antibody binding site is hypervariable

2 things about Ab: globular structure which is stable but also highly flexible allowing formation of finger-like loops that can be short or long as needed

Antigen binding sites are like fingers, can bind all kinds of structures

- antibodies see shapes (don’t care if its certain amino acid sequences, lipids, chemical, etc.) bc it depends on shapes

- structure versatile and flexible

antibody bound to antigen pic

Antibody make beta pleated sheets

- alpha helices give structure of antibody as fully folded primary protein

Pic shows antigen binding to light and heavy chain (circled in red)

formation of antigen bindng site of heavy chain (V_H domain)

Beta pleating is not variable

So variability lies in the alpha helices

Ig (immunoglobulin domain) is made up of the betas pleated sheets and alpha helical loops, and the alpha helical loops determines the variability in the binding site

CDR – complimentary determining regions (the loops, which are the alpha helical regions)

- each loop has number (1,2,3)

- protein structure complimentary to the antigen will bind

closer view of V and C regions of antibody structure

Hypervariable Regions of Ab V domains form discrete loops at one end of the folded domain

same for both light and heavy chain variable regions

variability is in that region based on reasoning from last slide

Certain parts of antibody sequence where most variability is (red parts)

• in loops, and makes up antibody binding site (see pic on right)

formation of the antigen binding site of heavy chain (V_H domain)

• this is a favorite protein fold

The diversity and structure of antibodies gives extreme felxibility/adaptability

antigen-binding sites are versatile

vary in shape and physical properties

Binding site can be diff shapes in order to be specific for the antigen

Ab binding sites can bind linear and discontinuous epitopes

Proteins folded into their shapes

Antibody binding sites can bind to antigen if it is a linear sequence of antigen protein, or even if it is discontinuous (if the specific shape made has the structure they recognize, bc the shape matters)

BIG contrast to T cell receptor – see proteins ONLY, and recognizes based on the linear peptide

What kind of molecules can antibodies bind to?

Is the antibody binding determined by sequence or shape?

almost any/any molecules

bc it only binds depending on the shape (binds to specifically shaped antigen)

lymphocyte development

Antigen gives instruction to B cell to expand clonally

Antigen to BCR is a bit more than this

If delete BCR from B cells, then no B cells exist if delete the gene

If B cell does not make heavy chain, this happens

Same true for T cells

cell membrane IgM (BCR) signaling guides B cell development

BCR signals cell to do changes intracellularly -> make antibodies/differentiate

TCR signaling guides T cell development

steps in B cell and T cell development

Progenitor -> B or T cells

- if it cannot make a little precursor receptor on the pre B/T cell, then the B/T cell dies

- must have the receptor to survive and develop

- receptor sends signals for development in cell (bc if not, cell dies)

- if have the receptor -> matures

Then goes on

If dev to be dangerous to our body -> it dies (negative selection)

If its fine -> positive selection and survives

heavy and light chain expression during B cell ( and T cell ) development

TCR – make TCRbeta chain first

• next make TCRalpha chain

BCR - make VH chain first

• next make VL chain

many receptors made will likely die due to no use – never finds its antigen

If react to own antigen and has strong signal, cells picked to die if there is strong reaction to antigens (negative selection)

positively selected for weaker antigen recognition -> go on to mature as B or T cell

Antigen receptor tickling – receptors always tickling cells they are on, keeps them alive??? I think this was a tangent so doesn’t matter???

Generation of a large B cell repertoir with diverse antigen receptors

antibody (immunoglobulin) repertoir diversity is 10^11

human and mouse genomes and gene stats

humans have 58 thousand genes

only 19950 code for proteins

how is it possible to make so many receptors??? How can the 10^11 different receptors be made (out of proteins, bc the chains are proteins) if we only have 19950 protein encoding genes?

prototypical gene expression

chromosome has DNA/genes -> mRNA -> polypeptide -> folded protein

our genome not large enough to contain 10^11 genes

There are about 20 thousand protein-coding genes in our genome.

How can 10¹¹ antibodies with distinct specificities be made?

Antibodies obtain their diversity through 2 processes. The first is called V(D)J (variable, diverse, and joining regions) recombination. During cell maturation, the B cell splices out the DNA of all but one of the genes from each region and combine the three remaining genes to form one VDJ segment.

generation of diversity question

susumu tonegawa

took DNA out of B cells

• ran DNA on electrophoresis, migrate, noticed differences in DNA bc of slower/faster movement in electrophoresis
• looked at light and heavy chains
• noted genome of B cells was diff between the B cells
• discovered that basically each cell carries new/different genes

now, how did the DNA change in each cell???

