9
HUMAN IMMUNOGLOBULIN GLYCOSYLATION
AND THE LECTIN PATHWAY OF COMPLEMENT
ACTIVATION
James N. Arnold1, Louise Royle2, Raymond A. Dwek2,
Pauline M. Rudd2, and Robert B. Sim1
1MRC Immunochemistry Unit
2Oxford Glycobiology Institute
Department of Biochemistry
University of Oxford
South Parks Road, Oxford OX1 3QU, UK
1. INTRODUCTION
Immunoglobulins are the major secretory products of the adaptive immune
system. They are glycoproteins which are found in all higher vertebrates (mammals,
birds, reptiles, amphibians, bony and cartilaginous fish, but not in jawless fish
(agnatha)) (Litman et al., 1999). In humans there are five classes IgG, IgM, IgA, IgE
and IgD. The immunoglobulins share similar structures (Fig. 1). Each immunoglobulin molecule is composed of two identical disulphide bridged class-specific heavy
chains, each disulphide bridged to a light chain of which there are two isoforms
named k and l. Both heavy and light chains are composed of regions called immunoglobulin domains. The immunoglobulin fold/domain is about 105–120 amino acids
long and is composed of b-sheet secondary structure (Amzel and Poljak, 1979).
The role of immunoglobulins is to bind to antigens via their N-terminal (variable
amino acid sequence) domains and to mediate effector
ff
functions, such as activation
of complement (Malhotra et al., 1995; Roos et al., 2001) or binding to receptors via
their constant (invariable sequence) domains (Mimura et al., 2000; Shields et al.,
2001). During immunoglobulin synthesis, rearrangement of gene segments and
somatic mutation creates variation in amino acid sequence in the N-terminal domains
(named VH and VL domains for Variable Heavy and Light chains respectively). The
light chains have one V domain and one constant sequence domain (CL). The
sequence of all l chain C domains is the same, and the sequence is homologous to
27
John S. Axford (ed.), Glycobiology and Medicine, 27-43.
© 2005 Springer. Printed in the Netherlands.
28
J. N. Arnold et al.
Figure 1. Immunoglobulin Structure
a) The structure of IgG showing the Variable Heavy (VH) and Constant Heavy (CH), Variable Light
(VL) and Constant Light (CL) domains. The diagram identifies the Fab, Fc and flexible hinge regions of
the molecule. This hinge varies in length between the different
ff
immunoglobulin classes and is replaced by
additional CH domain in IgE and IgM. The approximate positioning of the Asn-297 N-linkage site for
glycans is marked. b) Diagrammatic representation of IgG1, IgD, IgA1, IgE and IgM showing N- and Olinked glycan positions, and inter-chain disulphide bridges. The domains themselves contain intra-domain
disulphide bridges, although these are not marked. IgM circulates in the serum in both pentameric and
hexameric forms, in which the monomeric units are disulphide bridged together. Pentameric IgM contains
a single J chain but hexameric IgM does not (Weirsma et al., 1998).
Human Immunoglobulin Glycosylation
29
the C domain shared by all k chains. Heavy chains have 3 or 4 C domains. The
sequences of the C domains are class or subclass specific, i.e. all IgGs have identical
constant regions, as do all IgMs. Each clone of B lymphocytes secretes only one
immunoglobulin molecule, which has V regions unique to that particular B cell
clone. Total IgG isolated from human serum therefore contains 4 subclasses, each
with similar but distinct constant regions, and with 105–106 different
ff
V region
sequences.
IgM and IgD occur both as soluble forms (in serum) and membrane-bound
forms on B lymphocytes (Van Boxel et al., 1972). The membrane-bound forms have
an additional trans-membrane segment, C-terminal to the constant regions. IgA,
IgG, IgE are all soluble molecules: IgG is the most abundant in serum (10–15 mg/ml),
while IgA is the most abundant immunoglobulin overall. Most IgA is secreted
through epithelia into the mucous lining of the gastrointestinal and respiratory tract,
and into tears, saliva and milk (Norderhaug et al., 1999). The secreted form is
generally dimeric and contains an extra glycosylated polypeptide chain, SC (Secretory
Component) and glycosylated 16KDa J chain (Johansen et al., 2001; Royle et al.,
2003), which is also found in pentameric forms of IgM (Wiersma et al., 1998). The
single J chain is disulphide bridged to two C-termini of both IgM and IgA molecules
(Wiersma et al., 1998; Royle et al., 2003). IgA in serum is predominantly monomeric
but also forms dimers and higher polymers (Delacroix et al., 1982; Roos et al., 2001).
