Module 3 Antibody Structure and Function

Question 1

The structure of IgG

Answer 1

1. Comprised of heavy chain and light chain; linked by disulfide bonds
2. Constant region and variable region.

The variable part contains the antigen binding site and determine the specificity of the antibody.

Fab: fragment antigen binding
Fc: fragment crystallizable, mediate Effector functions by binding different immune system proteins

Hinge region: the unstructured regions of the heavy chain which can link the Fabs to Fc, function, as a flexible hinges within in the IgG molecule. These hinges allow the two fabs to adopt many different spatial orientation with respect to each other. This flexibility enables antigens spaced at different distances apart on the surface of pathogens to be bound by both fab arms of an IgG molecule.

Question 2

The different antibody isotypes?

Answer 2

Heavy chain: differences in the heavy chain C region defined the five main isotypes:

IgG / gamma / γ
IgM / mu / μ
IgD / delta / δ
IgA / alpha / α
IgE / epsilon / ε

Light chain has only two isotypes with no functional difference btw them:
Kappa / κ
Lambda / λ

Question 3

Three dimensional structure of IgG

Answer 3

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Question 4

Hypervariable region

Answer 4

CDR: complementarity determining region

FR: framework regions

FV: The hypervariable regions locate to three of the exposed loops that are present at the end of the domain furthest from the C region

Antigen binding site: pairing of a heavy chain and a light chain in an antibody molecule brings together the hypervariable loops from the Vh domain and the Vl domain

Question 5

What are epitopes?

Answer 5

The part of an antigen, to which a particular antibody bind is called antigenic determinant or epitopes

The epitopes are usually either carbohydrate or protein, because of the surface molecules of pathogens are commonly glycoproteins or polysaccharides.

Complex macromolecules, such as these will usually contain several different epitopes, each of which can be bound by different antibodies

Question 6

What are linear Epitope versus discontinuance Epitope?

Answer 6

Linear epitope: epitopes composed of several successive amino acids in a protein sequence

Discontinuance epitope: these epitopes are formed by two or more parts of the proteins antigen that are separated in the amino acid sequence, but come together in the folded protein structure

Question 7

What is the bond between an antigen and antibody?

Answer 7

The bond of antigen to antibody is based solely on noncovalent forces: hydrogen, bonds, hydrophobic interactions etc.

Question 8

How are monoclonal antibodies produced ?

Answer 8

Hybridomas: hybrid cells of mouse B cells and Myeloma cells

Question 9

IVIG

Answer 9

Intravenous immunoglobin

Question 10

Different types of mAb

Answer 10

See pic

Question 11

What is the difference between the gene encoding the C region vs V region

Answer 11

C gene: gene segments encoding C regions; the heavy chain locus contains C genes for all the different heavy chain isotypes

The gene segment encoding, the leader peptides, and the C regions consist of axons and intros that are ready to be transcribed

V regions cannot be transcribed, because they are encoded in a fragmented form of two (Vl) or three gene (Vh) segments; each segment being represented by an array of alternative forms that differ in nucleotide sequence. To be transcribed, these arrays have to be cut and spliced to produce a V-region exon that has just one form of each gene segment.

The 2 gene segments encoding the light chain V region:
V (variable gene segments)
J (joining gene segments)

The 3 gene segment in coding the heavy chain V region:
V
J
D (diversity gene segments)

Question 12

Somatic Recombination (First Diversity Creating Event)

Answer 12

1. Random recombination of gene segments creates diversity in the antigen binding sites
2. The V, D and J segments that recombine to make the exons encoding Vh and Vl are selected at random from the arrays of multiple V, D, and J segments.

Question 13

V(D)J recombinase

Answer 13

The enzymatic machinery of somatic recombination in B cells is called VJ Recombinase and involves numerous enzymes and other proteins. Almost all these proteins are also used by other human cells in mitotic recombination and DNA repair.

Two exceptions:
RAG1 and RAG2 protein (recombination activating genes), they are expressed only in developing B and T lymphocytes

RAG complex: Two RAG1 and two RAG2 proteins

Question 14

Junctional diversity (second diversity creating event)

Answer 14

Junctional diversity: Random incorporation of additional nucleotides during formation of the coding joints

This diversity is not encoded in the sequence of the germline DNA, but is generated by the sequential action of the enzymes that repairs a DNA breaks

P nucleotide / palindromic nucleotides

TdT: terminal deoxynucleotidyl transferase, it randomly adds nucleotides at end of the opened hair pins, generating further diversity

These newly added a nucleotides are N nucleotides, because they are non-templated, ie not encoded in the germline DNA

Question 15

Allelic exclusion

Answer 15

In a developing B cell, the process of immunoglobulin gene rearrangement is tightly controlled so that only one heavy chain and one light chain are finally expressed, from a phenomenon, known as Allelic exclusion

In naïve B cells alternative mRNA splicing produces IgM and IgD of the same antigen specificity

Question 16

What form is immunoglobulin first made?

