4- Antibody Structure/B Cell Diversity

What are antibodies?

immunoglobulins, made by effector B lymphocytes/plasma cells (activated B cells)

Where can antibodies be found?

secreted by plasma cells into

• plasma of blood

• lymph

• moved across epithelial surfaces to mucosal surfaces

• ex → mucus, tears, breast milk

What is the most common antigen?

proteins & carbs, found on pathogens or other substances

What distinguished B from T cells?

B cells binding to protein/carb antigens (CHECK)

What does vaccine protection rely on?

antibodies, want vaccines to make good antibody responses

What are effector functions?

neutralization of viruses & intracellular bacteria & opsonization/phagocytosis

What is antibody specificity?

can only bind to 1 antigen/small number of antigen

• 1 antigen detected by both arms, can be cross reactive

• 1 B cell is committed to making 1 type of antibody

• can recognize same antigen on 1 B cell, often repetitive components

• 2 binding sites = 2 places on 1 microbe or on 1 place on 2 microbes

if 1 thing is the same on multiple microbes, that B cell can bind to them

ex → surface proteins on small pox, cowpox, monkeypox are same, DNA & RNA viruses

What is an antibody repertoire?

all you individual antibody specificities, 10^9 specificities for diff Ab

• anything we interact w/ will make antibodies

• collection of all different unique antibodies w/in blood

What is clonal expansion?

massive proliferation of B cell w/ relevant specificity to infxn

• occurs in germinal center in lymph node

• multiple specificities can be activated at same time in diff germinal centers

What is the process of clonal expansion?

1. small number of B cells w/ a specificity

2. B cell recognizes an infxn (correct antigen)

3. B cell proliferates & differentiate into plasma cells 

What are the antibody isotypes?

diff classes of antibodies, MADGE

• IgM/Mu, IgA/alpha, IgD/delta, IgG/gamma, IgE/epsilon

Which antibodies have 4 H chains?

IgE, IgM

Which antibodies can act as B cell receptors?

IgM & IgD (DMs = let into your messages)

• can still be secreted

Which antibodies are exclusively secreted?

IgA, IgG, IgE (AGE)

What is IgG?

most abundant in serum, most versatile

Which antibody is made & secreted 1st?

IgM

T/F: All antibodies are immunoglobulins, but not all immunoglobulins are antibodies.

True, all antibodies are Igs

What is IgA?

important at mucosal surfaces

What is IgE?

predom used in parasitic infxns, also in allergy

What is the structure of antibodies?

2 heavy & 2 light chains of protein/polypep chains

• 1 arm = full light, some of heavy/amino terminal

• base = all heavy/carboxy terminal

How are the 2 heavy chains connected?

disulfide bonds in hinge region, allows movement & holds H/L

• lets Ab mols to bind w/ both arms to many diff arrangements of Ag on pathogens

What is the Fab region?

variable region, Light/heavy, binds antigen in pocket where H/L come together

• interacts w/ antigen

• each side has 2 pockets

What is the Fc region?

constant region, heavy

• binds host cells or Fc receptors if free floating

• interaction w/ host cell membs mediates a lot of interactions

• doesn’t interact w/ antigen

• encoded region of heavy chain encoded in genome, min of 5 for each diff Ab isotype

• bind to serum proteins & cell surface receptors

• interact w/ host cells (receptor interact, memb bind)

How does antibody binding occur?

disulfide bonds have flexibility, slight bend → interact w/ antigen on bact surface

• bacteria has diff surface proteins w/ diff shapes

• Ab has 2 binding sites → can bind 2 antigens at same time, both arms bind same type 

  • ex → both site binding a circle antigen

• hinge region gives Ab flexibility to bind antigens in diff areas

  • on same microbe or on 2 diff microbes but same antigen

  • distance b/w antigens

• 1 bacteria can have diff antigens, can have 3 diff antibodies for each diff antigen

What is the immunoglobulin fold?

regions of B sheets, Ab made up of Ig folds

• light chain = 2 Ig folds per chain

• heavy chain = 4 Ig folds per chain

What is the Ag binding site?

hypervariable regions of H chain V domain & L chain V domain

Which component of the L/H chain binds to the Ag?

