Antibody Structure & B-Cell Diversity Flashcards
What is the D region
The Diversity region, the second region on the heavy chain locus (Green Box)
- Only found on the heavy chain
Protein Coding Process
- RNA Transcription (Here is where introns are removed) creating mRNA
- In the ribosome mRNA is translated into a polypeptide chain
- Once this chain exceeds 40-50 amino acids it is then considered a protein
- After translation Polypeptide Glycosylation occurs in the Golgi and ER
- After translation Polypeptides are sorted and targeted by cells occurs in the ER
Summary:
RNA –> mRNA –> Protein
What kind of bond links the polypeptide chains
Disulfide bond
How does the Antibody’s structure effect its function
Determines how many domains the heavy chain has
(3 Domains = IgG, IgA, IgD,)
(4 Domains = IgM, IgE)
Antigen Binding Loops at the end are formed by helix to sheet transitions.
These fingerlike projections (Paratopes, antigen binding sites) are solvent accessible spaces allowing molecules to move through the finger loops and interact with the protein structure
Polypeptide vs Protein
1 Amino acid = Amino acid
2 or more Amino acid = Polypeptide
Polypeptides that are greater than 40-50 amino acids = Proteins
IgA Isotype
- Attributes
- Functions
- Properties
Attributes:
- Secreted as a dimer using the J chain
Functions:
- Neutralization
Properties
- Can penetrate through epithelial barriers
What links the chains on an antibody
Disulfide bonds
What determines the shape of an antibody
Heavy chains wobble in ER and fold upon themselves to form a tertiary structure (Through hydrophilic, hydrophobic, hydrogen bonds)
- Primary Structure: Amino acid structure
- Secondary Structure: Alpha helices, beta pleated sheets
- Tertiary Structure: Final antibody structure
Half life of different Ig Isotypes
IgG have the longest
IgM have the second longest
IgA have the third longest
IgE have the shortest
What Sections are joined together in Gene Recombination of Heavy Chains
Diversity Domain is joined with the Joined Domain
- This DJ Domain is joined with the Variable Domain
- Variable Domain is made up of three hypervariable regions
- The third hypervariable region is coded by a splice from both the Variable Domain and the Joined Domain
What contributes to the immense amount of diversity in Ig genes
Somatic Recombination
- V, D, J, C Domains
Somatic Hypermutation
- Rapidly dividing daughter cells that introduce random polymorphisms
What is a Paratope
Antigen Binding Site located on the Antibody
- Made up of Hypervariable Regions / Complementary Determining Regions
How do the variable and constant regions differ on the varying isotypes
Variable:
- Only differ by specificity, specific isotype does not impact variable region
Constant
- Different C-regions give rise to different effector functions
What chains of Ig are first to be transcribed, from what Isotype?
