 1679
Sugar-binding site
ERGIC-53-CRD
180o
MMCCFFDD22
N N
N
C C C
C
Fig. (8). Structure of the complex between ERGIC-53-CRD and MCFD2. Uncomplexed structures of rat ERGIC-53 (cyan) and human MCFD2 (pink) are
superimposed on the structure of the complex between human ERGIC-53 (yellow) and MCFD2 (green). The bound calcium ions are shown as spheres.
A C
GalNAcIII
OOOOHHHH6666
GalIV GaHHHHHHHHlOOOOOOOOI4444V3333CCCC55555555HHHH2222OOOOOOOO22222222HHHHHHHHOOOO1111OOOO4444HHHH3333OOOO3333HHHHCCCCOOOO8888CCCC55556666CCCCHHHH7777OOOO22222222OOOOOOOONNNN2222HHHHCCCCHHHHOOOO111111112222OOOO4444OOOOCCCC666633335555HHHH2222OOOOOOOOHHHH2222OOOOHHHH 1111OOOOHHHH4444OOOOCCCCHHHH333355552222OOOOHHHH2222OOOOOOOOHHHH1111OOOO CCCC1111HHHH2222HHHH HHHH2222 NNNN3333OOOOHHHH2222HHHH4444 5555 6666 7777 8888 999911110000 11111111
GGallNNAAcIIIIII NNeeuuAAcc HHHHHHHHOOOOOOOO8888HHHH7777 6666 4444OOOO 3333 GalII 6666GGllccI CCeerraammiiddee
NeuAc HHHHOOOOHHHH2222CCCCHHHHHHHHOOOO OOOOHHHH
9999 11110000 NNNN5555
GalII HHHH33331111CCCC1111CCCCOOOOHHHH
GlcI
B D
GalIV
GalNAcIII
GalIV
NeuAc
GalNAcIII
NNeuAAc
GalII GalII GlcI
GlcI
Fig. (9). Crystal structure of the complex between the GM1 pentasaccharide and cholera toxin (A) or simian virus 40 (B). GM1 is shown in blue. (C) Distance
constraints derived from the NOE data were employed for calculations pertaining to the 3D structures of the carbohydrate moieties of lyso-GM1 aqueous
micelles. Red and blue arrows show inter- and intraresidue NOEs, respectively. (D) Ten lowest-penalty structures are superimposed by fitting the ring atoms
of GalII, GalNAcIII and NeuAc.
shown to bind to a variety of gangliosides, though with different tion phase to form encounter complexes with A(cid:2), resulting in slight
affinities [102, 104, 105, 111]. Our NMR data suggest that the in- variations in the apparent K values.
a
teraction between A(cid:2)(1–40) and GM1 micelles involves multiple
To provide a more detailed understanding of the conformational
steps, including an initial encounter complex formation and a sub- state of A(cid:2)(1–40) accommodated in the ganglioside clusters, we
sequent accommodation process. It is therefore possible that the characterized the conformation of A(cid:2)(1–40) bound to lyso-GM1