IgE is the lowest abundance immunoglobulin, occurring as a monomer at <1 mg/ml.
IgD also occurs as a low abundance monomer in serum at <30 mg/ml, while IgM
is at high concentrations (~2.5 mg/ml). IgM occurs predominantly as pentamers
and hexamers, although a small amount of monomer also circulates (Sørensen
et al., 1999).
ff
classes of immunoglobulin are distinct in their major effector
ff
The different
functions. IgM is principally associated with complement classical pathway activation
via binding of C1q (Wiersma et al., 1998). IgG also activates complement via classical
(Duncan and Winter, 1988) and alternative pathways (Anton et al., 1989) and
mediates ADCC (Antibody Dependent Cell Cytotoxicity) (Sarmay et al., 1992). IgE
is associated with mast cell and basophil stimulation in allergic conditions. IgA in
secretions may act mainly to agglutinate (immobilise) or neutralise micro-organisms
(Lamm, 1997). No effector
ff
functions have been identified for IgD.
In addition to their enormous diversity of amino acid sequences and antigenbinding specificity, immunoglobulins display considerable diversity in the location
and number of glycosylation sites (both N- and O-linked) and great diversity in
glycan structure. The glycans attached to the immunoglobulins are important for
immunoglobulin solubility (Tarentino et al., 1974), subcellular transport and secretion (Gala and Morrison, 2002), conformation (Mimura et al., 2000), binding to Fc
receptors (Mimura et al., 2000), normal plasma clearance (Skockert, 1995) and
complement activation (Malhotra et al., 1995). This chapter discusses both the glycan
structures that are attached to the normal human serum immunoglobulins and their
potential roles in complement activation through the binding of the serum ‘recognition’ lectin, Mannan Binding Lectin (MBL), and the subsequent activation of the
lectin pathway of the complement system.
30
J. N. Arnold et al.
2. GLYCOSYLATION OF THE IMMUNOGLOBULINS
2.1. IgG
There are four subclasses of IgG, named IgG1–4, that differ
ff in their heavy chain
constant region sequence and disulphide bridging. The subclasses have distinctive
glycan pools (Jefferis
ff
et al., 1990).
All IgGs have a single N-linked glycosylation site on each heavy chain in the
CH2 domain at Asn-297 (Fig. 1). There are no conserved glycosylation sites in the
light chain or variable regions of the heavy chain. The glycan population attached
at Asn-297 contains three sets of glycoforms termed IgG-G0, -G1 and -G2 (Fig. 2b).
The IgG-G2 biantennary glycans occupying Asn-297 have two arms that both
terminate in galactose residues. This set of glycoforms accounts for approximately
16% of total IgG glycans. Approximately 35% are IgG-G1, which lack a terminal
galactose residue on one biantennary arm, exposing a GlcNAc residue. IgG-G0
glycans make up 35% and neither biantennary arm contains a galactose residue.
The final 14% of serum IgG glycans consist of IgG-G2 or -G1 glycoforms which
are sialylated. Within the glycans of IgG there is a diversity of structures caused by
the presence of bisecting GlcNAc residues (B in Fig. 2a) (approximately 30% of total
IgG1 glycan pool), core fucose (Fc in Fig. 2a) (approximately 70% of the total IgG1
glycan pool) and sialylation of the terminal 1,3 arm galactose residues (S in Fig. 2a)
(14% of total IgG1 glycan pool) (Butler et al., 2003)).
