IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 8. Sources of Diversity in Antigen-Recognizing Structures

HEAVY CHAIN CONSTANT REGION GENES

The same set of variable region genes is used to encode IMMUNOGLOBULINS of all classes. Isotype "switching" in a mature antibody-forming Cell simply involves changing the functional constant region Gene. This was discovered through the Analysis of Double myelomas, whose carriers' sera simultaneously contained Monoclonal Antibodies of two isotypes. For example, a patient with multiple myeloma was found to have IgM and IgG with identical light chains and V_H regions—the IgG differed from the IgM only by the replacement of the μ chains with γ chains. Similarly, IgM and SpD are often simultaneously present On the surface of a single lymphocyte, and despite their different isotypes, these membrane-bound immunoglobulins share identical antigen-binding sites.

All constant region genes are located on the chromosome downstream of the J segments. Mice have a single constant region gene for each immunoglobulin Class—IgM, IgE, IgD, and IgA (Cμ, Cε, Cδ, and Cα)—and for each of the IgG subclasses (Cγ1, Cγ2a, Cγ2b, and Cγ3) (Fig. 8.18). Except for Cδ, each C gene is preceded by a switch (S) sequence (Fig. 8.19).

Fig. 8.18. In mice, the constant region genes are located 8.5 kb downstream of the rearranged V-D-J segments. Preceding each C gene, except Cδ, are one or more switch regions (red circles) that repeat The nucleotide sequence preceding the 5' end of the Cμ gene. These switch regions allow any of the C genes to recombine with the V-D-J genes. Rearrangements of Cδ genes appear to occur via the same switch regions as in the case of Cμ genes, but during RNA Processing for IgD, the Cμ gene transcript is excised from the primary RNA transcript. In the bottom row, expanded diagrams of Cμ and Cγ2a Introduction/29.html">Gene Organization show the introns located between the exons for each domain (C1, C2, etc.). Cγ genes also possess a separate exon encoding the hinge region of the γ chain (H), and all these genes contain one or more additional exons required to encode the membrane-bound form of the immunoglobulin (M).

Fig. 8.19. 1. Primary immunization induces The formation of antibodies predominantly of the IgM isotype; secondary antigen administration stimulates The production of antibodies primarily of the IgG isotype. The Mechanism of isotype switching is schematically shown on the right. 2. In the primary response, the recombined V-D-J segment is transcribed together with the Cμ gene. Following intron removal during primary RNA Transcript Processing, mRNA for the secreted form of IgM is produced. During the "maturation" of the Immune Response, isotype-switching recombination occurs between the Sμ region and a downstream C gene (in this case, Cγ3). The DNA segment separating them forms a loop and is subsequently excised. As a result, the Cμ and Cδ genes are lost, and the two switch regions (Sμ and Cγ3) are joined.

The arrangement of C genes in the human IgH locus and their switching order are shown in Fig. 11.23.

Isotype switching can occur either through gene recombination or as a result of differential mRNA splicing

Isotype switching, which is crucial for the "maturation" of the immune response, may be preceded or accompanied by somatic Mutations in immunoglobulin genes. Initially, the entire segment of DNA including the recombined V_H gene as well as the Cμ and Cδ genes is fully transcribed; then, differential splicing of the primary transcript can yield two Different types of mRNA molecules—containing sequences complementary to the same V_H gene but different C genes (Cμ or Cδ). It has been suggested that much longer DNA segments are sometimes transcribed in this way, followed by differential splicing to form mRNAs for other Ig isotypes with identical V_H regions (Fig. 8.20). A similar process is observed in Cells simultaneously synthesizing IgM and IgE.

Fig. 8.20. Some B cells produce antibodies of several isotypes using a single long primary RNA transcript. Shown here is a transcript containing Cμ and Cδ sequences. The type of differential splicing that generates mRNA for the synthesis of either IgD (top) or IgM (bottom) depends on the placement of polyadenylation sites (which can vary). Additional polyadenylation sites within the same region of the RNA transcript determine whether the immunoglobulin will be translated in a membrane-bound or secreted form.

The most common mechanism of isotype switching is recombination between S regions (via DNA loop formation and subsequent excision), as a result of which another C gene takes a position next to the rearranged V-D-J gene (Figs. 8.19 and 11.23).

The formation of membrane-bound and secreted forms of immunoglobulins is determined by differential splicing of heavy chain RNA transcripts

The membrane-bound form of an immunoglobulin (the antigen-recognizing receptor) is identical to its secreted form (the antibody), except that it contains an additional C-terminal sequence within the heavy chain. Therefore, membrane-bound immunoglobulins are somewhat larger than their soluble counterparts. The additional Amino Acid Sequence spans The Cell membrane and "anchors" the molecule in The Lipid Bilayer. For example, in membrane-bound IgM, hydrophobic (lipophilic) residues are located between hydrophilic amino acid residues situated on both sides of the membrane (Fig. 8.21), presumably forming an α-helical chain segment within the membrane. Membrane-bound immunoglobulins exist exclusively as a basic four-chain unit; their molecules do not polymerize.

Fig. 8.21. Up to position 556, the C-terminal Amino acid sequences of the μ chains of secreted and membrane-bound IgM are identical. After residue 556, the μ chain of secreted IgM has 20 residues, whereas that of membrane-bound IgM has 41. In the secreted form, a carbohydrate unit is attached to the asparagine residue at position 563, and the Cysteine residue at position 575 participates in forming an interchain disulfide bond. Membrane-bound IgM contains a stretch of 26 hydrophobic amino acid residues (from 568 to 595) located between charged (hydrophilic) residues, which is capable of spanning the cell membrane with two turns of an α-Helix. A short positively charged tail segment of the heavy chain is immersed in the Cytoplasm.

The Synthesis of the two immunoglobulin forms is made possible by differential METABOLISM/31.html">Transcription of the germline C gene (Fig. 8.22). It is believed that the type of RNA transcript depends on the polyadenylation (poly-A) sequence, although the exact mechanism of this regulation remains unknown.

Fig. 8.22. Schematic representation of a portion of the gene elements encoding IgM. Exons for the Cμ3 and Cμ4 domains (H3 and H4), as well as for the transmembrane and cytoplasmic segments (M) of the membrane-bound IgM μ chain, are shown. An untranslated sequence (shown in gray) is present at the 3' end of exon H4 and the additional transmembrane segment M. S stands for stop codons. DNA Transcription can proceed via two pathways. If it terminates at S, the polyadenylated (poly-A) transcript is processed into mRNA for the secreted form of IgM. If transcription continues through the exons of the transmembrane segments, subsequent Processing of the transcript will remove the C-terminal amino acid codons and the H4 stop codon. As a result, the translated protein will have a different C-terminal sequence.