Antibody Diversity is Genetically Determined

Immunoglobulins are molecules providing immunity and protection from infections and microbial attacks. They have a typical Y shape. That is the reason they catch hold of microbes such as bacteria and viruses. 

The unique molecule of the microbes is known as an antigen. The antibody's Y structure has a region known as a paratope. It binds with the epitope of the antigen. Then it makes the process of elimination easier. Antibodies have a variety of functions. It is important to judge the greatness of an antibody based on its biological effect on a pathogen or the toxins produced by a pathogen. It is an important hallmark for many lab tests that an antibody binds to a particular antigen. Studying and knowing about antibodies is key to developing potential vaccines and therapeutics. Functions of the antibodies, such as virus neutralization, may help to inhibit pathogenesis.

The inhibitory results of antibodies on pathogenic organisms were recorded since the 1800s. Since then, many things have been known concerning the means that underlie the anti-microbial action of antibodies. However, these Y-shaped molecules often have numerous functions in vitro and in vivo. They either involve direct means or via interactions with FcRs or compliments. 

Modern means, such as knockout mice or antibodies engineered to repeal or improve functions have demonstrated promising results for more accurate investigations of antibody function. Yet, major questions about how an antibody plays roles in vivo remain unanswered, and multiple actions are possible to contribute to the anti-microbial influence.

The complexity of our immune system mainly depends on the immune cells such as the B cells, T cells, various antibodies, and other cells. As soon as a foreign molecule or a microbe enters the body, our immune system gets activated. The soldiers of the immune system, mainly the immune cells and the antibodies come forward and activate a cascade of mechanisms involved in killing the foreign particles and the microbes. They not only eliminate the microbe from the body but also remember it as an enemy so that when the same microbe re-enters next time, they easily eliminate it. The antibodies usually denoted as Y-shaped molecules, bind to the antigens and help in eliminating them. The antibodies exhibit diverse nature and their synthesis completely depends on certain gene expressions. These antibodies show receptors specific to the antigens. Although there are few genes in the human body, the antibodies have very diverse receptors. Hence, they identify many different types of antigens.

While distinguishing or recognizing foreign substances, our immune cells do not get confused with the cells inside the body. Hence, they differentiate the foreign cells and their own cells cleverly. The phenomenon of identifying a foreign antigen is known as immune recognition. Antibody structures exhibit complexity. Thus, they recognize a wide array of foreign antigens. Sequencing data reveals a specific amino acid sequence in the variable region and a few invariant sequences in the constant regions. Determination of an antibody involves specificity and sensitivity. The property of the antibody of being specific helps to determine the homologous and heterologous epitopes. The sensitive property of an antibody helps to recognize the antigen in between thousands of other substances. It is important to study the antibody structure before studying the diversity.

Image 1: Antibody diversity

Antibody (Immunoglobulin structure):

Antibodies are Y-shaped glycoproteins with four main polypeptide chains. The two long polypeptide chains with many amino acids are known as heavy chains. The remaining two chains consisting of short polypeptides are known as light chains. Each light chain gets connected to a heavy chain through a disulfide bond. The heavy and the light chains mainly consist of two distinct regions. The tips of heavy and light chains consist of the variable region (V). The remaining is known as a constant region (C). The variable region attaches to the antigen. A normal human being consists of one million antibodies with different antigen-binding specificities. Hence, the variable region consists of different amino acid sequences. Five types of heavy chains include γ, α, ε, δ, and μ. Two types of light chains are known as κ and λ. Types of antibodies in humans include IgA, IgD, IgE, IgG, and IgM respectively. 

Antibody diversity and Genetics:

A separate set of multigene families encode for heavy and light chains situated on different chromosomes. The light chain genes are present on the 2^nd and 22^nd chromosomes. The heavy chain genes are present on the 14^th chromosome. Several coding sequences known as gene segments get separated by the non-coding segments. On maturation of the B-cells, these genes get rearranged, thereby forming a functional immunoglobulin. Restriction mapping is a kind of physical mapping that shows specific sites for the restriction enzymes. They involve separation by lengths and are marked in numbers by bases. Hence, the study of DNA segments encoding the antibodies involves a restriction map.

