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ORGANIZATION AND EXPRESSION OF IMMUNOGLOBULIN GENESBy,Devika.RM Sc Student, Dept of Biotechnology Gulbarga University, Karnataka

Structure of Immunoglobulin

ORGANIZATION AND EXPRESSION OF IMMUNOGLOBULIN GENES

GENETIC MODEL COMPATIBLE WITH IMMUNOGLOBULIN STRUCTUREProperties of Antibodies

Vast diversity of antibody specificities. Presence in immunoglobulin heavy and light chains of a variable region at the amino terminal end and a constant region at the carboxy terminal end. The existence of isotypes with the same antigen specificities, which result from the association of a given variable region with different heavy chain constant region.

GERM LINE AND SOMATIC VARIATION MODELSTwo different sets of theories emerged to explain tremendous diversity of antibody structure.

How could stability be maintained in the constant (C) region while some kind of diversifying mechanism generated the variable (V) region?TWO GENE MODEL OF DREYER AND BENNETTIn 1965, W. Dreyer and J. Bennett suggested that two separate genes encode a single immunoglobulin heavy or light chain, one gene for the V region (variable region) and the other for the C region (constant region).These two genes must come together at the DNA level to form a continuous message that can be transcribed and translated into a single Ig heavy or light chain.

TONEGAWAS EXPERIMENT In 1976, S. Tonegawa and N. Hozumi found that separate genes encode the V and C regions of Immunoglobulins and that the genes are rearranged in the course of B-cell differentiation. Tonegawa was awarded Nobel prize for this work in 1987.

MULTIGENE ORGANIZATION OF IMMUNOGLOBULIN GENES Germ-line DNA contains several coding sequences, called gene segments, separated by non-coding regions.Gene segments are rearranged during B cell maturation to form functional Ig genes. Each multigene family has distinct features.The and light chain families contain V, J and C segments; the rearranged VJ segments encode the variable region of the light chains and the C gene segments encode the constant region.

The heavy-chain family contains V, D, J, and C gene segments; the rearranged VDJ genesegments encode the variable region of theheavy chain.Each V gene segment is preceded at its 5 endby a signal or leader (L) peptide that guides theheavy or light chain through the endoplasmic reticulum.Signal peptide is cleaved from the nascent light and heavy chains before assembly of the final Ig molecule.

CHAIN MULTIGENE FAMILYFunctional variable-region gene contains two coding segments a 5 V segment and a 3 J segment which are separated by a non-coding DNA sequence in unrearranged germ-line DNA. J for joining ( 39 bp gene segment). In mouse, multigene family contains 3 V , 4 J and 4 C gene segments. J 4 is a pseudogene or a defective gene that is incapable of coding proteins, denoted by . chain have 3 subtypes ( 1, 2 and 3). In humans, there are 31 functional V , 4 J and 7 C gene segments. It also contains many pseudogenes.

CHAIN MULTIGENE FAMILY In mouse, chain multigene family contains approximately 85 V , 5 J (one pseudogene) and 1 C gene segment. V and C encode variable region of the light chain and the J gene segment encode the constant region. There are no subtypes of light chains. In humans, approximately 40 V , 5 J and 1 C gene segments are present.

HEAVY CHAIN MULTIGENE FAMILY Leroy Hood proposed the existence of an additional gene segment in the heavy chain variable region. It was designated D for diversity because of its contribution to the generation of antibody diversity. The heavy chain multigene family on human chromosome 14 contain 51 VH, 27 DH , 6 JH and a series of CH gene segments. The CH gene segments consist of coding exons and non-coding introns. In humans and mice, the CH gene segments are arranged sequentially in the order C , C ,C ,C , C.

VARIABLE REGION GENE REARRANGEMENTS Variable region gene rearrangements occur in an ordered sequence during B-cell maturation in the bone marrow. The heavy chain variable -region rearrange first, then light chain. Process of variable region gene rearrangement produces mature, immunocompetent B cells which produce antibody with a binding site encoded by the particular sequence of its rearranged V genes.Rearrangements of the heavy chain constant region genes will generate further changes in the isotype expressed by a cell.

V-J REARRANGEMENTS IN LIGHT CHAIN DNAExpression of both and light chains requires rearrangement of the variable-region V and J gene segments.In humans, any of the functional V genes can combine with any of the four functional C- J combinations. In mouse, things are slightly more complicated. In human or mouse light chain DNA, any one of the V gene segments can be joined with any one of the functional J gene segments.Rearranged genes have a short leader (L) exon, a non-coding sequence (intron), a joined VJ gene segment, a second intron, and the constant region in order from 5 to 3 end.

