antibody add co-precipitating reagents spin down precipitate labeled antigen mixture complexed antigen resolubilize SDS gel sample autoradiograph separated proteins of the immune complex
mediated by acting as adapters that bind via their Fc region to Fc receptors on different cell types.
The three classes of Fc receptor for IgG are Fc␥RI, Fc␥RII, and Fc␥RIII
Three classes of cell surface receptor for IgG (FcγR) are defined in humans:
• FcγRI (CD64); • FcγRII (CD32); and • FcγRIII (CD16).
Each receptor is characterized by a glycoprotein α chain that binds to antibody and has extracellular domains homologous with immunoglobulin domains (Fig. 3.20) – that is, they belong to the immunoglobulin superfamily, as do receptors for IgA (FcαR) and IgE (FcεRI).
FcγRs are expressed constitutively on a variety of cell types and may be upregulated or induced by environ- mental factors (e.g. cytokines).
Biological activation results from cross-linking of the FcγR and consequent aggregation of immunoreceptor
tyrosine-based activation (ITAM) or immunoreceptor tyrosine-based inhibitory (ITIM) motifs in the
cytoplasmic sequences.
Phosphorylation of the ITAM motif triggers activities such as:
• phagocytosis;
• antibody-dependent cell-mediated cytotoxicity (ADCC);
• mediator release; and
• enhancement of antigen presentation.
Fc RECEPTORS ARE EXPRESSED BY GRANULOCYTES, MONONUCLEAR CELLS, AND MAST CELLS
Biological properties of human immunoglobulins
Fig. 3.18 The biological functions of different antibody classes and subclasses depend on which receptors they bind to and the cellular distribution of those receptors.
complement activation classical pathway alternative pathway lectin pathway Fc receptor binding FcγRI (monocytes)
FcγRIIa (monocytes, neutrophils, eosinophils, platelets)
FcγRIIb (lymphocytes)
FcγRIII (neutrophils, eosinophils, macrophages, LGLs, NK cells, T cells) FcεRI (mast cells, basophils)
FcεRII (monocytes, platelets, neutrophils, B and T cells, eosinophils)
FcαR (monocytes, neutrophils, eosinophils, T and B lymphocytes)
FcμR (T cells, macrophages) FcδR (T and B cells)
pIgR, poly-Ig receptor; mucosal transport FcRn, placental transport and catabolism products of microorganisms
SpA, staphylococcal protein A SpG, streptococcal protein G
*dependent on the allotype of FcγRIIa; LGLs, large granular lymphocytes; NK cells, natural killer cells
isotype IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgD IgE
varies with epitope density and antibody/antigen ratio varies with glycosylation status
++ +++ + + + – – – – – – + + + + – ±* ? – – – – – – – + + + +++ +++ + + + – – – – – – + – + – ++ – – ± – – – – – – + + + – – – – – – + – + + – – – – – – – – + – – + – – +++ – – – – – – + – + – – – – – – – – – – + – – – – – – – +++ ++ – – – – – –
In contrast, phosphorylation of ITIM blocks cellular activation.
Fc␥RI is involved in phagocytosis of immune complexes and mediator release
FcγRI (CD64; 72 kDa) binds:
• monomeric IgG1 and IgG3 with high affinity (108–109l/mol); and
• IgG4 with lower affinity (107–108l/mol).
It has a more restricted cellular distribution than the other FcγRs, but is expressed on all cells of the mononuclear phagocyte lineage, and is involved in the phagocytosis of immune complexes, mediator release, etc.
The α chain contains three immunoglobulin domains in the extracellular portion and is associated with a γ chain that bears an ITAM motif in its cytoplasmic part.
Fc␥RII is expressed as Fc␥RIIa and Fc␥RIIb
FcγRII (CD32; 40 kDa) is expressed as structurally and functionally distinct FcγRIIa and FcγRIIb forms with wide but differing cellular distribution.
The α chains have:
• low affinity (< 107 l/mol) for monomeric IgG1 and
IgG3;
• bind complexed (multivalent, aggregated) IgG with high avidity.
The FcγRIIa molecule:
• binds IgG1 and IgG3 only; and
• expresses an ITAM motif within its cytoplasmic tail. It may also be associated with a γ chain and cross-linking results in cellular activation.
Polymorphism in the FcγRIIA gene results in the presence of histidine or arginine at position 131 in the extracellular domains – the His131 allotype binds and is
activated by immune complexes of IgG2. The FcγRIIb molecule:
• expresses an ITIM motif within its cytoplasmic tail; and • when cross-linked, blocks cellular activation, partic-
ularly on B cells (see Fig. 11.5).
Fc␥RIII is expressed as Fc␥RIIIa and Fc␥RIIIb
FcγRIII (CD16; 50–80 kDa) is expressed as structurally and functionally distinct FcγRIIIa and FcγRIIIb, which are extensively glycosylated and have differing cellular distributions.
