Immunoglobulin's generation of diversity

Transcription

1 هللا مسب In this sheet, there are just the extra notes mentioned by the doctor, don't forget referring to slides. Immunoglobulin's generation of diversity Note that diversity is applied to T and B lymphocytes in addition to immunoglobulins. B and T lymphocytes are highly diversed, and the generation of diversity is similar in both, and the mechanisms are also similar. Slide 1&2 These are the proposed theories in the demonstration and explanation of diversity mechanism 1. The concept that was held in 50s is that one gene is transcribed into one mrna, which in turn translated into a single protein So one gene = one protein But the antibody is composed of two different proteins, so it can't be explained on the reason of one gene one protein The individual has the ability to produce limitless number of antibodies 10^10 to the 10^16 specificities can be produced by B- lymphocytes 2. the second proposed theory is the presence of genes in the germ line that are sufficient to produce all of these antibodies, means that each antibody is produced by each gene that is present In the germ line But, the human genome is composed only of 30,000 genes and that cannot explain the huge antibody specificity produced

2 Generation of diversity can't be explained based on number of genes present 3. Somatic mutation theory This theory is not valid, because there's a specific number of spontaneous mutations that occur (the rate of spontaneous mutations is 10^-8 to 10^-11) This theory cannot explain the diversity generated. There are number of genes that code for immunoglobulins, these genes undergo rearrangement and as a consequence of rearrangement and mutations that takes place, diversity is generated That's how the problems start to be illustrated Slide 3 Kappa light chain is encoded by clusters of genes present on short arms of chromosome 2. Lambda light chain is by genes on chromosome 22, and heavy chains by genes on chromosome 14. Note in the maturation of B lymphocyte, the heavy chain is produced first, then the light chain. Light chain gene rearrangement is a terminal step in the differentiation of B lymphocytes; once the light chain rearranges that means the B lymphocyte become mature. The cell expresses heavy chain before it's a mature B lymphocyte So maturity is determined by the light chain of immunoglobulin. Slide 4 The whole concept of rearrangement is demonstrated by tonegawa's, who demonstrated that the germ line DNA in immature B lymphocyte contains small segments.

3 He is the first who illustrates how antibodies produced. Note the gel electrophoresis where segments migrate according to size EXPLANATION 1. Size of DNA from germ line is small 2. in cases of mature antibody producing B lymphocytes like ' mveloma cells', the DNA coding for the antibody was large, indicating that it's composed of multiple of these segments, means that a REARRANGED PRODUCT WAS GENERATED. 3. The mechanism that explains that is by cutting and splicing to rearrange more than one segment coding for the antibodies. Slide 6 Light chain is composed of variable and constant domain. The rearranged product of light chain should be composed of these two domains If the variable domain of the light chain is composed of 95 amino acids, and the constant domain is composed of 100 amino acids,then the combined product ( rearranged one ) should be composed of 195 a. a. Actually the light chain (rearranged) has 208 a. a, means that there's an additional 13 a. a Question 1 From Where do the extra amino acids come from? There's an additional segment that adds to join the variable domain to the constant domain and that segment is called ' joining segment' Joining segment is present and is coded by a joining gene and it acts in the rearrangement process to connect the V region to C region within the light chain. Slide 7

4 In the heavy chain there's an additional segment called DIVERSITY SEGMENT, and it acts on the rearrangement process beside to the V, C, J regions This sequence adds diversity to the heavy chain In light chain two rearrangement processes take place, while in heavy chain three rearrangement processes are required. Slide 9 Diversity results from multiple gene germ line that operate for both light and heavy chains For the light kappa loci, the possibilities (number of kappa chains that can be produced) is 29*5; (145). For lambda chain, an individual can produce 30*4;( 120) different lambda genes. So, the total number of light chains that can be produced by an individual is 265 (145 kappas+120 lambdas). Slide 11 Regarding diversity region it can be read from 3 different starting points (3 different reading frames). 1. Frame 1, when we start reading all the nucleotides within the coding sequence 2. Frame 2, if we shift by one nucleotide 3. Frame 3, if we shift by 2 nucleotides Note that the frame will shift if we don't read the first or the second nucleotides, but if we don't read the first 3 nucleotides, the frame will not shift and remains the same. Thus DIVERSITY REGION contributes to the diversity of heavy chain; because it can be read in 3 frames.

5 Slide 12 Combinatorial diversity (possibilities of combinations) If we consider only heavy chain, there are: 40 V regions, 27 D regions, and 6 J regions By multiplying the possibilities are 5,520 And because D regions read in 3 frames, so 5,520*3 (16,560) possibilities Immunoglobulins are composed of two identical light chains and two identical heavy chains And there are 265 light chains for the kappa and lambda, thus 16,560*265. The possibilities taking into considerations as a result only to the presence of these genes is about 4 million, (4 million antibodies can be produced only depending on combinatorial diversity). Junctional diversity: when diversity joins the joining and those join the variable, joints are created there at the junction between coding sequences (joint indicates new sequences). Junctional diversity is the most significant in the diversity of immunoglobulins. The combinatorial diversity accounts for 4 million antibodies, and the immune system can generate up to 10^16 antibodies. So combinatorial diversity contribution is negligible (regarding the total diversity), and most of diversity is created by junctional diversity. In T lymphocytes, the junctional diversity is responsible for 10^9 different cells. Slide 14 The first one is the gene on chromosome 14 (germ line of heavy chain), the second is for kappa and the last for lambda.

