biol 485 quiz #4 (lectures 9-10)

where do the main mechanism of action of antibodies occur?

where do the main mechanism of action of antibodies occur?

extracellularly (blood, lymphatic fluid, interstitial tissue areas)

what are three mechanisms of action by antibodies?

neutralization, opsonization, complement activation

what is neutralization?

antibodies bind the bacterial/viral toxins and neutralize them so the bacteria/virus can’t find their receptor

what is opsonization?

antibodies bind bacteria/viruses, allowing other cells to opsonize them and destroy them

what is complement activation?

when antibodies coat an enveloped virus/bacteria, they are capable of activating complement near the surface of that virus/bacteria

what is the structure of an antibody?

a quaternary protein with 2 identical heavy chains & 2 identical light chains

how does antigen specificity occur?

by the interaction between light/heavy chain variable regions, called the Fab (fragment of antigen binding)

how does antibody effector activity occur?

the interaction of the constant regions of the heavy chain, called the Fc (fragment that crystalizes)

what part of the antibody is the specificity?

the arms

what part of the antibody determines the effector function?

the base

what is isotype switching/class switch recombination?

when you switch out the conserved domains of the antibody

once you make the antibody, which domain is fixed?

the variable domain

Ig proteins are consisted of what?

2 identical heavy chains, 2 identical light chains which can be either kappa or lambda

since we are diploid organisms, how many loci do we have for the heavy chain?

2 (one from dad, one from mom)

since we are diploid organisms, how many loci do we have for the light chain?

4 (one kappa, one lambda from mom; one kappa, ome lambda from dad)

what do B cells use recombination of gene segments in the BCR loci for?

to create diff possible antibody combinations

what gene segments do the light chain locus have?

variable (V), joining (J), and constant (C) regions

what gene segments do the heavy chain locus have?

variable (V), diversity (D), joining (J), and constant (C)

what type of recombination occur in the light chain?

VJ recombination

what type of recombination occur in the heavy chain?

VDJ recombination

what happens first in VDJ recombination?

D recombining with J

how is the light chain on the BCR made?

one V segment joins to one J segment through somatic recombination

which locus does light chain somatic recombination occur in?

either the kappa or the lambda locus

how is the heavy chain made?

one D will join with J. this DJ will join with one V, making a final VDJ, all using somatic recombination

what is the default conserved/constant region?

Mu

if the cell successfully recombines the kappa locus, what needs to happen to the lambda locus?

it needs to shut off b/c we don’t want diff light chain receptors

how many possible combinations do we have for the human heavy chain locus?

6,210

how many possible combinations do we have for the light chain kappa?

205

how many possible combinations do we have for the light chain lambda?

165

how many variations of antibodies do we have just from V(D)J recombination?

2.3 million

what is recombination directed by?

signal sequences

what flanks each antibody gene segment?

recombination signal sequences (RSS)

every V segment has an RSS _________________ to it

directly adjacent (downstream)

every J segment has an RSS __________

upstream

D segments are __________ by RSS

flanked

what conserved sequences do RSS have?

nonamer and heptamer

how are the nonamers and heptamers separated?

by either a 12 or 23 bp sequence

what are the two rules in order for RSS to occur?

“12/23” rule & opposite orientation rule

what is the direction of the RSS based off of?

where the heptamer is to the nonamer

if the V segment is facing forward and is 23 bp and the D segment is facing reverse and is 12 bp, can they recombine?

yes

if the V segment is facing forward and is 23 bp and the J segment is facing forward and is 12 bp, can they recombine?

no

what recognizes RSS sequences?

RAG1 and RAG2

what does RAG1 stand for?

recombination activating gene 1

what does RAG1 and RAG2 form upon binding to RSS sequences?

a tetramer

what does RAG1/2 bind to?

the RSS

what does the RAG1/2 complex do?

cleave DNA at the junction between the RSS and the variable coding region

what is the signal joint?

the location where the two RSS are joined

what is the coding joint?

the location where the two coding segments are joined

RAG1/2 makes a ______________ at the junction between the RSS and coding segment

single strand break

what causes a hairpin on the coding segment?

nucleophilic attack by the 3’-OH after the break

what happens when the hairpin is opened asymmetrically?

templated addition of palindromic nucleotides, called P nucleotides

what happens when the hairpin is opened symmetrically?

addition of nucleotides by TdT, called N nucleotides

what is junctional imprecision?

