Antibody Structure and Generation of B-cell Diversity

What are the Fab and Fc regions of the antibody?

Fab (fraction antigen binding) are the two arms; Fc (fraction crystallizable) is the one leg (identical on every antibody); formed from proteases that chop up the antibody

Describe the two process of the cleavage of antibodies into Fab and Fc regions

papain cleaves the hinge region and separates the Fab fragments from the Fc fragment; pepsin cleaves Fc region and makes Fab2 which are both Fab regions still attached together

What is the purpose of the hinge region?

flexibility accommodates for different bindings of different epitopes; however this region can be degraded but can be helped with carbohydrate guards

What is the purpose of the heavy chain constant domains?

mediates the function of the antibody

What two type of antibodies will a mature naive B cell present?

membrane IgM and membrane IgD (antigen specificity is identical bc variable domains are the same)

What is class switching?

how to get from one isotope to another; switches the heavy chains and variable regions do not change

How does IgE?

produced when in an environment with lots of parasites; will bind to mast cells and basophils and binds to antigen to cross link the two to cause the cells to degranulate and release toxic factors to the parasite

What does IgA do?

produced a lot in the gut; important role in barrier functions; binds to microbial flora and pathogens to prevent them from breaking through the epithelial barrier of the gut

Describe the tertiary structure of an antibody

four immunoglobulin domains for the heavy and two for the light chains; two heavy chains are disulfide bonded together and the immunoglobulin domains of the heavy and light chains are covalently bonded

What is the secondary structure?

light chain variable domain (110-120 amino acids) and light chain constant domains both have anti-parallel beta-pleated sheets with loop structures between each one

Explain the regions of variability in light chains in their primary structures

there are three complementary determining regions (CDRs) with high variability that make up the antigen binding sites; these three regions are close to each other in the primary structure; CDR3 will always have highest variability because

What is the purpose of having different epitopes for viruses?

to have the best chance of neutralizing the virus so it can detect all different kinds of epitopes

What is the advantage of having a repetitive epitope and not just different ones for a specific antigen?

will be a tighter bind and can recognize something similar on all pathogen molecules for more efficiency

What is the difference between linear and discontinuous eptiopes?

antibodies can either recognize a continuous line of amino acids on a protein (linear) or amino acids that are spread far apart (discontinuous)

What is important about discontinuous epitopes?

not reliable because it requires the virus/protein to fold in a certain way for the antibody to bind and that is unlikely

What are the noncovalent interactions the contribute to the strength of binding between the antibody molecule and the antigenic epitope?

electrostatic forces, hydrogen bonds, van der waals forces, hydrophobic forces, cation-pi interactions; the more similar the two are, the more strongly they will bind to each other

How do we generate various B cells and T cells?

in a loci that codes for immunoglobulin heavy and light chains, there are gene segments that can be randomly recombined to make different isotypes; there are the lambda and kappa light chains loci and one heavy chain locus

What are the V segments on the loci?

variable segments

What are the J segments on the loci?

joining segments

What is the C on the loci?

constant (there are three different lambda light chains which is why it has three constant regions and the rest have one)

What makes up the variable domain of the lambda light chain?

combination of one V gene segment (30) and one joining segment (4) (recombined upstream of constant region on chromosome 22)

What makes up the variable domain of the kappa light chain?

one V gene segment (35) and one J segment (5) (recombined upstream of the constant region on chromosome 2)

What makes up the variable domain of the heavy chain?

has D segments (diversity); recombines one V gene (40) with one D gene (23) and one J gene (6) (recombined upstream of constant region (there are 9 constant regions on heavy chain to encode for each isotype) on chromosome 14)

In heavy chains, what is the somatic recombination process?

take out all intervening DNA between V, D, and J segments; one D segment and one J segment are recombined together; then add a V gene to it

What happens when you are successful in recombining the genes on the heavy chain?

makes a heavy chain polypeptide that stops any more recombination on the heavy chain and starts light chain recombination

What happens once you have a rearranged DNA?

transcription occurs to make the primary RNA transcript; splicing occurs to get all introns out and you get mRNA; translation occurs to make a polypeptide chain to make the antibody

Describe the 12-23 base pair spacer rule on the light chain

on the lambda and kappa light chain loci, there are heptamers and nonamers that are conserved in each chain; on the lambda chain, there is a 23 spacer separating the two by the V segment; by the J segment, there is a 12 spacer between the heptamer and and nonamer; the same is flipped on the kappa light chain

Describe the 12-23 base pair rule on heavy chains

V segment - heptamer - 23 - nonamer —— nonamer - 12 - heptamer - D segment - heptamer -12 - nonamer ——— nonamer - 23 - heptamer - J segment

What is the purpose of the 12-23 base pair rule?

ensures that no more than one J and one D segment are recombined in any single chain; reasoning is because they have to have alternating spacers flanking each segments for them to bind together

What do RAG 1 and RAG 2 do?

recognizes heptamer and nonamers that flanks the gene segments and the 12-23 spacers; RAG will bind to flanking sequence of a J segment upstream, and then look for flanking sequence of the V gene segments downstream (bc they are opposite 12-23); makes a loop and cleaves the DNA and then glues together the V and J segments together to make a coding joint

What is the process of gluing the D and J segments (any segements work) back together after its cleaved?

RAG complex cleaves the heptamer from the gene segments which makes hairpins; RAG complex open hairpins by nicking one strand of the DNA, generating palindromic P- nucleotides; N-nucleotide addition by TdT(ONLY ON HEAVY CHAIN); both strands are paired and the unpaired nucleotides are removed by exonuclease; gaps are filled by DNA synthesis and ligation to form coding joint (P and N nucleotides adds more diversity)

What is Ig-a and Ig-b? (alpha and beta)

heterodimeric structure that interacts with antigen receptor; when the antigen receptor binds to BCR, it undergoes a conformational change and it leads to signalling

What is the purpose of Ig-a and Ig-b?

when a BCR is mature and expressed on the surface of an immature, naive B cell (IgM), it can bind antigens, however, it can’t do anything else

How is IgM and IgD produced?

once the VDJ segment is produced on heavy chain locus, a B cell will first create an mRNA transcript where the VDJ segment is spliced from the constant regions; once the B cell reaches the spleen, the IgD is made and expressed on the B cell

How is it that a B cell can express both IgM and IgD at the same time?

differential RNA splicing

How does IgM go from transmembrane to secreted?

when a B cell differentiates into a plasma cell, it will use RNA splicing to change to the C-terminus of the antibody (SC region for secreted and transmembrane region for transmembrane)