membrane-embedded receptor.5 Between species, the membrane/cytoplasmic tail region is the most highly conserved portion of the CH domains, which befits its role as a link to the intracellular signal transduction pathways that ultimately regulate B-cell function.
and individual antigenic determinants. Epitopes recognized within the V portion of the antibodies used for immunization that identify individual determinants are termed idiotypes (Fig. 5.3), whereas epitopes specific for the constant portion are termed isotypes. Recognition of these isotypes first allowed grouping of Igs into recognized classes. Each class of Ig defines an individual set of C domains that corresponds to a single H chain constant region gene. For example, IgM utilizes µ H chain C domains and IgE utilizes ε C domains.
FIG. 5.2. Ribbon Diagram of a Complete Immunoglobulin (Ig)G1 Crystal (1 hzh in the protein data bank [PDB] from Data of Harris et al.202). The major regions of the Ig are illustrated. The heavy-chain constant regions (green) also include the hinge (yellow) between the first two domains. Cγ2 is glycosylated (also seen in yellow). The heavy- and light-chain variable regions (red and dark blue, respectively) are N terminal to the heavy- (green) and light-chain (light blue) constant regions. Complementarity determining region loops in the heavy- and light-chain variable regions (yellow and white) are illustrated as well.
FIG. 5.3. Electron micrographs (top; ×350,000) and interpretive diagrams (below) of murine mAb hybridoma group A carbohydrate (HGAC) 39 (specific for the cell wall polysaccharide of Streptococcus pyogenes) in complex with anti-idiotypic mAb Fab fragments. HGAC 39 is represented in the diagrams as an open figure, and the Fab anti-Id probes are represented as solid figures. The Fab arms of the antibody targets and probes are drawn to indicate their rotational orientation as planar (oval with open center), intermediate (bone shape with or without central opening), or perpendicular (“dumbbell shaped”). Different complexes illustrate the range of Fab-Fab angles made possible by segmental flexibility. 1, anti-IdI-1 Fab; 2, anti-IdI-2 Fab; 39, HGAC 39; 3a, anti-IdI-3a Fab; K, anti-Cκ Fab; X, anti-IdX Fab. IdI designates an individual idiotope, and IdX, a crossreactive idiotope. Antibody complexes were stained with 2% uranyl formate as described by Roux et al.108 Reproduced from Proceedings of the National Academy of Sciences (from Roux et al.108 with permission).
Papain digested IgG into two Fab fragments, each of which could bind antigen, and a single Fc fragment. Nisonoff developed the use of pepsin to split IgG into an Fc fragment and a single dimeric F(ab)2 that could cross-link antigens.23 Edelman broke disulfide bonds in IgG and was the first to show that IgG consisted of two H and two L chains.24
TABLE 5.1 Definitions of Key Immunoglobulin Structure Nomenclature
FIG. 5.4. The Immunoglobulin Domain. A: A typical variable domain structure. Note the projection of the C-C′ strands and loop away from the core. B: A typical compact constant domain structure. C: The cysteines used to pin the two b-sheets together are found in the B and F strands. D: The folding pattern for variable and constant domains.2
FIG. 5.5. Sequence Conservation and Hypervariability within a Heavy Chain Variable (V) Domain. The primary sequence the V domain can be divided into four regions of sequence conservation, termed framework regions (FRs), and three regions of hypervariability, termed complementarity determining regions (CDRs). A schematic of the genomic origin of the variable domain is shown at the top of the figure. The classic separation of the sequence into FR and CDR by Kabat et al.31 is shown below the gene structure. The letter designation for individual β strands is given beneath the Chothia and Lesk nomenclature,32 which focused more on structure. The positions of each of the four invariant residues of the VH chain (FR1 Cys22, FR2 Trp36, FR3 Cys92, and FR4 Trp103) are shown as darkened circles on the Chothia and Lesk model. The ImMunoGeneTics information system® designation has attempted to rationalize sequence variability with structure and is the current nomenclature of choice.33
FIG. 5.6. The Structure of an Fab. The antigen-binding site is formed by the heavy (H) and light (L) chain B-C, C′-C″, and F-G loops. Each loop encodes a separate complementarity determining region (CDR). The location of CDRs H1, H2, H3, L1, L2, and L3 are shown. The opposing H and L chain C-C′ strands and loop help stabilize the interaction between VH and VL. This C-C′ structure is encoded by the second V framework region, FR2. The inclusion of this structure permits the variable (V) domains to interact in a head-to-head fashion. The E-F strands and loop are encoded by the FR3 region and lie directly below the antigen-binding site. The A-B strands and loop encode FR1 and lie between the CH1 and CL domains and the rest of the Vs. The beta sheet strands of the CH1 and CL domains rest crosswise to each other. The illustration is modified.203
H chain can combine with any one L chain, thus 211 V, D, and J gene segments can provide approximately 1 × 107 different H:L combinations.