Ig gene pieces (segments) are arrayed in the chromosomes and are randomly recombined to create novel Ig genes

V – variable genes

D – diversity genes

J - joining genes

Turned out – assembled heavy chain – combination of 1 V, 1D, and 1J together

randomly combine a V, a D, and a J together

resulting recombined region is used to generate mRNA -> and then create protein

germline organization of the B cell receptor gene Heavy Chain locus

only in B cells, are these recombined to make a new gene

this shows the gene

variable region of the gene (V sections, D sections, and J sections), then the rest is constant region

cont.

variable region of the gene will create the part of the protein which is the antigen binding site

this is how variable region is in IgH

germline organization of B cell receptor gene light chain locus

Ig K

Ig gamma

System shopping for pieces + putting together to make new combinations

Ex: wardrobe of 30,000 outfits

- by mixing outfits, there are 1.5x10^6 outfits total

Done at chromosomal level

- only done by immune B and T cells

- other cells don’t do this

how do you make a new gene in each b cell clone that never existed and cannot be inherited to the offspring?

How this happens

Picks 1V, 1D, and 1J `and puts together

Order in process bc of DNA sequences surrounding the small V, D, J regions

- the red and orange

- red sequences cannot combine with red, and orange cannot combine with orange (keeps it orderly and correct)

- specific sequences cannot recombine with the same one (ex: 23 must be next to 12)

= so red must be next to orange

= makes order to rearrangement of genes

cont.

All this is to make variable binding site of antibody

recombination activating gene (RAG) 1 & 2

Gene in lymphocytes RAG 1 and 2

– imagine as a scissor that cuts DNA in right place (where the red/orange triangles were) to join them together (does the gene cutting for recombination)

before the VDJ segments can join together

Hardly any exons in these genes, not typical for human genes (human genes usually have a lot of exons)

V(D)J recombination occurs in the context of
what is known as 12/23 rule

according to rule - gene segments to be recombined different recognition sequences (RSS)

• ex: one segment has 23 RSS and the other has 12 RSS
• important feature of the regulation of VDJ recombination that makes the process orderly

essentially, 23RSS must connect with 12RSS (so that the order ends up being VDJ)

Same idea – rule of can combine only red – orange or orange – red

In light chain, V and J can combine

generation of B cell diversity by recombination and expression of Ig genes

Basically show if u pick V1,D2, and J13

Making 2 diff genes in the 2 scenarios

potential Ig repertoire

shows possibilities

VDJ gene recombination to make functional antibody genes (video)

Ig genes componsed of separated segments of DNA that become joined together by process called somatic recombination to make functional gene

• V (variable) gene segment
• D (diversity)
• J (joining) segment

gene segments that recombine have specific sequence motifs (recombination signal sequence, RSS)

protein complex with product of RAG 1 & 2 genes - bind to RSS motifs RANDOMLY among the gene copies (ex: out of all V randomly pick a V)

• pinch/bring gene segment together and clip genes in between = so the desired gene segments are now adjacent (are ligased together so they connect now) = recombination process completed
• cleavage creates a hairpin DNA at end of gene segments
• more proteins make complex with RAG protein complex, to join the 2 RSS ends (RAG complex is holding the cut out portion of the gene) = create closed circle of DNA -> no further role in process

video is NOT on test, too specific

- can test to see if there are enough lymphocytes in someone (can test for immunodeficiency) by testing for circular piece at end??? This may not be correct

Need immune system for action against outside things (not just for solely bodily function)

light chains have only V and J segments

light chains have only V and J segments

What makes the random recombination of V, D, and J gene segments an orderly process?

12/23 rule?

BCR vs. TCR diversity generation by VDJ recombination

B cell dev steps coincide with VDJ recombination

I dont understand this

Takaways

• unique structure of antibodies makes them versatile in their binding ability to antigen
• antibodies have Variable (V) regions (antigen binding) that make it possible to bind nearly unlimited number of antigens
• recombination activating genes (RAG) 1 & 2 are required to create antibody variable genes
• diverse VH regions are generated by a unique mechanism of genomic DNA rearrangement by random selection of V, D, and J gene segments (like exons)
• VH + VL together create vast and diverse binding specificities of antibodies

Antibodies have stable antibody structure

RAG genes required to make antibody variable genes

Together – heavy and light create receptor that together bind to antigen

- 3 fingers from light, 3 from heavy make …?

extra material after this in lecture slides

necessary? maybe review them later? i dont think he went over it