S2 I&i Exam 4: Ig & Tcr Structure

Front 1

Differentiate btwn precipitation & agglutination of antigens by antibodies (Abs)

Back 1

precipitation:
SOLUBLE antigen

agglutination:
INSOLUBLE/particulate antigen

*in both cases large bound antigen cannot stay suspended & settles

Front 2

What occurs when an Ab binds an Fc receptor?

Back 2

phagocytosis of antigen


(Fc portion of Ab molecule binds to Fc receptor on phagocytotic cell)

Front 3

where are Fc receptors expressed?

Back 3

macrophages
neutrophils
eosinophils
dendritic cells
NK cells

Front 4

Differentiate btwn the 3 main Fc receptor types

Back 4

Fcg receptors: FcgRI, FcgRII, and FcgRIII

Fca receptors

Fce receptors: FceRI and FceRII]
on mast cells, eosinophils

Front 5

Part of the H chain & the entire L chains each form an arm (N terminal) of the Y shaped Ig, the remaining portion of the 2 H chains (C termini) forms the body of the Y. The body is connected to each arm by ___________ bonds.

Back 5

Disfulfide

Front 6

T/F

Ig can be membrane bound on B lymphocytes OR soluble as one component of the serum in blood.

Back 6

True!


soluble Ig = antibody

Front 7

What site of the Ig is formed by the paired V (variable) regions of L and H chains?

Back 7

Antigen-binding site

Front 8

Ab molecules have a relatively unstructured portion in the middle (btwn body & arms), forming the flexible _______________.

Back 8

Hinge region

Front 9

Papain (plant protease) cleaves IgG AT the hinge region, what 3 fragments does this produce?

Back 9

2 Fab fragment (fragment antigen binding)
^corresponding to the arms

1 Fc fragment (fragment crystallization)
^corresponding to the body/stem of Y shaped Ab

Front 10

Pepsin (gut protease) cleaves IgG BELOW the hinge region (below level of disulfide bonds), what 2 fragments does this produce?

Back 10

1 F(ab)2 fragment
^corresponds to both arms joined by disulfide bond

1 Fc fragment
^rest of stem, unstable--> broken down

Front 11

What is the term for the classification of different Ig molecules based mainly on AA sequence ?

Back 11

Isotype

Front 12

Do isotypes depend on heavy chains, light chains, or both?

The differences are best defined in the (constant/variable) regions of these molecules.

Back 12

Both
(H chain has more influence)

Constant

Front 13

IgA, IgD, IgE, IgG, and IgM (5 diff isotypes) are defined based on differences in (L/H) chains.

Back 13

H

Front 14

Kappa and lambda are defined based on differences in (L/H) chains.

Back 14

L



**L chains is formed by ONE C domain

Front 15

a) IgA, IgD, and IgG are made up of how many Heavy chain C domains?

b) How many Heavy chain C domains are in IgM and IgE?

Back 15

a) 3

b) 4 (the extra domain is the elongated hinge region)

Front 16

What is the term for identifiable allelic variations in the constant regions of the heavy or light chain genes?

(for example: G3m(b0) is a variant of IgG3 characterized by a phenylalanine at position 436 of gamma3 chain. not present in all people)

Back 16

Allotype

Front 17

What is the term that refers to unique features of the antigen binding site (comprising both H & L chain V/hypervariable regions) that determine the antigen specificity of a given Ig molecule

Back 17

Ideotype

Front 18

Differentiate btwn private & public ideotypes

Back 18

private- w/i an individual, different ideotypes (ex numerous B cell variants)

public- 2 individuals share same idiotypic determinant--> ideotype is cross reactive

Front 19

Which Ig is predominantly present as a pentamer in the serum?


Which Ig is most abundant?

Back 19

IgM



IgG1

Front 20

IgA monomer weighs abour 160kDa, while what form of IgA weighs about 390 kDa?