CDR loops/hypervariable regions

What are CDR loops?

complementarity determining region loops, make up hypervariable region

• ends of L/H chain that bind antigen

• 3 loops in each H/L chain

• differences in aa sequence, residues interacting w/ microbe, w/in binding site

Why are antigen binding sites hypervariable?

differences in aa sequences let them bind many diff antigens

What is an antigen?

something an Ab binds

What is an epitope?

actual aa of antigen that Ab binds w/ CDR loops in hypervariable region

What changes in reinfection?

epitope changes when reinfected w/ something already vaccinated against

T/F: An antigen can have only 1 epitope.

False, antigen can have multiple epitopes, & be bound by multiple diff Ab

How are antigens multivalent?

multiple Abs can bind diff epitopes on the same antigen, bind more than once

• multiple Abs can bind multiple of same epitope on antigen

• BCRs do multivalent interactions due to steric hindrance

Why are multivalent interactions necessary?

so Ab can do neutralization & opsonization

How are antigen binding sites shaped?

invaginations of different shapes

• can have a region on site sticking out, sticks into antigen pocket

• Ab + Ag match shapes for binding 

What are linear & discontinuous epitopes?

can bind epitopes/aa sequences in dis/continuous manner

What are linear epitopes?

continuous, bid to aa next to e/o in aa sequence & folded protein

What are discontinuous epitopes?

more common, aa that aren’t next to e/o in aa sequence, but are in folded protein

What are monoclonal Abs?

pool of Abs specific to an epitope of an Ag

• only 1 specificity → all Abs identical

• most Ab treatments given are monoclonal → specific to pathogen

• don’t exist in wild, lab made

What are polyclonal Abs?

many diff Abs w/ diff specificities, how Abs are really found

• multiple plasma cells w/ Abs

What is an antiserum?

polyclonal, serum w/ all Abs of recognized over life

• treatments for people w/ B cell disorder that don’t make Abs

• used to provide passive immunity to infections and diseases.

What is unique about Ig genes?

• organized differently, number of diff Ag made is limitless

• only expressed in B cells

• instead of 1 complete gene, Ig H & L chain loci have gene segments w/ alt versions of part of V regions

  • express Ig gene = need to recombine 

  • DJ remixing tracks for a better music flow

T/F: For the Ig gene to be expressed, segments must be rearranged to assemble functioning gene, only happens in developing B cells.

True, an Ig gene is only functional this way, and this only happens in B cells

When will a membrane bound Ig appear at the B cell surface?

→ when rearrangements done & complete H/L chains made

Where are Ig genes found?

H chain locus

k L chain locus

lambda L chain locus

What segments make up the variable region?

V → variable

D → diversity (H chain only)

J → joining

• Heavy = VDJ

• Light = VJ

What are V regions encoded by?

VL & VH segments that undergo rearrangement during B cell development, facilitating diverse antibody specificity.

What are the 2 types of gene segments that encode the L chain V region?

variable & joining gene segments

What makes the H chain locus unique?

additional set of diversity/D gene segments

What is somatic recombination?

cutting & resplicing of VDJ segments in B cells to generate diverse antibody specificities

• makes single gene segment of L/H to make DNA sequence for Ig chain’s V region

How does somatic recombination occur for the L chain?

single recombination b/w V & J segments

How does somatic recombination occur for the H chain?

1. DJ segment

2. DJ segment to V segment for VDJ

What are the 2 options for light chains?

K & lambda light-chain loci, only use 1 at a time, never both 

Is VDJ recombination random or calculated?

random process, allows endless variety unless all options are chosen

What is the lambda light chain locus?

all variable segments 1st, 5’ end, then alternating joining & associated constant region

What is the K light chain locus?

all variable segments (5’ end) then all joining segments then all constant regions

What is the difference between the lambda & K light chain loci?

no difference in function, K just is done first, if K fails, go to lambda

What is the heavy chain locus?

all variable segments (5’ end) then all diversity, then all joining then all constant regions

What determines the class of Ab?

constant region of heavy chain

Which light chain locus has more options/segments?

K light chain locus

Which segment has the most number of segments total?

variable in general, specifically H chai

Which chain has the most constant & joining segments?

heavy chain

What is the general order of increasing number of segments?

lambda least < K < H

EXCEPTION → K < lambda < H for constants 

What is the RSS?

recombination signal sequence

• heptamer, nonamer & spacer in between

Why is the RSS important for VDJ recombination?

signals to recombine, can’t do a recombination w/o it

• V → 3’ 

• D → 5’ & 3’ (2 RSS)

• J → 5’ 

What is a heptamer?

always touches segment

What is a nonamer?

on inside, face other segment

What is unique about heavy chain recombination?

