Exam 2 -- Antigen Recognition by BCRs and TCRs

TRUE or FALSE — Innate and Adaptive Immunities are very different

FALSE !! — They are extremely related and intertwined systems

How are antibodies shaped?

Y-Shape!

TRUE or FALSE — BCRs and TCRs bind and recognize antibodies?

FALSE — Antigens

What’s the chain situation on antibodies?

2 Heavy and 2 light chains

What are the 5 Ig classes?

IgG, IgM, IgD, IgA, IgE

Structure of Antibody 1

1

Structure of Antibody 2

2

What is the N-Terminus of an antibody responsible for?

Antigen binding

What is the C-Terminus of an Antibody responsible for?

Effector Functions

What are the two kinds of ligtht chains?

Lambda ( λ ) and Kappa ( κ )

What are the five kinds of heavy chains?

IgM / Mu (μ) — IgD / Delta (δ) — IgG / Gamma (γ) — IgA / Alpha (α) — IgE / Epsilon (ε)

How long is the total IgG?

Heavy Chain = 50 kDa // Light Chain = 25 kDa

Total chain length = 150 kDa

TRUE or FALSE — Antibodies can exist with both a Lambda ( λ ) and Kappa ( κ ) light chain

FALSE !!!!! ONLY ONE!!!!!!!

TRUE or FALSE — Antibodies can exist with multiple types of heavy chains

FALSE !!!!! ONLY ONE!!!!!!!

How many Disulfide bonds connect an Antibody?

4 - 2 connecting heavy chains and 2 connecting light chains

:: 4 Disulfide bonds connect the antibody chains

Antibodies are very stable!

Into what two pieces can Papain proteylytic cleavage break antibodies?

Fab and Fc

What is the Fab Fragment?

Fragment Antigen Binding

What is the Fc Fragment?

Fragment Crystallization - Effector Domain / Constant Region / C-Terminus

What’s the difference between the Fab and Fc ?

Fab - Many different antigen binding spots, high variability, non-crystalizable

Fc - All the same region, crystalizable

Why is Fab non-crystalizable?

High-variability

Into what two pieces can Pepsin cleave antibodies?

F(ab’)₂ and pFc’

What is F(ab’)₂ ?

Two Fab’s bound/cleaved together

What is pFc’?

Pieces of Fc

Are antibodies cleaved by Pepsin crystallizable?

NO!!!!!

Are Polyclonal Antibodies derived from ONE or MULTIPLE B-Cells?

Multiple

Are Monoclonal Antibodies derived from ONE or MULTIPLE B-Cells?

One

:: Polyclonal Antibodies are derived from multiple B-Cells

Polyclonal antibodies are not genetically identical, and can recognize many epitopes

:: Monoclonal antibodies are derived from one B-Cell

Monoclonal antibodies have the exact same amino acid sequence, are crystalizable, and recognize the exact same epitope

TRUE or FALSE — Polyclonal antibodies are crystalizable

FALSE !!

TRUE or FALSE — ALL PARTS of monoclonal antibodies are crystalizable

TRUE ! - They are genetically identical - even monoclonal-derived p(Fab’)₂

Monoclonal Antibody shorthand

mAb

What is Recombinant Antibody Technology?

A means of genetically altering antibodies to combine variable regions, allowing for rapid production of Abs

What parts are included in a recombinant antibody?

Just the heavy and light variable chain regions (Ab-binding sites)

TRUE or FALSE — Recombinant antibodies are still able to bind antigens

TRUE !

Benefits of Recombinant Antibody Technology

Quick/easier creation of antibodies

Therapeutic benefits (smaller, easier entry into tissue, more specific targeting, tumor therapy against custom (cancer, toxin, protein) antigens)

TRUE or FALSE — Antibody arms are extremely rigid

FALSE — Ab arms are joined by a flexible hinge region

Picture of the Immunoglobin Domain

Picture of the Immunoglobin Domain

How is the immunoglobin domain shaped?

2 anti-parallel beta sheets held together by disulfide bonds

Forming a Beta Barrel

“Immunoglobin Fold”

What part of an antibody is the immunoglobin domain?

The heavy and light variable chains

When dealing with Antigen-Antibody binding, what is the framework region?

AA sequences that form the boarder structural framework of the domain

AA portions that make up the bulk of the immunoglobin domain - Beta barrels

Pretty constant amongst Ab’s

When dealing with Antigen-Antibody binding, what is the Hypervariable region?

Highly variable AA regions that allow Abs to bind a multitude of Ags

How are Hypervariable regions organized?

3 Discrete loops in the folded structure of an antibody

What’s another name for the Hypervariable Reason?

The Complementary Determining Region (CDR)

What are CDR’s (Complementary Determining Regions) ?

More specific hypervariable regions that complement the antigen that they bind

Still more variable than Framework regions, but more specific than hypervariable regions

How many CDR’s (Complementary Determining Regions) are contained within an antigen binding site?

6

How many CDR’s (Complementary Determining Regions) are contained within an antibody?

12

How many CDR’s (Complementary Determining Regions) are contained within a heavy chain?

3

How many CDR’s (Complementary Determining Regions) are contained within a light chain?

3

How do antibodies bind antigens?