Back 20

Secretory IgA

Front 21

What are the 3 major functions of Ab?

Back 21

Neutralization
Opsonization
Complement activation

Front 22

What is the process of coating the surface of pathogen or other particle with a particular molecule that makes it more readily ingestible by phagocytes?

Back 22

Opsonization

(ex: antibody & complements opsonize pathogens to be phagocytosed by neutrophils and macrophages)

Front 23

What is a group of serum proteins that, when activated, participate in a controlled enzymatic cascade which destroy pathogenic organisms by formation of a membrane attack complex (MAC)?

Back 23

Complement

Front 24

What is it called when Ab binds to bacterial, viral or protein (ex: toxin) in such a wway as to inactivate them and prevent their causing harm?

Back 24

Neutralization

Front 25

The differences in the aa sequences of the V domains between different Ab molecules are concentrated within particular regions, which are called what?

Back 25

Hypervariable regions

Front 26

The hypervariable region of the H and L chains are brought together to form a hypervariable surface called what?

Back 26

Antigen binding site

Front 27

What are the much less variable regions flanking the hypervariable regions called?

Back 27

Framework regions

Front 28

What is another name for the hypervariable loops?

Back 28

Complementarity-determining regions (CDRs), because they provide a binding surface that is complementary to that of the antigen

Front 29

What term is used in relation to a protein antigen to refer to the "normal" 3D structure where many nearby aa's are not necessarily contiguous in sequence.

Back 29

Native

Front 30

What is a protein called that has been removed of its 3D structure and is simply a connected string of aa's?

Back 30

Denatured

Front 31

What is a term for a small chemical complex that determines the specificity of an antigen-Ab interaction?

Back 31

Epitope (antigenic determinant)


*epitope= specific part of the Ag that the Ab binds

Front 32

Viral capsids are often composed of repeating protein subunits, and would therefore be ___________.

Back 32

multivalent

Front 33

a) What is the term for the number of different antibodies (in terms of antigen specificity) an individual is capable of making?

b) What is this equivalent to?

c) What has the size of a human's been estimated to be?

Back 33

a) Antibody repertoire

b) B cell repertoire (the number of different B cell clones that exist prior to immunization & express membrane Ig molecules with distinct specificities)

c) >1 billion

Front 34

a) What is an antibody produced by an individual clone of a B cell?

b) What can it be used for?

Back 34

a) Monoclonal antibodies


b.) used to generate large # of antigen specific antibodies for in vivo therapy or in vitro diagnosis purposes

Front 35

How are monoclonal antibodies produced in the lab?

Back 35

-from hybridoma cells = B cells fused to myeloma/tumor cells using PEG
-cells cultured w/ HAT
-A (aminopterin) inhibits de novo synthesis
-H (hypoxantine) & T (thymidine) necessary for nucleotide synthesis allows fused cells to replicate
-remaining myeloma lack HGRPT--> die (along w/ B cells from mouse spleen)
-fused cells w/ desired Ab (from B cell) are selected & rest are discarded = monoclonal antibody
(myeloma portion allows fused cell to continuously replicated= tons of Abs)

Front 36

Define germline DNA.

Back 36

Un-rearranged Ig gene DNA found in germ cells, all non-B cells, and the earliest B cell progenitor cells. The DNA that is inherited

Front 37

Name the position that a gene occupies in a chromosome.

Back 37

Locus

Front 38

a) Where is the kappa light chain locus?
b) How many functional V-kappa gene segments?
c) J-kappa segments?
d) C-kappa segments?

Back 38

a) Chromosome 2
b) 40
c) 5
d) 1

Front 39

a) Where is the lambda light chain locus?
b) How many functional V-lambda gene segments?
c) J-lambda segments?
d) C-lambda segments?

Back 39

a) Chromosome 22
b) 30
c) 4
d) 4

Front 40

a) Where is the heavy chain locus?
b) How many functional V-H gene segments?
c) D-H segments?
d) J-H segments?
e) C segments?

