Lecture 4 - Structure and Genetics of Antibodies

structure of an IgG antibody molecule

they consist of two identical heavy chains and two identical light chains, a hinge region and an antigen binding site, the domains of an Ig molecule have similar structures

heavy and light chains (structure)

they are composed of constant and variable regions

antigen binding site

it is formed by the heavy and light chain pair of one variable region

hinge region

it is located within the constant region of the heavy chain, it confers flexibility to antibody arms, it allows spatial flexibility in antigen-binding being that it is a polypeptide with no defined region

Ig domains

barrel shaped structure in which B strands running in opposite directions (antiparallel) pack together to form two B sheets held together by a disulfide bond, it is a compact structure for variable and constant domain of the light chain

light chain

consists of one variable domain and one constant Ig domain

heavy chain

consists of one variable domain and 3 constant domains (IgG

Ig domains

very important + present in multiple molecules (very common), they are also present in the TCR and many other proteins

how was Ig discovered?

tiselius and kabat demonstrated that most antibodies are in the y-globulin fraction of serum proteins when analyzed with electrophoresis, they compared serum from rabbits immunized with ovalbumin and serum from rabbits immunized with OVA but pre-treated with OVA to remove anti-OVA antibodies (immunoprecipitation), they found that when they did this most of the proteins in the immunoprecipitated rabbit were gone, showing that they migrate in the y fraction

papain

allows proteolytic cleavage by cutting above the disulfide bond, disconnecting the two Fab regions from each other and the Fc region, 2 fragments with same weight, retaining ability of antigens to bind to it so Fc region is the antigen binding site

pepsin

allows proteolytic cleavage by cutting below the disulfide bond, this causes the two Fab arms to stay together so you maintain an intact antigen binding site, the pFc region has no function

hypervariable regions (HVs or CDRs)

They are the region of the heavy chain that binds the antigen/complementarity-determining regions, there are 3 in the heavy chain

framework regions

these are less variable compared to the HVs, flanking them

where are HVs located?

they are found in discrete loops of the folded structure, they form loops that link tgt some particular B strand in the flattened ribbon structure. In the folded structure of the V domain, the HV loops, also called CDRs are brought tgt to form antigen binding regions. In the complete structure, the pairing of a heavy chain and a light chain brings tgt the HVs from each chain to create a single HV surface that forms the antigen binding site at the tip of each arm.

how do antibodies bind antigens?

they do so via contacts in CDRs that are complementary to the size and shape of the antigen, the vicinity of the interaction is important and the antigen binding pocket is the finger protruding from the CDR

conformational/discontinuous epitope

might be formed by amino acids far apart in the primary sequence of a protein, destroyed if the 3D structure of the protein is destroyed, actual amino acid residues are distant in the anitbody and an epitope is created by folding of the protein

linear/discontinuous epitope

formed by a linear sequence of adjacent amino acids in the primary sequence of a protein. Not destroyed if the 3D structure of the protein is destroyed

how is an epitope lost?

by denaturation so even if you don’t have a native conformation the antigen can bind,

inaccessible epitope

Ig binds to epitope in denatured protein only

accessible epitope

Ig binds to epitope in both native and denatured protein

immunogens

molecules that stimulate immune responses

hapten

molecule that has a very simple structure and does not have to induce immune response, its antigenic so it can recognize the antigen but it is not immunogenic, ex; DNP

conjugate

it is a protein that is a very good immunogen, so when added to a hapten it can help induce an immune response to a particular antigen (ex; when KLH is in complex with DNP)

what makes a good immunogen?

large molecule, the right dose, subcutaneous administration, complex structure, distinct from the host molecule, adding adjuvants and MHC

route of immunogen administration

subcutaneous is the most effective followed by intraperitoneal and least effective is intravenous

adjuvants

helps in actually triggering the immune response, they are agents that promote slow release and local inflammation enhancing the immune response by stimulating the innate system

MHC

mediates effective antigen presentation of protein antigens to T-cells for T cell activation

noncovalent forces

allow antibodies to bind to conformational shapes on the surface of antigens

electrostatic forces

attraction between opposite charges

hydrogen bonds

hydrogen shared between electronegative atoms (N, O)

van der waals forces

fluctuations in electron clouds around molecules polarize neighboring atoms oppositely

hydrophobic forces

hydrophobic groups interact unfavorable with water and tend to pack together to exclude water molecules, the attraction also involves van deer waals forces

cation-pi interaction

noncovalent interaction between a cation and an electron cloud of a nearby aromatic group

affinity

the strength of the binding between a single antigen-binding site of an antibody and an epitope of an antigen

dissociation constant (Kd)

measures the affinity of the antibody, it indicates how easy it is to separate an antigen-antibody complex into its components by changing their concentration (Kd = AbAg/Ab-Ag complex)