FcγRIIIa is a transmembrane protein (like FcγRI, FcγRIIa, and FcγRIIb), whereas FcγRIIIb is GPI (glycosyl phosphatidyl inositol) anchored (see Fig. 3.20).
The α chains of FcγRIIIa:
• have a moderate affinity for monomeric IgG (approx- imately 3× 107l/mol); and
• may be associated with γ/ξ and/or β chains bearing ITAM motifs.
FcγRIIIa is expressed on monocytes, macrophages, NK cells, and some T cells.
The FcγRIIIb form:
• is selectively expressed on neutrophils; and • has a low affinity for monomeric IgG (<107l/mol).
It appears that engagement of FcγRIIIb can result in transmembrane signaling through its association with other membrane proteins bearing signaling motifs.
Immunoglobulins in the serum of the fetus and newborn child
Fig. 3.19 IgG in the fetus and newborn infant is derived solely from the mother. This maternal IgG has disappeared by the age of 9 months, by which time the infant is synthesizing its own IgG. The neonate produces its own IgM and IgA; these classes cannot cross the placenta. By the age of 12 months, the infant produces 80% of its adult level of IgG, 75% of its adult IgM level, and 20% of its adult IgA level.
Ig (mg/ 100 ml) 1200 1000 800 600 400 200 150 100 50 0 0 2 4 6 8 0 2 4 6 8 10 12 months 20% 75% 80% birth Ig (% of adult level) 60% maternal IgG total antibody infant IgG IgM IgA Fcγ receptors
Fig. 3.20 Receptors for Fcγ in humans belong to the immunoglobulin superfamily, and have either two or three extracellular immunoglobulin domains. Motifs (ITAM, ITIM) on the intracellular segments or on associated polypeptides are targets for tyrosine kinases involved in initiating intracellular signaling pathways. GPI anchor FcRI (CD64) FcRIIa (CD32) ITAM ITAM ITIM ITAM ITAM ITAM FcRIIIa (CD16) FcRIIIb FcRIIb /, / / or β
POLYMORPHISM IN THE FcγRIIIA AND FcγRIIIB GENES: FcγRIIIB-NA1 AND FcγRIIIB-NA2 FORMS – Polymorphism in the FcγRIIIA gene results in the presence of phenylalanine (Phe) or valine (Val) at position 158 in the extracellular domains; the Val158allotype is associated with
more efficient NK cell activity.
Polymorphism in the FcγRIIIB gene results in multiple amino acid sequence differences, which affect the extent of glycosylation.
The consequent FcγRIIIb-NA1 and FcγRIIIb-NA2 forms are reported to differ in functional activity.
Q. What factors determine whether a particular IgG subclass will have a particular biological function (e.g. the ability to opsonize a bacterium for phagocytosis by a macrophage)? A. The distribution of Fc receptors between different effector cells, the ability of each subclass of antibody to bind to these Fc receptors, and the affinity of binding.
IgG Fc interaction sites for several ligands have been identified
Application of site-directed mutagenesis, X-ray crystallo- graphy and nuclear magnetic resonance spectroscopy has allowed elucidation of the molecular topography of IgG Fc interaction sites for several ligands that bind over- lapping non-identical sites at the CH2/CH3 interface, for
example:
• staphylococcal protein A; • streptococcal protein G; • FcRn; and
• a monoclonal rheumatoid factor Fab fragment. The interactions between maternal IgG and the MHC class I molecule-like FcRn expressed on the intestinal epithelium of the neonatal rat have now been studied at high resolution (Fig. 3.21) and are believed to mimic closely the binding of the human placental counterpart, hFcRn, with maternal IgG. Titration of IgG histidine residues in the binding site for FcRn may explain its: • pH sensitivity;
• binding at pH 6.5 (the pH within vacuoles); and • dissociating at pH 7.4 (the pH of blood).
Given the symmetry of the Fc region, the fragments used in the experiments above are functionally divalent and may form multimeric complexes.
If monomeric IgG were divalent for FcγR and C1q, however, it would not function properly because circu- lating monomeric IgG could form activating multimers. The interaction sites for these ligands (e.g. FcγR and C1q) have been ‘mapped’ to the CH2 domain next to the hinge.
The crystal structure of an IgG Fc/FcγRIII complex reveals an asymmetric interaction site embracing the CH2 domains of both heavy chains, thereby ensuring
monovalency.
Site-directed mutagenesis indicates that common amino acid residues engage each of FcγRI, FcγRII, and FcγRIII, but care should be taken in extrapolating from isotype to isotype and from species to species.
For example, Glu318, Lys320, and Lys322 of the CH2
domain is widely quoted as the interaction motif for C1q activation, but this result was obtained for mouse
IgG2b with guinea pig complement. This finding does not apply to the activation of human complement by human IgG1 – even though human IgG1 expresses the same Glu-x-Lys-x-Lys motif.
