Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins...
-
date post
23-Jan-2016 -
Category
Documents
-
view
217 -
download
0
Transcript of Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins...
![Page 1: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/1.jpg)
Antibody Diversity
![Page 2: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/2.jpg)
Problem…the immune system makes over one billion different
antibody proteins• In 1950’s: central dogma stated DNA—to RNA—to
protein• One gene for each protein• Required millions of genes just for the immune
system• Does not seem possible, but most scientists thought
it might be• Today we know the human genome is less than
30,000 genes• So, what is really going on???
![Page 3: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/3.jpg)
Current theory must account for the following known properties of
antibodies
![Page 4: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/4.jpg)
B lymphocyte development
![Page 5: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/5.jpg)
B lymphocyte development (2)
![Page 6: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/6.jpg)
Germ-line vs somatic-variation theories
• Germ-line: stated that each antibody had its own gene….nothing special, but required billions of genes to account for numbers of antibodies
• Somatic-variation: some mutation and recombination created vast number of genes for antibody formation
• This introduced a new concept: targeted mutation or recombination of DNA: is it possible??
• Paradox: how could stability be maintained in C region and diversity exist in V region?
![Page 7: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/7.jpg)
Dreyer and Bennett• In 1965 proposed radical theory to account for
diversity of antibodies• Each antibody was coded for by two separate
genes• One for the variable region• One for the constant region• Combined at the DNA level and expressed
single mRNA• Suggested 1000’s of variable region genes and
only one constant region gene• Close, but no cigar• Most biomedical scientists did not like this idea
and rejected it
![Page 8: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/8.jpg)
Tonegawa’s demonstration• 1976—used restriction enzymes and DNA probes to show that
germ cell DNA contained several smaller DNA segments compared to DNA taken from developed lymphocytes (myeloma cells)
![Page 9: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/9.jpg)
Genes for immunoglobulin proteins are found on different chromosomes
![Page 10: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/10.jpg)
Multigene organization of Ig genes
![Page 11: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/11.jpg)
Kappa light chain rearrangement
![Page 12: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/12.jpg)
Heavy chain rearrangement
![Page 13: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/13.jpg)
Mechanism of variable region rearrangements
• Each V, D and J segments of DNA are flanked by special sequences (RSS—recombination signal sequences) of two sizes
• Single turn and double turn sequences (each turn of DNA is 10 base pairs long)
• Only single turn can combine with a double turn sequence
• Joining rule ensures that V segment joins only with a J segment in the proper order
• Recombinases join segments together
![Page 14: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/14.jpg)
P and N region nucleotide alteration adds to diversity of V region
• During recombination some nucleotide bases are cut from or add to the coding regions (p nucleotides)
• Up to 15 or so randomly inserted nucleotide bases are added at the cut sites of the V, D and J regions (n nucleotides_
• TdT (terminal deoxynucleotidyl transferase) a unique enzyme found only in lymphocytes
• Since these bases are random, the amino acid sequence generated by these bases will also be random
![Page 15: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/15.jpg)
N nucleotide addition at joining segments: the addition of random
bases
![Page 16: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/16.jpg)
Randomness in joining process helps generate diversity by creating
hypervariable of antigen binding site
![Page 17: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/17.jpg)
Imprecise joining generates diversity
![Page 18: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/18.jpg)
Some rearrangements are productive, others are non-productive: frame shift
alterations are non-productive
![Page 19: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/19.jpg)
Allelic exclusion: only one chromosome is active in any one lymphocyte
![Page 20: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/20.jpg)
7 means of generating antibody diversity
![Page 21: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/21.jpg)
Diversity calculations
![Page 22: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/22.jpg)
Somatic hypermutation adds even more variability
• B cell multiplication introduces additional opportunities for alterations to rearranged VJ or VDJ segments
• These regions are extremely susceptible to mutation compared to “regular” DNA, about one base in 600 is altered per two generations of dividing (expanding) lymphocyte population
![Page 23: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/23.jpg)
Combination of heavy and light chains adds final diversity of
variable region• 8262 possible heavy chain combinations
• 320 light chain combinations
• Over 2 million combinations
• P and N nucleotide additions and subtractions multiply this by 104
• Possible combinations over 1010
![Page 24: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/24.jpg)
Location of variability occurs within CDR regions of V domains (antigen
binding sites)
![Page 25: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/25.jpg)
Class switching among constant regions: generation of IgG, IgA and IgE with same
antigenic determinants—idiotypes
![Page 26: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/26.jpg)
Synthesis, assembly and secretion of
immunoglobulins
![Page 27: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/27.jpg)
Regulation of Ig gene transcription• Each lymphocyte rearranged gene has regulatory sequences that
control gene expression• Promoters: initiation sites of RNA transcription• Enhancers: upstream of downstream that transcription from the
promoter sequence• Silencers: down-regulate transcription in germline cells• Gene rearrangement brings enhancer and promoter regions close
together and eliminates silencer regions allowing transcription
![Page 28: Antibody Diversity. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein.](https://reader035.fdocuments.in/reader035/viewer/2022081603/56649d4e5503460f94a2d864/html5/thumbnails/28.jpg)
Understanding of immunoglobulin structure and formation has opened
up a new world of possibilities
• Monoclonal antibodies
• Engineering mice with human immune systems
• Generating chimeric and hybrid antibodies for clinical use
• Abzymes: antibodies with enzyme capability