(Ab)

concentration of free antibody

(Ag)

concentration of free antigen

(Ab-Ag complex)

concentration of the antigen-antibody complex

smaller Kd

means affinity is stronger/higher, this is ideal for complex formation, in typical humoral immune responses it ranges from 10^-7 to 10^-11

avidity

the overall strength of binding between a multivalent antibody and its corresponding multivalent antigen, it considers all binding interactions in a complex

monovalent

this is a very sparse epitope and the avidity is very low because only one binding site available

bivalent

this is what we see in an IgG antibody. There are two binding sites available, so the avidity of the interaction is high, and this is much higher than the affinity

polyvalent

this is what we see in an IgM antibody where there are 2+ binding sites so in this case because it’s a pentamer there are 10 binding sites. Because IgM is the first antibody class formed during a primary immune response, there is no somatic hypermutation, so affinity is low, but it compensates with avidity being very high

how are different antibody classes distinguished?

by their heavy chain constant regions (does not apply to light chains), they also differ in the distribution of carbohydrate groups

IgM and IgE

lack a hinge region but each contains an extra heavy chain domain

IgD

major role as membrane bound Ig

IgE

normally very low serum levels but they are elevated in allergic reactions

secretory Ig

IgA and IgM can be transported across epithelial barriers into the gut lumen

placental transfer

the neonatal Fc receptor carries IgG across the placenta and prevents IgG excretion from the body, it recycles it so IgG ½ life is longer

neutralization

high affinity IgG and IgA antibodies can neutralize toxins and block infectivity of viruses and bacteria

complement activation

antigen/antibody complexes activate the classical complement pathway by binding to C1q (IgM is a better activator of complement than IgG)

opsonization

Fc receptors recognize antibodies (IgG, IgA) bound to pathogens and promote phagocytosis

parasite defense (and allergies)

mast cells and basophils bind IgE antibodies via the high affinity Fce receptor, secrete inflammatory mediators

dimeric IgA

it is transported into the gut lumen through epithelial cells at the base of the crypts, it binds to the layer of mucus overlying the gut epithelium, in the gut it acts to neutralize pathogens and their toxins, it contains a secretory component that protects IgA from proteolysis

IgA structure

it has two functionally identical subclasses (IgA1 and IgA2), there are 3 Ca domains in the IgA heavy chain, the J chain forms the dimer, it is the most abundant Ig in secretion

plgR

poly Ig receptor, it allows binding of IgA dimer + transportation into the lumen

IgM

it is a pentamer (J chain formed it) and the largest antibody isotype, it generates the primary antibody response, the most important antibody gene for new chain is the closest to gene segments for the variable region, they are low affinity but high avidity due to pentameric form, it is the best antibody for activating complement

how does IgM activate complement?

the first way is pentameric IgM molecules bind to antigens on the bacterial surface and adopt the staple form/bound to the membrane, then C1q binds to one bound IgM molecule leading to the activation of C1r which cleaves and activates the serine protease C1s

how does IgG activate complement?

the IgG molecules bind to antigens on the bacterial surface and C1q binds to at least 2 IgG molecules, this binding activates C1r which cleaves and activates the serine protease C1s

IgG

it is the most abundant class in serum, it has a longer half life than other antibody classes, it is the predominant antibody in secondary responses due to its high affinity from somatic hypermutation there are 3 Cy domains in their heavy chains and 4 different classes (IgG1 - IgG4)

how do the IgG subclasses differ?

they differ by length of hinge regions and number of disulfide bonds

which IgG subtype is the best for placental transfer?

IgG1 followed by IgG3, IgG2 and lastly IgG4

which IgG subtype is the best for complement activation?

IgG3 but importantly IgM is the most favorable antibody for complement activation, but this is followed by IgG1, IgG2 and lastly IgG4

which IgG subtype is the best for macrophage binding/opsonization?