IgM–antigen complexes are very efficient activators of the classical complement system, but the mechanism by which IgM binds C1q appears different from that of IgG. The conformational change from a ‘star’ to a ‘staple’ conformation upon binding to multivalent antigen is thought to unveil a ring of occult C1q-binding sites that are not accessible in the star-shaped configuration (see Fig. 3.9).
Glycosylation is important for receptor binding to IgG
N-linked glycosylation of the IgG Cγ2 domain is essential for the binding and activation of FcγRI, FcγRII, FcγRIII, and C1q. The oligosaccharide is of a complex type, generating multiple glycoforms, and forms multiple non- covalent interactions with the polypeptide such that it appears to be sequestered within the protein structure and so inaccessible for direct interactions with the FcγRI, FcγRII, FcγRIII, and C1q ligands.
Fidelity of glycosylation has become an important issue in the production of monoclonal antibodies for therapy because glycosylation is a species- and tissue-specific post- translational modification (see Method Box 3.1).
Chinese hamster ovary (CHO) cells and mouse myeloma cells can produce appropriately glycosylated human IgG heavy chains, though the range of glycoforms is restricted. Recent studies have shown that the glyco- forms thus produced do not express optimal effector functions (such as killing of tumor cells by ADCC; see
Fc RECEPTORS ARE EXPRESSED BY GRANULOCYTES, MONONUCLEAR CELLS, AND MAST CELLS
Neonatal rat intestinal Fc receptor FcRn
Fig. 3.21 Principal interactions between neonatal rat intestinal FcRn and the Fc of maternal IgG (derived from milk) are illustrated by ribbon diagrams of FcRn (domains α1, α2, α3, andβ2m are shown in red, light green, purple, and gray,
respectively) and of Fc (CH2 and CH3 domains are shown in blue and yellow). The main contact residues of the FcRn (α1 domain, 90; α2, 113–119 and 131–135; β2m, 1–4 and 86) are
depicted as space-filling structures. (Reproduced from Ravetch JV, Margulies DH. New tricks for old molecules. Nature 1994;372:323–324) 3 2 CH2 CH3 2m 1
Chapter 10). These production cell lines, and other possible production platforms, are being engineered to produce optimal human glycoforms by transfecting in human glycosyltransferases and ‘knocking-out’ non- human glycosyltransferases.
The two classes of Fc receptor for IgE are FcRI and FcRII
Two classes of Fc receptor for IgE (FcεR) are defined in humans (Fig. 3.22):
• the high-affinity FcεRI (45 kDa), which is expressed on mast cells and basophils and is the ‘classical’ IgE receptor; and
• the low-affinity FcεRII (CD23; 45 kDa), which is expressed on leukocytes and lymphocytes.
The α chain of FcεRI is a glycoprotein and has two extra- cellular domains homologous to immunoglobulin domains; it is therefore a member of the immunoglobulin superfamily.
The low-affinity FcεRII is not a member of the immunoglobulin superfamily, but has substantial struc- tural homology with several animal C-type lectins (e.g. mannose-binding lectin [MBL]).
Cross-linking of IgE bound to FcRI results in histamine release
The high-affinity receptor (FcεRI) is present on the surface of mast cells and basophils as a complex with a β (33 kDa) and two γ (99 kDa) chains to form the αβγ2
receptor unit (see Fig. 3.22).
FcεRI binds IgE with an affinity of approximately 1010l/mol such that, although the serum concentration of
IgE is very low, the receptors are permanently saturated. Cross-linking of the IgE bound to these receptors results
in the activation and release of histamine and other vasoactive and inflammatory mediators.
FcRII is a type 2 transmembrane molecule
FcεRII is the low-affinity (CD23) receptor and is a type 2 transmembrane molecule (i.e. one in which the C termini of the polypeptides are extracellular, see Fig. 3.22). The two forms of human CD23 are:
• CD23a, which is expressed in antigen-activated B cells and influences IgE production; and
• CD23b, expression of which is induced in a wide range of cells by IL-4.
CD23a and CD23b differ by six or seven amino acids in their cytoplasmic N termini and contain different signaling motifs that modify their functions.
IgE receptors bind to IgE by different mechanisms
Decades of research and controversy surround the identification of the interaction site on IgE for the high- affinity FcεRI receptor.
Recent crystal structures of a Cε2Cε3Cε4 fragment and Cε3Cε4–FcεRI complex seem to have resolved the issue, and there is a striking structural homology between this site and the site on IgG Fc that binds FcγRIII.
One distinctive feature is that, unlike IgG binding to FcγRIII, binding of IgE to its receptor does not appear to depend on glycosylation of the immunoglobulin.
These studies should be interpreted with caution because the antibody fragments were not produced in mammalian cells.
Possible models for the interaction of IgE with the FcεRI and FcεRII receptors are illustrated in Fig. 3.22. The low-affinity IgE Fc receptor, FcεRII (CD23), is a C- type lectin and is therefore sensitive to glycosylation status.