Glycobiology, 2015, vol. 25, no. 9, 920–952
doi: 10.1093/glycob/cwv037
Advance Access Publication Date: 1 June 2015
Review
Review
Antibody recognition of carbohydrate epitopes†
Omid Haji-Ghassemi2, Ryan J Blackler2, N Martin Young3,
and Stephen V Evans1,2
2
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8P 3P6, and 3Human
Health Therapeutics, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6
1
To whom correspondence should be addressed: e-mail: [email protected] (SVE)
†
The atomic coordinates and structure factors for all structures discussed are available in the Protein Data Bank, Research
Collaborators for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org) (see Table I).
Received 12 February 2015; Revised 12 May 2015; Accepted 24 May 2015
Abstract
Carbohydrate antigens are valuable as components of vaccines for bacterial infectious agents and
human immunodeficiency virus (HIV), and for generating immunotherapeutics against cancer. The
crystal structures of anti-carbohydrate antibodies in complex with antigen reveal the key features of
antigen recognition and provide information that can guide the design of vaccines, particularly
synthetic ones. This review summarizes structural features of anti-carbohydrate antibodies to over
20 antigens, based on six categories of glyco-antigen: (i) the glycan shield of HIV glycoproteins; (ii)
tumor epitopes; (iii) glycolipids and blood group A antigen; (iv) internal epitopes of bacterial lipopolysaccharides; (v) terminal epitopes on polysaccharides and oligosaccharides, including a group of
antibodies to Kdo-containing Chlamydia epitopes; and (vi) linear homopolysaccharides.
Key words: Anti-carbohydrate antibodies, carbohydrate recognition, Chlamydia, HIV-1 gp120, lipopolysaccharide, monoclonal
antibody, X-ray crystal structures
Introduction
Many of the concepts that define modern molecular immunology stem
from studies of antibody recognition of carbohydrate antigens, chiefly
carried out by pioneers such as Drs Michael Heidelberger and Elvin
Kabat on pneumococcal polysaccharides, dextrans and blood group
antigens. However, the structural characterization of carbohydratespecific antibodies was then hampered by the lack of homogeneous
functional antibody species. Nevertheless, work on heterogeneous
preparations by Kabat and others established many key characteristics, e.g., that the combining site could accommodate up to six residues
and could take the form of a pocket or groove (Kabat 1978). The
general structure of antibodies (Figure 1) was established by protein
sequencing and crystallography in the 1970s, and fully elucidated
by the structures of two whole IgGs determined by McPherson and
colleagues (Harris et al. 1998). The discovery that mouse myelomas
can secrete homogeneous IgA and IgM anti-carbohydrate antibodies
(Potter and Leon 1968) led to a major advance in understanding antibody–antigen recognition with the solution of two crystal structures of
Fab fragments: the phosphocholine-binding IgA M603 (Satow et al.
1986) and the galactan-binding IgA J539 (Suh et al. 1986). The
M603 Fab structure included phosphocholine bound in a small pocket
in the centre of the binding site and since phosphocholine can be a
dominant epitope when it occurs on polysaccharides (Young et al.
2013), M603 may be considered the first anti-carbohydrate-antibody
crystal structure.
The specificities of myeloma immunoglobulins were determined
empirically, but the introduction of hybridoma technology by Kohler
and Milstein (1975) made available monoclonal antibodies to a wide
range of defined carbohydrate antigens. The resulting developments in
understanding the antibodies produced in response to carbohydrate
antigens have been comprehensively reviewed by Brorson et al.
(2002). On the structural side, the first hybridoma Fabs solved with
bound antigen fragments were Se155-4 (Cygler et al. 1991), an antibody to a Salmonella lipopolysaccharide (LPS), and BR96 (Jeffrey
et al. 1995), an antibody to a human tumor antigen, the Lewis
Y antigen (Ley). Their binding sites showed the grooves and cavities
© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
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Antibody recognition of carbohydrate epitopes
Table I. PDB codes for antibody structures discussed in this review in order of appearance
Antibody
Antigena
Liganded
PDB code
Unliganded
PDB code(s)
Organism
or antigen
Antigen class
2G12
PG9
PG16
PGT121
PGT124
PGT128
PGT135
chBR96
hu3S193
291-2G3-A
chR24
Man9GlcNAc2
V1/V2 domain of gp120
V1/V2 domain of gp120
Self N-glycan
V3 domain of gp120
V3 domain of gp120
Gp120 core
Fucα1,2Galβ1,4(Fucα1,3)GlcNAcβ1,3Gal (Ley)
Fucα1,2Galβ1,4(Fucα1,3)GlcNAcβ1,3Gal (Ley)
Galβ1,4(Fucα1,3)GlcNAcβ (Lex)
Unliganded
1OP5
3U4E
4DQO
4FQC
4R2G
3TYG
4JM2
1CLY
1S3K
1UZ8
1OM3
3U36
3MUG
4FQ1, 4FQQ
4R26
HIV-1 glycoprotein
HIV-1 glycoprotein
HIV-1 glycoprotein
HIV-1 glycoprotein
HIV-1 glycoprotein
HIV-1 glycoprotein
HIV-1 glycoprotein
Tumour epitopes
Tumour epitopes
Tumour epitopes
Tumour epitopes
P3
237mAb
L363
BGA
Unliganded
ERGT(GalNAc)KPPPLEELS
Mouse CD1d receptor and C20:2 αGal ceramide
Unliganded
3IET
3UBX
HIV-1
HIV-1
HIV-1
HIV-1
HIV-1
HIV-1
HIV-1
Ley antigen
Ley antigen
Lex antigen
GD3
glycosphingolipid
NeuGc-GM3
Tn antigen
Gal ceramide
Blood group A
Se155-4
Abeα1,3Manα1,4Rhaα1,3Gal
1MFC
SYA/J-6
Rhaα1,2Rhaα1,3Rhaα1,3GlcNAcα1,2Rao
1M7I
1M71
Sh. flexneri Y
F22-4
→2Rhaα1,2Rhaα1,3(Glcα1,4)Rhaα1,3GlcNAcβ1→
3BZ4
3C5S
Sh. flexneri 2a
Ab52
Unliganded
3UJT
F. tularensis
N62
Unliganded
4KPH
F. tularensis
WN1 222-5
3V0V
LPS core
LPT3-1
GlcNα1,2Glcα1,2Glcα1,3(Galα1,6)Glcα1,3(Hepα1,7)
(P4)Hepα1,3(P4)Hepα1,5(Kdoα2,4)Kdoα2,6(P4)
GlcNβ1,6GlcNα(1P)
GlcNAcα1,2Hepα1,3(Glcβ1,4)Hepα1,5Kdo
4C83
S-20-4
α1,2 2-O-Methylperosamine disaccharide (Ogawa)
1F4Y
1F4W
S25-2
Kdo2,8Kdo2,4Kdo
3SY0
1Q9K, 1Q9L
S25-2
Kdo
3T4Y
Chlamydia
S54-10
Kdo2,4Kdo2,4Kdo
3I02
Chlamydia
S73-2
Kdo2,8Kdo2,4Kdo
3HZV
Chlamydia
S67-27
Kdo2,8-7-O-Me-Kdo
3IKC
Chlamydia
S64-4
Kdo2,8Kdo2,4Kdo2,6GlcN4P1,6GlcN1P
3PHO
Chlamydia
S25-26
Kdo2,8Kdo2,4Kdo-O-allyl
4M7J
CS-35
Araβ1,2Araα1,5(Araβ1,2Araα1,3)Araα1,5Araα
3HNS
J539
Unliganded
2FBJ
Galactan
Yst9.1
Unliganded
1MAM
B. abortus
mAb735
α2,8NeuAc (sialic acid) octamer
a
4JM4
1UCB
1UZ6
1BZ7
3IU4
1JV5
3V0W
Salmonella B
N. meningitidis
4M7Z, 4M93,
4MA1
Chlamydia
Chlamydia
Mycobacteria
3WBD
Different nomenclature is used for antigens consisting of protein/peptide and carbohydrates.
Polysialic acid
Tumour epitopes
Tumour epitopes
Glycolipid
Human ABO(H) blood
group A
Internal epitopes of
bacterial LPS
Internal epitopes of
bacterial LPS
Internal epitopes of
bacterial LPS
Internal epitopes of
bacterial LPS
Internal epitopes of
bacterial LPS
Internal epitopes of
bacterial LPS
Internal epitopes of
bacterial LPS
Terminal bacterial
epitopes
Terminal bacterial
epitopes
Terminal bacterial
epitopes
Terminal bacterial
epitopes
Terminal bacterial
epitopes
Terminal bacterial
epitopes
Terminal bacterial
epitopes
Terminal bacterial
epitopes
Terminal bacterial
epitopes
Homopolysaccharide
epitopes
Homopolysaccharide
epitopes
Homopolysaccharide
epitopes
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O Haji-Ghassemi et al.
Fig. 1. Schematics of antibody isotypes IgG, IgD, IgA, IgE and IgM showing the heavy (dark gray) and light (lighter gray) chains organized into domain dimers.
Shown for IgG is the antigen (black) bound by the N-terminal variable light (VL) and variable heavy (VH) chain domains. The inset shows the main chain trace of
the six CDRs that form the antigen-binding site in antibody S25-26 (PDB: 4M7J). White hexagons represent the N-glycans found on fragment crystallizable heavy
chains and gray hexagons represent O-glycans. The secretory IgA antibody is made up of two monomeric IgA molecules, a joining chain (J-chain) and a five-domain
secretory component. The IgM antibody is secreted as a pentamer that includes a joining chain and numerous N-glycan sites.
that had been predicted by Kabat (Wilson and Stanfield 1995). These
antibodies represent the two major types of carbohydrate antigen that
have subsequently been structurally investigated.
Immunology of carbohydrate antigens
The numerous possible combinatorial linkages, modifications and
relative degree of flexibility of many carbohydrates require antibodies
to utilize a variety of strategies in their recognition. Details of the genetic events that lead to the observed binding site diversity of antibodies
are described in several reviews (Dudley et al. 2005; Maizels 2005;
Jung et al. 2006).
In summary, the majority of the observed immunoglobulin array
stems from the formation of a nascent B-cell lymphocyte, where
genes coding for one variable heavy (VH) and one variable light (VL)
domain are constructed from a limited repertoire of inherited germline gene segments. These consist of V (variable), D (diversity) and J
( joining) gene segments for the heavy chain, and VJ segments for
the light chain that are located on a separate chromosome and can
be either kappa or lambda type. The recombination events can be
quite variable, and substitutions are often incorporated between the
gene segments that result in productive and non-productive
immunoglobulins. V(D)J recombined genes encoding VH and VL
domains are further paired with constant (C) gene segments that determine antibody isotype. Following translation, the antibody polypeptides are modified at glycosylation sites, particularly in the
constant-regions (Figure 1) where these carbohydrate moieties are
involved in modulation of effector functions (Mimura et al. 2001;
Jefferis 2009). The initial result in nascent B-cells is a class M immunoglobulin glycoprotein of specific sequence, and each B-lymphocyte will
display multiple copies of its particular membrane-bound IgM antibody on its surface.
The final genetic source of antibody diversity relies on T-cell help.
If the antigen is T-cell-dependent like many proteins and peptides,
T-cell co-stimulation of the B-cell can induce somatic hypermutation
of the antibody genes to produce daughter cells with mutant immunoglobulins of potentially higher affinity (Sharon 1990; Li et al. 2004;
Teng and Papavasiliou 2007).
Generally speaking, carbohydrates are classified as T-cell independent antigens (Murphy et al. 2012), where B-lymphocytes are activated without the presentation of antigen fragments via MHC
molecules to T-lymphocytes (Vos et al. 2000). However, the inability
of most carbohydrate antigens to recruit T-cell help results in a B-cell
response lacking affinity maturation and weighted toward the
923
Antibody recognition of carbohydrate epitopes
Figure 1 Continued
production of IgM and IgG2 in human and IgM and IgG3 in mouse
(Greenspan et al. 1988; Scott et al. 1988; Stein 1992; Ullrich 2009; Wigelsworth et al. 2009). Furthermore, the anti-carbohydrate immune response usually produces antibodies with “V-region restriction” where a
relatively limited set of germ-line gene segments will generate antibodies
against a broad range of epitopes (Pascual et al. 1992; Brorson et al.
2002; Nguyen et al. 2003; Brooks, Müller-Loennies, et al. 2010; Blackler
et al. 2012).
To overcome this restricted response, glycoconjugate antigens have
been developed in which carbohydrate antigens or fragments thereof
are coupled to proteins, and the protein moieties can then recruit T-cell
help. The intracellular processing of carbohydrate antigens has been
found to involve degradation by means of reactive oxygen and nitrogen species (Duan and Kasper 2011), and this knowledge has led
to improved designs for a second generation of glycoconjugate
vaccines (Buskas et al. 2008; Astronomo and Burton 2010; Avci
et al. 2011; Costantino et al. 2011). However, two exceptions to the
T-independent paradigm have been found: Polysaccharides that carry
both negatively and positively charged substituents, i.e., zwitterions,
can interact with MHCII species (Avci and Kasper 2010). The oxidative breakdown of polysaccharide antigens can also produce species
capable of this type of interaction (Velez et al. 2009). Secondly,
some glycolipid antigens are presented by MHC homologs CD1a, b,
c and d, to various families of T-cell receptors (TCRs; Icart et al.
2008).
Affinities of anti-carbohydrate antibodies
The observed affinities of anti-carbohydrate antibodies are typically
lower by factors of 103–105 than antibodies specific for protein or
peptide antigens (Krause and Coligan 1979; MacKenzie et al. 1996;
Brorson et al. 2002). This is compensated by their initial expression
as decavalent IgM and their observed class switching bias toward
IgG3 in mice and IgG2 in humans, which tend to self-associate
through their constant regions to form multivalent networks (Greenspan
et al. 1988; Cooper et al. 1991). The multivalent nature of these antibody clusters results in a marked increase in avidity (Edberg et al.
1972; Greenspan and Cooper 1992; Yoo et al. 2003) and reflects an
evolved mechanism for the recognition of multivalent or densely displayed carbohydrate antigens. The surface clustering of multivalent antibodies can only occur when there are correspondingly large numbers of
antigen molecules present, which serves to distinguish between cells that
924
display many copies of the antigen (such as bacteria) and normal cells
that display just a few. This ability of antibodies to distinguish chemically
identical epitopes depending on their environment is termed “context dependent recognition” (Ramos and Moller 1978; Wylie et al. 1982; Caoili
2010), and is particularly relevant for tumor antigens.