outer carbohydrate branches play specific roles during the associa-
and GM1 micelles by employing 920 MHz NMR spectroscopic
1680 Current Pharmaceutical Design, 2011, Vol. 17, No. 17 Kamiyaet al.
GalIV
A B
GalNAcIII
(cid:2)(cid:2),(cid:2)(cid:3)(cid:3) (cid:2)(cid:2),,(cid:2)(cid:3)(cid:3)
IIII,II IIIIII,IIII
(cid:5)(cid:6)(cid:4) (cid:5)(cid:6)(cid:4)
ees (cid:3)(cid:4) ees (cid:3)(cid:4) NeuAc GalII GlcI
gr gr
e e
dd dd
/ /
(cid:3) (cid:7)(cid:3)(cid:4) (cid:3) (cid:7)(cid:3)(cid:4)
GalIV
(cid:7)(cid:5)(cid:6)(cid:4) (cid:7)(cid:5)(cid:6)(cid:4)
(cid:7)(cid:5)(cid:6)(cid:4) (cid:7)(cid:3)(cid:4) (cid:3)(cid:4) (cid:5)(cid:6)(cid:4) (cid:7)(cid:5)(cid:6)(cid:4) (cid:7)(cid:3)(cid:4) (cid:3)(cid:4) (cid:5)(cid:6)(cid:4) GalNAcIII
(cid:2)(cid:2)//deggrees (cid:2)(cid:2)/deggrees
(cid:2),(cid:2)(cid:3) (cid:2),(cid:2)(cid:3)
IV,III A,II
(cid:5)(cid:6)(cid:4) (cid:5)(cid:6)(cid:4)
NeuAc
ese (cid:3)(cid:4) ese (cid:3)(cid:4) GGaallIIII GGllccII
e e
r r
g g
e e
d d
(cid:3)/(cid:7)(cid:3)(cid:4) (cid:3)/ (cid:7)(cid:3)(cid:4)
GalIV
(cid:7)(cid:5)(cid:5)(cid:6)(cid:6)(cid:4)(cid:4) (cid:7)(cid:7)(cid:5)(cid:5)(cid:6)(cid:6)(cid:4)(cid:4)
(cid:7)(cid:5)(cid:6)(cid:4) (cid:7)(cid:3)(cid:4) (cid:3)(cid:4) (cid:5)(cid:6)(cid:4) (cid:7)(cid:5)(cid:6)(cid:4) (cid:7)(cid:3)(cid:4) (cid:3)(cid:4) (cid:5)(cid:6)(cid:4) GalNAcIII
(cid:2)/degrees (cid:2)/degrees
GalII
GlcI
NeuAc
Fig. (10). Comparison of the glycosidic torsions and 3D structures of the carbohydrate moieties of lyso-GM1 aqueous micelles (green) with those of GM1
obtained by crystallographic analyses [PDB code: 2CHB (magenta); 3CHB (blue); 3BWR (yellow)]. (A)(cid:2),(cid:3) maps of glycosidic torsions. (cid:2) = O5-C1-On-Cn,
(cid:3) = C1-On-Cn-Cn-1. (B) Two structures are superimposed by fitting the ring atoms of GalII, GalNAcIII and NeuAc.
methods in conjunction with 2H, 13C and 15N-labeling of A(cid:3)(1–40) method could also be a powerful tool for screening compounds that
[112]. The secondary chemical shifts of the polypeptide backbone act as (cid:2)-helix stabilizers of A(cid:3), and therefore, could be anti-AD
indicated that A(cid:3)(1–40) forms discontinuous (cid:2)-helices at the seg- drug candidates.
ments His14–Val24 and Ile31–Val36 upon binding to the gangliosidic
micelles, leaving the remaining regions disordered. Furthermore, by SUMMARY AND PROSPECTIVES
using the TROSY-based saturation transfer method, we demon- Accumulating 3D structural data of the carbohydrate–protein
strated that A(cid:3)(1–40) is accommodated on the hydropho- complexes provides the basis for biomolecular engineering and the
bic/hydrophilic interface of the ganglioside cluster exhibiting an design of drugs that modulate molecular recognition processes. The
up-and-down topological mode in which the 2 (cid:2)-helices and the carbohydrate chains adopt different conformations upon binding to
C-terminal Val39–Val40 dipeptide segment are in contact with the different (or even the same) lectins. Such conformational multi-
hydrophobic interior, whereas the remaining regions are exposed to plicities, along with lower affinities, in the interactions with pro-
the aqueous environment (Fig. 11). These findings suggest that the teins are unique but difficult properties to deal with in the carbohy-
GM1 clusters offer a unique platform for binding coupled with drate–protein interaction systems, in both experimental and theo-
conformational transition of A(cid:3) molecules, thereby restricting their retical approaches. To quantitatively understand the energetics of
spatial rearrangements to promoting specific intermolecular interac- such interaction systems, several issues must be addressed, includ-
tions leading to cross-(cid:3)-sheet formation. This raises a novel me- ing the characterization of dynamics and hydration of carbohy-
dicinal strategy to suppress (cid:3)-structure formation by stabilizing the drates, proteins and their complexes under various pH and ionic
(cid:2)-helical structure of A(cid:3) on the ganglioside clusters. Indeed, it has conditions.