IgM and IgD Constant regions are the first to be transcribed
What is the J Region
The Joining region, the second region on the light chain locus, and the third region on the heavy chain locus (Yellow Box)
What directs the RAG Enzyme Recombination Process
Recombination Signal Sequences (RSS)
- Two Types
1. Heptamer
2. Nonamer
Separated by 12 and 23 base pair spacers (12/23 Rule)
- Provides the right amount of space for the RAG enzyme to insert itself
RAG always cuts at the Heptamer
IgG Isotype
- Attributes
- Functions
- Properties
Attributes
- Long half life
Functions:
- Great at everything
- Neutralization
- Opsonization
- Activating complement system
- Sensitizing cells for killing by NK cells
Properties:
- Transport across placenta
- Transport across extravascular spaces
What creates diversity in antibody proteins
Lymphocytes rearrange the DNA before RNA transcription
NOT from the variations in enzymes splicing of introns
What is the C region
The Constant region, the third region on the light chain locus, and the forth region on the heavy chain locus (Blue Box)
- Codes for the domains of folded amino acids structures in the antibody’s constant region
Domains are linked together like a traditional gene, no DNA recombination, are ready to be transcribed
- Enzymes still have to splice off the introns
What Isotype is secreted first
IgM is secreted first as a pentamer
What are the different Antibody Isotypes
Heavy Chain:
IgG (Gamma), IgA (Alpha), IgM (Mu), IgE (Epsilon), IgD (Delta)
Light Chain:
Kappa and Lambda
How many chains on an antibody
4 polypeptide chains
- 2 heavy chains (On the inside and make up the main body too)
- 2 light chains (On the outside)
What are the seven changes that a Ig gene will experience
- Assembly of the V-region
- Through Somatic Recombination - Generation of Junctional Diversity
- Due to imperfect rearrangement nucleotides are inserted and removed - Assembly of Transcriptional Elements
- Transcription is activated, IgM and IgD are expressed through differential splicing
- Reversible - Membrane Ig or Secreted Antibody are produced depending on the presence of a Hydrophobic MC Region
- Reversible - Somatic Hypermutation
- Isotype Switching
- Recombination involving downstream constant domain
What codes proteins
Segments DNA (genes) code for proteins
- Specifically Exons are the coding segments
- While Introns are the non-coding (intervening) segments
IgM Isotype
- Attributes
- Functions
- Properties
Attributes:
- Very large pentamer
- Relatively fast half life for quick immune response
Function:
- Activates Complement System
Property:
- Can transport across epithelium but have to squeeze through due to their large size
Gene Segment Recombination for Light Chain vs Heavy Chain
Light Chain
- V is joined to J
Heavy Chain
- D is joined to J
- DJ is joined to V
Differences in the different Antibody Isotypes
IgM and IgE have 4 domains compared the standard 3 domains
Each isotype have differences in glycosylation
- Extra bumps for antigenicity
- Affects their function
Immunoglobulin vs Antibody
Antibodies are the excreted form of Immunoglobulins
Immunoglobulins of one specificity are expressed on the surface of B cells
How are surface Ig expressed
- Separate Ig molecules enter the ER and self-assemble into Ig molecules
- These Ig molecules also contain a Hydrophobic Membrane Coding Region
- The newly created immunoglobulin is then tethered to the cell membrane and expressed as a B receptor
- Ig associates with disulfide-linked transmembrane proteins (Ig-alpha and Ig-beta)
- Signals to the B-cell that the surface Ig has bound an antigen
What is a Hypervariable Region
Variable region
- Lots of variability in the amino acids found here
- Provides complementary to the shape, charge, hydrogen, hydrophobicity, polarity, hydrophilicity
What Sections are joined together in Gene Recombination of Light Chains
Variable Domain is Joined with the Joined Domain
- Variable Domain is made up of three hypervariable regions
- The third hypervariable region is coded by a splice from both the Variable Domain and the Joined Domain
What is the process of Recombination Switching
- IgM and IgD are co-expressed depending on mRNA splicing of the primary RNA transcript
- Further looping of the constant region leads to the switching to a different isotype
- Either IgA, IgE, IgG is produced
What is an Epitope
Antibody Binding Site located on the Antigen
- Usually carbohydrates, proteins, or both
- Usually are macromolecules (Protein, Carbohydrates, Lipids)
- Usually multivalent
What is the V Region
Variable Region, the first region (Red Boxes) on the locus
- Contains many different exons that can be chosen to determine the specificity
Lamda Light: 30 Variabilities
Kappa Light: 40 Variabilities
Heavy: 65 Variabilities
What is a Complementarity Determining Regions
Provide a binding surface complementary to the antigen
Define Germline Configuration
The genome found in every cells, in Ig cells the Germline Configuration is comprised of many Ig gene loci
What is a Framework Region
Non-variable region,
- Is hydrophobic so it pushes away from the solvent accessible space
- Binds by disulfide bonding
Variable Region Domain
Single Variable Domain
- With one on the light chain (VH) and one on the heavy chain (VL)
Which Ig isotypes are transcribed first
- How are the other Isotypes transcribed
Depending on how the exons and introns are cut and splice either IgM or IgD are transcribed and are co-expressed
- The rest are transcribed by looping of the constant region
IgE Isotype
- Attributes
- Functions
- Properties
Attributes
- Bit larger due to having an extra domain
Functions:
- Immediately taken up by mast cells
- Allergic response
Properties
What is a Domain on an antibody
Immunoglobulin domains are made up of subunits of 100-110 amino acids joined together as a polypeptide
What is Aby Affinity Maturation
Antibody becomes more specific for the antigen that is triggering it
- Caused by Somatic Hypermutation choosing a more specific Ig for the specific antibody
Constant Region Domain
Single Non-Variable Domain
- Little to no amino acid diversity
What is polypeptide sorting and targeting by cells?