V-D-J REARRANGEMENTS IN HEAVY CHAIN DNAThis process requires two separate rearrangement events within the variable region. DH gene segment first joins to a JH segment; the resulting DHJH segment joins a VH segment to generate a VH DHJH unit that encodes the entire variable region.Rearrangement produces a short L exon, an intron, a joined VDJ segment, another intron and a series of C gene segments.

MECHANISM OF VARIABLE REGION DNA REARRANGEMENTSRecombination signal sequences direct recombination

ENZYMATIC JOINING OF GENE SEGMENTS V-D-J recombination takes place at the junctions between RSSs and coding sequences is catalyzed by enzymes collectively called V(D)J recombinase. In 1990, David Schatz, Marjorie Oettinger and David Baltimore first reported the identification of two recombination-activating genes, RAG-1 and RAG-2. The RAG-1 and RAG-2 proteins and the enzyme terminal deoxynucleotidal transferase (TdT) are the only lymphoid specific gene products that had been shown to be involved in V-(D)-J rearrangement.

PRODUCTIVE AND NON-PRODUCTIVE REARRANGEMENTS Imprecise joining of rearranged segments may result in out of phase joining and the triplet reading frame for translation is not preserved. In such a non-productive rearrangement, the resulting VJ or VDJ unit will contain numerous stop codons which interrupt translation. When gene segments are joined in phase, the reading frame is maintained. In such a productive rearrangement , the resulting VJ or VDJ unit can be translated in its entirety, yielding a complete antibody.

ALLELIC EXCLUSION A B cell is diploid, it expresses the rearranged heavy chain genes from only one chromosome. The process by which this is accomplished is called allelic exclusion. It ensures that functional B cells never contain more than one VHDH JH and one VLJL unit. It is essential for the antigenic specificity of the B cell.

GENERATION OF ANTIBODY DIVERSITYSeven means of antibody diversification have been identified in mice and humans. Multiple germ-line gene segments Combinatorial V-(D)-J joining Junctional flexibility P-region nucleotide additionN-region nucleotide addition Somatic hypermutation Combinatorial association of light and heavy chains

1.MULTIPLE GERM LINE 2.COMBINATORIAL V-D-JGENE SEGMENTS JOINING In human germ line DNA, 51 VH, 27 D, 6 JH, 40 V , 5 J , 31 V and 4 J segments are present. In mouse, about 85 V , 134 VH ,4 functional JH , 4 functional J ,3 functional J and an estimated 13 DH but only 3 V gene segments are present.They contribute to the antigen-binding sites in antibodies. In humans, the ability of any of the 51 VH gene segments to combine with any of the 27 DH segments and any of the 6 JH segments allows a considerable amount of heavy-chain gene diversity to be generated (51276=8262 possible combinations). 40 V5 J=200 and 30V 4 J=120 possible combinations. Total antibody combining diversity in human to well over 1010 .

3.JUNCTIONAL FLEXIBILITY Increases the enormous diversity generated by means of V, D, and J combinations.Can lead to many nonproductive rearrangements, but it also generates several productive combinations that encode alternative amino acids at each coding joint, thereby increasing antibody diversity.

4.P-ADDITION 5.N-ADDITION

6.SOMATIC HYPERMUTATIONResults in additional antibody diversity that is generated in rearranged variable-region gene units.Also causes individual nucleotides in VJ or VDJ units to be replaced with alternatives, thus possibly altering the specificity of the encoded immunoglobulins. Usually occurs within germinal centersTargeted to rearranged V-regions located within a DNA sequence containing about 1500 nucleotides, which includes the whole of the VJ or VDJ segment.Largely randomAffinity maturation: following exposure to antigen, those B cells with higher affinity receptors will be selected for survival because of their greater ability to bind to the antigen this takes place in germinal centers.

7. ASSOCIATION OF HEAVY AND LIGHT CHAINSCombinational association of H and L chains also can generate antibody diversity. In humans, potential no: of heavy and light chain combinations is 2,644,240 (8262 heavy chain 320 light chain). This is higher than the actually generated amount because not all VH , D or VL gene segments are used in the same frequency.

CLASS SWITCHING AMONG CONSTANT REGION GENES

EXPRESSION OF IMMUNOGLOBULIN GENES

DIFFERENTIAL RNA PROCESSING OF HEAVY CHAIN PRIMARY TRANSCRIPTS Immunoglobulin can exist in either membrane- bound or secreted form. Processing can yield different mRNAsThis processing explains the production of secreted or membrane bound forms of a particular immunoglobulin and the simultaneous expression of IgM and IgD by a single B cell.