Answer 16

Immunoglobulin is first made in a membrane bound form

Question 17

The composition of B cell receptor?

Answer 17

IgM/or other isotypes & Igα and Igβ

Igα and Igβ are required for the immunoglobulin to be transported to the cell surface and are needed for intracellular signaling

Iga and Igβ can interact with intracellular signaling proteins and have binding sites for the C region on the immunoglobulin

Question 18

What are the differences between membrane associate immunoglobulin and soluble form of immunoglobulin?

Answer 18

The key difference is at is a carboxyl terminal part of the heavy chain.

Membrane-bound form has a hydrophobic sequence, which helps with anchoring in the membrane

Soluble form / antibody has a hydrophilic sequence

Question 19

Somatic Hypermutation (Third Diversifying event)

Answer 19

1. Happens after antigen activation
2. Single nucleotide is substitutions / point mutations almost randomly, and add a high rate throughout the rearranged V regions of heavy chain, and light chain genes.
3. Happens to the CDR3 of the HV
4. Enzyme needed: somatic hypermutation is dependent on the enzyme, activation induced cytidine deaminase (AID), made only by proliferating B cells

AID converts cytosine in single strand of the DNA to uracil

Question 20

Purpose somatic hypermutation?

Answer 20

Increase BCR binding affinity

Question 21

Affinity maturation

Answer 21

After somatic hypermutation, some of these mutant immunoglobulins have a substitutions in the antigen binding site that increase its affinity for the antigen.

B cells bearing these mutant high affinity immunoglobulin receptors compete most effectively for binding to an antigen and are preferentially selected to mature into antibodies secreting plasma cells.

Question 22

Isotype Switching ie class switching

Answer 22

Isotype switching produces immunoglobulin with a different constant region but identical, antigen specificity

Further DNA recombination events enable the rearranged V-region exon to be used with other heavy chain C genes.

Enzyme required: AID

Happens after antigen activation

Question 23

Hyper-IgM syndrome

Answer 23

Lack of enzyme AID and immunoglobulins don’t go through somatic hypermutation and isotype switching and can only produce low affinity IgM not other types of antibodies

Question 24

What types of diversifying events in B cell development

Answer 24

Before antigen activation:

1. Somatic recombination
2. Junctional diversity (TdT)

After antigen activation: (AID)
1. Somatic hypermutation / affinity maturation
2. Isotype switching

Question 25

What different effector functions different type of immunoglobulins have?

Answer 25

Avidity: the overall strength of binding to multiple epitopes of an antigen is called avidity of the antibody

Question 26

FcRn

Answer 26

1. Cellular receptors that bind immunoglobulin Fc regions and called Fc receptors
2. Function:
   (1) Protect IgG from degradation, reason for antibodies much longer half-life than other plasma proteins

(2) Transports antibodies from the bloodstream into extracellular spaces of tissues

Question 27

Transcytosis

Answer 27

Receptor mediated transport of a macromolecule from one side of a cell to the other is called transcytosis

Question 28

IgE function?

Answer 28

IgE provides a mechanism for rapid ejection of the parasites and pathogens from the body

Question 29

Acquired immunity from mothers

Answer 29

Before and after birth, mother is provided their children with protective antibodies

Question 30

How does IgM initiate classical complement pathway?

Answer 30

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Question 31

Fcγ

Answer 31

1. A Fc receptor, enables effector cells to bind IgG and be activated by IgG bound to pathogens
2. FcγRI is specific for IgG and constitutively, expressed by monocytes, macrophages and dendritic cells; specific to myeloid cells; Major function is to facilitate the uptake and degradation of pathogens by phagocytes and professional antigen presenting cells

Question 32

How does Fc receptors function?

Answer 32

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Question 33

Family of Fc receptors that bind to IgG

Answer 33

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Question 34

Fc receptor for NK cells?

Answer 34

FcγRIII, an activating IgG receptor, is the only Fc receptor expressed by NK cells

Question 35

ADCC

Answer 35

Antibody dependent cell mediated cytotoxicity

Question 36

Summary of different Fc receptors

Answer 36

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