Back 40

a) Chromosome 14
b) 65
c) 27
d) 6
e) 1

Front 41

T/F

Even though the number of genes are limited, Ig V-region sequences are constructed from gene segments, which can "mix and match" to make a large number of combinations to make >10 billion different sequences

Back 41

True!

Front 42

Antibody gene segments for V region are rearranged to construct V-region sequences. What is this called?

Back 42

Somatic recombination

Front 43

How do the 1st and 2nd hypervariable regions differ from the 3rd hypervariable region?

Back 43

The 1st and 2nd are completely within the V segment, while the 3rd is determined by the junction between the V and J segments.

Front 44

Construction of the H-chain V region involves joining which gene segments?

Construction of the L-chain V region involves joining which gene segments?

Back 44

V, D (diversity), & J
(First D and J join, then V joins the combined DJ sequence)

V & L

Front 45

A functional L-chain gene consists of what 3 exons?

What are they separated by?

Back 45

L (leader peptide), V, & C


separated by introns (non coding regions)

Front 46

What determines the sequence variability of the 3rd hypervariable regions of the H-chain?

Back 46

Difference in D gene segments (27) and its junction with V and J gene segments

Front 47

Is somatic recombination the same process or different for TCR vs BCR/Ig/Ab?

Back 47

Same

Front 48

Each gene segment (V, D, J, and C) is flanked by specific sequences used by the recombination machinery. What are they called?

Back 48

Recombination signal sequence (RSS)

Front 49

What is the RSS composed of?

Back 49

1. A conserved 7 base segment (heptamer)
2. A non-conserved spacer (either 12 or 23 bases)
3. A conserved 9 base segment (nonamer)

*RSS provides recognition sites for VDJ recombinase (enzyme that cuts & rejoins DNA)

Front 50

What are the main enzymes/proteins involved in the joining and that recognize the RSS's?

Back 50

RAG-1 and RAG-2

(RAG = recombination activating gene)

Front 51

A nonsense mutation (no function) in the RAG gene leads to what?

A missence mutation (some function) in the RAG gene leads to what?

Back 51

nonsense= No T or B cell fxn = SCID (severe combined immunodeficiency disease)
-present w/ multiple infection including thrush, opportunistic infection (cough, pneumonia)

missense= some T cell fxn= Ommen syndrome
-multiple infection including red rash, failure to thrive

Front 52

What is the rule of 12/23?

Back 52

An RSS with a 12 base spacer is only joined with an RSS with a 23 base spacer.

Front 53

RAG-1 & RAG-2 proteins interact w/ each other & with _____________ to form a RAG complex

Back 53

HMG (high mobility group proteins)

Front 54

What keeps 2 variable regions from being joined together?

Back 54

The two RAG complexes bind to different RSS flanking segments
One binds to the 23 bp V segment, the other to the 12 bp J gene segment of another RSS flanking segment
--> thus the segments will be joined V with J

Front 55

The interaction of what brings the ends of the segments (V,D,J,C) to be joined in close proximity?

Back 55

the 2 RAG complexes w/ with the 2 RSS

Front 56

After the ends of the segments (V,D,J,C) are joined together in close proximity, the ends of the DNA are then cleaved to create what?

Back 56

a single-stranded hairpin loop at the end of each segment (V,D,J,C) + a broken area at the 2 heptamer sequences

Front 57

T/F

The hairpins are "opened up" often symmetrically.

Back 57

False!

The hairpins are "opened up" often asymmetrically.

Front 58

RAG-mediated cleavage of the hairpin generates what?

Back 58

palindromic P nucleotides

Front 59

What enzyme randomly adds up to 20 NON-templated nucleotides in between the segments formed by opening up of the hairpin loop?
What are these nucleotides called?

Back 59

TdT (terminal deoxynucleotidyl transferase)

N nucleotides

Front 60

After DNA-dependent protein kinase & other proteins binds to the hairpin & broken ends of RSS, what joins the V & J ends & the RSS ends?