IgG1 is the most abundant subclass of IgG in the serum. IgG2 and IgG3 have
a preferred linkage of the galactose residues to the a1,3 arm mannose, whereas IgG1
has preferential linkage of galactose to the a1,6 arm mannose. IgG4 is reported to
contain predominantly fully galactosylated structures (Jefferis
ff
et al., 1990).
There is considerable amino acid sequence diversity in the variable regions, and
N-linked glycosylation sites can occur in the variable regions. These are relatively
rare. A recent survey of heavy chain variable region cDNA sequences showed that
only 7 out of 75 (9.3%) had a potential N-linked glycosylation site in the variable
region (Zhu et al., 2002). The glycans that occupy these sites are predominantly
sialylated structures, with a high incidence of bisecting GlcNAc residues (Youings
et al., 1996.; Wormald et al., 1997).
2.2. IgM
IgM is found predominantly in the serum as a pentameric structure disulphide
bridged at the CH3 domains and at the tail piece (a flexible region following the
CH4 domain) and believed to form a ring structure. Pentameric IgM also has a J
chain that contains a single N-linked glycosylation site. IgM can also adopt a
hexameric structure that contains no J chain (Wiersma et al., 1998). IgM heavy
chain (m chain) has five N-linked glycosylation sites at Asn-171, Asn-332, Asn-395,
Asn-402, and Asn-563. Asn-402 and Asn-563 have been shown to be occupied by
oligomannose structures (Chapman and Kornfeld, 1979; Wormald et al., 1991). The
other N-linked glycosylation sites on each m chain in normal human serum IgM are
occupied predominantly by complex biantennary glycans. The most predominant
glycan is FcGlcNAc A G S (26% of total glycan pool). Sialylated structures
2 2 2 1
Human Immunoglobulin Glycosylation
31
Figure 2. Glycan Structure and IgG Glycoforms.
a) Shows the general nomenclature used to describe sugar residues, bond angles and sugar linkages of the
ff
glycan structures that occupy glycoproteins. b) Shows the predominant glycan structures that
different
occupy the Asn-297 site in IgG. The glycans shown may also vary by the presence of absence of a core
Fucose and/or bisecting GlcNAc.
32
J. N. Arnold et al.
Figure 3. IgA Glycosylation Types.
IgA has two subclasses, IgA1 and IgA2, and both have N-linked glycosylation at Asn-263 and Asn-459.
IgA1 contains nine potential O-linked sites in the hinge region, of which five or six have been shown to
be occupied. *The sixth O-linked site occupies one or more of Ser-224, Thr-233, Ser-238 or Ser-240 (Tarelli
et al., 2004). IgA2 has no potential O-linked sites in its hinge region. IgA2 is subdivided into IgA2m(1)
which has two additional N-linked glycosylation sites in the CH1 domain and CH2 domain, and IgA2m(2)
which has these additional N-linked sites but also a third additional CH1 domain N-linked glycosylation site.
(61.8%), core fucosylated (65%) and bisected structures (38%) are present in the
total glycan pool (J.Arnold unpublished data).
2.3. IgA
IgA has two conserved N-linked glycosylation sites, at Asn-263 in the CH2
domain and Asn-459 located in the 18 amino acid tail piece on each a chain. There
are two subclasses of IgA designated IgA1 and IgA2. IgA2 has two forms that
contain two (IgA2m(1)) or three (IgA2m(2)) extra conserved N-linked glycosylation
sites respectively (Fig. 3).
IgA occurs in several different
ff
oligomeric forms, and is present both in serum
and in secretions. Serum IgA and Secretory IgA (SIgA), have distinct populations
of glycan structures.
The 23 amino acid hinge region in IgA1 contains nine potential O-linked sites
of which five have been shown to be occupied (Mattu et al., 1998: Baenziger and
Kornfeld, 1974b). These sites are Thr-228, Ser-230, Ser-232, with Thr-225 and Thr-236
Human Immunoglobulin Glycosylation
33
Figure 4. Core I Structures, Neutral, Mono-, and Di-Sialylated.