The lower affinities observed for carbohydrate-specific antibodies,
and other carbohydrate-binding proteins such as lectins, derive from
the binding not being driven only by enthalpic factors, and emphasise
the relative importance of entropic considerations (Bundle and Young
1992; Bundle et al. 1998; Engström et al. 2005). The general lack of
rigidly defined structures in many carbohydrates would require entropically unfavourable immobilization of these otherwise flexible segments upon antibody binding. However, attempts to demonstrate
this experimentally have had mixed results; a rigid antigen analog of
a Salmonella epitope tethered between the O-6 atoms of the Gal and
Man residues was bound equally well as the free form (Bundle et al.
1998), while a similar analog of a Shigella flexneri epitope showed enhanced binding affinity (McGavin and Bundle 2005). Additionally,
the extension of a ligand by the addition of a sugar unit can lead to
ambiguity in the interpretation of binding data, as the fixing of the
anomeric oxygen atom in one conformation may increase affinity
without the added sugar necessarily contacting the antibody surface.
In contrast, if the new sugar unit has cooperative interactions with the
protein and enthalpic contributions that offset the loss of conformational entropy, the overall Ka will be similar to that of the shorter substrate (Kabat 1960; Jennings 2012). Such misinterpretation can be
minimized by studying each oligosaccharide in its methyl glycoside
(MAG) form, which also avoids α/β mutarotation equilibrium that
may complicate ligand modelling in structural studies (Bundle 1989;
Yates et al. 1996; Bundle et al. 2012).
The hydrophilic nature of carbohydrates increases the possibility
that water molecules have to be displaced or trapped during complex
formation, both of which have distinct entropic consequences, making
the net thermodynamic contribution of each interaction difficult to
model. Generally, a greater desolvation of receptor and ligand corresponds to higher affinity, as the inherent entropic penalty of carbohydrate binding is offset (Woods 1998; Fadda and Woods 2010). While
the role of the water molecules that solvate both the carbohydrate ligand
and its receptor was considered in detail in the 1990s (Chervenak and
Toone 1994; Lemieux 1996), this area is still not fully understood,
particularly the role of hydrophobic interactions (Snyder et al. 2011).
The overall low binding of carbohydrates is usually accompanied by
relatively fast kon and koff rates, summarized for lectins in Scharenberg
et al. (2014). Kinetic data have been reported for only a few of the
anti-carbohydrate antibodies described in this review. Mutants of
the anti-blood group A single-chain fragment variable (scFv), blood
group A (BGA; Thomas et al. 2002) had kon rates of 2.4–5.4 × 104 M−1
s−1 and koff rates of 3.7–6.4 × 10−2 s−1 (KD values 0.8–2.1 µM) for
the trisaccharide antigen, which are similar to those for the binding of
Glc1Man9GlcNAc2 by calreticulin, 3.9 × 104 M−1 s−1 and 8 × 10−2 s−1
(Patil et al. 2000). Antibodies to charged carbohydrate antigens, such
as the Kdo trisaccharide recognized by anti-Chlamydia antibodies, can
have KD values <1 µM, with both kon and koff rates that are faster, e.g.,
2 × 105 M−1 s−1 and 0.12 s−1 for S25-2 (Müller-Loennies et al. 2000).
Finally, the presence of –COOH or –CH3 groups on the carbohydrate can lead to higher affinities by permitting ionic or hydrophobic
interactions, e.g., for antibodies that recognize charged Kdo [All
carbohydrate nomenclature used are spelled out unless they are specified in the essentials of Glycobiology 2nd edition abbreviations list
(Varki et al, 2009)] species (Müller-Loennies et al. 2000) and methyl
groups on the Vibrio cholera Ogawa antigen (Villeneuve et al. 2000).
O Haji-Ghassemi et al.
Antibody structure determination
To date, only a few structures of intact immunoglobulins have been
determined (Harris et al. 1998; Saphire et al. 2003). The flexibility
of the hinge region of an intact immunoglobulin (Harris et al. 1998)
makes crystallization difficult. Structural studies are therefore generally carried out on Fab fragments, generated by limited proteolysis of the
immunoglobulins with papain or pepsin. However, crystallizing Fabs
with bound ligand is made difficult not only by the low affinities of
anti-carbohydrate antibodies but also by the packing of Fab molecules
in crystals where the constant region of one Fab often lies across the
binding site of a neighbouring one, blocking the access of ligands.
This mode of crystal contact is unfortunately only too common in
Fab crystallizations (Davies et al. 1990). Nevertheless, nearly all the
structures described here were obtained with Fabs from mouse hybridoma proteins, mostly IgG2a, or in the case of anti-HIV, from human
hybridomas obtained from lymphocytes of resistant individuals.
Structures of antibodies against carbohydrates deposited in the protein
data bank can be accessed using SAbDab database (Dunbar et al. 2014)
(http://opig.stats.ox.ac.uk/webapps/abdb/web_front/Welcome.php) or
Glyco3D database (Pérez et al. 2015) (http://glyco3d.cermav.cnrs.fr/
home.php).
Recombinant expression of smaller antigen-binding fragments,
such as scFvs, in Escherichia coli can provide access to binding fragments from IgMs that cannot be obtained in useful quantities by proteolysis (Patenaude et al. 1998). It also enables concomitant studies
by site-directed mutagenesis. Fragments consisting of only VH or VL
domains, called single domain antibodies (sdAbs), are even smaller
molecules that can be solved directly with NMR techniques (Vranken
et al. 2002), though carbohydrate-specific sdAbs are rare (Stijlemans
et al. 2004; El Khattabi et al. 2006; Behar et al. 2009).
Modelling of carbohydrate-antibody systems
Because many anti-carbohydrate antibodies could not be crystallized
or could only be solved without bound ligand, modelling studies have
often been necessary. In a series of landmark papers, Chothia and
co-workers first demonstrated that the conformation of individual
complementary determining loops (CDRs) can be grouped into just
a few “canonical forms” based on the loop length and position/identity
of key residues (Chothia and Lesk 1987; Chothia et al. 1989, 1992;
Tramontano et al. 1990), and this work has since been expanded
(Al-Lazikani et al. 1997; Morea et al. 1997, 1998; Abhinandan and
Martin 2008; North et al. 2011). Structural prediction based on this information is limited by the difficulty in modelling the relative orientation of each CDR or even of the heavy and light chain domains
(Reczko et al. 1995), and the highly variable nature of the CDR H3
(Kuroda et al. 2008). Further, contribution of solvent is generally
ignored in such studies. On-line prediction packages are available that
compare the query sequence to antibodies of known structure and select
the closest homologs for each CDR (Marcatili et al. 2008; Sircar et al.
2009); the most effective application is reported to be Rosetta Antibody
(Almagro et al. 2011).
The prediction of structures of antigen complexes is even more
challenging, but the number of in silico binding and docking studies
has been rapidly expanding with advances in theory and computing
power (Woods 1998; Agostino et al. 2009, 2010; Fadda and Woods
2010; North et al. 2011). These methods have grown increasingly
powerful, particularly when combined with NMR experiments.
These are carried out in solution and can provide detailed and
highly relevant information about the changes in the conformations
of carbohydrate ligands as they bind (Peters and Pinto 1996;
925
Antibody recognition of carbohydrate epitopes
Haselhorst et al. 2009; Oberli et al. 2010). The most common
experiments focus on 1H nuclear Overhauser effect transfer resonances
and saturation transfer difference (STD)-NMR, which are sensitive to
protein–carbohydrate interactions and to changes in protein and carbohydrate conformations upon binding (Kogelberg et al. 2003; Theillet
et al. 2009; Roldós et al. 2011). Suitable on- and off-rates for the
antigen–antibody interaction are critical for successful use of these
methods, which otherwise may result in poor signal-to-noise ratios,
thus making it difficult to distinguish the free from the bound state
(Oberli et al. 2010). A striking example of what can be achieved by
combining modelling, NMR and docking approaches is the complex
structure prediction of an antibody with a β1,2Man antigen from
Candida albicans (Johnson et al. 2012).
Figure 1, including domain-swapped structures and unusually long
CDR loops. Structural work on antibodies to glycan epitopes on
gp120 began with characterization of the binding of high-mannose
oligosaccharides, then progressed through designed fragments of
gp120 bearing two N-glycans (which can be mannose-rich or hybrid
types), then complete gp120, and finally to a trimer of the envelope
protein with antibody PGT 122. The latter showed both its interactions with the glycans and the relationship of the gp41 and gp120
subunits (Julien et al. 2013). The antibodies described here use three
different types of structure to access distinct glycopeptide epitopes on
gp120, which overlap around a “hotspot” at Asn332 (Kong et al.
2013).
The domain-swapped antibody, 2G12
Types of carbohydrate antigen
It has been claimed that the only absolute rule in biology is that cell
surfaces are festooned with carbohydrates (Gagneux and Varki
1999): glycoproteins and glycolipids in the case of eukaryotic cells,
and capsular polysaccharides and LPS in the case of bacterial cells.
These antigens are the most common classes studied in carbohydrate–antibody complexes. The eukaryotic carbohydrates include
tumor antigens and glycoprotein structures that viruses acquire from
their host cells’ biosynthetic machinery. The relative abundances of
some mammalian surface carbohydrates have been observed to
change when a cell becomes malignant, and these modified sugars,
often on gangliosides, have been extensively investigated as potential
targets for immunotherapy (Ragupathi 1996). These glycans are of
course self-antigens and the immune responses to them can therefore
lead to antibodies with remarkable structural properties.
The main antibodies that have been studied structurally (Table I) are
directed towards bacterial carbohydrate epitopes found on the LPS and
on mycobacterial lipoarabinomannans (LAMs). There is some crossover between eukaryotic and bacterial carbohydrate antigens because
some bacteria evade immune surveillance by using antigenic mimicry,
i.e., their polysaccharide antigens resemble carbohydrates normally
found in human tissues. An example of this phenomenon occurs with
the capsular polysaccharides of group B meningococci and E. coli K1
strain, which consist largely of homopolymers of α2,8-linked sialic
acid (Frosch et al. 1985; Silver et al. 1988). The same structure occurs
as a developmental antigen in fetal mammalian cells (Rosenberg et al.
1986; Rutishauser and Jessell 1988) and in neural cell adhesion molecule (Finne et al. 1983), and so is poorly immunogenic (Jennings
and Lugowski 1981; Bartoloni et al. 1995).
The human monoclonal antibody 2G12 broadly neutralizes T-cell lineadapted strains of HIV-1 through activation of both antibody-dependent cell-mediated cytotoxicity and complement system (Buchacher et al.
1994; Trkola et al. 1996), with a strong dependence on the multiplicity
of N-linked glycans on the surface of the glycoprotein gp120 (Trkola
et al. 1996).
The structure of 2G12 Fab complexed with Man9GlcNAc2
(Figure 2A) reveals arguably the most remarkable example of high
avidity antibody binding to carbohydrate antigens (Calarese et al.
2003, 2005), where the VH domains of two neighbouring Fabs
“swap” to form a Fab dimer (Kunert et al. 1998). The swapping of
the VH domains produces an additional antigen-combining site at
the VH/VH interface that allows 2G12 to bind a cluster of glycans present on the surface of gp120 separated by as much as 35 Å. The critical
nature of the domain swap was demonstrated using mutagenesis
studies of recombinant full-length 2G12 antibody, which showed
that a single mutation Ile19 (Figure 2A) to Arg on the VH domain resulted in a non-domain-swapped variant of 2G12 that was able to
bind Manα1,2Man motifs presented on an ELISA plate, but unable
to recognize Manα1,2Man clusters in cells expressing the HIV-1 envelope trimer (Doores et al. 2010). The Manα1,2Man ends of the three
arms of the Man9 structure are the main sites recognized by 2G12
(Figure 2B), particularly the D1 and D2 arms (Calarese et al. 2005).
2G12 displays an intriguing similarity to the binding mechanism
of the cyanobacterial lectin, cyanovirin, which is also capable of neutralizing HIV-1 through binding to the D1 arm of Man9GlcNAc2 on
surface of gp120 (O’Keefe et al. 2000) and has a domain-swapped
form similar to that of 2G12 (Botos et al. 2002).
Anti-HIV antibodies with long CDR H3
Antibodies to glycan epitopes on HIV
A major goal of HIV vaccine development has been directed towards
induction of broadly neutralizing monoclonal antibodies that recognize epitopes in the envelope proteins (McGaughey et al. 2004;
Pantophlet and Burton 2006; Pinter 2007; Haynes et al. 2010), with
increasing focus on the glycoconjugate epitopes of HIV-1 (Wang
2006). The variable region of gp120 has long been a challenge for
structural studies due to its sequence heterogeneity and its abundant
glycosylation with high-mannose structures (Man 9 GlcNAc 2 ).
Referred to as the glycan shield, it displays self-antigens of the host
and acts as a physical barrier to protect the HIV envelope trimer
against immunoglobulin recognition (Cao et al. 1997; Rusert et al.
2011). The antibodies described in these studies were obtained from
three HIV-1-infected patients. Notably, they have structures that differ
significantly from the usual form of immunoglobulin shown in
There are a number of conventional (non-domain swapped) broadly
neutralizing antibodies in addition to 2G12 that display specificity
for mixed N-linked glycans on gp120 of HIV-1 (Pejchal et al.
2011). Monoclonal antibodies PG9 and PG16 both bind the V1/V2
region to neutralize a broad range of HIV-1 strains with high efficacy,
however, the variable nature of gp120 further complicates structural
analysis.
To overcome the structural heterogeneity of gp120 and aid crystallization trials, McLellan and colleagues generated glycopeptide antigen mimics that retained the minimal scaffold of the V1/V2 region.
Complexed structures of V1/V2 scaffolds from two different HIV-1
strains with PG9 were subsequently determined to 2.19 and 1.80 Å
resolution. The structures revealed another unusual binding mechanism in which a very long CDR H3 of 26 residues with a charged “hammerhead” tip inserts into the glycan shield, fitting neatly between two
N-linked glycans to contact the protein surface of gp120 (Figure 2C;
926
O Haji-Ghassemi et al.