Back 60

DNA ligase IV

Front 61

The DNA btwn the V & J is excised by exonuclease enzyme as the ____________ & the V & J segments are joined to form the _________

What is the final product?

Back 61

signal joint

coding joint

new double-stranded DNA

Front 62

What heavy chain segment is composed of the junction between the V and J segments?

Addition of P & N nucleotides at the junction results in what?

Back 62

HV3


junctional diversity
(^additional diversity of 3rd hypervariable region)

Front 63

a) What limits expression of Ig genes to only one copy of a chromosome (thus Ag specificity)?

b) Following what, the completed gene can be expressed on the cell surface as a functional protein?

Back 63

a) Allellic exclusion


b) Somatic recombination

Front 64

What does the membrane coding sequence encode?

Back 64

Encodes the transmembrane and cytoplasmic portion of Ig

Front 65

Near the membrane coding sequences of IgM and IgD, there are sites for what?

Back 65

Polyadenylation
(Delta & meuw polyadenylation sites, which ever remains = IgD or IgM)

(occurs along with cleavage = RNA splicing--> mRNa)

Front 66

a) (IgD/IgM) can be secreted or membrane bound, but (IgD/IgM) is only found as a membrane bound form.

b) The different forms are created by what?

Back 66

a) IgM (secreted or bound), IgD (bound only)


b) Alternate splicing, similar to the splicing that gives rise to IgM and IgD
(if membrane associated chain is spliced out--> secreted IgM is produced)

Front 67

Secreted IgM is pentameric. What is the advantage/disadvantage of this form?

Back 67

advantage- has 10 binding sites--> binds more Ag

disadvantage- limited effector function

Front 68

Why does alternative splicing lead to both IgM and IgD being expressed on naive B cells?

Back 68

Because the Cm and Cd segments are in close proximity in the germline--> thus splicing of same gene in different way/location will produce either IgM or IgD
(NOT = class switching)

Front 69

Ig class switching occurs in B cells at what time?

Back 69

At the time of INITIAL activation with antigen

Front 70

The class that the Ig is switched to is heavily determined by what?

Back 70

the local environment of the B cell at activation (cytokines, receptor interactions, etc)

Front 71

What is the name for clusters of highly repetitive sequences, flanking the 5' end of C gene segments, that mediate recombination?

Back 71

Switch regions

(the switch region flanking the mu gene interacts with a switch region flanking one of the other C genes (for IgE, IgA, & IgG)

Front 72

After the 2 different switch regions interact, the _________ is excised & only the C gene for the new class remains--> mRNA for new Ig isotype is formed

What from the original IgM remains in the new Ig?

Back 72

mu & delta gene (and any other intervening C genes)


same VDJ= Antigen specificity remains the same, whereas, the effector function is changed.

Front 73

Somatic hypermutation occurs in mature B cells in secondary lymphoid organs well (before/after) somatic recombination.

What does it lead to?

Back 73

After


point mutations in the V regions which gives rise to mutant Ig molecules on B surface

Front 74

T/F

Somatic hypermutation occurs following B cell activation by a T-dependent antigen.

Back 74

True!

Front 75

What enzyme converts some cytidine residues to uracils?

Back 75

AID (activation induced deaminase)

Front 76

a) During replication, the uracils can be changed to what?

b) What else can happen to the uracils?

Back 76

a) Thymidines, generating a C->T mutation

b) Can be excised as errors (via UNG) & replaced with random nucleotides

Front 77

Is the rate of mutation in Ig V regions higher or less than the genome wide rotation rate?

Back 77

The rate of mutation in Ig V regions can be 10^3-10^4 times the genome wide mutation rate. This allows for increasing antibody affinity for the antigen

Front 78

Mutated antibodies as surface Ig are selected for increasing affinity. Therefore, what happens to those cells with Ab that got weaker in affinity?