6 The gene organization of these 3 germ lines: For the heavy chain (the leading sequence then variable then diversity then joining and finally the constant) For the kappa (the leading sequence for each of the variable segments then the joining and then the constant) For the lambda chain (it's leader for each variable and constant with joining; each constant is preceded by a joining segment) Slide 15 In a B cell a rearranged primary transcript is generated as a consequence of rearrangement of one of the variable segment with one of the joining, then is rearranged with the constant domain of the light chain,creating a spliced mrna which codes for V,J,C regions. Slide 16 There's only 33% chance of success for the rearrangement to be in frame coding for proteins. In 67% of cases (two thirds) the cell fails to rearrange the primary transcript. B cell here rearranges kappa chain. In this example, the cell undergo non-productive rearrangement (in 67% of cases), and it will be given another chance to rearrange another product, also it has 33%chance of success in this second trial. Now if the second trial fails and the kappa chain rearrangement fails, the cell will be given another chance for rearrangement of lambda chain (the same mechanism 33%success, 67% failure) NOTE that the maturation of cells don't continue if they fail to rearrange. Slide 17 This is an illustration of heavy chain rearrangement

7 Remember that the first rearrangement process takes place between diversity and joining Somatic recombination occurs at the level of DNA which can now be transcribed and then translated into proteins (heavy chain production) NOTE that this process of rearrangement takes place early in the course of B lymphocyte differentiation. Slide 18 Primary transcript of RNA is generated as a consequence of joining rearranged to diversity then to variable ten the whole constant domains are spliced into this product. Number of constant domains in IgM and IgE is 4, while it's 3 in IgG, IgA, and IgD. The constant regions are produced with the hinge region in between. This hinge region coding sequence is present on the germ line and is spliced with constant domains. Slide 19 This illustrates B cell development. This process is not as simple as that ; synthesis of heavy chain takes place with the chains rearranged in the cytoplasm, then it's expressed with a surrogated light chain ( temporary light chain) until the light chain is synthesized. So heavy chain splicing (rearrangement) takes place until the light chain synthesis completed. Heavy chain starts before the light chain because there's diversity region, which spliced to joining. Slide 21 We have two forms of antibodies; cell surface and secreted antibodies.

8 They are different with respect to function, but they have the same structure. Slide 22 How can B cell produce the same protein and that protein is either expressed on the cell or secreted from the cell??? It has shown that the constant domain of the heavy chain has additional optional exons. These polyadenylated sites are associated with sequences that determine whether an antibody will be expressed on the surface or secreted into the serum or blood. The constant domain of IgM is associated with two polyadenylated sites, which determine either the antibody will be secreted or membrane bound, how?? If splicing takes place with secretary polyadenylation sequence (pas) coding for secreted antibody, the antibody will be secreted. And if splicing takes place with the membrane polyadenylated site (pam) coding for membrane bound IgM, the antibody will be membrane bound, and IgM will be expressed on the membrane of B lymphocytes. Slide 23 & 24 these two antibodies differ in structure and function ; the membrane bound antibody possess a transmembrane segment and even short cytoplasmic part,whereas the secreted form doesn't contain this transmembrane segment,and also it's smaller in size. Slide 27 both of secreted and membrane bound antibodies are produced similarly ; they're produced in RER and translated by ribosomes, then they're carried within a vesicle to Golgi apparatus ( where glycosylation takes place), the vesicle will later transported and become membrane bound or it secretes its own contents outside the cell.

9 Slide 29 Immunoglobulins have in an intervening sequence that are in between and are associated with coding sequences. And it was found that always a sequence of seven nucleotides known as "heptamers" are associated with the protein sequences and these sequences are separated from nine-nucleotide sequences known as "nonamer" by a 12 or 23 nucleotides. So each of the protein sequences whether they're variable, diversity, or joining have seven nucleotides separated by 12 or 23 from a 9 nucleotide sequence. This rule applied for kappa, lambda, and heavy chains, so it's applied for all B lymphocytes. The 12 represents one turn of DNA helix, 23 represents two turns That's mean heptamer and nonamer are separated by one or two turns Slide 30 Recombination signal sequences are heptamers and nonamers, so called because recombination cannot take place without recognition of these sequences (that's why they are always present), and the enzymes RAG1 and RAG2 (recombination activating gene 1&2) that are responsible for recombination must recognize these signals (the intervening sequences) Slide 32 RSS is the determining factor in the direction (orientation) of rearrangement) So always rearrangement takes place from 23 to a 12 or from 12 to a 23, but not from 12 to 12 or from 23 to 23(the space must be 12 or 23). Slide 33 The direction of rearrangement is as follow: 1. Joining to diversity then variable to diversity.

10 * NOTE that it cannot take joining to variable, because joining and variable will be 23 to 23, so the mechanism of rearrangement is governed by the spacer. It's called 12/23 rule or one turn /two turns rule. Heptamers are associated with the coding sequences and nonamers are spaced by 12 or 23. Slide 36 If we have in reality the heptamer rearranges to heptamer or nonamer rearranges to nonamer, maintaining the one or two turn spacer, after this rearrangement it will be excised (removed),resulted in the production of rearranged product that has the sequence of VARIABLE then DIVERSITY then JOINING PRODUCT FIRST DIVERSITY JOINS JOINING THEN THE DIVERSITY-JOINING JOINS VARIABLE. SORRY IF THERE'S ANY MISTAKEs, I'VE TRIED MY BEST TO MAKE EVERYTHING CLEAR. DONE BY: ALAA SHQEIRAT

11