V & J always have same spacer to prevent recombination deleting diversity segment 

• diversity segment has same spacer (12) on 5’ & 3’ RSS

What is RAG 1/2?

recombinating activating genes, enzyme doing VDJ

• only expressed in developing B & T cells

• 1 binds to 12 spacer, other to 23 spacer 

How does RAG ½ initiate VDJ recombination?

loops segment of DNA to be recombined

• scrunched = heptamer & nonamer w/ 12 spacer 

• longer = heptamer & nonamer w/ 23 spacer

What spacers are needed for enzyme conformation?

12 & 23 spacer, only way to do recombination

• NEVER do 12 & 12 or 23 & 23 

What is the unanimous 2nd step in VDJ recombination?

RAG ½ cleaves b/w segment & heptamer

What are the products of VDJ recombination?

coding joint & signal joint

What is the coding joint?

V & J/VDJ segments come together, are ligated

What is the signal joint?

unwanted DNA, will be degraded, DNA b/w heptamers of coding joint, ligated at heptamers 

• all DNA still in germ line cells → pass on all possibilites of B/T cell segments

What enzyme is unique to heavy chain rearrangement?

TdT, which adds N-nucleotides/noncoded/random to the ends of the DNA during recombination

• must add in 3s for a codon otherwise frameshift mutation

T/F: Junctional diversity is unique to the heavy chain only.

False, junctional diversity occurs in both heavy and light chains during B cell receptor formation.

What happens to unpaired N nucleotides?

exonuclease will delete them 

What adds in base pairs for N & P nucleotides?

NHEJ complex 

What is junctional diversity?

increase diversity in aa sequence of H & L chain CDR3s

• mechanism increaseing diversity w/ random incorporation of additional nucleotides to coding joint

• insert residues into CDR loops for antigen diversity of Abs

T/F: Junctional diversity, although random, is still encoded in germline DNA.

False, generated by enzyme action (TdT & RAG)

Which enzymes are unique to VDJ recombination?

RAG ½ & TdT

What is splicing?

get rid of intro regions of pre-mRNA & left w/ exons

• where constant regions are brought in

• no loss of DNA, can go back & make og mRNA

What is alternative splicing?

use alternative exons not usually expressed

Which Ab is the first to be made?

IgM

• unless signal to do alt splicing & make IgD

Which Abs are expressed o surface of a B cell?

IgM & IgD, BCR can use wither as constant regions 

• use alt splicing to choose IgM & IgD

When is the constant region chosen?

after VDJ recombination, chosen by splicing/alt splicing

How is IgM expressed?

normal splicing for

• Cu → constant for IgM

• MC → transmembrane segment, makes membrane bound

• dominant, trim out introns

• sequence at bottom adds transmembrane region to be embedded in PM

How is IgD expressed?

alt splicing chooses

• Cdelta → IgD constant region

• AAA → transmembrane region for IgD

What components make up BCR?

IgM/D, Iga & IgB

How does signaling occur with a BCR?

Iga & IgB, heterodimer & signaling domain of BCR, have immunoglobulin fold

• no variability, same across all B cells 

What does the BCR do?

sample diff antigens until activated, once activated improves the B cell

What happens in the BCR when the antigen binds?

activate signal cascade, send down through Iga & IgB, leading to B cell activation

• send down to turn on transcription factors to proliferate & differentiate 

Where does signaling start at in the BCR?

tails of Iga & IgB, long cytoplasmic tails for signaling

What is somatic hypermutation?

B cell activated by Ag, further diversification of V domain sequence

• made VDJ just not mature yet

• reengage AID, reeval variable sequence

  • H & L mini change

  • constants don’t change

What is unique about somatic hypermutation?

unique to BCR, T cells don’t get better over time

• induce prolif of B cells, each has slightly diff Ig

• only in B cells that have seen antigens/been activated by them

What mediates somatic hypermutation?

AID enzymes, activation induced deamination

• induces many mutations in variable region

• C → U conversions

• DNA Pol sees U, gets confused, repaired w/ random nucleotide by NHEJ = mutation

What is affinity maturation?

The process by which B cells produce antibodies with increased binding affinity for an antigen through somatic hypermutation and selection.

• select for mutations that bind antigen better

• make all mutations, test to see which one aid in Ag binding 

• don’t change specificity, just changing bind & affinity

What is the benefit of a booster vaccine?

re-expose us to get Ab that that binds better, affinity maturation