This is called _____________

Back 78

Undergo cell death



affinity maturation

Front 79

The TCR is composed of (a) how many polypeptide chains of roughly equal length, which are created by gene segments that are combined by (b) ______________ (similar to B cells) to form a functional gene.

Back 79

a) two (alpha & beta)

b) rearrangement
(B cell has heavy & light instead of alpha & beta)

Front 80

Each chain of a TCR consists of what 3 domains?

Which domain from each chain from the antigen binding site?

Back 80

Variable (V), constant (C), & a membrane anchoring domain

Variable chain from alpha & beta chain from antigen binding site

Front 81

T/F

TCRs are always secreted, never membrane-bound.

Back 81

False!

TCRs are always membrane-bound, never secreted

Front 82

T/F

Both T & B cells contain hypervariable CDR loops on their V domain & undergo isotype switching

Back 82

FALSE

B cells ONLY undergo isotype switching, T cells cannot
- BOTH have CDR

Front 83

a) The TCR requires help from four ________ membrane proteins to be transported from the ER to the cell surface.

b) Three of these proteins are collectively called the CD3 complex. What are they?

c) What's the 4th protein?

Back 83

a) Invariant

b) CD3-gamma, CD3-delta, and CD3-epsilon

c) zeta chain

Front 84

Without _______, the TCR cannot be transported to the plasma membrane and is eventually degraded.

Back 84

CD3


*CD3 critical for activation of T cell

Front 85

The _________, present at the end of the CD3 domain is critical for intracellular signalling & acts w/ the zeta homodimer in T cell replication & cytokine production

Back 85

ITAM
(immune receptor tyrosine-based activation motif)

Front 86

In TCR genes, which locus (alpha or beta) is similar to an Ig L-chain locus, containing sets of V & J segments?
which is similar to an Ig H-chain locus, containing V, D, & J segments?

Back 86

Alpha-chain (light)


Beta-chain (beta)

Front 87

Which TCRs show more diversity for antigen alpha-beta or gamma-delta?

Back 87

gamma-delta


*part of innate, lacks specific antigen receptor, doesnt require MHC presentation

Front 88

a) What percent of circulatory T cells are gamma-delta?

b) Where might gamma-delta T cells be the predominant cell type?

Back 88

a) 1-5%

b) in some epithelial tissues (especially in the intestines)

Front 89

Which locus of TCR is on chromosome 14 WITHIN the alpha-chain locus between V-alpha and J-alpha gene segments?

Back 89

The delta chain locus



(gamma locus is on chromosome 7)*

Front 90

T/F

DNA rearrangement within the alpha-chain locus will result in deletion of the delta-chain locus (of TCR).

Back 90

True!

Front 91

What are 4 similarities between Ig and TCR rearrangements?

Back 91

1. Similar recombination mechanisms, including RAG-1 and RAG-2. (and TdT)

2. Both undergo productive and non-productive rearrangements

3. Both have junctional diversity, including P and N nucleotide addition

4. Both exhibit allelic exclusion= if one allele forms a functional chain, the other is removed to prevent multiple antigen receptors-> specificity

Front 92

What are 5 differences between Ig and TCR gene rearrangements?

Back 92

1. TCR loci have fewer V segments
2. TCR genes show greater junctional diversity
3. TCR genes do not exhibit somatic hypermutations
4. TCR genes do not undergo isotype switching
5. TCR always membrane bound, never secreted

Front 93

What Ig is the most potent opsonin AND the only Ig that can be transported across the placenta?

Back 93

IgG

(IgG1 is the most common involved in these fxns)

Front 94

What Ig is involved in the sensitization of mast cells?

Back 94

IgE

Front 95

What is the only Ig that can be transported across epithelium?

Back 95

IgA (dimer)

Front 96

A B cell expressing both IgM & IgD is (mature/immature)

Back 96

MATURE!!!