Fig. 2. Antibodies to HIV gp120. The variable VL chains are shown in white and VH chains are shown in red or cyan. The CDR H3 loop is shown in yellow, carbohydrate
antigens are in green, water molecules as cyan spheres, and hydrogen bonds to carbohydrate antigen as yellow dashed spheres. CDRs are labelled L1, L2, L3, H1, H2
and H3. HIV glycoprotein gp120 is always magenta. Contacts between CDR H3 and the protein backbone of gp120 are highlighted as black dashes. (A) 2G12 (PDB:
1OP5) “domain-swapped” Fab dimer in complex with branching Man9GlcNAc2 oligosaccharide of gp120. Mutation of 2G12 H19 Ile residue (magenta) at the
interface between the two heavy chains to Arg abolishes domain exchange and results in single Fab fragments lacking the third binding site. (B) Stereo diagram
of 2G12 combining site, showing the hydrogen bonding to the D1, D2 and D3 arms of the branched Man9GlcNAc2 oligosaccharide antigen. (C) Stereo ribbon
diagram of PG9 (PDB: 3U4E) antibody variable domains bound to the variable regions 1 and 2 (V1/V2) of gp120. CDR H3 forms a T-shaped loop that inserts
itself between the two N-linked glycosylation sites, making contacts to the protein backbone of V1/V2 scaffold. (D) Stereo diagram of PG9, highlighting the
hydrogen bonding to the two N-linked glycan arms of Asn156 and Asn160 on gp120. Hydrogen bonds between CDR H3 and the protein backbone of gp120 are
highlighted as black dashes. (E) Stereo ribbon diagram displaying complex of PG16 (PDB: 4DQO) variable region with the V1/V2 domain of gp120. (F) Stereo diagram
of PG16 highlighting the hydrogen bonding to the two N-linked glycan arms of Asn160 and Asn173 on gp120. (G) Stereo ribbon diagram of PGT121 (PDB: 4FQC)
antibody variable domains bound to “self-complex” in which one Fab binds to the complex biantennary glycan of a second Fab (H) stereo diagram of PGT121
highlighting the hydrogen bonding to the complex biantennary glycan. (I) Stereo ribbon diagram of PGT124 (PDB: 4R2G) antibody variable domains bound to
the variable region 3 (V3) domain of gp120. (J) Stereo ribbon diagram of PGT128 (PDB: 3TYG) antibody variable domains bound to the variable region 3 (V3)
domain of gp120. (K) Stereo diagram of PGT128 highlighting the hydrogen bonding to the two N-linked glycan arms of Asn331 and Asn301 on V3 domain of
gp120. (L) Stereo ribbon diagram of PGT135 (PDB: 4JM2) antibody variable domains bound to core region of gp120. (M) Stereo diagram of PGT135 highlighting
the hydrogen bonding to the two N-linked glycan arms of Asn332 and Asn392 on gp120. This figure is available in black and white in print and in color at
Glycobiology online.
Antibody recognition of carbohydrate epitopes
Figure 2 Continued
927
928
O Haji-Ghassemi et al.
here utilize multiple glycans for high-affinity interaction. The authors
reason that this mechanism has evolved from distinct affinity maturation events; whereby PGT 124 is selected for efficient recognition
of the HIV trimer by minimizing binding to surrounding heterogeneous N-glycans (Garces et al. 2014). Despite the smaller recognized
surface of this antibody, its neutralizing potential is comparable with
other anti-HIV antibodies (Sok et al. 2014).
Anti-HIV antibodies with additional CDR insertions
Figure 2 Continued
Sattentau 2011). In this way, PG9 overcomes sequence variability inherent to the V1/V2 protein by recognition of the protein backbone
rather than to the variable amino acid side chains (Figure 2D). The
CDR H3 contacts not only two N-glycans but also forms parallel
β-sheet like bonds to a β-strand of the antigen. The tip of the loop is
highly unusual in that it includes sulfated Tyr residues, which can form
salt-bridges to cationic residues on the antigen. Because the CDR H3
penetrates so deeply, it contacts the inner Asn-linked GlcNAc as well
as terminal Man residues of the Man5GlcNAc2 (Asn160) (Figure 2D).
Interactions with a second N-glycan at Asn156 or Asn173 are also
formed by Tyr100K of CDR H3 and the inner GlcNAc. The strong
evolutionary conservation of the two glycans in gp120 allows PG9
to recognize some 80% of HIV-1 strains (McLellan et al. 2011).
A second antibody PG16 was solved in complex with the V1/V2
scaffold to 2.44 Å resolution, which showed many shared binding
characteristics of PG9 (Figure 2E), including binding the Asn160 glycan, the H-bonding to the peptide backbone and the charged interactions of the sulfated Tyr on a 26-residue CDR H3 (Pancera et al.
2013). However, the second N-glycan at Asn173 was of the complex
type, with one Neu5Ac-Gal-GlcNAc arm on the Man5GlcNAc2 core
(Figure 2F). Interactions with the α2,6-linked Neu5Ac dominated
PG16’s binding, contributing half the surface area and most of the
H-bonds to the antibody. The residues that form the contacts are different in PG9, explaining its preference for Man5GlcNAc2. Three
PG16-specific amino acids: ArgL94, SerL95 and HisL95A form the
complementary surface that allow for the recognition of the terminal
sialic acid (Figure 2F), whereas in PG9 the three amino acids are
ThrL94, ArgL95 and ArgL95A. Replacement of the three CDR L3
residues of PG9 with that of PG16 resulted in a chimeric antibody
that displayed specificity for both mannose rich and complex-type
N-glycans; leading to enhanced HIV-1 neutralization (Pancera et al.
2013).
Another example of an antibody with a long CDR H3 of 22 residues, PGT 121 was solved in a “self-complex” in which one Fab was
bound to the complex biantennary glycan of a second Fab (Figure 2G;
Mouquet et al. 2012). This crystal form was obtained during an attempted co-crystallization with a non-sialylated biantennary glycan.
The sialic acid was again α2,6-linked and formed the bulk of the contacts with the antibody, which were exclusively from the VH domain
(Figure 2H). Recently, the structure of a closely related antibody
PGT 124 was solved in complex with gp120 (Garces et al. 2014).
PGT 124 facilitate binding to HIV trimer via a single glycosylation
site (Figure 2I), while all other gp120-specific antibodies discussed
Monoclonal antibodies PGT 127 and PGT 128 share the preference
shown by 2G12 for the Manα1,2Man ends of the D1 and D2 arms
of high-mannose glycans (Figure 2J), but are structurally distinct
(Pejchal et al. 2011). As well as 17-residue long CDR H3s, they
have six residue insertions in their H2 CDRs that are critical for glycan
recognition. Structures with Man9 were obtained at higher resolutions
(1.65 and 1.29 Å) than for any other anti-gp120 antibodies. Hence the
H-bonding network and the involvement of water molecules were well
defined. The recognition was through a surface defined by CDRs H2,
H3 and L3; key residues included four Trp (Figure 2K), two being
from the CDR H2. The buried surface areas were high, 748 Å2 for
PGT 128, consistent with the higher viral neutralization titers of
these antibodies compared with PG9 (648 Å2) (Kong et al. 2014). A
structure was also obtained with an engineered gp120 outer domain
bearing two N-linked glycans, for which the Ka was 2.2 × 107 M−1.
Both glycans and part of the peptide were bound, with a
Man9GlcNAc2 structure in the previous Man9 site and a Man5GlcNAc2 portion of an N-glycan in a second site. The latter is unusual
in being formed not only by the longer CDR H2 but also by residues
72–75 of the framework region 2 or sometimes referred to as the
CDR H4 loop. At the same time, CDR H3 contacts the peptide backbone of the antigen.
Another unusual CDR has been found in PGT 135, which has a
five-residue insertion in CDR H1 along with an 18 residue long
CDR H3 (Kong et al. 2013). Its structure was solved in unliganded
form and with an assemblage of other molecules, namely gp120
core, CD4 and an anti-CD4 Fab (Kong et al. 2013). No large conformational changes were seen between the two forms. The two long CDRs
protrude from the antibody surface far enough to match the length of
the glycans, and reach side chains of the protein (Figure 2L). The total
size of the peptide-glycan epitope is very large, 1334 Å2, with 70%
contributed by the glycans (Kong et al. 2014). Endoglycosidase treatment of a PGT 135-gp120 complex showed protection of N-glycans at
three sites, though the Fab contacts two of the glycans at Asn392 and
Asn332 (Figure 2M) more than the third at Asn386. The Asn392 and
Asn332 glycans interact with opposite faces of CDR H3 in a bifurcated manner, from the ends of the D2 and D3 arms down to
the GlcNAc residues. PGT 135 recognizes the opposite face of the
Asn332 glycan compared with PGT 128 and consequently there are
fewer H-bonds and more apolar interactions (Figure 2M), including
ones with a sequence of seven hydrophobic residues of CDR H3.
Antibodies to tumor carbohydrate epitopes
Some of the most intriguing and potentially clinically important
studies of anti-carbohydrate antibodies stem from the observation
that some cell-surface oligosaccharides are tumor antigens, such as
the Lewis x and y antigens, their sialylated counterparts, and gangliosides. Gangliosides are glycosphingolipids, which are ceramide-linked
oligosaccharides with at least one terminal sialic acid residue. The
modulation of ganglioside type and concentration has been associated
with the growth and differentiation of tissues, and with carcinogenesis
929
Antibody recognition of carbohydrate epitopes
(Hakomori and Kannagi 1983; Hakomori 1989, 2001; Bitton et al.
2002; Birkle et al. 2003; Xu et al. 2005). Hence, cancer vaccines
based on glycolipids are receiving increasing attention (Wandall and
Tarp 2007; Durrant et al. 2012) including some completely synthetic
ones (Buskas et al. 2008; Wilson and Danishefsky 2013).
One of the best-studied tumor-related antigens is a single GalNAcα residue O-linked to Ser or Thr, referred to as the Tn antigen (Ju et al.
2011; Richichi et al. 2014). The Tn antigen is present in many different types of cancer and generally not present in normal adult tissues
(Ju et al. 2011). Consequently, it offers a perfect target for immunotherapy and has been the subject of many antibody studies (Takahashi
et al. 1988; Numata et al. 1990; Baldus et al. 1992; Hakomori 2001;
Brooks, Schietinger et al. 2010; Yuasa et al. 2012).
The Ley-specific antibodies, BR96 and 3S193
The Ley surface antigen is expressed at high levels in a variety of
tumor cell lines, and as a result antibodies against this structure
have been intensively investigated in hopes of coupling the fragment
variable (Fv) and Fab fragments with toxic substances or radiochemicals as “magic bullets” that selectively target and kill malignant cells
(Hellström and Hellström 1991; Oldham and Dillman 2008). Its
structure is Fucα1,2Galβ1,4(Fucα1,3)GlcNAcβ1,3Gal-R.
The monoclonal antibody BR96 specific to the Ley antigen showed
promising results during phase I clinical trials; the humanized Fab conjugated with the anti-cancer drug doxorubicin was rapidly internalized by cancer cells, resulting in tumor clearance in nude mice
(Hellström et al. 1990; Garrigues et al. 1994). While phase II trials
were underway, the crystal structures of both the murine and humanized Fab structures were solved in complex with a nonoate methyl
ester glycoside derivative of Ley, to 2.80 and 2.50 Å, respectively
(Chang et al. 1994; Jeffrey et al. 1995). The BR96 antibody binds
the Ley tetrasaccharide from its non-reducing end (Figure 3A) in a
large cavity 10 Å deep and 12 Å wide. This is formed mainly by aromatic residues (Figure 3B) and binding energy is postulated to stem
from hydrophobic effects. The buried surface area was 422 Å2, most
of it from the VH domain whose CDR H3 also formed most of the
H-bonds. Though specific contacts are made to all four sugars, the
affinity of the Fab is only 2 × 105 M−1 (Yelton et al. 1995). A comparison with the unliganded form of the antibody, determined at 2.6 Å
(Sheriff et al. 1996), showed evidence for an induced fit since CDRs
L1, L2 and H2 changed conformations on binding. However, CDR
H3 which makes the major contacts with the antigen did not change
conformation significantly.
A randomized phase II study of BR96-doxorubicin conjugate
completed in 1999 resulted in significant gastrointestinal toxicities
in patients with metastatic breast cancer, probably due to the presence
of Ley antigen at lower levels in the gastrointestinal tract of normal epithelial cells. As a result of these studies, BR96–doxorubicin conjugate
was not approved for human use.
A second Ley-specific antibody 3S193 has been generated using mice
immunized with human adenocarcinoma cells (Kitamura et al. 1994). It
displayed no cytotoxicity to O, A, AB and B human blood group containing erythrocytes (Kitamura et al. 1994; Scott et al. 2000), making it
a suitable therapeutic candidate. A 1.9 Å resolution crystal structure
of the humanized 3S193 has been determined (Ramsland et al. 2004).
Nucleotide sequence comparison between BR96 and 3S193 revealed
that they share V-genes and not surprisingly, the 3S193 antibody
formed a similarly shaped pocket (Figure 3C) dominated by hydrophobic residues (Figure 3D). Despite these similarities, there were nine
amino acid differences compared with BR96 in the CDR regions,
seven of which were located on CDRs H2 and H3. Further, the
orientation of the main chain backbone of CDR L1 allows humanized
3S193 to form a hydrogen bond to the Ley-specific Fuc residue through
Asn28 (Figure 3D), whereas this residue is further away in BR96. In contrast to the BR96 structure, there are numerous water molecules in the
combing site of 3S193 that participate in bridging interactions.
The Lex-specific antibody 291-2G3-A
The Lex antigen is not only expressed on tumor cells but also occurs on
the surface of parasitic worms such as schistosomes. The structure of
291-2G3-A Fab was solved with and without the trisaccharide
Galβ1,4(Fucα1,3)GlcNAcβ (Van Roon et al. 2004), with a Ka measured for this hapten of 9.3 × 104 M−1. The liganded structure was obtained to 2.05 Å by soaking the unliganded crystals with the Lex
trisaccharide, which was possible due to solvent channels in the crystal
allowing access to the combining site (Van Roon et al. 2004). There is
a shallow binding pocket 15× 13 × 10 Å (Figure 3E), in which the trisaccharide can contact all six of the CDRs, with a buried surface of
302 Å2. As with many carbohydrate-specific antibodies, several
hydrophobic side chains are oriented towards the hydrophobic face
of the pyranose ring and the methyl group of N-acetylglucosamine
MAG, providing the driving force needed for binding (Figure 3F).
Unlike many antibodies, however, 291-2G3-A involves all six CDRs
in binding, forming highly specific interactions with Lex antigen. Comparison between bound and unbound states indicated a lock and key
binding mechanism.
The ganglioside GD3-specific antibody, R24
Murine IgG3 antibody R24 is specific for the ganglioside GD3,
Neuα2,8Neuα2,3Galβ1,4Glcβ-ceramide, and displays one of the
most sophisticated mechanisms for carbohydrate recognition by an
antibody. GD3 is a normal differentiation antigen and is observed in
low concentrations on cell surfaces; however, GD3 can be found in
high concentrations on the surfaces of melanomas, soft tissue sarcomas and tumors (Dippold et al. 1980; Hamilton et al. 1993). R24
has been shown to recognize membrane surfaces containing high concentrations of GD3 while ignoring lower concentrations, making R24
a potential venue for cancer therapy (Urmacher et al. 1989; Helfand
et al. 1999).