FIG. 5.7. A comparison of two human Clan I VH sequences that belong to different VH families (modified4). Shown is a comparison of the deoxyribonucleic acid and amino acid sequences of the V5-51 and V1-2 gene segments. Each line depicts a divergence in a nucleotide or amino acid at that position. Shown at the bottom is a replacement/silent site substitution analysis by interval. Random mutation tends to exhibit an R/S ratio of 2.9. The smaller the ratio, the greater the preservation of sequence. The intervals identified by the arrows predict the family and clan of origin.
intervals: CDRs-H1, -H2, and -H3; and CDRs-L1, -L2, and -L3.31 The CDR sequences of V gene segments tend to be enriched for codons where mutations maximize replacement substitutions.56 This includes the RGYW motif that facilitates somatic hypermutation.57,58 While evolution appears to favor CDR1 and CDR2 sequences that facilitate codon diversity, it also appears to preserve specific loop structures.
FIG. 5.8. Evolutionary Relationships among Vertebrate VH Families. The sizes or relative placements of the evolutionary connecting lines are not to scale. VH sequences from all mammalian species analyzed to date can be placed into one of these three clans (modified4).
CDR-H3 loop is enriched for tyrosine and glycine,31,69 and relatively depleted of highly polar (charged) or nonpolar (hydrophobic) amino acids, although the precise pattern depends on the species of origin.70 This pattern of amino acid utilization is established early in B-cell development, prior to the expression of Ig on the surface of the cell (Fig. 5.9)49,71,72,73 and reflects evolutionary conservation of JH and DH gene segment sequences. In particular, although the absolute sequence of the DH is not the same, the pattern of amino acid usage by RF is highly conserved. Of the six potential RFs, RF1 by deletion is enriched for tyrosine and glycine. RF2 and RF3 by deletion are enriched for hydrophobic amino acids, as they are by inversion. RF1 by inversion tends to encode highly polar, often positively charged, amino acids.69 Various species use different mechanisms to bias for use of RF1 by deletion, to limit use of hydrophobic RFs, and to restrict or prevent use of RFs enriched for charged amino acids. Forced rearrangement into RFs with charged amino acids yields an altered repertoire enriched for charge and depleted of tyrosine and glycine.71
FIG. 5.9. Both in the absence or presence of N-addition, the preference for tyrosine and glycine in complementarity determining region-H3 begins early and intensifies with B-cell development. VH 7183DJCµ transcripts were cloned and isolated from fractions B (pro-B cells) through F (mature B cells) from the bone marrow of 8- to 10-week-old terminal deoxynucleotidyl transferase-sufficient and terminal deoxynucleotidyl transferase-knockout BALB/c mice.73 The amino acids are arranged by relative hydrophobicity, as assessed by a normalized Kyte-Doolittle scale.204,205 Use is reported as the percent of the sequenced population. The number of unique sequences per fraction is shown.
preferred range of canonical structures. CDR-H3, the focus of junctional diversity, lies at the center of the antigen-binding site. The conformation of its base tends to fit within three basic structures. The loop varies greatly in sequence, yet still maintains a bias for the use of tyrosine and glycine. Thus, diversity increases with proximity to the tip of the antigenbinding site but appears to be held within regulated limits.
FIG. 5.10. Location and Generation of Complementarity Determining Region-H3. A: A cartoon of the classic antigen-binding site (modified4). Due to its central location, most antigens bound to the antibody will interact with complementarity determining region-H3.