The neutral, mono- and di-sialylated Core I O-linked glycan structures, that have been identified on serum
IgA1 and also IgD hinge regions. The nomenclature is explained in Fig. 2.
partially occupied (Mattu et al., 1998). Recently a sixth occupied O-linked site at
one or more of Ser-224, Thr-233, Ser-238 or Ser-240 (Tarelli et al., 2004) has been
identified. No O-linked glycans have been identified on IgA2.
2.3.1. Serum IgA
Serum IgA consists mainly of IgA1. IgA1 and IgA2 contain similar N-linked
glycan structures (Endo et al., 1994; Royle et al., 2003). Over 80% of the glycans
are di-galactosylated bi-antennary complex glycans. Less than 10% are tri- and
tetra-antennary structures (Mattu et al., 1998). Sixty four percent of the glycan
structures are sialylated and 95% of these are linked a2–6 to galactose. The predominant glycan is GlcNAc A G S (24%). The glycan pool has 36% of the glycans
2 2 2 2
containing a core fucose residue and 25% containing a bisecting GlcNAc residue.
The oligosaccharides attached to the Fab in IgA2 differ
ff from those that occupy the
Fc in for example, the presence of triantennary structures and outer-arm fucose
residues such as GlcNAc A G FS which accounts for 3.7% of total Fab glycan
2 3 3 3
pool (Mattu et al., 1998).
The O-linked glycans on the heavy chain of IgA1 have been identified (Mattu
et al., 1998, Field et al., 1994 and Rudd et al., 1994)). The hinge is predominantly
occupied by mono-sialylated core I structures (37%) and neutral core I structures
(31%) (Mattu et al., 1998) (Fig. 4).
2.3.2. Secretory IgA
SIgA is a dimer, held together with a J chain (which has one N-linked site) and
Secretory Component (SC). The SC is the extracellular portion of the epithelial
polymeric Ig receptor (pIgR), and is required for transcytosis of the IgA across the
epithelium to the mucosal surface. The SC has seven N-linked glycosylation sites.
SIgA contains both IgA1 and IgA2 populations.
SIgA is present in mucosal secretions such as colostrum and milk and can bind
to microorganisms, their metabolic products and toxins, preventing their attachment
34
J. N. Arnold et al.
to the epithelium and facilitating their excretion. This process is known as immune
exclusion (reviewed by Lamm, 1997).
The N-linked glycans from the heavy chain of colostrum SIgA consist of approximately 15% sialylated structures (solely a2–6 linked sialic acids), with over 75% of
structures containing a bisecting GlcNAc and 50% being core fucosylated.
Oligomannose structures account for 12% of the N-linked glycan pool. There is a
lack of glycan processing of the N-linked glycans on the a-chain of SIgA, as only
20% of structures are fully galactosylated, and 66% have an exposed terminal
GlcNAc residue. The major structures occupying SIgA heavy chain are
FcGlcNAc A B (30%), GlcNAc A B (21%) and FcGlcNAc A BG (8%) (Royle
2 2
2 2
2 2
1
et al., 2003).
There is a large diversity of O-linked glycan structures on the heavy chain of
SIgA1, which contains over 50 different
ff
structures of up to 15 residues in size (Royle
et al., 2003), in contrast to the restricted pool of structures present on serum IgA1.
The glycans occupying the J chain single N-linked site are predominantly
sialylated biantennary structures (75%). Fifty percent of all structures are core
fucosylated, and 50% of the neutral structures contain a bisecting GlcNAc residue
(Royle et al., 2003). Interestingly no bisecting GlcNAc is present on the sialylated
structures (Royle et al., 2003).
The seven N-linked sites of the SC are occupied by a large diversity of structures,
many of which are not found on the immunoglobulin heavy chains, for example,
outer-arm fucosylated glycans. The presence of these structures may be explained
partially by the fact that epithelial cell glycosylation machinery glycosylates the SC,
whereas the plasma cell glycosylates the immunoglobulin. The SC N-linked glycans
are predominantly bi-antennary structures. Tri-antennary (11.7%) and tetra-antennary (<1%) structures are also present (Royle et al., 2003). Over 70% of the glycans
are sialylated, predominantly mono-sialylated structures, and over 65% of the glycans
contain a core fucose (Royle et al., 2003).