The crystal structure of unliganded R24 was determined at 3.1 Å
resolution for the mouse Fab and at 2.5 Å for a chimeric form with
human constant domains (Kaminski et al. 1999). A prominent pocket,
8.5 × 12 × 8 Å, is formed by the H1, H2 and H3 loops, which is large
enough to accommodate the terminal sialic acid residue of GD3
(Figure 3G). R24 binds GD3 very weakly in solution, yet it has been
observed to bind to cell-surface GD3 with higher affinities than can be
explained by bivalent IgG binding. Part of this binding can be
explained by the natural tendency of murine IgG3 antibodies to associate through their respective constant regions to form multivalent antibody clusters (Greenspan et al. 1988; Berney et al. 1991; Greenspan and
Cooper 1992). In addition, R24 has been shown to bind other molecules
of R24 through their antigen-binding domains via “homophilic binding”, which occurs while R24 is binding membrane-anchored GD3
(Kaminski et al. 1999) (Figure 3H). This means that R24 contains an
idiotope not only for GD3 (IDGD3) but also an idiotope to other molecules of R24 through homophilic binding (IDHOM). The large number
of cooperative interactions forming on the surface increases the likelihood of complement activation and recruitment of other immune effector cells for the eventual lysis of the cancerous cell.
Site-directed mutagenesis studies have shown that IDGD3 and
IDHOM are distinct sites, as mutants could be generated which
930
O Haji-Ghassemi et al.
Fig. 3. Electrostatic surface potentials are colored red and blue for negative and positive charges, respectively, and white color represents neutral residues.
Carbohydrate antigens are shown as green, water molecules are shown as cyan spheres, and hydrogen bonds to carbohydrate antigen are shown as yellow
dashed spheres. CDRs are labelled L1, L2, L3, H1, H2 and H3. GlcNAc, N-acetyl-β-D-glucosamine; Fuc, α-D-fucose; Gal, β-D-galactose; Mag,
N-acetyl-β-D-glucosamine MAG; GalNAc, N-acetyl-α-D-galactosamine. (A) Electrostatic surface potential of chimeric BR96 (PDB: 1CLY) variable domains in
complex with nonoate methyl ester derivative of Lewis Y (Ley) antigen. (B) Stereo diagram showing the interactions between chimeric BR96 and the Ley
antigen. (C) Electrostatic surface potential of humanized 3S193 (PDB: 1S3K) Fv in complex with Ley antigen. (D) Stereo diagram showing the interactions
between the humanized 3S193 Fab and the Ley antigen. (E) Electrostatic surface potential of 291-2G3-A (PDB: 1UZ8) Fv in complex with the Lex trisaccharide
Galβ1,4Fucα1,3GlcNAcβ antigen. (F) Stereo diagram showing the interactions between 291-2G3-A Fab and the Lex trisaccharide antigen. (G) Transparent surface
depiction of chimeric R24 (PDB: 1BZ7) Fv. The variable VH chain ribbon is colored red and the VL chain ribbon is colored blue. CDR loops are highlighted. (H)
Schematic model of the formation of an R24 mAb network on the surface of membrane expressing large amounts of ganglioside GD3 (red). Molecules of R24
are able to bind GD3 antigen through their GD3 idiotope (IDGD3) and simultaneously bind other molecules of R24 through their homophilic idiotope (IDHOM). (I)
Electrostatic surface potential of 237mAb (PDB: 3IET) Fv bound to mouse O-glycopeptide; ERGT(GalNAc)KPPPLEELS. Peptide portion is colored purple. (J)
Stereo diagram showing the interactions between 237mAb and O-glycopeptide antigen, highlighting hydrogen bonds to the GalNAc residue. (K) Stereo ribbon
diagram displaying L363 (PDB: 3UBX) Fv bound to mouse CD1d receptor ( purple) and C20:2 αGal ceramide (green). The variables VL chain and VH chain are
shown as white and red ribbons, respectively. (L) Stereo diagram showing the interactions between L363 antibody and αGal ceramide. (M) Transparent surface
depiction for the Fv region of a blood group A-specific antibody BGA (PDB: 1JV5). The variable VH chain ribbon is colored red and the VL chain ribbon is colored
blue. CDR loops are highlighted. This figure is available in black and white in print and in color at Glycobiology online.
Antibody recognition of carbohydrate epitopes
Figure 3 Continued
931
932
removed IDHOM without disrupting antigen binding. The location of
these amino acid residue mutations suggested that CDR H2 was the
location of IDHOM (Chapman et al. 1990). This was confirmed in
the crystal structure of the unliganded Fab from R24, which showed
that homophilic binding proceeded through a β-strand interaction between the H2 loops of adjacent R24 molecules (Kaminski et al. 1999).
Further, R24 self-association through its murine IgG3 effector regions
results in the formation of multivalent arrays on the cell surface displaying high concentrations of GD3, which explains why R24 ignores
cell surfaces with GD3 oligosaccharides present in normal levels. R24
provides one of the best examples of antibody recognition of antigen
in a context-specific manner.
The ganglioside NeuGc-GM3-specific antibody, P3
The P3 mAb was generated by immunizing mice with the ganglioside
N-glycolyl-GM3 (NeuGcα2,3Galβ1,4Glcβ-ceramide), which is often
present on the surface of malignant cells (Vazquez 1995). P3 binds a
range of N-glycosyl-containing gangliosides as well as sulfatides, and an
anti-idiotype mAb of P3, named 1E10, has been used successfully to produce antibodies against these tumor-associated antigens in clinical trials
(Vazquez et al. 1998; Alfonso et al. 2002; Guthmann et al. 2006; Neninger et al. 2007; Hernandez et al. 2008). To prevent cross-reactivity with
the acetylated variant of the antigen (NeuAc, lacking only an oxygen
atom compared with the NeuGc), a chimeric P3 (chP3) was produced,
which is remarkably specific to the NeuGc-GM3/GM2 gangliosides.
Modelling and mutagenesis analysis of the CDR regions of P3
revealed surface complementary in shape and charge to a NeuGc residue, with polar contacts to positively charged arginine residues located
in this groove (Perez et al. 2001; Lopez-Requena, De Acosta, et al.
2007). A single mutation of an Arg residue in CDR H1 abolished binding to the ganglioside antigen (Lopez-Requena, Rodriguez, et al. 2007).
As with R24 antibody, the liganded structure of chP3 is not available; however, the unliganded Fab structure of chP3 was solved to
1.75 Å resolution (Talavera et al. 2009). It has a long CDR H3 loop
that protrudes from the middle of the site, separating the VL and VH
domains, and hence there is no central cavity. Unfortunately, CDR
H3 was not accurately modeled in the crystal structure posing further
complications for binding prediction. In order to use docking stimulations one needs to overcome the diversity of conformations available
for CDR H3 and the flexibility of the ligand. Talavera’s group used
nine different conformations for the CDR H3 backbone and a set of
criteria to predict the complex structure. These criteria included the
mutational and binding assay data for chP3 and the energy and conformational restrictions of the ligand. As a result, they were able to
narrow down 1800 possible different liganded structures to only one
conformation, which fit their binding and mutational data. The model
displays a hydrophilic pocket with all functional groups of NeuGc
participating in interactions with the antibody. The hydrophilic nature
of the binding site would also likely involve water bridged interactions,
which are harder to predict during docking simulations and were therefore excluded from this study. Moreover, the model complemented earlier binding data with derivatives of NeuGc-GM3, which anticipated
that the carboxylate group of NeuGc would be facing the guanidinium
group of Arg31 VH chain, forming a salt bridge with this residue
(Moreno et al. 1998).
The Tn antigen-specific antibody, 237mAb
The Tn antigen and related T-antigen are among the most studied cancer carbohydrate antigens, and the Fab from 237mAb, an IgG2a κ,
was the first glycan-specific anti-Tn antibody to be solved. Its structure
O Haji-Ghassemi et al.
was determined with a dodecameric GalNAc-peptide, ERGT(GalNAc)KPPPLEELS, a segment of a tumor-associated glycoprotein, at
2.2 Å resolution, and with free GalNAc at 2.6 Å (Brooks, Schietinger,
et al. 2010). The antibody is not observed to bind the unglycosylated
peptide, and the GalNAc residue dominates the interaction with
glycopeptide (Figure 3I and J). The Ka for the glycopeptide is 7.1 ×
106 M−1, a high value for a carbohydrate ligand but not unusual for
a peptide. The site takes the form of a shallow groove for the peptide
moiety, with the GalNAc accommodated in a central pocket formed
by germ-line residues of CDRs H2, H3 and L1. The buried surface
area was 532 Å2. Every hydroxyl group forms at least one H-bond directly to the antibody, without involvement of water bridges. Overall,
there are 8 H-bonds from the VL domain and 11 from the VH. Thus
specificity for the tumor antigen is achieved by a combination of interactions with both the glycan and peptide moieties.
Other mammalian glycan epitopes
The αGal ceramide-specific antibody, L363
Antibodies that mimic TCR binding are useful to study or modulate
T-cell function by specifically blocking TCR activation (Mareeva
et al. 2008; Dahan et al. 2011; Dahan and Reiter 2012). When the
glycolipid α-galactosyl ceramide (αGal ceramide) is presented to
T-helper cells though CD1d receptor of invariant natural killer
T-cells, the latter become activated and secrete cytokines for increased
anti-tumor activity (Van Kaer 2004; Hong and Park 2007). This property of αGal ceramide has spurred investigators to study the interaction of the TCR with the CD1d molecule using antibodies against
the αGal ceramide in its presented form (Yu et al. 2005). One antibody
called L363 was found to bind αGal ceramide glycolipid only when
bound to CD1d, resembling biding to the TCR (Yu et al. 2012).
The binding affinity (dissociation constant, KD) was measured to be
in the nanomolar range by surface plasmon resonance (SPR).
The structure of L363 Fab bound to mouse CD1d-αGal Ceramide
glycolipid analogue determined to 3.1 Å resolution (Figure 3K) reveals
contacts to the antigen similar to the TCR, though the individual
CDRs do not superimpose well with the loops of the TCR (1–3α
and 1–3β) (Yu et al. 2012). Binding to the CD1d-glycolipid complex
requires both L and H chains, where contacts to glycolipid occur only
with the L-chain (Figure 3L) and the H-chain forms a surface complementary to the CD1d, forming similar contacts as the TCR. A crucial
contact occurs between the mouse CD1d receptor and Leu99 of the
TCR 3α loop, which is replaced by the Trp104 residue in CDR H3
of L363 antibody. The structure highlights the significant recognition
of an αGal residue in conjunction with key residues of CD1d receptor
in order to achieve mimicry of the TCR, though in contrast to the
TCR, L363 antibody is not able to induce the structural changes required to bind glycolipids from Borrelia and Streptococcus species.
The blood group A-specific antibody, BGA
It is well known that the immune response to blood group trisaccharide antigens is strong, and a mismatched blood transfusion can lead
to massive intravascular hemolysis and eventual death. However,
the A and B blood group antigens differ only in the substitution of a
hydroxyl group for an N-acetyl group on the terminal sugar residue.
The A and B trisaccharide antigens are, respectively: GalNAcα1,3
(Fucα1,2)Galβ-O-R and Galα1,3(Fucα1,2)Galβ-O-R, where R is a
carbohydrate moiety of a glycolipid or glycoprotein. The recognition
of these epitopes is therefore extremely specific. The ABO blood
groups have been shown to contain “micro-epitopes”, where different
933
Antibody recognition of carbohydrate epitopes
antibodies recognize unique orientations and conformations of the
antigens. It has been proposed that residues that have a direct role in
antibody–antigen contact with one antibody may perform a steric role
in stabilizing a specific conformation in recognition by another antibody (Obukhova et al. 2011).
The structure of the Fv portion of an anti-BGA antibody has been
determined (Thomas et al. 2002) at 2.2 Å resolution, but complexes
with the trisaccharide epitope fragment could not be obtained. The Fv
was obtained from an scFv expressed from a synthetic gene based on the
sequence of the anti-A monoclonal IgM, AC1001 (Chen et al. 1987). Its
affinity for the antigen is low, Ka 3.4 × 103 M−1, but scanning of a glycan micro-array confirmed its specificity for the A trisaccharide. The
antibody has a V-shaped pocket 11 Å deep (Figure 3M), 4.7 Å wide
at the top and 2.8 Å wide at its base. This is well able to accommodate
the key terminal GalNAc and docking experiments have shown how the
GalNAc can specifically bind within it (Woods et al. 2013).
More recent docking and molecular simulations have produced a
model for the recognition of the blood group A trisaccharide by BGA
(Makeneni et al. 2014), which satisfactorily explained its specificity.
A histidine residue is a notable feature of the site, and antigen binding
was shown to be pH dependent. It increases at lower pH, which is the
opposite behavior to that of the anti-Salmonella antibody Se155-4.
Antibodies to internal epitopes of bacterial
heteropolysaccharides
This group of antibodies recognizes LPS antigens of Gram-negative
bacterial pathogens, including epitopes from four O-chains and two
cores. O-Chains are highly antigenic, and often contain immunodominant branching sugar repeating units. In the biosynthesis of O-chains,
whether linear or branched, repeating units of two to five sugars are
assembled as blocks and transferred to the growing chain (Raetz
and Whitfield 2002). The epitope recognized by an antibody may
encompass parts of more than one repeating unit, and it can be necessary to synthesize oligosaccharides that span through two units to establish which segment constitutes the epitope.
In addition to the antibodies considered here, two further examples should be noted. The original antigen for the phosphocholinebinding myeloma protein M603 is not known, but is probably of
this class of antigen. The phosphocholine is bound in a centrally located pocket in the binding site, leaving a large surface surrounding
it available for interaction with a carrier glycan (Satow et al. 1986).
The structure of a human Fab to the capsular polysaccharide of
Streptococcus pneumoniae type 23F has been deposited in the PDB
as 4HIJ. It is the first example of an antibody structure for a Grampositive antigen. The immunodominant moiety is a rhamnose branch,
but further details await publication.