Through tons of noncovalent, reversible interactions

TRUE or FALSE — Antibodies bind antigens reversibly

TRUE !

What’s unique about camelid and shark Abs?

All heavy chains

Camelid - Smaller (2 Fc, 1 Fab)

Shark - Bigger (5 Fc, 1 Fab)

How do BCRs differ from Igs?

Surface bound - short transmembrane domain

Igβ and Igα related subunits help with signaling

How do TCRs differ from Igs

Resembles a membrane-bound Fab fragment

Cα, Vα, Vβ, and Cβ

What are the two kinds of TCR heterodimers?

α:β and γ:δ

Which TCR heterodimer is more common?

α:β

What’s special about γ:δ TCR heterodimers (prevalence)

Much more uncomming (5-15% of all T-Cells)

What makes up an α:β TCR’s antigen-binding site?

CDRs 1, 2, and 3

Their disulfide bonds

TRUE or FALSE — For proper presentation, TCR epitopes must be present on a protein’s surface

FALSE !!

TRUE or FALSE — TCR epitopes must be linear/continuous peptide sequences

TRUE !

Why do TCR epitopes need to be linear, but do not need to be present on protein surfaces?

Because proteins are chopped up prior to presentation, and small fragments are what’s presented in MHC molecules

What are the two ways antibody epitopes can appear

Linear/Continuous and Conformational/Discontinuous

What kind of antibody epitope conformation is ALWAYS recognized by TCRs?

Linear/Continuous ONLY

What kind of antibody epitope conformation is ALWAYS recognized by BCRs/Abs/Igs?

Conformational or Discontinuous (typically, not a requirement)

In what form do TCRs recognize Antigens?

In the form of a peptide-MHC molecule complex

TRUE or FALSE — Abs/Igs have a wide range of epitopes

TRUE ! Their epitopes are not always proteins!

What are the domains of an MHC I protein? (# / makeup)

3 Domains

α chain, β-2 Microglobin, Peptide-Binding cleft

1 Transmembrane domain (MHC I)

What are the domains of an MHC II protein? (# / makeup)

4 Domains

2 α chains, 2 β chains, and the peptide binding cleft

2 Transmembrane domains (MHC II)

Why is MHC turnover important?

Recency of peptides displayed allows immune cells to recognize more recent pathogen infections

Describe the MHC I peptide binding cleft

Closed, holds shorter peptides

Stabilizing noncovalent interactions in the middle + held in on the ends

Presents cytosolic/endogenous peptides

Describe the MHC II peptide binding cleft

Open on the ends, holds longer peptides

Stabilizing noncovalent interactions in the MIDDLE of the peptide

Presents exogenous peptides

:: MHC I molecules have closed sides of the the presentation cleft, and many stabilizing interactions at the sides of their binding cleft

:: MHC I molecules bind smaller amino acids (~9 sequences) and have higher specificity

What are anchor residues?

Structurally related amino acids in a MHC I molecule’s binding cleft

Typically Phenylalanine (Phe/F) and Tyrosine (Tyr/Y)

What do anchor residues do? (MHC I)

Proteins bind anchor residues at their amino and carboxyl ends

They bind at the amino/carboxyl ends because MHC I molecules are closed on the ends

:: MHC II presentation clefts are open on the sides, and have many stabilizing interactions in the middle of their binding cleft

:: MHC II molecules can bind longer amino acids (~13-17 sequences) and have higher specificity

TRUE or FALSE — There’s a lot of variation within MHC binding clefts

FALSE !! — Anchor residues correspond with common core sequences present within bound peptides

What specific sequences do peptides contain to mediate MHC II binding

Core sequences

In the middle of the protein, noncovalently binds the middle of the binding cleft

How do TCR’s bind the MHC I complex?

CDR (Complement Determining Region) 3α and 3β bind the center of the peptide

CDR 1 and 2 α (of the TCR) contact the Amino terminus

CDR 1 and 2 β (of the TCR) contact the Carboxylic Acid terminus

In what context do TCR's bind the MHC II peptide?

TCR’s understand the protein in the context of the MHC II molecule

Presentation of peptide on the MHC molecule matches to the cognate/matching TCR

:: MHC molecules are highly polymorphic, there are hundreds of alleles of MHC class 1 and 2 genes in humans

:: MHC molecules can bind a vast array of proteins!

(Due to the differences in their protein binding clefts)

The CD4+ T-Cell Co-Receptor has 4 Ig-Like domains

The CD4+ T-Cell Co-Receptor has 4 Ig-Like domains

Where are the main polymorphic areas in MHC molecules?

The peptide binding cleft!

The CD8+ T-Cell Co-Receptor is a dimer

The CD8+ T-Cell Co-Receptor is a dimer

MHC I :

CD8+ - Cytotoxic T-Lymphocytes (CTLs)

MHC II :

CD4+ - Regulatory / Helper T-Cells (Tregs/Th)

What cells express MHC I cells?

All nucleated cells

NOT RED BLOOD CELLS!!!! No nucleus

What cells express MHC II cells?

Antigen-presenting cells

Dendritic Cells, Macrophages, B-Cells

Epithelial cells of the thymus

Where do T-Cell CD8+ and CD4+ co-receptors sit when interacting with MHC molecules?

Next to the MHC molecule