2.4. IgD
IgD has three N-linked glycosylation sites in the Fc at Asn-354, Asn-445,
Asn-496 (Takahashi et al., 1982). Asn-354 in the CH2 domain is occupied solely by
oligomannose structures (GlcNAc Man ) (Mellis and Baenziger, 1983a; Arnold
2
5-9
et al., 2004) which represent 34% of the total glycans. The predominant oligomannose
structure is GlcNAc Man . Glucosylated mannose structures (GlcNAc Man Glc ,
2
8
2
9
1
GlcNAc Man Glc and GlcNAc Man Glc ) are also present (Arnold et al., 2004;
2
8
1
2
7
1
Mellis and Baenziger, 1983a). The other 66% of glycan structures have been shown
to terminate in galactose or sialic acid. These glycans occupy the two CH3 domain
N-linked glycosylation sites Asn-445 and Asn-496. At these two CH3 N-linked sites
71% of the oligosaccharides are sialylated; both mono- (53%) and di-sialylated
(47%) glycans have been identified. Twenty nine percent of glycans terminate in
galactose residues, 50% contain core fucosylation and 50% of the glycans contain
a bisecting GlcNAc (Arnold et al., 2004) at these two sites.
The hinge region of IgD contains several potential O-linked glycosylation sites.
In an IgD myeloma protein IgD:WAH, O-linked glycans occupy Ser-106 and Thr-126,
-127, -131 and -132, although it is uncertain if Thr-131 and –132 are both occupied
Human Immunoglobulin Glycosylation
35
(Mellis and Baenziger, 1983b; Takahashi et al., 1982). Another myeloma IgD:NIG-65
contains seven O-linked glycosylation sites; the five identified in IgD:WAH and also
Ser-110 and Thr-113 (Takayasu et al., 1982). The O-linked glycans present on the
hinge region are solely Core I structures: di-, mono-sialylated and neutral structures
(Fig. 4) (Arnold et al., 2004; Mellis and Baenziger, 1983b).
2.5. IgE
IgE has seven N-linked glycosylation sites in the e chain at Asn-140, Asn-168,
Asn-218, Asn-265, Asn-371, Asn-383, Asn-394 (Dorrington and Bennich, 1978). The
Asn-394 N-linked glycosylation site is occupied solely by oligomannose structures
(Dorrington and Bennich, 1978; Baenziger and Kornfeld, 1974b). The predominant
oligomannose structure is GlcNAc Man (8.3% of the total glycan pool). The other
2
5
six exposed glycosylation sites on each e chain are occupied predominantly with
sialylated glycan structures (46% mono- 42% di-sialylated structures), 12% galactose
terminating structures, 68% core fucosylated and 14% bisected structures (Arnold
et al., 2004).
3. MANNOSE BINDING LECTIN (MBL) AND THE LECTIN
PATHWAY
A
OF COMPLEMENT ACTIVATION
3.1. MBL
MBL (Fig. 5) is a glycoprotein, also known as Mannan/Mannose Binding
Protein and is member of the collectin family of proteins (Malhotra et al., 1994).