1.EXPRESSION OF MEMBRANE OR SECRETED IMMUNOGLOBULINDifference depends on differential processing of a common primary transcript.Mature naive B cells produce only membrane-bound antibody and differentiated plasma cells produce secreted antibodies. The secreted form has a hydrophilic sequence of about 20 amino acids in the C terminal domain; This is replaced by a sequence of about 40 amino acids containing a hydrophilic segment that extends outside the cell, a hydrophobic transmembrane segment and a short hydrophilic segment at C terminal that extends into cytoplasm.

2.SIMULTANEOUS EXPRESSION OF IgM AND IgD Controlled by differential RNA processing .If the heavy chain transcript is cleaved and polyadenylated at site 2 after the C exons, then the mRNA will encode the membrane form of the u heavy chain.If polyadenylation is instead further downstream at site 4, after the C exons, then RNA splicing will remove the intervening C exons and produce mRNA encoding the membrane form of the heavy chain.

SYNTHESIS, ASSEMBLY, AND SECRETION OFIMMUNOGLOBULIN

REGULATION OF Ig-GENE TRANSCRIPTIONThree regulatory sequences in DNA regulate transcription of immunoglobulin genes:1. Promoters: promote initiation of RNA transcription in a specific direction. Located about 200 bp upstream from transcription initiation site. 2. Enhancers: nucleotide sequences located some distance upstream or downstream from a gene that activate transcription from the promoter sequence in an orientation-independent manner.3. Silencers: nucleotide sequences that down-regulate transcription, operating in both directions over a distance.Each VH and VL gene segment has a promoter located just upstream from the leader sequence.These promoters contain a TATA Box, which serves as the site for the binding of no: of proteins that initiates transcription.

EFFECT OF DNA REARRANGEMENT INHIBITION OF Ig GENE ON TRANSCRIPTION EXPRESSION IN T-CELLS Rate of transcription of a rearranged VLJL or VH DH JH unit is as much as 10,000X the rate of transcription of unrearranged VL or VH segments. Oncogenes: genes that promote cellular proliferation or prohibit cell death. These can often translocate to the immunoglobulin heavy or light chain loci.Expression of these genes is elevated significantly under the influence of an Ig enhancer. T-cell receptor (TCR) undergoes V-(D)-J rearrangement to generate functional TCR genes. Occurs by the similar recombination mechanism of Ig . Complete Ig gene rearrangement of H and L chains occurs only in B cells and complete TCR-gene rearrangement is limited to T cells.

ANTIBODY GENES AND ANTIBODYENGINEERING Possible to design and construct genes that encode immunoglobulin molecules in which the variable regions come from one species and the constant regions come from another.A. CHIMERIC AND HYBRID MONOCLONAL ANTIBODIESTo make an antibody one needs to clone recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse antibody gene and the constant-region exons from a human antibody gene.The antibody encoded by such a recombinant gene is a mouse-human chimera, also known as humanized antibody.

Its antigenic specificity is determined by the variable region and is derived from the mouse DNA.Its isotype is determined by the constant region and is derived from the human DNA.Heteroconjugates (or bispecific antibodies): hybrids of two different antibody molecules.Constructed by chemically crosslinking two different antibodies or synthesized in hybridomas consisting of two different monoclonal-antibody-producing cell lines that have been fused.

B. MONOCLONAL ANTIBODIES CONSTRUCTEDFROM IG-GENE LIBRARIESUse of PCR to amplify the DNA that encodes antibody heavy chain and light chain Fab fragments from hybridoma cells or plasma cells. A promoter region and EcoRI restriction site are added to amplified sequence and the resulting constructs are inserted into bacteriophage , yielding separate H and L chain libraries. Random joining of H and L chains yield numerous novel H-L constructs.This procedure generates a diversity of antibody specificities; clones containing these random combinations of H + L chains can be rapidly screened for those secreting antibody to a particular antigen.

MICE HAVE BEEN ENGINEERED WITH HUMAN IMMUNOGLOBULIN LOCIOne can knockout the heavy and light chain immunoglobulin loci in mouse embryonic stem cells and introduce very large DNA sequences containing human heavy and light chain gene segments.Advantage of this process: completely human antibodies are made in cells of the mouse B-cell lineage, from which antibody-secreting hybridomas are readily derived by cell fusion.Therefore, this process offers a solution to the problem of producing human monoclonal antibody of any specificity desired.

SUMMARY

REFERENCE

IMMUNOLOGY-Janis Kuby, 5th edition ESSENTIALS IN IMMUNOLOGY-Lecture by Prof. Anjali.M.Karande, IIS, Bangalore

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