Salmonella typhimurium-specific antibody, Se155-4
The first high-resolution three-dimensional description of antibody
recognition of any carbohydrate was monoclonal antibody Se155-4,
an IgG1 λ1, in complex with a fragment of the O-chain from Salmonella typhimurium serogroup B (Cygler et al. 1991). The Fab from
Se155-4 was successfully crystallized with a dodecasaccharide ligand
obtained by phage degradation of the O-chain from S. typhimurium,
which contains the repeating tetrasaccharide →2(Abeα1,3)Manα1,4Rhaα1,3Galα1→ with a protruding abequose. The antibody
combining site utilizes a combination of groove- and cavity-type recognition. Unambiguous electron density for the central trisaccharide
segment showed, as expected, that the abequose was the dominant
element (Figure 4A) with a contact surface contribution of 121 Å2
out of 255 Å2 in a central pocket ∼8 Å deep and 7 Å wide, formed
chiefly by Trp residues and a Phe together with backbone segments.
There are three His residues in the site, including one that forms a
critical water bridge at the base of the abequose-binding pocket
(Figure 4B), which explained the marked pH-dependence of binding
(Bundle, Eichler, et al. 1994). CDRs H1, H3, L1 and L3 contribute
to the pocket. The heptasaccharide complex suggested that the full
antigen would be bound almost perpendicular to the VH : VL interdomain surface, though most groove-type sites follow this interface
(Cygler et al. 1993). An intramolecular hydrogen bond seen in the trisaccharide antigen in the crystal structure could not be detected by
NMR in the free trisaccharide, which is a strong indication that binding requires a change in conformation of the antigen by rotation
around the glycosidic bonds (Bundle, Baumann, et al. 1994).
Antibody interactions with the epitope include numerous hydrophobic contacts and microcalorimetry has shown that there is a considerable positive entropic contribution to the binding of various ligands,
which was attributed to the favorable desolvation of the hydrophobic
protein surface (Sigurskjold and Bundle 1992). Being a dideoxy
sugar, abequose is considerably more hydrophobic than most carbohydrates, and almost all of the side chains interacting with it are aromatic.
This is reflected by the relative contributions of enthalpy and entropy
to the overall binding, which vary greatly with temperature, and the
Ka reaches a maximum of 1.6 × 105 M−1 at ∼290 K. These findings emphasize the complexity of associations between carbohydrate ligands
and proteins generally, and how a full thermodynamic study can provide much greater insight than a simple measurement of Ka.
Se155-4 was also the first structurally characterized carbohydratespecific antibody for which Fab and scFv fragments expressed from
synthetic genes in E. coli were used to characterize the effects of
amino acid residue mutations around the combining site (Brummell
et al. 1993; Zdanov et al. 1994). For example, serial mutation of
each of the four solvent exposed residues (Gly100, His101, Gly102
and Tyr103) on CDR H3 did not abolish binding. Substitutions
involving the contact residue HisH101 with carboxylate or amide
side chain groups resulted in binding affinities close to wild type,
while substitutions of GlyH102 resulted in a significant decrease in affinity. There were no instances of rational site-directed mutagenesis in
which the affinity for the antigen improved.
Bacteriophage display was used to produce a variety of altered antibody fragments (Deng et al. 1994). The mutants with improved binding
were found to have CDR H2 side chains mutated to smaller ones, e.g.,
Ser to Ala, and Ala to Gly, suggesting that improvement in binding may
have involved removal of steric clashes rather than enhanced positive
interactions. A second round of phage display in which the mutations
were limited to CDR residues generated an antibody with improved
affinity that had a single Met to Ile replacement of a residue behind a
binding-site Trp, which altered slightly the shape of the Abe pocket.
Shigella flexneri O-chain polysaccharide-specific
antibody, SYA/J-6
The Sh. flexneri variant Y contains a linear tetrasaccharide repeat
→2Rhaα1,2Rhaα1,3Rhaα1,3GlcNAcα1→ (Carlin et al. 1984; Hanna
and Bundle 1993). A murine mAb, SYA/J-6 was generated in response
to immunization with bacterial cells of Sh. flexneri variant Y (Carlin
et al. 1984, 1986). The affinity of this antibody for a pentasaccharide
in which the tetrasaccharide repeating unit is extended by a Rha at the
reducing end was 3.8 × 104 M−1 (Vyas et al. 1993, 2002).
The structures of SYA/J-6 Fab were initially determined as complexes with the above pentasaccharide and Rha-2-deoxy-Rha-GlcNAc
trisaccharide (Vyas et al. 1993). However, only two sugar rings were
934
O Haji-Ghassemi et al.
Fig. 4. Electrostatic surface potentials are colored red and blue for negative and positive charges, respectively, and white color represents neutral residues.
Carbohydrate antigens are shown as green, water molecules as cyan spheres and hydrogen bonds to carbohydrate antigen are shown as yellow dashed
spheres. The variable VH chain ribbon is colored red and the VL chain ribbon is colored blue. CDRs are labelled L1, L2, L3, H1, H2 and H3. Abe, 3,6-dideoxy-Dxylo-hexopyranose (abequose); Rha, α-L-rhamnose; Gla, α-D-galactose; Man, α-D-mannose; GlcNAc, N-acetyl-β-D-glucosamine; Glc, α-D-glucose; Bgc,
β-D-glucose; GlcN, α-D-glucosamine; Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid. (A) Electrostatic surface potentials of Se155-4
(PDB: 1MFC) Fv in complex with the Abeα1,3Manα1,4Rhaα1,3Gal tetrasaccharide repeating unit of S. typhimurium. (B) Combining site of Se155-4 antibody
showing important residues involved in the recognition of the Abeα1,3Manα1,4Rhaα1,3Gal antigen. (C) Electrostatic surface potentials of SYA/J-6 (PDB: 1M7I).
Fv in complex with Shigella flexneri Y-variant O-chain pentasaccharide. ABCDA* monomers represent Rhaα1,2Rhaα1,3Rhaα1,3GlcNAcα1,2Rao, where the
terminal sugar on the reducing end (A*) represents methyl-α-L-rhamnopyranoside (Rao). (D) Stereo diagram showing the interactions between SYA/J-6
antibody and the Sh. flexneri Y-variant O-chain polysaccharide antigen. (E) Electrostatic surface potentials of F22-4 (PDB: 3BZ4) Fv bound to two repeats of the
Sh. flexneri serotype 2a pentasaccharide unit. AB(E)CD monomers represent the repeating unit →2Rhaα1,2Rhaα1,3(Glcα1,4)Rhaα1,3-GlcNAcβ1→. (F) Stereo
diagram showing the helical nature of the Sh. flexneri serotype 2a decasaccharide antigen. (G) Stereo diagram showing interactions between F22-4 and the
decasaccharide antigen. (H) Transparent surface depiction of mAb Ab52 (PDB: 3UJT) Fv. CDR loops are highlighted. (I) Transparent surface depiction of mAb
N62 (PDB: 4KPH) Fv, highlighting the V-shaped-binding site. (J) Electrostatic surface potentials of WN1 222-5 (PDB: 3V0W) Fv in complex with E. coli serotype
R2 dodecasaccharide core antigen. Phosphates are depicted in orange. The lipid A backbone residues of the LPS antigen were not visible in electron density
and therefore not included in the model. (K) Stereo diagram showing the interactions between WN1 222-5 and the E. coli serotype R2 dodecasaccharide core
antigen. (L) Electrostatic surface potentials of LPT3-1 (PDB: 4C83) Fv in complex with N. meningitides inner core GlcNAcα1,2Hepα1,3(Glcβ1,4)Hepα1,5Kdo. (M)
Stereo diagram showing the interactions between LPT3-1 antibody and the N. meningitides inner core. Hydrophobic interactions are highlighted with black
dashes. This figure is available in black and white in print and in color at Glycobiology online.
located at the time, the GlcNAc and its preceding Rha. The Rha is
completely buried within the binding site and the GlcNAc is 75% buried. The second Rha appears to be mainly solvent exposed. Since then,
the structures have been improved to 2.50 Å for the pentasaccharide
and 2.30 Å for the trisaccharide, with unambiguous density for
both antigens (Vyas et al. 2002). The antibody CDRs display high
complementarity with the pentasaccharide (Figure 4C), making a
total of eight hydrogen bonds and other contacts to all five carbohydrate residues (Figure 4D) along a deep groove (25 Å long, 10 Å deep
at the center and 12 Å wide), with the largest number of polar contacts
to the GlcNAc residue. Its Ka was 2.5 × 105 M−1 whereas the trisaccharide reached 1.7 × 106 M−1. The deep groove allowed the trisaccharide to achieve higher affinity where the central Rha residue is
completely buried. In contrast, the ends of the tetrasaccharide repeat
are solvent exposed. In a similar manner to the Sh. flexneri antibody
F22-4, peptide mimics have also been produced for the Y-type antigen
and solved as a complex with the antibody (Vyas et al. 2003). There
were many water molecules mediating the peptide’s interaction with
the Fab, which may account for its affinity being only 2-fold higher
than that of the pentasaccharide.
Shigella flexneri serotype 2a-specific antibody, F22-4
The Sh. flexneri serotype 2a is defined by the repeating branched
pentasaccharide unit →2Rhaα1,2Rhaα1,3(Glcα1,4)Rhaα1,3GlcNAcβ1→ (Phalipon et al. 2006). A number of oligosaccharide
fragments of this antigen have been synthesized (Belot et al. 2004;
Antibody recognition of carbohydrate epitopes
935
Figure 4 Continued
Wright et al. 2004; Phalipon et al. 2006). The development of an
effective vaccine against this pathogen has proven difficult, with
only a few glycoconjugates with these antigens observed to induce protective antibodies (Phalipon et al. 2006). Of five mAbs produced
from immunization with homologous bacteria, an IgG1κ antibody,
F22-4, displayed reactivity to various Shigella 2a O-antigen (O-Ag)
constructs, including the pentasaccharide repeating unit and a
decasaccharide. Inhibition assays with both the penta- and decasaccharide revealed high avidity binding (μm range) and subsequent isothermal calorimetry yielded a Ka of 1 × 106 M−1 (Theillet et al. 2009).
Crystal structures of F22-4 complexed with both penta- and decasaccharide units of the O-Ag have been recently determined to 1.8 and
2.0 Å, respectively (Vulliez-Le Normand et al. 2008). The binding site is
a shallow groove (Figure 4E) 20 Å × 15 Å × 8 Å along the VH/VL boundary and a large area of 1125 Å2 is buried upon antigen binding. The
structures show nine adjoining residues of the antigen forming a helical
structure on the antibody surface (Figure 4F), with six residues making
contact with antibody (Vulliez-Le Normand et al. 2008). The structure
suggests that a minimal Shigella O-Ag epitope of two repeating units is
required for optimal complementarity and an effective glycoconjugate
936
O Haji-Ghassemi et al.
Figure 4 Continued
vaccine. Like Se155-4, F22-4 uses a groove-type binding with a cavity to
accommodate the branched residue with a complex pattern of 11
H-bonds and 14 coordinated water molecules (Figure 4G). Since the
Shigella serotype Y and serotype 2a O-Ag have similar features, it is
not surprising that antibodies raised against these structures share identical germ-line gene segments; however, the shorter CDR H3 of F22-4
(four amino acids) forms a deeper pocket for the branching Glc residue
than SYA/J-6 antibody (nine amino acids). Additionally, a phage library
was used to pan F22-4 against peptides that could bind and mimic the
Shigella O-Ag. A decapeptide that could bind F22-4 with high affinity
was identified and although the crystal structure revealed an entirely
helical conformation of this peptide in the bound state, immunological
mimicry was not achieved (Theillet et al. 2009). The solution NMR
structure of the peptide alone revealed that the helical nature of the peptide was only maintained in the central region, which may account for
the lack of mimicry.
Francisella tularensis-specific antibodies, Ab52 and N62
Francisella tularensis is a facultative intracellular pathogen requiring
low infection dose upon inhalation with high mortality rates,
making it a potential agent for bioterrorism (Conlan 2011).
Its O-Ag consists of a tetrasaccharide repeat comprised of highly
modified sugars such as galactosaminuronic acid amide, and is
the dominant epitope of this pathogen; the repeating structure
is –2Qui4NFoβ1,4GalNAcANα1,4GalNAcANα1,3QuiNAcβ1–
(Gunn and Ernst 2007). Among a panel of antibodies, the IgG2a
κ monoclonal Ab52 displayed highest avidity for this internal
epitope spanning two tetrasaccharide O-Ag repeats. It conferred
protection to BALB/c mice when injected with a lethal dose of live
virulent F. tularensis (Roche et al. 2011; Lu et al. 2012).
The unliganded Fab structure of Ab52 was recently determined
at 2.1 Å; experiments to obtain complexes with a dimer of the tetrasaccharide repeating unit were not successful (Rynkiewicz et al.
2012). The binding site has the form of a large canyon between the
VH and VL domains with a central pocket (Figure 4H). The CDR
H3 is unusually short, making it an ideal candidate for molecular
docking simulations. Docking experiments performed included the
evaluation of a range of O-Ag repeat lengths and arrangements,
and the conformation of CDR H3 was predicted using the same method as for chP3 Fab above. Further, the simulation is subjected to
grid-based ligand docking with energetics or glide algorithm, which
systematically approximates conformational, orientational and positional space of the docked ligand, accompanied by a scoring function
for the predicted affinity (Friesner et al. 2004). Though the minimal
epitope was thought to be two repeats of the tetrasaccharide O-Ag,
glide scores showed that the terminal A and D′ sugar residues were
poorly accommodated in the binding site. Thus, an epitope consisting
of a V-shaped hexasaccharide was proposed to bind Ab52 Fab.
937
Antibody recognition of carbohydrate epitopes
The structure of an Fab of N62, an antibody specific for the terminal epitope of this antigen, has recently been solved by the same
group (Lu et al. 2013) at 2.6 Å resolution without a ligand. N62
and Ab52 are the first pair of antibodies to be solved to internal and
terminal epitopes of the same antigen. N62 has a cavity 8 × 10 × 9 Å
(Figure 4I) lined with aromatic residues. Docking experiments suggested that it would accommodate a terminal Qui4NFo residue with
the GalNAcAN contacting the rim of the cavity above it. The larger
contact area provided by N62 burying a terminal residue compared
with recognition of the internal epitope by Ab52 explains the higher
affinity of N62 for its epitope, 1.9 × 105 M−1. Vaccines designed to
present terminal epitopes rather than internal ones may therefore
lead to antibodies that are more protective.