Collectins are large oligomeric proteins with multiple lectin domains and collagenous
regions. MBL is synthesized in the liver and secreted into the blood stream. MBL
is an important component of the innate immune system, which binds calciumdependently to sugars that have hydroxyl groups on the carbon-3 and carbon-4
orientated in the equatorial plane of the pyranose ring (Weis et al., 1992). This gives
MBL affinity for mannose, fucose and N-acetyl glucosamine (GlcNAc) (Turner et al.,
1996). This specificity allows MBL to bind to sugar arrays on the surfaces of
microorganisms, including bacteria, viruses and fungi (Holmskov et al., 1994), but
not to human glycoprotein glycans, the structures of which generally terminate in
galactose or sialic acid. MBL has a structure and function similar to that of C1q,
the recognition molecule that initiates the classical pathway of complement. MBL
binds to sugar residues via the Carbohydrate Recognition Domain (CRD) (lectin)
heads. The affinity of a single CRD for carbohydrate is very weak (10−3M) (Iobst
et al., 1994). Multiple CRD binding leads to a much greater avidity. Levels of MBL
in human serum vary greatly between individuals (Turner, 1996), from below 50ng/ml
to above 10ug/ml. The variation of MBL levels is caused by several identified
polymorphisms in the coding sequence and promoter regions of the MBL gene
(Madsen et al., 1995). The coding sequence polymorphisms disrupt the Gly-X-Y
repeat that is found in the collagenous region destablilising the collagen triple helix
formation (Sumiya et al., 1991: Lipscombe et al., 1992), and consequently heterozygotes have low levels of MBL in the blood. Low levels of MBL have been linked to
severe and recurrent infections in children (Summerfield et al., 1997).
36
J. N. Arnold et al.
Figure 5. Structure of MBL.
MBL is composed of identical 25kDa polypeptides that form a trimer through the formation of a triple
helix of the collagen-like regions that is the basis of the MBL subunit (or monomer). This subunit can
then disulphide bridge at its N-terminus to form higher order structures. MBL circulates in the serum
mainly as a hexameric molecule (i.e. six subunits, 18 polypeptide chains). The collagen-like region is
attached to a Carbohydrate Recognition Domain (CRD) which binds to sugar arrays that have hydroxyl
groups on the carbon-3 and carbon-4 orientated in the equatorial plane of the pyranose ring (Weis
et al., 1992).
MBL participates in the host defense response through two major pathways.
Firstly, it acts directly as an opsonin, promoting phagocytosis of foreign material to
which it has bound. There are several candidate receptors through which this process
may be mediated. The main candidate receptor is cell surface calreticulin (Sim et al.,
1998; Ogden et al., 2001), but there is also evidence for the participation of complement receptor 1 (CR1: CD35) (Ghiran et al., 2000). The second pathway through
which MBL functions is by triggering the lectin pathway of complement activation
via MBL associated serine protease-2 (MASP-2) (Vorup-Jensen et al., 2000; Hajela
et al., 2002).
3.2. MASPs
MBL circulates in the serum bound to the serine protease pro-enzymes, MASPs,
of which three have been identified to date; MASP-1, MASP-2 (Matsushita et al.,
1992; Thiel et al., 1997) and MASP-3 (Dahl et al., 2001). The MBL-MASP complex
was shown to be capable of consuming the complement components C2 and C4
(Ikeda et al., 1987). It is now generally accepted from recombinant protein work
that MASP-2 is solely responsibly for the cleavage of C2 and C4 to produce C4b2a
(Vorup-Jensen et al., 2000). This provides MASP-2 with a function similar to that
of C1s in the C1 complex. The biological roles for MASP-1 and MASP-3 are
currently unknown. MASP-1 has been shown to cleave ‘dead’ C3 (C3 in which the
thiolester bond has hydrolyzed) at a slow rate. Cleavage of physiological ‘live’ C3
(C3 in which the thiolester bond is intact) occurs at a very slow rate, suggested to
Human Immunoglobulin Glycosylation
37
Figure 6. The Complement System.
The lectin and classical pathways rely on cleavage of complement protein C4, forming C4b, to which C2
binds and is cleaved, that leads to the formation of C4b2a, a C3 convertase that activates C3. C3 is cleaved
into C3a and C3b, which is further cleaved to form the iC3b opsonin. Activation of C3 leads to the
formation of the membrane attack complex which causes cell lysis. The alternative pathway relies on
preformed C3b, or C3(H O) which forms spontaneously at a slow rate. C3b binds factor B, which is
2
cleaved by Factor D to form another C3 convertase, C3bBb. The C3 convertases are inactivated by decay
accelerating factor, Factor H, C4b-binding protein and complement receptor I, which speed up the dissociation of the convertase. C3b and C4b when bound by cofactors such as Factor H are cleaved by Factor
I and inactivated. The C3 convertases have naturally short half lives in the circulation.
be too slow to be physiologically important (Hajela et al., 2002). MASP-1 also
cleaves Factor XIII (plasma transglutaminase) and fibrinogen, two substrates of
thrombin, potentially implicating MASP-1 in localized coagulation (Hajela et al.,
2002).