recently been reported that compounds such as
In addition, the assembly state of glycoconjugate clusters is
N1-decanoyl-diethylenetriamine that bind and stabilize the (cid:2)-helical
very important for characterizing carbohydrate–protein interactions
region of A(cid:3) attenuate fibril formation and consequent toxicity in
[114, 115]. It is conceivable that the size and curvature of the gan-
the Drosophila model of AD [113]. The NMR spectroscopic gliosidic micelles along with the A(cid:3) density could determine the
Carbohydrate–protein Interaction Systems as Therapeutic Targets Current Pharmaceutical Design, 2011, Vol. 17, No. 17 1681
1
D A E F R
H 1.0
D o
S ati
Hydrophilic GGY1E0V 20 SN K GA30 G GV y rIntensity 0.1
Hyydropphobic H HQ K LV F FA E DVG II G MV 40 VOHH NH HO
LL
(cid:4)-helix I (cid:4)-helix II
Fig. (11). Schematic drawing of A(cid:3)(1–40) lying on the hydrophobic/hydrophilic interface of ganglioside clusters (adapted from reference [112] with modifica-
tion]. The amino acid residues exposed to the hydrophilic and hydrophobic milieus are represented by closed and open circles with single-letter codes. Map-
ping of nonexchangeable hydrogens showing the PRE effects observed for lyso-GM1 micelles in the presence of a C-terminally spin-labelled A(cid:3)(1–40) on the
lowest-penalty 3D model of the carbohydrate moiety. The linear colour gradient (from red to white) indicates the intensity ratios of the CH peaks of lyso-GM1
before and after radical quenching [102]. The sugar hydrogen atoms coloured in red and the ceramide hydrogen atoms located within the red ellipse area ex-
hibit lower peak intensity ratios, indicating that they are in close spatial proximity of the C-terminus of A(cid:3)(1–40).
occurrence of A(cid:3)–A(cid:3) interactions leading to polymerization events and M. Y.-U. are recipients of a Japan Society for the Promotion of
[116]. Moreover, A(cid:3) fibril formation triggered by ganglioside clus- Science Research Fellowship for Young Scientists.
ters has been shown to be significantly enhanced in the presence of
cholesterol [117, 118]. Single amino acid substitutions of A(cid:3) and ABBREVIATIONS
(cid:2)-synuclein, an intrinsically disordered protein identified as the HA = Hemagglutinin
major component of the Lewy bodies and Lewy neurites of Parkin-
Gal = Galactose
son’s disease, alter their interaction properties with specific gangli-
oside clusters [25, 26, 119]. Furthermore, the structural basis of the Glc = Glucose
carbohydrate–carbohydrate interactions within and between glyco- Man = Mannose
conjugates clusters remains largely unexplored [92, 120]. GlcNAc = N-acetylglucosamine
To address these issues, it is imperative to apply advanced ex- GalNAc = N-acetylgalactosamine
perimental methodology employing ultra-high-field NMR spec-
NeuAc = N-acetyl neuraminic acid
troscopy and other biophysical methods using appropriately de-
signed systems, along with computational simulation for larger Sia = Sialic acid
sizes and longer timescales using accurate force fields for carbohy- LSTc = Lactoseries tetrasaccharide c
drates [121]. Such a combination of experimental and theoretical
FV = Coagulation factor V
approaches would offer fundamental insights into the molecular
processes involved in carbohydrate–protein interactions and yet FVIII = Coagulation factor VIII
putative carbohydrate–carbohydrate interactions leading to novel F5F8D = Combined factor V and factor VIII defi-
concepts for designing drugs targeting neurodegenerative and cur- ciency
rently incurable disorders.
Con A = Concanavalin A
ACKNOWLEDGEMENTS CRD = Carbohydrate recognition domain
We wish to acknowledge Dr. Yoshiki Yamaguchi (RIKEN), Dr. ER = Endoplasmic reticulum
Yasuo Suzuki (Chubu University), Dr. Katsuhiko Yanagisawa (Na- ERGIC = ER-Golgi intermediate compartment
tional Institute for Longevity Sciences), and Dr. Tomoshi Kameda
ERGIC-53 = 53-kDa membrane protein of the ER–Golgi
(AIST) for useful discussions and collaborations on some topics
intermediate compartment
shown in this paper. We thank Dr. Soichi Wakatsuki (High Energy
Accelerator Research Organization) and Dr. Tadashi Satoh VIP36 = Vesicular integral protein of 36 kDa
(RIKEN) for providing the atomic coordinates of the 3D structural VIPL = VIP36-like
model for binding between VIP36-CRD and high-mannose-type
MCFD2 = Multiple coagulation factor deficiency 2
glycans. This work was supported, in part, by the Nanotechnology
FAC = Frontal affinity chromatography
Network Project and Grants in Aid for Scientific Research
(17046017, 18023032, 20023033, 20107004, 21370050, 21870052, A(cid:3) = Amyloid (cid:3)
22020039, 22790076) from the Ministry of Education, Culture, GA(cid:3) = GM1-bound A(cid:3)
Sports, Science and Technology of Japan, and by the CREST pro-
AD = Alzheimer’s disease
ject from the Japan Science and Technology Agency. Y. K, H. Y.