The inner core LPS-specific antibodies, WN1 222-5
and LPT3-1
LPS cores are attractive targets for antibody therapeutics and vaccines
given their higher conservation relative to O-chains. WN1 222-5, an
IgG2a κ, reacts with core region of LPS and has been shown to neutralize a broad range of pathogenic Gram-negative serovars even in the
presence of the O-chain (Di Padova, Brade, et al. 1993; Di Padova,
Gram, et al. 1993). Furthermore, WN1 222-5 has been shown to reduce the inflammatory cascade of septic shock in vivo, likely due to the
hindering of LPS uptake and subsequent transfer to the Toll-like receptor 4-myloid differentiation factor 2 (TLR4-MD2) complex (Pollack
et al. 1997).
The ligand used in the structural work on the Fab (Gomery et al.
2012) was a dodecasaccharide obtained from the core of E. coli R2
type, and is the largest bacterial oligosaccharide solved as an antibody
complex (Figure 4J). Its structure is GlcNα1,2Glcα1,2-Glcα1,3
(Galα1,6)Glcα1,3(Hepα1,7)(P4)Hepα1,3(P4)Hepα1,5(Kdoα2,4)Kdo
α2,6(P4)GlcNβ1,6GlcNα1-P where Hep is L-glycero-D-mannoheptose and Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid; the Ka
for this antigen was 3.1 × 107 M−1, a very high value for a carbohydrate–protein interaction. The complex was solved at 1.73 Å resolution and the free antibody at 2.13 Å. The binding site is an open
groove along the VH–VL interface with a protruding CDR H2 that
moves upon binding the antigen, resulting in a total of 525 Å2 of buried surface area. Seven of the sugars formed contacts to the antibody
(Figure 4K), including the five residues of the compact inner core region plus two from the adjacent outer core. The other three sugars
of the outer core were also well resolved, but the di-GlcN phosphate
of the lipid A portion was disordered. The majority of the binding interactions, 12 of 13 H-bonds, were with the VH domain. The ligand
also displayed 10 intramolecular bonds. The basis for TLR4 mimicry
by WN1 222-5 is centered on the recognition of three key residues,
Hep I, II and III, which form the majority of the epitope; these residues
lie in the same conformation observed in the TLR4-LPS-MD2 structure (PDB code: 3FXI) (Park et al. 2009). Significantly, the attachment
site for the O-chain on the GlcIII residue is not part of the epitope, but
is solvent exposed and allows WN1 222-5 to overcome the heterogeneity associated with the O-chain. There are several phosphates around
the core, but only the phosphate on the second heptose showed a significant charge interaction, with Arg52 of CDR H2.
A second anti-inner core mAb, LPT3-1 (IgG2b) has recently been
determined to 2.69 Å resolution in complex with Neisseria meningitidis lipooligosaccharide (Parker et al. 2014). Neisseria meningitidis is
typically comprised of an inner core tetrasaccharide GlcNAcα1,2Hepα1,3(P4)Hepα1,5Kdo, where the terminal α1,2GlcNAc is unique to
N. meningitidis (Yang et al. 2012). Common substitutions on this
conserved inner core include β1,4Glc on HepI, and ethanolamine
phosphate substitution at 3-OH and/or 6-OH positions on HepII
(Weynants et al. 2009; Yang et al. 2012). As with WN1 222-5, the
lipid A carbohydrate moiety did not participate in binding and was
only partially ordered in the complex structure of LPT3-1. The antibody displays groove type binding, with a total buried accessible
surface area of 300 Å2 (Figure 4L), of which only 23 Å2 is provided
by the light chain (Parker et al. 2014). TyrH50 and TrpL92 residues
help in the formation of a hydrophobic pocket which accommodates
the methyl group on the terminal GlcNAc residue. There are a total of
nine hydrogen bonds, with the GlcNAcα1,2 Hep disaccharide forming
most of these interactions (Figure 4M). The unliganded structure is not
available for LPT3-1, and therefore conclusions cannot be made
whether an induced fit mechanism is utilized during binding.
Antibodies to terminal epitopes on bacterial
and fungal oligosaccharides
In addition to the anti-tumor antibodies, structures have been solved
for antibodies that recognize non-reducing terminal epitopes on a
variety of other glycan antigens, including bacterial polysaccharides,
and fungal and human oligosaccharides. These epitopes often induce
antibodies of higher affinity, perhaps because their presentation
allows the antibody sites to develop larger contact surfaces, as noted
for the F. tularensis antibodies above. However, they can also help to
evade the immune response by mimicking host antigens, e.g., the
blood group B epitope displayed by a Salmonella LPS (Perry and
MacLean 1992).
The V. cholerae O1-specific antibody, S-20-4
Analysis of protective antibodies against the pathogenic V. cholerae
serogroup O1 led to the conclusion that the Ogawa serotype displays a
specific antigenic determinant (Apter et al. 1993), with ∼90% of the
binding energy attributed to the terminal monosaccharide residue
(Wang et al. 1998). The Inaba antigen consists of an α1,2-linked perosamine polysaccharide (perosamine being 4,6-dideoxy-4-amino-D-Man),
N-acylated with 3-deoxy-L-glycero-tetronic acid, while the Ogawa antigen bears an additional O-methyl group at the C2 of the terminal sugar
(Apter et al. 1993; Ito et al. 1994; Villeneuve et al. 2000). Only weak
cross-reactivity to the Inaba antigen has been reported for Ogawa
serotype-specific antibodies (Liao et al. 2002).
Crystal structures of an IgG1κ antibody, S-20-4, complexed with
mono- and disaccharides of the Ogawa antigens were solved to 2.3
and 2.8 Å, respectively (Villeneuve et al. 2000). The structures revealed
a central hydrophobic pocket defined by Trp and other aromatic residues that accommodate the methylated terminal perosamine residue
with 420 Å2 of buried surface area (Figure 5A). The affinity of this
antibody is relatively high, 3.9 × 105 M−1 for the monosaccharide
and 1.2 × 106 M−1 for the disaccharide, which has been attributed
to specific interactions with the methyl group. Intriguingly, the structure showed that the Inaba antigen would fit in the combining site of
S-20-4 with identical contact sites as the Ogawa antigen (Figure 5B).
Thus, the low cross-reactivity towards the Inaba antigen implies that
dehydration of the hydrophobic pocket must be the driving force for
binding to the Ogawa antigen.
Antibodies against chlamydial LPS antigens
Members of the bacterial family Chlamydiaceae possess an unusual
truncated LPS consisting of lipid A linked to short oligomers of the
inner core sugar αKdo that are joined by 2,4 and/or 2,8 linkages
938
O Haji-Ghassemi et al.
Fig. 5. Electrostatic surface potentials are colored red and blue for negative and positive charges, respectively, and white color represents neutral residues.
Carbohydrate antigens are shown as green, water molecules are shown as cyan spheres and hydrogen bonds to carbohydrate antigen are shown as yellow
dashed spheres. The variable VH chain ribbon is colored red and the VL chain ribbon is colored blue or white. CDRs are labelled L1, L2, L3, H1, H2 and H3. Kdo,
3-deoxy-D-manno-oct-2-ulosonic acid. (A) Electrostatic surface potentials of S-20-4 (PDB: 1F4Y) Fv showing the hydrophobic pocket accommodating the methyl
group of the α1,2 2-O-methylperosamine disaccharide (Ogawa antigen). (B) Stereo diagram of S-20-4 antibody in complex with an α1,2-linked
2-O-methylperosamine (4-amino-4,6-dideoxy-D-mannose) disaccharide whose amino groups are acylated with 3-deoxy-L-glycero-tetronic acid. Black dotted
lines represent hydrophobic contacts (∼3.00 Å) between Ala H98 and the C2 methyl group. (C) Electrostatic surface potential diagram of S25-2 (PDB: 3SY0) in
complex with Chlamydiaceae-specific Kdo2,8Kdo2,4Kdo trisaccharide epitope. KdoIII represents the terminal residue, while KdoI is normally linked to the lipid
A carbohydrate backbone. (D) Stereo diagram of S25-2 (PDB: 3T4Y) in complex with Kdo monosacharride. The subsequent images do not display these
conserved interactions. (E) Stereo diagram of S25-2 (PDB: 3SY0) in complex with Kdo2,8Kdo2,4Kdo showing interactions with the second and third Kdo
residues. (F) Stereo diagram of S54-10 (PDB: 3I02) in complex with Kdo2,4Kdo2,4Kdo showing interactions with second and third Kdo residues. (G) Stereo
diagram of S73-2 (PDB: 3HZV) in complex with Kdo2,8Kdo2,4Kdo showing interactions with first and third Kdo residues. (H) Stereo diagram of S67-27 (PDB:
3IKC) in complex with Kdo2,8-7-O-Me-Kdo showing CDR L3 and CDR H3, with side chains that form complementarity to the unnatural 7-O-Me addition. (I)
Stereo diagram of S64-4 (PDB: 3PHO) in complex with Kdo2,8Kdo2,4Kdo2,6GlcN4P1,6GlcN1P. The GlcN1P residue was not modelled due to poor electron
density. (J) Electrostatic surface potential diagram of S25-26 (PDB: 4M7J) in complex with Kdo2,8Kdo2,4Kdo-O-allyl trisaccharide. (K) Stereo diagram showing
interactions between mAb S25-26 and the Kdo2,8Kdo2,4Kdo-O-allyl trisaccharide. (L) Electrostatic surface potential of the CS-35 (PDB: 3HNS) Fv in complexed
with the hexa-α-D-arabinose Araβ1,2Araα1,5(Araβ1,2Araα1,3)Araα1,5Araα (Araf6) antigen. The intact LAM structure is attached to the methyl group of arabino
residue A. The combining site is highly complementary to the reducing terminal residue A and to the non-reducing end E. Poor electron density was observed
for residues F and D. (M) Stereo diagram showing the interactions between CS-35 antibody and the Araf6 antigen. This figure is available in black and white in
print and in color at Glycobiology online.
Antibody recognition of carbohydrate epitopes
Figure 5 Continued
939
940
(Brade and Rietschel 1984; Brade et al. 1987). These carbohydrate
structures were used to generate a large number of mAbs that fall
largely into two distinct V-gene restricted families named after their
prototypic clones, the S25-2 and S25-23 types. The S25-2 type displayed a striking range of specificities and cross-reactivities, while
the S25-23 type bound exclusively to the Chlamydiaceae-specifc
Kdo2,8Kdo2,4Kdo trisaccharide antigen (Brade et al. 1987, 1994,
2000; Kosma et al. 1990, 1999; Holst et al. 1991; Muller et al.
1997; Müller-Loennies et al. 2000).
Crystal structures of the S25-2 family of antibodies display a pocket
of conserved V-gene sequence that binds terminal Kdo residues
(Figure 5C) via several hydrogen bonds (Figure 5D) (Nguyen et al.
2003; Brooks et al. 2008). Significantly, a bifurcated salt bridge between an arginine of CDR H2 and the carboxyl group of the terminal
Kdo was the first observation of a charged residue interaction in carbohydrate binding by an antibody (Nguyen et al. 2003). Additional Kdo
residues of di- and trisaccharide chlamydia antigens as well as synthetic unnatural antigens are accommodated in the combining sites of
S25-2 family mAbs by a flexible groove composed mainly of an Asn
in CDR H2 and an Arg in L1 (Figure 5E), where the mutation in
CDR H2 of AsnH53 to Lys allows additional interactions to Kdo
oligosaccharides and increases avidity for all antigens (Blackler et al.
2011).
S25-2 family mAbs with different CDR H3s display varying levels
of cross-reactivity to the 2,8- and 2,4-linked Kdo oligosaccharides.
Whereas S25-2 and the similar S25-39 display higher avidity for the
terminal 2,8 linkage, S45-18 and S54-10 (each using a different
CDR H3 sequence) extend a phenylalanine residue from H3 into
the binding pocket where it forms favorable stacking interactions
against the terminal 2,4 linkage (Figure 5F) (Nguyen et al. 2003;
Brooks, Müller-Loennies, et al. 2010). S73-2 also preferentially
binds the 2,4 terminal linkage, with Kdo2,4Kdo2,4Kdo bound in
the same orientation as S45-18 and S54-10, but shows weak crossreactivity for the 2,8-terminal linkage, where Kdo2,8Kdo2,4Kdo
adopts a “bent” conformation with Kdo2 bound in the conserved
pocket (Figure 5G) (Brooks, Müller-Loennies, et al. 2010). S67-27
displays a long backward-leaning CDR H3 that allows the synthetic
modified Kdo antigen Kdo2,8-7-O-Me-Kdo to bind with higher
avidity than natural antigens through extra surface contact with
CDR H3 (Figure 5H) (Brooks, Müller-Loennies, et al. 2010).
These structures suggest the S25-2 V-gene combination can provide recognition of a wide range of antigens of Kdo-like structure
through a conserved cross-reactive binding site that is tuneable by
CDR H3 to refine specificity, provide redundant specificity with unique binding mechanisms, or enable binding to previously unencountered antigen modifications. Immunization with Kdo antigens also
produced antibodies of alternate light chain V-gene descent. S64-4 displays less cross-reactivity than the S25-2 family antibodies but much
higher avidity for Kdo2,8Kdo2,4Kdo2,6GlcN4P1,6GlcN1P. The
structure revealed a similar binding pocket for terminal Kdo with a
more open binding site above that requires a large planar antigen conformation to achieve significant complementarity (Figure 5I; Evans
et al. 2011). This mAb demonstrates the value of different V-gene
combinations to recognize additional epitopes of the same antigen
to enable tighter binding of larger ligands.
The S25-23-type antibodies use a different set of heavy chain V, D
and J genes to confer strict recognition of Kdo2,8Kdo2,4Kdo containing antigens, with no observed cross-reactivity towards the Kdo monoor disaccharide, or even the Kdo2,4Kdo2,4Kdo trisaccharide (Brade
et al. 1997; Müller-Loennies et al. 2000). ITC of S25-23 showed
high affinity with KD values of 6.54 × 10−8 and 9.90 × 10−8 for the
O Haji-Ghassemi et al.
Kdo2,8Kdo2,4Kdo tri- and Kdo2,8Kdo2,4Kdo2,6GlcN4P-1,6GlcN1P
pentasaccharide, respectively (Haji-Ghassemi et al. 2014). Recently,
the crystal structures of an S25-23-type antibody, S25-26 were reported
to high resolution in liganded and unliganded forms (Haji-Ghassemi
et al. 2014).