RAG1/2 cutting doesn’t give a clean joining but nucleotides are added and/or lost to give even more variability at the junctions of VD, DJ, or VJ

what type of gene can junctional imprecision lead to?

a dead gene due to an early stop codon

what are the mechanisms to generate antibody diversity in B cells?

multiple gene segments, heavy chain/light chain combinatorial diversity, P nucleotide addition, exonuclease trimming, nontemplated N nucleotide addition

what is heavy chain/light chain combinatorial diversity?

the same heavy chain can combine with diff light chains and vice versa

what is P nucleotide addition?

templated nucleotides addition between coding joints, resulting from asymmetrical cleaving of hairpin structures

what is exonuclease trimming?

sometimes occurs at coding joints in which nucleotides are lost and reading frames are changed

what is nontemplated N nucleotide addition?

adding in random nucleotides between coding joints; mediated by TdT activity

what is V(D)J recombination?

somatic cell recombination occurring in B cells at the chromosomal level

what does every V segment have upstream of it?

a promoter

why can’t a V segment promote transcription without being recombined first?

it needs to be in close proximity to the enhancer in order for transcription to occur. this only occurs when V is recombined with J since the Js are next to the enhancer

what are the 5 distinct classes of antibody (isotypes)

IgA, IgD, IgE, IgG, and IgM

where are the conserved (C) gene segments located on the heavy and light chain loci?

downstream of the V(D)J gene segments

when are the C segments added?

after V(D)J recombination, after transcription of the DNA into RNA, at the step of mRNA splicing

which C segments will the heavy chain splice with?

the most downstream segment (C mu)

where does V(D)J recombination occur?

in the bone marrow

what do B cells recombine first?

the heavy chain locus

what is a pre-BCR?

heavy chain dimer with surrogate light chains

what does the pre-BCR allow?

for the B cell to test if heavy chain recombination was successful, & proper signaling results in proliferation of the B cell

what happens if the heavy chain recombination was successful?

a single light chain will recombine (either on locus of kappa or lambda)

what results from successful recombination of the heavy and light chains?

mature BCR complex

what does allelic exclusion ensure?

that each developing B cell synthesizes only one heavy and one light chain

what does production of a functional pre-BCR signal?

an end to heavy chain recombination

what does production of a non-functional pre-BCR lead to?

VDJ recombination of the other heavy chain locus

what happens if the B cell does not produce a functional pre-BCR after attempting both loci of the heavy chain?

apoptosis

what does production of a functional mature BCR signal?

an end to light chain recombination

what does production of a non-functional mature BCR lead to?

recombination of a diff light chain locus

what happens if the B cell doesn’t produce a functional mature BCR after attempting both the kappa and lambda alleles?

apoptosis

list the useful categories of MHCI

every nucleated cell, internal proteins, CD8+ T cells, cytotoxic T cells

list the useful categories of MHCII

pAPCs, external proteins, CD4+ T cells, T helper cells

how are T cells distinguished from B cells?

by the expression of the T cell receptor that will detect non-self in the context of a self protein called MHC

what can activate immune cells?

having a non-self peptide MHCI or not having MHCI at all

what does MHC stand for?

major histocompatibility complex

which cells present MHCI?

every nucleated cell

where do MHCI cells sample proteins from?

internally

where do professional antigen sample proteins from?

externally

professional APCs can also __________, when they take in external antigen and present it on MHCI

cross-present

what combination of MHC and peptide give a “no danger here” signal?

MHCI + self peptide

how many points of contact between pAPC and T cell do TCR signaling complex have?

3

what does the TCR recognize?

both the peptide and the MHC that the peptide is presented in

where does the TCR touch the MHC?

on the outside

what are the coreceptors of a TCR?

CD4 and CD8

what do CD4 and CD8 bind to?

the MHC

what does the binding of CD4/CD8 to MHC cause?

increased avidity of the binding

what else is required for T cell activation?

a second signal provided by CD28 coreceptor binding to CD80 or CD86 on the pAPC

what does it mean if CD80 or 86 is on?

that the non-self peptide that is being presented came from an inflamed environment

what does the TCR rely on for signal transduction?

an associated CD3 complex

what does the CD3 complex consist of?

3 dimers: δε pair, γε pair, ζζ (zeta-zeta) pair

what interaction does not initiate a signal during T cell activation?

CD4/CD8 coreceptor binding MHC