The DNA segments encoding a variable region get separated from the DNA segment encoding a constant region by an intermediate region encoding a joining segment. The heavy chain studies revealed the presence of one more region known as the D region (for diversity) placed between the V and J regions. A non-coding region separates each of the coding regions. A single type of gene gets expressed for each V, D, J, and C region in a single antibody molecule. Various DNA coding regions naturally recombine to produce a diverse antibody. 

Image 2: Antibody structure

Various regions	Total Genes	The number of genes expressed
Variable region (V)	86	1
Diversity region (D)	30	1
Joining region (J)	9	1
Constant region (C)	11	1

Table: The expression of the genes in various regions

Splicing and recombination:

Naturally, DNA recombination occurs in the coding segments. For forming a heavy chain, splicing of any one variable region on a D region occurs followed by a J region. This process of splicing is known as V-D-J joining. Then the constant portion of the heavy chain undergoes splicing. The transcription and translation processes follow these events. RNA processing involves the splicing of the introns or the non-coding regions. When the antigen stimulates an immune response, the heavy chain DNA undergoes a further rearrangement in which V, D, and J combine with any C gene segment. The V, D, J, and C regions for a heavy chain DNA are known as V_H, D_H, J_H, and C_H respectively. The process is known as class switching. Exactly how the process occurs remains unclear. However, there are flanking regions situated upstream of the C_H region consisting of multiple copies of short repeats (GAGTC and TGGGG). Mutations also help to increase the genetic variation of the antibodies.

Image 3: V-D-J rearrangement

Antibody diversity differs in various species:

The creation of most of the immunoglobulin genes is similar in humans and mice. There is a combinatorial repertoire of immunoglobulin genes. The combinatorial rearrangements of VDJ gene segments are germ-line based.   It occurs primarily in the bone marrow. In organisms such as birds, rabbits, sheep, cattle, and others, the primary repertoire gets generated in the gut-associated lymphoid tissue (GALT). Many species do not undergo combinatorial VDJ rearrangements. Chickens involve a limited repertoire of functional VDJ genes. Hence, the B cells migrate to the Bursa of Fabricius and undergo rapid proliferation and diversification mediated by gene conversion. In rabbits, gene conversion and somatic hypermutation occur in the GALT in the specialized microenvironments of the appendix. The sheep and the cattle diversify their antibodies in the Peyer’s patch. The jawed vertebrates including the cartilaginous fish undergo VDJ rearrangements. Jawless fish such as Hagfish and Lampreys lack adaptive immunity. These fish use completely different genes known as variable lymphocyte receptors (VLRs) to generate an immune response. These VLRs are leucine-rich repeats capable of producing somatic diversity. Not only animals and fish but also plants and insects possess immunoglobulin genes.

Gene conversion is not an ordinary process. It extensively diversifies the rearranged genes. Gene conversion is a special case of somatic mutation, thereby known as somatic hypermutation. The word somatic hypermutation indicates the occurrence of a high-class mutation in the variable regions of the immunoglobulin genes. Thus the gene sequence portions get modified assuming the corresponding sequence of the donor gene. The donor gene acts as a template gene. This type of somatic mutation is known as templated somatic mutation. Once the antigen enters the immune system, it gets exposed to millions of antibodies. But only a few types of antibodies have a sufficient affinity to trigger an immune response. Depending on the affinity binding, antibodies with sufficient affinity interact with the antigen.

AgDscam genes in mosquitoes:

Insects such as mosquitoes possess genes encoding immunoglobulins. However, they lack specific mechanisms such as somatic hypermutation and recombination. Hence, mosquitoes have a different mechanism. The researchers at John Hopkins discovered AgDscam, a way in which mosquitoes combine their immunoglobulin domains of a single gene. AgDscam indicates Anopheles gambiae Down’s syndrome cell adhesion molecule. It produces a variety of pathogen-binding proteins. It follows the process of alternative splicing guided by immune signal transduction pathways. Hence, it helps to increase the binding capacity of various pathogen binders.

