IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 6. Antibodies and Cellular Receptors Therefor

ANALYSIS OF ANTIBODY STRUCTURE AND FUNCTION

Preparation of proteolytic fragments for the analysis of antibody Structure and function

The plant protease Papain cleaves the IgG molecule in the hinge region between the Cγ1 and Cγ2 domains into two identical antigen-binding fragments, Fab (from antigen-binding fragment), and one crystallizable fragment, Fc (from crystallizable fragment). The Study of these fragments has significantly contributed to the elucidation of antibody structure and function, as it made it possible to separate the Fab region, which interacts with the antigen, from the Fc region, which is responsible for effector Functions, such as Complement fixation, binding to monocytes, or placental transfer.

Prolonged papain Digestion causes the release of an Fc' fragment from the Fc fragment, consisting of two incomplete Cγ3 domains. Some of the main enzymatic Cleavage sites of the human IgG1 molecule are shown in Fig. 6.21.

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Fig. 6.21. Pepsin cleaves the heavy chain of human IgG1 at positions 234 and 333 to yield F(ab')₂ and pFc' fragments. Further pepsin digestion leads to The breakdown of the central portion of the molecule into low-molecular-weight Peptides. Papain cleaves the IgG1 molecule in the hinge region of the heavy chains (at position 224) into two Fab fragments and one Fc fragment. Repeated exposure of the Fc fragment to papain results in the Cleavage of the peptide bond at positions 341 and 433, releasing Fc'.

Another enzyme frequently used in Structural and functional studies is pepsin. It cleaves the IgG molecule into two large fragments: F(ab')₂, which entirely encompasses both Fab parts joined at the heavy chain hinge regions, and pFc', which corresponds to the two Cγ3 domains of the molecule.

The IgG molecule is also cleaved by many Other Enzymes. For instance, brief Trypsin Treatment of acid-treated Fc fragments allows the Isolation of the Cγ2 domain for subsequent structural and functional comparison with other subfragments of the IgG molecule, particularly with pFc'.

Binding of diverse Antigens by Antibodies is mediated by hypervariable sequences within the antigen-recognition sites

The Amino Acid Sequence of certain short segments within the variable region of heavy and light chains is extremely variable. In light chains, these segments, designated as hypervariable, are located near positions 30, 50, and 95 (Fig. 6.22). They are also referred to as complementarity-determining regions (CDRs), because it is they that form the antigen-binding sites of the antibody molecule. The sequences between the hypervariable segments are termed framework regions (FR). Each light and heavy chain V region contains three CDRs (CDR1–CDR3) and four FRs (FR1–FR4). The Molecular Basis of hypervariability is discussed in Chapter 8.

Fig. 6.22. Variability is calculated by comparing the sequences of many individual light chains as The ratio of the number of different amino acid residues found at a given position to the frequency of the most common residue. The regions of highest variability, three of which belong to the V_L domain, are designated as hypervariable. Additional residues were found in some sequences (at positions indicated by arrows), but they were omitted here (to improve comparability). Dark yellow highlights the hypervariable regions (CDR), red marks the most hypervariable positions, and yellow indicates the four framework regions (FR). (Illustration kindly provided by Prof. E.A. Kabat.)

The variable regions of light and heavy chains are spatially folded such that the hypervariable regions lie close to one another, forming the antigen-binding structure On the surface of the molecule. Such segments are most frequently located at the turns of the peptide chain (see Fig. 6.6).

Numerous regions responsible for effector functions have been identified in the antibody structure

While the localization of antigen-binding sites was established very rapidly, the sequences responsible for most effector functions resisted precise localization for a long time. Some preliminary data were obtained from Experiments on the inhibition of antibody functional activity by their subfragments generated via enzymatic cleavage; however, progress remained slow until the advent of Site-Directed Mutagenesis, which enables the selective substitution of various amino acid residues in a known peptide sequence and thus the Determination of the Functional Significance of specific residues.

This method was first applied to study The Mechanism of complement activation by IgG antibodies. By that time, it was already known that C1q (a subcomponent of C1) interacts with the Cγ2 domain of IgG. Using site-directed mutagenesis, researchers successfully identified the C1q-binding site, which involves the side chains of three amino acid residues in the Cγ2 domain: Glu-318, Lys-320, and Lys-322. This sequence appears to be typical for the interaction sites between IgG molecules and C1q.

