Module 3 Antibody Structure and Function Flashcards
The structure of IgG
- Comprised of heavy chain and light chain; linked by disulfide bonds
- 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.
The different antibody isotypes?
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
Three dimensional structure of IgG
See pic
Hypervariable region
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
What are epitopes?
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
What are linear Epitope versus discontinuance Epitope?
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
What is the bond between an antigen and antibody?
The bond of antigen to antibody is based solely on noncovalent forces: hydrogen, bonds, hydrophobic interactions etc.
How are monoclonal antibodies produced ?
Hybridomas: hybrid cells of mouse B cells and Myeloma cells
IVIG
Intravenous immunoglobin
Different types of mAb
See pic
What is the difference between the gene encoding the C region vs V region
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)
Somatic Recombination (First Diversity Creating Event)
- Random recombination of gene segments creates diversity in the antigen binding sites
- 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.
V(D)J recombinase
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
Junctional diversity (second diversity creating event)
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
Allelic exclusion
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
What form is immunoglobulin first made?
Immunoglobulin is first made in a membrane bound form
The composition of B cell receptor?
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
What are the differences between membrane associate immunoglobulin and soluble form of immunoglobulin?
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
Somatic Hypermutation (Third Diversifying event)
- Happens after antigen activation
- 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.
- Happens to the CDR3 of the HV
- 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
Purpose somatic hypermutation?
Increase BCR binding affinity
Affinity maturation
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.
Isotype Switching ie class switching
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
Hyper-IgM syndrome
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
What types of diversifying events in B cell development
Before antigen activation:
- Somatic recombination
- Junctional diversity (TdT)
After antigen activation: (AID)
1. Somatic hypermutation / affinity maturation
2. Isotype switching
What different effector functions different type of immunoglobulins have?
Avidity: the overall strength of binding to multiple epitopes of an antigen is called avidity of the antibody
FcRn
- Cellular receptors that bind immunoglobulin Fc regions and called Fc receptors
- 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
Transcytosis
Receptor mediated transport of a macromolecule from one side of a cell to the other is called transcytosis
IgE function?
IgE provides a mechanism for rapid ejection of the parasites and pathogens from the body
Acquired immunity from mothers
Before and after birth, mother is provided their children with protective antibodies
How does IgM initiate classical complement pathway?
See pic
Fcγ
- A Fc receptor, enables effector cells to bind IgG and be activated by IgG bound to pathogens
- 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
How does Fc receptors function?
See pic
Family of Fc receptors that bind to IgG
See pic
Fc receptor for NK cells?
FcγRIII, an activating IgG receptor, is the only Fc receptor expressed by NK cells
ADCC
Antibody dependent cell mediated cytotoxicity
Summary of different Fc receptors
See pic