Thus, in conclusion, antibody diversity occurs through gene recombination. The process of gene recombination varies from organism to organism. Humans involve enzyme-mediated VDJ recombination, gene conversion, and somatic hypermutation. The other organisms may involve slightly different mechanisms. Hence, these genes play an important role in building the immune system.
References:

[1] Kuby Immunology

[2] Immunology, Pathak, and Palan

[3] Genetics, 9th Edition (Multicolour Edition), Verma P.S. & Agarwal V.K.

[4] Principles of genetics, 8th ed, Gardner, M. J. Simmons, D. P. Snustad
[5] Insect Infection and Immunity: Evolution, Ecology, and Mechanisms, Jens Rolff, Stuart Reynolds

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Splicing of Intronic Sequences

The intervening sequences or the introns do not code for any proteins. However, they are present in the gene separating the coding sequences from each other. They were discovered recently, a few decades back. Till now we only know a few types of intronic sequences and their locations. Some belong to nuclear pre-RNAs whereas others belong to organelle pre-RNAs. Complete maturation of RNA is tough to achieve without the removal of intronic sequences. Hence, without splicing of introns, RNA does not get translated into a protein. The pre-mRNA consists of an alternative arrangement of introns and exons. After a particular exonic sequence, an intronic sequence starts, followed by exons and introns again. Also, a mRNA consists of many introns with lengthy sequences covering the majority of the mRNA transcript. Thus, they take up a considerable length of the transcript, and thus they get spliced or excised. The exons, on the other hand, have specific sequences coding for amino acids. Hence they need to be ligated together.

There are total eight types of introns known till now. The eukaryotic nuclear pre-RNAs consist of the GU-AG introns and the AU-AC introns. A specific class of introns known as a group I introns occur in the eukaryotic nuclear pre-rRNA as well as organelle RNAs. The group II and group III introns occur only in the organelle RNAs. Next type of introns is known as Twintrons. They are a simple type of introns consisting of one intron embedded in the other intron. Complex Twintrons consist of multiple embedded introns. They are also present in the organelle RNAs. Nuclear pre-tRNAs consist of pre-tRNA introns. The above types of introns belong to eukaryotic RNAs. However, the group I and group II introns also belong to prokaryotic RNAs. There are only a few pieces of evidence regarding the archeal introns. The spliceosomes help in carrying out this important process. They consist of pre-mRNAs bound to small nuclear ribonucleoprotein particles known as snRNPs or snurps. They are small nuclear RNAs with protein complexes. Five principal snRNAs include U1, U2, U4, U5, and U6, and are associated with proteins to form snRNPs.

Events before the initiation of splicing:

A complex known as the commitment complex initiates the splicing activity. It comprises U1SnRNP. The U1 binds to 5’ splice site by RNA-RNA base pairing. Certain proteins act as binding factors. The examples include SF1, U2AF35, and U2AF65. These binding proteins help to make protein-RNA contacts with the branch site, the polypyrimidine tract and the 3’ splice site respectively. Another important complex known as pre-spliceosome complex consists of the above commitment complex along with another snRNP known as U2-snRNP. This snurp attaches to the branch site. The synergy of U1 snRNP and U2 snRNP brings the 5’ splice site near the branch point. Hence, the commitment complex and the pre-spliceosome complex assist the spliceosome. A spliceosome is a protein-RNA complex that removes the introns. A spliceosome complex forms when U4/U6 snurps and the U5-snRNP attach to the pre-spliceosome complex. It leads to the movement of 3’ splice site closer to the 5’ site and the branch point. Now, the two esterification reactions occur. The reaction gets catalyzed by the U6-snRNPs. An important group of proteins known as splicing factor proteins or SR proteins assists in the splice site selection. The word “SR” indicates a C-terminal domain containing serine and arginine.

Steps involved in splicing

1.     U1 snRNP binds to 5’ splice junction of the intron. It occurs due to the base pairing of the U1 snRNA in the snRNP at the 5’ splice junction.