In the case of IgM, the mechanism of complement activation is likely different. Free circulating IgM in a star-like conformation is apparently unable to activate complement, but acquires this capability upon antigen binding. According to Feinstein et al. (1986), upon binding to a polymeric or cross-linked antigen, the F(ab')₂ units of IgM deviate from the plane of their initial position, causing the pentamer to adopt a "crab-like" configuration readily discernible by Electron Cell/15.html">Microscopy (see Fig. 6.11). These conformational changes presumably expose a ring of C1q-binding sites that are hidden in the star-like configuration of the pentameric IgM molecule due to the close apposition of adjacent monomers. The C1q-binding site (presumably residues His 430, Asp/Gly 432, and Pro 436) is located within the Cμ3 domain, and its structural localization is analogous to that of the putative site of the same Specificity in the Cγ2 domain.

IgG molecules interact with a variety of cellular Fc receptors. Studies utilizing site-directed mutagenesis have shown that the high-affinity FcγRI receptor on monocytes binds to a structural motif of the γ chain centered around a leucine residue at position 235, located between the Cγ2 domain and the hinge region.

More recently, the mechanism of interaction between maternal milk IgG and FcRn (an MHC class I-like receptor) expressed on the neonatal rat intestinal epithelium was elucidated (see Fig. 6.23); it is thought to resemble the mechanism by which human maternal IgG binds to hFcRn, the placental analogue of FcRn. The Fc site that binds FcRn is located at the junction of the C_H2 and C_H3 domains, overlapping with the interaction site for staphylococcal protein A. Three or four Histidine residues likely play the primary functional role in this region: they apparently govern IgG binding to FcRn at pH 6.5 (the pH of milk entering the intestinal lumen) and its release at pH 7.5 (Blood pH).

Fig. 6.23. Key interactions between the FcRn of the neonatal rat intestinal epithelium (α1, α2, and β₂m domains colored in red, green, purple, and gray, respectively) and the Fc portion of maternal milk IgG (C_H2 and C_H3 domains colored in blue and yellow). The major contact residues of FcRn—residue 90 in the α domain, 113–119 and 131–135 in the α2 domain, and 1–4 and 86 in the β₂m domain—are depicted as globular structures. (Reproduced with permission from Ravetch, Margulies, 1994)

Introduction/32.html">Genetic Engineering techniques have been employed to identify the sites of the IgE molecule that bind to FcεRI on mast Cells or FcεRII on B cells. The synthesis of recombinant peptides corresponding to various segments of the ε-chain sequence, along with an assessment of their inhibitory effect on IgE-cell receptor interactions, demonstrated that IgE binding to FcεRI is likely mediated by a 76-residue peptide between Cε2 and Cε3 (Fig. 6.24), whereas FcεRII presumably recognizes a structure formed by The amino acid residues of the Cε3 domains of both ε chains (Fig. 6.25).

Fig. 6.24. Proposed localization of the mast cell FcεRI receptor-binding site in human IgE. The Cε2, Cε3, and Cε4 domains are arranged in this order from top to bottom. The binding peptide (shown in white) apparently begins with a glutamine residue at position 301 and ends with an Arginine residue at position 376. (Reprinted by permission from Helm et al., 1988.)

Fig. 6.25. Proposed localization of the B-cell FcεRII receptor-binding site in human IgE. The Cε2, Cε3, and Cε4 domains are arranged in this order from top to bottom. Residues from Lysine-367 to valine-370 inclusive (in the Cε3 domain) of both ε-chains are presumed to be primarily responsible for binding (indicated by an arrow on one side and highlighted in white on the other). (Reprinted by permission from Vercelli et al., 1989.)

Some data have also been obtained on the topography of interactions between the Fc region of an IgG molecule and Protein A of Staphylococcus aureus. The binding site is presumably located in the junction region between the Cγ2 and Cγ3 domains of IgG.

Questions for Discussion

■ Antibody molecules vary structurally in their so-called constant regions. How is this diversity manifested, and what advantage does it confer upon the Organism?

■ It is hypothesized that IMMUNOGLOBULINS phylogenetically evolved from a single ancestral protein-like precursor corresponding to a single domain. How did Gene duplications, by generating a multidomain protein, contribute to The formation of a functionally competent molecule?

■ A large family of diverse Fcγ receptors has been identified. Based on their General structural layout, how could immunoglobulin-receptor interactions be enhanced?

■ How do molecular biology techniques help map the structural sites of immunoglobulins responsible for effector functions? How much more effective are they compared to the Methods used in the past?

Antibodies and Their Cellular Receptors