IgG1 and IgG3

constant region

confers functional specialization on the antibody

how did Tonegawa discover that V genes rearrange in B cells?

he took DNA from B cells and non-B cells/germline DNA, he cut and hybridizes the DNA segments with radioactive probes and found that rearrangement brings 2 distant regions of DNA segment close together (only saw this in B cell not germline DNA)

λ light chain locus

it is less variable than the k light chain (29-32 V segments), and the number of joining regions is equivalent to the number of constant regions (4-5 each)

k light chain locus

this is more variable than the λ light chain locus (31-35 V segments) however it only has 5 J segments and 1 constant region

heavy chain locus

it differs from the light chain due to the presence of diversity segments (23), it also has a greater number of variable segments (38-46), joining (6) and constant regions (9)

Ig variable region structure

it has 3 CDRs/HVs and it is encoded within a single V region exon which is formed by the joining of VDJ gene segments, the first 2 CDRs are contained within the V gene segment while the 3rd is at the junction between V and J gene segments

how are light chain V regions constructed?

they are constructed from gene segments, first the germline DNA undergoes somatic recombination so the D-J region is rearranged and the exon is there to send protein to the surface of the cell, then recombination occurs again so either the V-J or V-DJ regions are joined and the DNA is rearranged, this DNA then undergoes transcription creating the primary transcript and then polyadenylation of the transcript marks the end of it so that the polypeptide chain is formed producing the mature mRNA post translation

how is heavy chain V region construction different from light chain?

somatic recombination occurs twice, the first time joins the D and J segments together and the second joins the V and DJ segments together creating the full rearranged DNA segment

recombination signal sequences (RSS)

they are flanking DNA sequences that guide the rearrangement of V, D and J gene segments, this involves a 12bp RSS and a 23bp RSS

12/23 rule

this rule states that a gene segments with a 12bp will only recombine with a segment that has a 23bp RSS

nonamer and heptamer

these regions are conserved in recombination

RAG

enzyme that allows recombination

joining segments with the same transcriptional orientation

this is the more common mechanism for recombination where the intervening DNA is looped out and lost from the chromosome, it results the formation of an excision circle (signal joint) and a coding joint, the loop gets excised from the chromosome taking the two RSS regions with it

joining segments with opposite transcriptional orientation

this is the less common mechanism where alignment of the RSSs requires a coiled configuration, thus joining the ends of the two heptamer sequences now results in the inversion and integration of the intervening DNA into a new position on the chromosome, coiled region is retained in an inverted orientation

enzymatic steps of RAG dependent VDJ rearrangement (light chain)

in the cleavage step, the endonuclease activity of RAG makes single strand cuts in the DNA backbone precisely between each coding segment and its RSS, at each cutting point this creates a 3-OH group which then reacts with a phosphodiester bond on the opposite DNA strand to generate a hairpin leaving a blunt double stranded break at the end of the RSS, the proteins keep the ends of hairpin DNA protected

RAG1

operates cut between each coding segment and its RSS (single cut)

RAG genes

they are expressed only in developing B and T cells, the RAG knockout mice fail to develop B or T lymphocyte and mutations in RAG1 or RAG2 mutations are a cause of SCID (severe combined immunodeficiency, lack B and T lymphoytes)

artemis/DNA-PK

this opens the hairpin by cutting it at several positions creating sticky DNA, which generates palindromic (P) nucleotides that diversifies the joints between gene segments during immunoglobulin gene rearrangment

TDT

terminal deoxynucleotidyl transferase, it modifies the cut end of the DNA hairpin by adding random extra N nucleotides creating diversity,

DNA ligase (IV:XRCC)

this ligates DNA ends forming an imprecise coding joint or in signal joints it would be precise

P nucleotides

they are in the VJ junction due to the asymmetric nicking of a hairpin end, its palindromic meaning the DNA is read the same way from either direction, only found in the coding joint

N nucleotides

they are added by the enzyme Tdt which is only expressed in developing B and T cells, only found in the coding joint

recombinational inaccuracy

creates a huge level of diversity in CDR3, this is the variable addition and subtraction of nucleotides at the junctions between gene segments, 2/3 of the rearrangements are non-productive/out of frame leading to in frame stop codons (TAA, TAG or TGA in the J or C region), so greater diversity trade off for loss of efficiency

IgM and IgD

they are derived from the same pre-mRNA transcript and are both expressed on the surface of mature B cells, this is possible because they have different polyadenylation sites

transmembrane and secreted forms of Ig

they are derived from the same heavy chain sequence by alternative RNA processing