3.3. Complement and the lectin pathway of complement activation
The complement system (Fig. 6) is a major part of the innate immune response
that eliminates foreign and altered-self cells by opsonisation and lysis. It is the body’s
first line of defense against infectious agents. The complement system recognizes
foreign matter through proteins with specific binding affinities to potential Pathogen
Associated Molecular Patterns (PAMPs) including lipopolysaccharide, lipoproteins,
peptidoglycan, lipoarabinomannan and oligosaccharide and charge arrays. The binding of ‘recognition’ proteins MBL and C1q leads to the activation of the complement
cascade which is controlled and propagated through serine proteases and regulated
directly by a serpin, C1-inhibitor (Cooper, 1985) that binds and inactivates these
cascade triggering proteases. There are three routes of complement activation; the
classical, alternative and lectin pathways. The classical pathway is triggered by the
C1 complex. The C1 complex is composed of C1q and 2 each of the serine proteases
C1r and C1s (Arlaud et al., 1987).
38
J. N. Arnold et al.
Figure 7. MBL and the Immunoglobulins
A summary of the interaction of MBL with the immunoglobulins.
MBL is the recognition molecule of the lectin pathways of complement activation. Binding of MBL to a target activates MASPs. Activated MASP-2 cleaves the
complement protein C4, forming C4b, to which C2 binds and is also cleaved by
MASP-2, leading to the formation of C4b2a, a C3 convertase that activates C3. C3
is cleaved into C3a and C3b, which is further cleaved to form the iC3b opsonin.
Activation of C3 leads on to the formation of the membrane attack complex (MAC)
that causes cell lysis.
4. THE INTERACTION OF MBL WITH THE IMMUNOGLOBULINS
The immunoglobulins contain populations of glycans, some of which terminate
in mannose or GlcNAc which are potential binding ligands for lectin-like recognition
proteins of the innate immune system, such as MBL, macrophage Mannose Receptor
and the surfactant proteins SP-A and SP-D. The known interactions of MBL with
immunoglobulins are summarised in Fig. 7.
The glycans of IgG have restricted motion because of the terminal galactose
residues attached to the glycan structures. The IgG CH2 domain has a hydrophobic
area on the peptide surface of each heavy chain. Galactoses attached to the a1,6
arm of the glycan interact with this region, and this together with >80 other
interactions such as hydrogen bonding and van der Waals interactions holds the
glycan in contact with the protein surface, which also prevents further processing to
attach terminal sialic acid (Wormald et al., 1997). The glycans therefore have limited
Human Immunoglobulin Glycosylation
39
mobility. In IgG-G0, the glycans do not have terminal galactose residues, but
terminal GlcNAc residues. These glycans are more mobile, as the glycan-protein
interactions are not sufficient to hold the glycan anchored to the protein surface
(Wormald et al., 1997). MBL has been shown to bind to the terminal GlcNAc
residues of the IgG-G0 glycans (Malhotra et al., 1995). IgG-G0 glycoforms have
been shown to increase dramatically in Rheumatoid Arthritis (RA) (Parekh et al.,
1985). This increase has been shown to correlate with disease activity (Rook et al.,
1991). Garred et al. (2000) correlated MBL levels with disease onset and progression
in RA patients. This was consistent with the suggestion by Malhotra et al. (1995)
that activating the lectin pathway of the complement system could be a potential
route to additional inflammation in RA.
IgD has the same domain structure as IgG, however the glycans found at the
N-linked site homologous to that in IgG (Asn-297 in IgG and Asn-354 in IgD) are
solely oligomannose structures. Although these are potential ligands for MBL, MBL
does not bind IgD (Arnold et al., 2004). The oligomannose glycans at Asn-354 are
inaccessible to MBL because the complex glycans occupying Asn-445 on the CH3
domain block the access to the oligomannose glycans (Arnold et al., 2004).