In contrast to the S25-2 family of mAbs, S25-26 does not have a
specific pocket for the recognition of a single Kdo residue, but rather
has an extended groove along the heavy chain that spreads the trisaccharide across the combining site to allow recognition of all three residues in a linkage/length-dependent manner (Figure 5J). This
mode allows for hydrogen bonds (of which there are 13 total) to be
evenly distributed among each of the three Kdo residues (Figure 5K),
instead of the majority of binding occurring through the terminal
Kdo residues as in the S25-2-type antibodies. Further, S25-26
relies more heavily on water to form a complementary surface to
antigen with a total of 16 water bridge interactions from 10 water
molecules (Figure 5K). The groove-type recognition of S25-26 allows
for specific interactions to the first and terminal Kdo carboxyl
groups, which in turn depends on the glycosidic linkage length
and stereochemistry, explaining specificity toward the longer
Kdo2,8Kdo2,4Kdo trisaccharide over the Kdo2,4Kdo2,4Kdo counterpart and the lack of observed cross-reactivity to Kdo mono and
Kdo2,8Kdo disaccharide.
Finally, the structures of three unliganded forms as well as the
liganded form of S25-26 revealed density for N-glycosylation present
on Asn 85 of the variable heavy chain, adjacent to CDR H3. Analysis
of the glycan revealed a heterogeneous mixture with a common
root structure that contained an unusually high number of terminal
Gal–Gal moieties, which have been implicated in allergic responses
to therapeutic mAb treatment (Chung et al. 2008). One of the
unliganded structures of S25-26 showed significant order of the
glycan with appropriate electron density for nine residues. Interestingly, all S25-23 type (of which five have been characterized)
antibodies possess the identical glycosylation site, and there is preliminary evidence that partial cleavage of the N-linked sugars
results in increased affinity of Fab towards antigen (unpublished
data).
The clear conclusion from this collection of structures is how the
immune system has evolved to provide redundant and adaptable protection against important bacterial carbohydrates using a relatively
small number of germ-line genes.
Mycobacterial LAM-specific antibody, CS-35
Because of their prominent role in pathogenesis of mycobacterial
infections, surface glycolipids such as LAMs have been attractive targets for vaccination (Strohmeier and Fenton 1999). A large portion of
LAMs contain α1,5-linked D-arabinofuranose (Araf ) units, with
branching points consisting of two arrangements: linear tetraarabinoside, Araβ1,2Araα1,5Araα1,5Araα- and the related hexaarabinoside Araβ1,2Araα1,5(Araβ1,2Araα1,3)Araα1,5Araα- (Nigou
et al. 2003; Briken et al. 2004).
Several antibodies specific for LAMs have been identified from
mycobacterial infections, of which mAb IgG CS-35 is the most extensively studied (Verbon et al. 1990; Kaur et al. 2002; Arias-Bouda et al.
2003). Binding assays with synthetic oligosaccharides indicated CS-35
has highest avidity for the hexasaccharide branched terminal structure
(Ka 1 × 105 M−1) and to a lesser extent for the linear tetrasaccharide
structure (Kaur et al. 2002). The antibody also loses avidity upon
the addition of mannose residues capping the non-reducing terminal
arabinosyl end, also known as ManLAMs.
941
Antibody recognition of carbohydrate epitopes
Crystal structures of CS-35 Fab complexed with the tetra- and
hexa-arabinosyl residues were solved to 1.8 and 2.0 Å resolution, respectively (Murase et al. 2009). The CDRs formed a triangular cavity
with high complementarity to the non-reducing ends of the Y-shaped
branched hexasaccharide epitope (Figure 5L), which wraps around
TyrH98, and a groove complementary to the reducing end of the
LAM structure (Figure 5M). All six CDRs contact the ligand and
509 Å2 of surface is buried. There are few direct H-bonds between
the hexasaccharide ligand and the antibody, but there is an extensive
network through water molecules, three of which are internal to the
ligand. As with the other antibodies specific to terminal epitopes,
CS-35 mAb displays the highest specificity to the terminal sugar residues (Figure 5M), evident from the clear electron density for these residues as well as the Ara at the branching point.
Candida albicans mannobiose-specific antibody, C3.1
Candida species are the most common cause of candidiasis, a fungal
infection that occurs in immunocompromised patients, and the leading causative agent is C. albicans (MacCallum 2010). A few di- and
trimannoside conjugates have elicited protective capabilities in animal
models (Cutler 2005; Xin et al. 2008; Fidel and Cutler 2011). The
best characterized mAb against the homopolymeric β-mannans of
C. albicans is the protective mAb C3.1 (Han et al. 2000). Unlike earlier
findings of Kabat with anti-dextran antibodies (Kabat 1960), C3.1
displayed reduced affinity as the size of the mannose oligomers increased past the trisaccharide unit.
STD NMR, chemical mapping, ELISA and computational methods
using 1,2-β-linked mannose oligosaccharide fragments (Johnson et al.
2012) showed that oligosaccharides bound the antibody in conformations similar to the free ligand. The antigenic determinant is composed
of β1,2Man disaccharide (Ka 3.7 × 104 M−1), with a marginal increase
in affinity to the trisaccharide (Ka 5.5 × 104 M−1), and a decrease in affinity of the tetrasaccharide, (Ka 1.1 × 104 M−1). Based on the minimal
epitope requirements of C3.1, a disaccharide protein-conjugate was designed and it conferred protective immunity against C. albicans in rabbits
(Bundle et al. 2012; Johnson and Bundle 2013).
Modelling of C3.1 Fv using a structural homolog and the knowledge that it possesses a short (six residues) CDR H3, permitted the
construction of a model that showed high complementarity to the
natural C. albicans trisaccharide antigen. Further, the model revealed
a groove type binding site, consistent with antibodies against internal
homopolymeric epitopes.
Antibodies to homopolysaccharides
There are a number of areas where structural progress has been slow.
This is particularly true where there are several identical overlapping
antigenic determinants along the polysaccharide chain, and until
recently, only unliganded structures had been obtained for antibodies
to this type of antigen.
Early immunological studies of these antigens were facilitated
by the relative ease with which defined oligosaccharide fragments
can be obtained by acid hydrolysis. Experiments on antibodies specific
for dextrans with Glc-oligosaccharides, in conjunction with similar
experiments using oligopeptides, gave the first estimate for the
size of the combining site of antibodies (Kabat 1957; Newman and
Kabat 1985; Padlan and Kabat 1988). However, the apparent simplicity is deceptive, and the interpretation of binding data to long chain
polysaccharides is not always straightforward. This is due to the mixture of complexes that can be formed with their oligosaccharides, with
different occupancies of the subsites. For example, a trisaccharide
may occupy subsites one through three, or subsites two through
four. In addition to the numerous internal determinants along the
chain, the non-reducing terminus can be a potent antigenic site, and
while antibodies to the internal epitopes can also bind the terminal
sugars, the reverse is generally not true.
Further, modelling of antibody–antigen complexes based on the
structure of the antigen-free protein has been made particularly difficult by the fact that it cannot easily be determined in which direction
the linear antigen will be bound in the site.
The galactan-specific myeloma protein, J539
J539 was one of the first anti-carbohydrate mouse myeloma proteins
described and it is specific for galactans consisting of β1,6-linked galactose residues (Manjula et al. 1975). This antibody was induced by
treatment of BALB/c mice with mineral oil (Jolley et al. 1973). Several
other myeloma proteins (e.g., T601, X24, X44, S10 and T191) share
similar galactan specificity and these proved to be formed from the
same VH and VL genes with limited heterogeneity (Manjula et al.
1977; Rudikoff et al. 1980; Glaudemans 1987). The estimated Ka values for tri- and tetrasaccharide fragments were 1.5 and 3.4 × 105
M−1, respectively (Jolley et al. 1974). Measurements with other fragments and analogues led to a model in which there were four subsites
within a surface binding cleft, with most of the binding energy deriving from interaction with the non-reducing terminal unit (Glaudemans
et al. 1986; Glaudemans 1991).
The Fab fragment structure of the antibody was solved and refined
to 4.5 Å by Navia et al. (1979), and improved successively to 2.6 Å
resolution (Suh et al. 1986) and to 1.9 Å resolution (PDB entry:
2FBJ). The crystals were obtained only in the absence of oligosaccharide, and soaking experiments were unsuccessful due to the tight packing of the Fab fragments. The X-ray structure showed a large pocket
(Figure 6A) connected to two grooves whose walls are defined by Trp
and Tyr residues (Suh et al. 1986). Fitting of a pentasaccharide ligand
on the basis of only van der Waals contacts was attempted, but it
was complicated by the fact that the chain direction could not be established (a problem that was also encountered with modelling of the
Brucella A system) and by the flexibility of the β1,6 linkage. While a
consistent picture of the interactions of the sugars with the binding-site
has been obtained, the physical locations of the postulated subsites
remain unknown.
Brucella abortus O-chain polysaccharide-specific
hybridoma, Yst9.1
Species of Brucella produce major cell wall polysaccharide antigens
based on the unusual sugar N-formyl-perosamine (4,6 dideoxy4-formamido-D-Man) (Caroff et al. 1984; Freer et al. 1995). In the
B. abortus A polysaccharide, the perosamine residues are α1,2 linked
with a small percentage of α1,3. In the B. mellitensis M antigen, every
fifth linkage is an α1,3 (Bundle, Cherwonogrodzky, et al. 1987;
Bundle, Cherwonogrodzky, Perry, et al. 1987; Peters et al. 1990).
Recently, the M structure was re-investigated and the repeat unit
was shown to have three α1,2 linkages to each α1,3 (Kubler-Kielb
and Vinogradov 2013). The α1,2 linkage along with the D-manno
configuration leads to the A antigen having a helical form with the
α1,2 linkages forming a central axis, and the sugar rings perpendicular
to it. The angle between successive formamido groups is 216°, giving
rise to a five-residue conformational repeat along the helix (Kihlberg
et al. 1991). Among panels of monoclonal antibodies raised against
the two antigens, one termed Yst9.1, raised to the homogeneous
form of the A antigen produced by Yersinia enterocolytica, had the
942
O Haji-Ghassemi et al.
Fig. 6. Electrostatic surface potentials are colored red and blue for negative and positive charges, respectively, and white color represents neutral residues.
Carbohydrate antigens are shown as green, water molecules are shown as cyan spheres and hydrogen bonds to carbohydrate antigen are shown as yellow
dashed spheres. The variable VH chain ribbon is colored red and the VL chain ribbon is colored blue or white. CDRs are labelled L1, L2, L3, H1, H2 and H3. (A)
Transparent surface depiction for the Fv region of galactan-specific antibody J539 (PDB: 2FBJ). (B) Transparent surface depiction for the Fv region of B. abortus
O-chain polysaccharide-specific antibody Yst9.1 (PDB: 1MAM). (C) Stereo ribbon diagram of two mAb735 (PDB: 3WBD) Fv fragments in complex with α2,8-linked
N-acetylneuraminic acid octasaccharide fragment. Fv fragments bind the octasialic acid in a bent U-shaped manner. CDR H3 which is highlighted in yellow. (D)
Stereo diagram showing interactions between the mAb735 Fv fragments and the octasialic acid residue. (E) Electrostatic surface potential depiction of mAb735
variable domains bound to trisialic acid fragment. This figure is available in black and white in print and in color at Glycobiology online.
Antibody recognition of carbohydrate epitopes
greatest specificity for the A antigen (Bundle et al. 1984, 1989). Investigation with SPR revealed an avidity of 1.9 × 107 M−1 for the IgG,
which is considerably higher than most carbohydrate-specific IgM
immunoglobulins (Young et al. 1999).
A model of the antibody–antigen complex was proposed by
Oomen et al. (1991). When compared with the subsequent crystal
structure of the unliganded Fab, determined at 2.45 Å resolution
(Rose et al. 1993), the model matched in many aspects except for a
3 Å translation of the V domains with respect to each other. The packing of the Fab molecules was such that their binding sites were occluded by neighboring molecules, so soaking experiments with
oligosaccharides were unsuccessful. The binding site forms a large
groove 20 Å long, 10 Å deep and 15 Å wide (Figure 6B), lined with
aromatic residues. This crevice is broader than that of J539, as
would be required for the greater width of the A antigen with its
sugar rings perpendicular to the helix axis, compared with the extended form of the α1,6 galactans. The groove is clearly long enough
to accommodate at least a pentasaccharide and the bulk of the interaction appears to be hydrophobic in nature. A central depression in the
groove could accommodate a formamido group. A series of pentasaccharide analogs were synthesized in which N-formamido groups were
selectively replaced with hydroxyl groups, i.e., N-formamido perosamine became D-Rha (Kihlberg and Bundle 1991; Kihlberg et al. 1991).
However, binding studies of them could not be interpreted into a consistent set of subsites (D.R. Bundle, personal communication). A structure of an antigen complex is needed to elucidate the exact binding
mechanism to this unique carbohydrate ligand.
The polysialic acid-specific antibody, mAb735
The bacteria N. meningitides and E. coli capsular type K1 are known
agents of human bacterial meningitis. Both bacteria display capsular
polysaccharide composed largely of α2,8-linked N-acetylneuraminic
acid (also called group B meningitis polysaccharide or GBMP)
(Jennings et al. 1985; Michon et al. 1985). Several antibodies specific
for GBMP have been reported, most notable of which is mAb735 that
recognizes homopolymers of GBMP (Frosch et al. 1985). As the binding sites of immunoglobulins had for years been established to be no
longer than 8–10 saccharide residues, it was surprising to observe that
mAb735 would only effectively bind GBMP polymers many times that
length. Given that the antibody could not directly interact with potentially hundreds of required saccharide residues, the answer had to lie in
the behavior of longer-chain polysaccharides.
NMR studies of the polysaccharide showed that it adopted extended helical conformations in solution with the carboxylate groups
protruding on the “inside” of the helix and N-acetyl groups protruding on the “outside” (Michon et al. 1987; Wessels et al. 1987; Wessels
and Kasper 1989; Brisson et al. 1992). The preponderance of the helix
form increases with the number of oligosaccharide residues, which explains the preference of mAb735 for longer polymer lengths. Thermodynamics studies revealed two mAb735 Fabs binding per 41-residue
polymer with an affinity of ∼3 × 105 M−1, which suggested an epitope
of 10–20 residues, whereas oligosaccharides of 9–15 residues were
bound an order of magnitude weaker by the IgG (Evans et al. 1995).
The Fab crystal structure of unliganded mAb735 was determined
at 2.8 Å resolution (Evans et al. 1995) and revealed a combining site with
complementarity in shape and charge to the predicted helical conformation of the antigen. Attempts to obtain crystals with PSA were not successful. A set of positively charged residues is clustered to one side of a
shallow groove, while the other half is mainly hydrophobic. This would
allow ionic interactions to occur with the negatively charged carboxyl
943
groups at one end of the antigen helix, while the twist of the helix
would bring the N-acetyl groups towards the hydrophobic residues.