2.     U2 snRNP binds to a sequence known as branch point sequence located at the 3’ splice site junction.

3.   U4/U6 snRNP and a U5 snRNP interact with each other and bind to the U1 and U2 snRNPs resulting in looping of the intron. Hence, they bring two ends closer.

4.     The U4 snRNP gets dissociated.

5.     Then forms the active spliceosome.

6.    The snRNP cleaves the introns from the first exon at the 5’ splice junction. The 5’ end of the intron gets bound with an adenine nucleotide in the branch point sequence. Hence, the structure formed out of this activity is known as a lariat.

7.   The intron gets excised by cleavage at the 3’ junction. Thus, the first and the second exons get ligated together.

Image: Splicing procedure

Splicing studies in Tetrahymena

The process of splicing in the Tetrahymena is known as self-splicing. It occurs in the pre-rRNA. Following are the steps:

1.     The cleavage occurs at the 5’ splice junction accompanied by an addition of G- nucleotide to the 5’ end of the intron.

2.     The cleavage at the 3’ splice junction releases the intron. Then the two exons get spliced.

3.     Next, the intron starts circularizing by a 3’ nucleotide bonding to an internal nucleotide, thereby producing a lariat.

4.     The last step involves cleavage of the lariat to produce a circular RNA and a linear molecule.

Types of the aberrant splicing:

Sometimes aberrations happen even in simple processes such as splicing. There are two forms of aberrant splicing. The first one is known as an exon skipping. In this, the aberrant splicing results in loss of one exon from the mRNA. The other form of aberrant splicing involves selection of a cryptic splice site. In this type of selection, there is a loss of an important part of an exon from the mRNA. It alternatively results in the selection of an intron to be retained.

Alternative splicing

It is a crucial finding in the field of genomics. Alternative splicing enables a single transcript processed in a related but a different mRNA. It leads to the synthesis of a variety of proteins.

1.     Alternative splicing in Drosophila:

It occurs during sex determination and gene regulation. The beginning of the cascade involves sex-specific alternative splicing of the Sxl pre-mRNA. A full length functional Sxl protein gets synthesized in females because the third exon gets skipped in the females, unlike males. The Sxl blocks the 3’ splice site in the first intron TRA pre-mRNA. It encodes a functional TRA protein. In males, the fourth exon of DSX pre-mRNA gets skipped. It encodes a male-specific protein known as DSX protein. The mechanism for encoding female specific protein involves a different strategy.

2.     Alternative splicing in humans:

A gene in humans known as an SLO gene comprises 35 exons in all. It encodes for a membrane protein regulating the entry and exit of the potassium ions in and out of the cells. There is an involvement of the total of eight exons. These genes are active in the inner ear. They determine the auditory properties of the hair cells on the basilar membrane of the cochlea. About 40-60% of human genes have alternative splice forms. Hence, alternative splicing is very important in the human genome and its functional complexity.

Trans-splicing in C. elegans:

The exons present in different RNA molecules get spliced in the trans-splicing process. C. elegans consists of SLRNA. It is known as a spliced leader RNA. It is almost 100 nucleotides in length. Here, a fork forms instead of a lariat. Some genes in C. elegans get transcribed in pairs from a single promoter.

Eukaryotic pre-rRNAs and pre-tRNA splicing:

Eukaryotes consist of autocatalytic pre-rRNA intron sequences. These introns belong to the group I family of introns and found in mitochondrial and chloroplast genomes. They occur in pre-rRNA and pre-mRNA. The first reaction involves a free nucleoside. The second transesterification involves attack of the 3’-OH to the phosphodiester bond at 3’ splice site causing cleavage and release of an exon. For releasing the introns from the pre-tRNA, introns do not involve trans-esterification reaction. An endonuclease cuts the two splice sites. 

References:
[1] Molecular Biology of the Cell, Bruce Alberts
[2] Molecular Biology of RNA, David Elliott, Michael Ladomery
[3] IGenetics, Peter Russell, second edition
[4] Gene Regulation: A Eukaryotic Perspective, David S. Latchman

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