MBL has been shown to interact with certain polymeric types of IgA but not
SIgA (Roos et al., 2001; Royle et al., 2003). MBL binds to polymeric and dimeric
forms of IgA with the highest avidity, but MBL does not bind to monomeric serum
IgA (Roos et al., 2001). The glycans in IgA with which MBL is interacting have not
been identified although it has been inferred from models that all the glycans on
IgA (but not SIgA) are exposed and could potentially bind (Mattu et al., 1998).
SIgA contains a large array of glycans terminating in GlcNAc residues (Royle
et al., 2003). However these structures are masked from lectin binding by the SC
which wraps around the IgA heavy chains. The SC itself contains predominantly
sialylated complex glycans (Royle et al., 2003). The SC structure blocks access of
MBL to the IgA glycans, although it has been suggested that these may be revealed
when SC binds to pathogens (Royle et al., 2003).
IgE has a different
ff
domain structure from IgG, IgD and IgA. The hinge peptides
are replaced by immunoglobulin domains which form a rigid dimer. The crystal
structure of the Fc and CH2 hinge domain showed an asymmetrically bent quaternary structure, where the CH2 domain bends over one side of the Fc (Wan et al.,
2002). Oligomannose structures occupy Asn-394 (homologous site to Asn-297 in
IgG and Asn-354 in IgD) (Dorrington and Bennich, 1978; Arnold et al., 2004). MBL,
however, does not bind IgE (Arnold et al., 2004). The access to these oligomannose
glycans is prevented because of the CH2 hinge domain which completely blocks
access to the oligomannose glycans from one side. The CH2 hinge domain is proposed
to ‘flip’ between two bent quaternary conformations with the CH2 hinge domains
on either side of the Fc domain, preventing access to the oligomannose glycans from
both sides of the Fc (Arnold et al., 2004).
IgM is found in the serum as a pentamer and a hexamer. The IgM monomer
unit has a very similar structure to that of IgE, with an Ig domain replacing the
hinge region. IgM, however, contains oligomannose glycans at two N-linked glycosylation sites, located at Asn-402, homologous to the Asn-394 in IgE (and Asn-297 in
IgG and Asn-354 in IgD) and at Asn-563 at the C-terminus (Wormald et al., 1991).
It has been shown that immobilised human IgM does not bind MBL on microtitre
40
J. N. Arnold et al.
plates (Roos et al., 2003). The oligomannose glycans at Asn-402 are predicted to be
inaccessible on the basis that it is similar in structure to IgE, where the CH2 domain
‘flips,’ between two bent quaternary conformations (J. Arnold and M. Wormald,
unpublished data). The accessibility of the tail piece oligomannose glycans is currently
unknown and under investigation. It may be the case that the structural change that
occurs upon IgM binding to antigen (referred to as the staple form of IgM), may
present the oligomannose sugars to MBL for binding. There have been reports (see
Fig. 7) of human IgM binding to rat MBL (Koppel and Solomon, 2001) and human,
bovine and murine IgM binding to rabbit MBL (Nevens et al., 1992) (Fig. 7). In the
latter case it appears that MBL may be binding only a small subpopulation of
human IgM (J.Arnold, unpublished data).
5. CONCLUSIONS
The glycans attached to the immunoglobulins have a great diversity in structure,
location and number. The predominant complex glycan structures are biantennary,
which are variably galactosylated and sialylated. There is also a high proportion of
structures that contain either or both a bisecting GlcNAc and/or core fucose residue,
in different
ff
percentages between the immunoglobulins.
Glycan structures that could act as potential ligands for MBL have been
identified on all the immunoglobulins. In human serum only IgG-G0 and polymeric
and dimeric IgA have been shown to bind MBL and initiate the lectin pathway of
complement (Malhotra et al., 1995; Roos et al., 2001). In other immunoglobulins
small quantities of GlcNAc-terminating glycan structures have been identified in the
glycan pool. These structures may define small subpopulations of the immunoglobulins to which MBL could bind.
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