Recently, a structure at a resolution of 1.8 Å was reported for an Fv
form of mAb735 in a complex with an octasaccharide fragment of
polysialic acid (Nagae et al. 2013). The hapten was bound in a unique
manner, such that a U-shaped conformation at the interface between
the binding sites of two Fv molecules was formed (Figure 6C), with
two internal H-bonds bridging the arms and many associated water
molecules. This conformation is unlikely to represent the situation in
solution. Residues 2–4 and 6–8 contact the 2 Fvs, respectively, and 11
water molecules were involved in the H-bonding (Figure 6D). The area
buried per trisaccharide element was 406 Å2 (Figure 6E) and the highest Ka measured by isothermal calorimetry was for a pentasaccharide,
1.5 × 103 M−1. CDR H3 formed most of the H-bonds, but all the
CDRs except L3 contacted the antigen and there was no conformational change on binding. There were no charge interactions between
amino acid side chains and the carboxylates of the octasaccharide.
Knowledge of the ability of high-molecular-weight GBMP to
form extended helical structures in solution has formed the basis for
new vaccines against human bacterial meningitis (Jennings 1997;
Pon et al. 1997; Alfonso et al. 2002; Mond and Kokai-Kun 2008).
Replacement of the acetyl groups by N-propyl ones led to an antigen
with enhanced vaccine potential. An effect of GBMP chain length on
immunogenicity was seen in immunization trials in mice using
polysaccharide-tetanus toxoid conjugates with long NPrGBMP or
short (NPrSia)4 glycans. The mAbs raised against the long NPrGBMP
had greater bactericidal activity in vitro (Pon et al. 1997). Two antibodies to this antigen, 13D9 and 6B9, have been solved crystallographically at 2.45 and 2.06 Å, respectively (Johal et al. 2013). The
structure of 13D9 revealed a CDR H2 that lies in a non-canonical
conformation, which docking studies show is a critical feature in
accommodating the extended NPrPSA antigen. A proposed model of extended NPr-PSA decasaccharide bound to 13D9 was consistent with
STD-NMR experiments. Interestingly, chain direction was established
due to the unfavorable binding energy calculated for one direction versus
the other. Further, 13D9 displayed superior protective properties against
meningitis and its convex surface was again consistent with binding of the
antigen in an extended helical conformation. In contrast, the 6B9 site had
a groove into which a shorter epitope would fit, but this size of epitope is
not protective. Hence, the clinical effectiveness of GBMP-based vaccines
will depend on their ability to induce antibodies that recognize extended
helical GBMP epitopes on bacterial cell surfaces.
General features of anti-carbohydrate antibodies
The antigens for the antibodies covered here differ widely in size, from
monosaccharides to dodecasaccharides, and in some cases include
charged groups such as carboxylates and phosphates. Therefore,
only broad conclusions about recognition factors can be drawn.
Soon after the structures of Se155-4 and BR96 were reported, Wilson
and Stanfield (1995) compared them with the classic predictions made
by Kabat about groove and cavity sites. They found that his predictions were generally confirmed by the structures, but the sites were
more complex. The H-bonds were formed by Asp, Asn, Glu, Gln
and Arg residues and the peptide backbone, including bidentate
bonds and coordinated water molecules. The aromatic residues Trp
and Tyr lined the grooves and bound to the hydrophobic faces of
the sugars. The variety of antigens reviewed here is a far wider one,
but Wilson and Stanfield’s conclusions remain valid. With regard to
the types of amino acid that occur in binding sites, the same residues
are common in the sites of other carbohydrate-binding proteins such
944
O Haji-Ghassemi et al.
as lectins; however, antibodies do not use calcium ions as C-type lectins and legume lectins do.
CDR H3 is the most important of the CDRs (Xu and Davis 2000),
reflecting its unique genetic origin from the D gene segment. Its length
and composition (Table II) helps to control the overall shape of the site
and it contributes disproportionately to the binding of the antigen.
There is a considerable spread in lengths of the CDR H3, from 2 to
13 residues, plus the exception of PG9 at 26 residues, with the commonest length being 6 or 7 residues. There is no apparent correlation
between the size of the carbohydrate epitope and the length of the
CDR H3, and while the majority of the antibodies show cleft-shapedbinding sites for the antigen, the CDR H3 length and sequence cause
wide variations in this feature.
It is interesting to compare the CDR H3 compositions to those of
all six CDRs combined from a large set of mouse antibodies, and to
their framework regions (Table III; Padlan 1990). Compared with
the full CDR set, the anti-carbohydrate CDR H3 set (213 residues)
possesses far more Gly (17.4% vs. 6.8%) but far less Ser (3.3% vs.
14.7%). Aromatic residues form 23.5% of the H3 set compared
with 18.7% of the CDR set and only 9.8% of the framework set; in
the H3 CDRs, there is more Phe (8.5 vs. 3.4%) but similar amounts of
Trp and Tyr. Asp and Glu both occur more often than their amide
forms and are unequal in abundance in the H3 set (∼4 : 1). Though
these four residues were found to be important for H-bonding to
antigens (Wilson and Stanfield 1995), they only comprise 17.3% of
the H3 residues. Notably Asn is only 4.2% compared with 8.3% in
the full CDR set. Charged residues comprise 23% of the total
Table II. CDR H3 sequences of carbohydrate-binding antibodiesa
Antigen
Antibody
CDR H3 sequence
F. tularensis
Sh. flexneri 2a
F. tularensis
nPro-polysialic
Tn antigen
Lex antigen
V. cholera
Polysialic acid
B. abortus
nPro-polysialic
Chlamydia
Mycobacteria
LPS core
Salmonella B
Sh. flexneri Y
Galactan
Human ABO(H) blood
group A
N. meningitidis
Ley antigen
Gal ceramide
Chlamydia
NeuGc-GM3
GD3 glycolipid
Chlamydia
HIV-1 gp120
Chlamydia
HIV-1 gp120
Ab52
F22-4
N62
6B9
237mAb
291-2G3-A
S-20-4
Mab735
Yst9.1
13D9
S25-26
CS35
WN1 222-5
Se155-4
SYA/J-6
J539
BGA
GF
PM
YRF
LRAV
GKVRN
ETGTRF
HFYAVL
GGKFAM
DPYGPA
SRGRTL
DETGSWF
FGNYVPF
QGRGYTL
GGHGYVG
GGAVGAM
LHYYGYN
QYGNLWF
LPT3-1
BR96
L363
S25-2
P3
R24
S54-10
2G12
S73-2
PG9
MRITTDWF
GLDDGAWF
AAGIRWAWF
DHDGYYERF
SGVREGRAQWF
GGTGTRSLYYF
DMRRFDDGDAM
KGSDRLSDNDPF
DINPGSDGYYDAL
EAGGPDYRNGYNYYDF
YDGYYNYHYM
a
The sequences shown span from VH residue 95–100, numbered according to
the PDB entries.
and the Arg content of H3 is higher than the CDR and framework
sets (8.0% vs. 3.8% and 3.4%, respectively). Lys is very low in H3
(1.4% vs 4.4% and 5.44%), Thr is less common (4.2% vs 6.6%
and 9.37%) and Cys is completely absent. As would be expected for
external loops, the hydrophobic amino acids Leu, Val, Ile, Met and
Pro together are only 14% of all CDR H3 residues. Hence, judging
by their H3 compositions only, carbohydrate antigens appear to induce antibodies with CDR compositions that are different from
those for other antigens. But it is the higher Phe, Arg, Asp and Gly contents that are distinctive, and the H-bonding residues Asn, Ser and Thr
usually associated with carbohydrate binding are actually less common. It has been shown that the D segments are most often incorporated into VH genes in reading frames which favor Gly and Tyr residues
(Abergel and Claverie 1991). This is consistent with the observed Gly
content but not as much with the Tyr.
However, when the H-bond forming residues in the structures
solved with bound carbohydrate are tallied up, a total of 94 residues,
a different picture emerges. Overall, the VH domain predominates
with almost twice as many H-bonding residues than the VL, 61 vs.
33, divided equally among its CDRs; L3 is a little higher at 24. This
distribution differs from the earlier analysis of Padlan (1994) in
which the six CDRs were more equal in their contributions. The
most abundant H-bonding residue is Tyr (14 occurrences), followed
by Asn (12) and Trp (11). Also common are Arg (10), His (9) and
Glu (8). Further, the distribution of these residues among the six
CDRs is strikingly uneven. Arg and Asn chiefly occur in the CDRs
L3 and H2, 4–5 times each; Tyr and Trp in L3 (9) and H1 (11); His
in L1 and H3, four times in each; and Glu in H2 (6). Given that Tyr
and Trp are most important in forming the binding-site cavities and
consequently making van der Waals contacts with an antigen, their
abundance is to be expected.
Another aspect of recognition that deserves attention is the buried
surface area of antigen binding, which varies considerably for each antibody. The buried surface areas of the various HIV-1-specific antibodies
have been reviewed elsewhere (Kong et al. 2014). As a further example,
Table III. Combined CDR H3 compositions calculated from Table II,
compared with those of all six CDRs and of framework regions in the
VL and VH domains of a large set of mouse antibodies (Padlan 1990)
Amino acid
CDR H3
CDR H3 (%)
CDR set (%)
FR set (%)
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
Total
14
17
9
20
0
3
5
37
5
3
10
3
7
18
7
7
9
7
25
7
213
6.5
8.0
4.2
9.4
0
1.4
2.3
17.4
2.3
1.4
4.7
1.4
3.3
8.5
3.3
3.3
4.2
3.3
11.7
3.3
5.29
3.76
8.32
5.78
0.05
3.43
2.41
6.75
2.83
3.03
4.45
4.40
2.27
3.37
3.48
14.70
6.61
2.18
13.13
3.78
(6298)
6.65
3.36
1.06
3.48
2.40
5.94
4.33
10.44
0.34
4.23
8.72
5.44
1.39
3.32
3.94
12.12
9.37
2.52
3.97
6.98
(17,142)
945
Antibody recognition of carbohydrate epitopes
Table IV. Buried surface area between Fab and antigen of chlamydial
antibodies
Antibody (PDB code)
Total for antigen:
from light/heavy (Å2)
Antigen
S25-2 (3T4Y)
S25-2 (3SY0)
S54-10 (3I02)
S73-2 (3HZV)
S67-27 (3IKC)
S67-27 (3IJY)
S64-4 (3PHO)
147: 82.0/64.8
286: 154/132
315: 144/171
311: 174/137
295: 145/150
274: 132/142
382: 207/175
S25-26 (4M7J)
331: 47.2/284
Kdo
Kdo2,8Kdo2,4Kdo
Kdo2,4Kdo2,4Kdo
Kdo2,8Kdo2,4Kdo
Kdo2,8-7-O-Me-Kdo
Kdo2,8Kdo
Kdo2,8Kdo2,4Kdo2,
6GlcN4P1,6GlcN1P
Kdo2,8Kdo2,4Kdo
Calculated with AreaIMol (Lee and Richards 1971) in CCP4 suite (Bailey
1994) using 1.4 Å probe radius and standard van der Waals radii. All solvent
molecules were excluded for the calculations.
the areas of the chlamydial LPS-binding antibodies discussed in this review are summarized in Table IV. Generally, the S25-2 type antibodies
in complex with ligands larger than a disaccharide occlude ∼300 Å2,
with near even contributions from light and heavy chains. However,
S64-4 forms a complementary surface that encloses 382 Å2 of the pentasaccharide ligand, owing to a different light chain that forms additional contacts to the lipid A carbohydrate backbone. In contrast, the heavy
chain-dependent S25-26 antibody forms a shallow groove (when
compared with the S25-2 type), yielding a binding surface area of
331 Å2. The higher affinity of S67-27 for the synthetic disaccharide
Kdo2,8-7-O-Me-Kdo compared with Kdo2,8Kdo is consistent with
the 21 Å2 increase in buried surface area for the 7-O-Me.
Water molecules play multiple roles in carbohydrate binding and
they are of critical importance in the overall energetics. Though the
number of water molecules identified depends on the resolution of
the structure, many of the water networks around the reviewed ligands
are extensive, particularly for the larger oligosaccharides. Notable
examples include the mAb 735 Fv structure and the Sh. flexneri antibody
F22-4. The network can adjust to aid the binding of related oligosaccharides, as seen in the case of the mycobacterial antibody CS-35, and
this flexibility may come into play when a whole polysaccharide antigen
is being bound rather than a fragment of it. The set of antibodies includes examples of critical water molecules forming part of H-bonding
networks deep within the site, e.g., Se155-4, as well as waters H-bonding
to the periphery around it. Where unliganded structures are available, it
is apparent that the hydroxyls of the glycan ligand often displace bound
water molecules. But simultaneously with these waters being returned to
the bulk solvent, other water molecules are being captured that were not
observed in the unliganded structures. Hence, assessing the overall entropy changes associated with waters during binding is far from simple.
Another factor in binding energetics is the presence of internal H-bonds
in the bound forms of some antigens, some involving water molecules
such as the mycobacterial antibody CS-35. These help to form the antigens into compact shapes, but NMR experiments suggest that they may
not be present in the free solution state.
A further energetic factor is induced fitting of the antibody site to
the antigen. The prevalence of this factor is hard to assess because free
and bound forms are not available for most of the antibodies, and
intermolecular contacts in the crystals can also cause movements
of the CDRs. But for some antibodies there are definite major shifts
of the CDRs consistent with induced fitting to the antigen, including
the anti-LeY antibody BR96 and the anti-Chlamydia antibodies. Similar changes were seen with antibodies to peptides and to DNA (Wilson
and Stanfield 1993). In contrast, there were no changes seen in the
mAb 735 Fv structure with octasialic acid compared with the unliganded Fab one.
In summary it is clear that antibodies against carbohydrates show
distinct features; however, there are no “rules” governing their behavior
and for each generalization that is made, there will be one or more antibodies that show contradictory behavior. For example, the much longer
epitope requirement by the mAb735 and the pH sensitivity of Se155-4
are contrary to the behavior of other systems. Collectively, however, they
give a generally consistent view of how recognition operates in these systems at the oligosaccharide level and there are indications that the recognition of the complete polysaccharide has additional features.
Funding
This work was supported by the Natural Sciences and Engineering
Research Council of Canada.
Conflict of interest statement
None declared.
Abbreviations
Abe, 3,6-dideoxy-D-xylo-hexopyranose; CDRs, complementary determining loops; Fv, fragment variable; Ley, Lewis Y antigen; scFvs, singlechain fragment variables; sdAb, single domain antibody; SPR, surface
plasmon resonance; STD, saturation transfer difference; VH, Variable
domain of the heavy chain; VL, Variable domain of the light chain.
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