**1. Introduction**

The immune system of vertebrates is characterized by genes of the Ig-superfamily (IGSF) that encode the immunoglobulin (Ig) genes, genes that encode the T cell receptor (TCR), a portion of the structure of the genes encoding the major histocompatibility molecules (MHC), Ig cell surface and transport receptors, some families of cytokines and chemokines as well as numerous other proteins important to the immune system. IGSF genes also encode proteins in sponges, coelenterates and flatworms (Blumbach et al., 1998; Miller & Steele, 2000; Ogawa et al., 1998). While not a topic for this chapter, we acknowledge that the IGSF genes are not the only family of genes used to generate an antibody repertoire in vertebrates. The VLR-based receptors of jawless fishes that belong to the LRR family of receptors, have had a parallel evolution (Herrin & Cooper, 2010).

Figure 1 illustrates the signature features of proteins encoded by the IGSF genes. Highly diagnostic is the so-called "-barrel" or "Ig fold". Anti-parallel -pleated sheets form the staves of the barrel that are joined at each end by flexible polypeptide chains. These flexible polypeptides on the face of a heavy chain variable region domain (VH; Fig. 1A) contain three combinatorial determining regions (CDRs). The variable light chain domain (VL; not shown) also contributes three CDRs. CDRs from both VH and VL domains coalesce to form the antibody binding site (Fig. 1B). A striking feature of IGSF genes that encode the variable region domain of Igs is the degree of polygeny such that duplicated VH genes alone can occupy > three megabases (Matsuda et al., 1990). There are three such variable region loci in mammals: VH, Vand V. The former encodes the variable heavy chain domain (Fig. 1A) while V and V encode the light chains variable region domains. All three loci are independent (non-linked) although a few orphan human VH genes can be found in other linkage groups (Matsuda et al., 1990). Popular textbooks suggest that this polygeny explains why antibodies can recognize >1010 different antigens. It is argued that if each specific antibody required a completely separate gene, more DNA would be needed than exists in the mammalian genome. To reduce the need for so many different germline encoded antibody binding sites, a system of somatic gene segment recombinations and later, somatic hypermutation (SHM) or somatic gene conversion (SCG), evolved.

The complete antibody molecule (and other proteins encoded by IGSF genes) is often composed of a tandem series of -barrel domains as illustrated in Fig. 1B. Each domain in such multi-domain molecules differs slightly in structure and correspondingly, in function.

Immunoglobulin Polygeny: An Evolutionary Perspective 115

their redundancy value. This could explain why individuals with major deletions of certain

**2.1 Translocon organization of gene segments characterizes higher vertebrates**  Figure 2A shows the organization of the light and heavy chain loci. Each locus can be divided into subloci that, from 5' to 3', are known as the V, D, J and C regions. The light chain loci are similar but lack D subloci. As discussed above, the V, D, and J regions encode the antibody binding site for the heavy and light chain, and are comprised of a large number of duplicated gene segments that vary among species (Table 1). The VH and VL gene segments are the largest (~ 300 nucleotides) and encode both framework regions (FR) and CDR1 and CDR2. The FR regions encode the -pleated sequences of the -barrel (Fig. 1A). Displayed in linear fashion FR1, CDR1, FR2, CDR2 and FR3 comprise a VH (or VL) gene (Fig. 4 and 5). The 3' portion of the JH segment (after the tryptophan codon; Fig. 8) encodes

FR4 while CDR3 results from the recombination of V-D-J or V-J (see Section 4).

C

n=70 n=7

Switch C� C C3 C1 C2 C1 ψC C2 C4 C1 C

Switch � C Exons

Switch C C1 C C1 C13

Fig. 2. The translocon organization of Ig genes of mammals. A. Organization of the variable region gene segments of the human heavy chain (VH), kappa ) and lambda () loci. Brackets indicate the number (n) of gene segments of a particular type. Switch regions are depicted with diagonal strips. B. Organization of the constant region of the heavy chain locus of human and

rabbit. The site of intralocus segment duplication in humans is indicated.

Duplicon 1 Duplicon 2

n=13

Remainder of C-region see Part B, below

C genes remain healthy (see Section 6.3).

N~100 n=30 n=9

Heavy VH DH JH

n=66 n= 5

Kappa V J

Lambda V JC

**2. Organization of the Ig Loci** 

**B**

**A**

**Human**

**Rabbit**

�

In this article we will focus on the genes encoding the VH domain of Ig (Fig. 1A) as well as those encoding the so-called "constant regions" (C) of IgG, the mammalian flagship antibody isotype. We use as examples the sequences of duplicated VH genes in swine and bats (opposite extremes) and the duplicated C genes encoding the subclasses of swine IgG, as evidence to suggest how this polygeny occurred.

Fig. 1. Duplication/diversification of Ig genes resulted in macromolecules with repeating units. A. The variable heavy chain domain (VH) with its characteristic -barrel or Ig fold. The dark polypeptides connecting the " barrel staves" contain the CDR regions. B. Complete Igs are multidomain molecules comprised of many Ig fold domains. The CDRcontaining peptides occur only in the VH and VL domains. The remaining C-domains comprise the constant region of the Ig. The "monomeric Ig" shown in Fig. 1B is bivalent, with two identical VH/VL pairs that contain the antigen binding sites.

In the interests of those who are not immunologists, we describe the different Ig-loci, how they vary among vertebrates and the processes involved in the generation of the antibody repertoire (Section 2). Section 3 discusses the gene duplication phenomenon which resulted in the polygeny that characterizes the vertebrate Ig genome, while Section 4 reviews the somatic processes that lead to the synthesis and secretion of antibodies in higher vertebrates. Section 5 discusses the selection processes involved in gene usage. Finally, we provide data from studies in fetal/neonatal piglets, newborn rabbits and the chicken, to support the view that only a small number of the many duplicated V-region Ig genes are actually used. We provide examples in which only one or a few VH genes are needed to generate the antibody repertoire so long as the machinery for somatic recombination and somatic mutation is in place. Based on these examples and comparing them to antibody repertoire development in lower vertebrates, we hypothesize that the extensive polygeny in the Ig loci of higher vertebrates exists as an evolutionary vestige but is retained because of its redundancy value. While the recent duplication/diversification of C allows for specialized effector function, IgG in rabbits did not diversify, yet few would argue against the success of this mammalian order. Thus, many of the C duplicons in other mammals may also have been retained for their redundancy value. This could explain why individuals with major deletions of certain C genes remain healthy (see Section 6.3).