Based on transcription each protein will have a different function
- They can be secreted
- They can be inserted into the phospholipid bilayer and expressed on the cell membrane
What non-covalent bindings contribute to antigen-antibody binding
Ionic Bonds
Hydrogen Bonds
Van der Waals Attractions
Hydrophobic Attraction
What do lymphocytes have that allow for Gene Segment Reccombination
Recombination Activating Genes (RAG)
- RAG-1 and RAG-2 are found only in Lymphocytes (B-Cells and T-Cells)
Other enzymes found in all nucleated cells are also used to repair DNA breaks
What is Allelic Exclusion
Lymphocyte has chosen to stick with one variable region. No further variable rearrangement can occur
- Lymphocyte will only produce one type of Ig
However, Ig can rearrange further downstream heavy chains to choose a future different isotype
Why is Glycosylation important in the immune response?
Changes the antigenicity when protein is glycosylated
Important because Mammalian Polypeptides are glycosylated.
- While bacteria (prokaryotes) can not glycosylate
What is an Antibody Isotype?
Different classes of antibody’s
- Genetic Variations in the constant regions of the heavy chain and light chain
In the seven steps of a Ig Gene which steps are reversible
Co-expression of IgM and IgD
Synthesis changes from membrane Ig to secreted antibody
IgD Isotype
- Attributes
- Functions
- Properties
Attributes:
- Nothing too notable
Functions:
- Instructor of self and non-self
Properties:
- Nothing too notable
Aby Functional Regions
- How it was found?
- Names of fragments
- Function of fragments
Plant Protease Papain cleaves antibody into 3 fragments
Gut Protease Pepsin degrades Fc, antibody can now no longer tag molecules
2 Fab (Important for antigen binding)
1 Fc (Important for opsonization, tagging molecules for disposable)
Is Immunoglobulin diversity generated before or after encounter with an antigen
Occurs before
How are Ig genes different from other genes
Except in B-Cells Ig genes are fragmented
- The B-cell can then rearrange the fragments to create a gene segment that is then transcribed and translated
How do antibodies bind antigens
Molecular Complementarity
- Shape, Charge, Polarity, Hydrophobicity
Non-Covalent Binding
Define Gene segment
Ig genes are fragmented, each fragment section is a gene section or a Ig gene locus
Surface Ig Form vs Secreted Ig Form
Derived by the same process of enzyme recombination
- Just the Surface Ig contain a Hydrophobic Membrane Coding Region
What Loci make up the Germline of an Ig Gene
- 1 Set of Fragmented Genes that code for Lamda light-chain locus
- 1 Set of Fragmented Genes that code for
Kappa light-chain locus - 1 Set of Fragmented Genes that code for 1 heavy-chain locus
All of these Fragmented Genes are brought together to transcribe an mRNA and translate a protein
What does a multivalent antigen do
Has multiple different kinds of epitopes that kind bind many different kinds of antibodies
Serum Levels of each Ig Isotype
IgG, then IgA and IgM, then small amounts of IgE
What are the regions on a polypeptide chain of an antibody
Variable Region (N-terminus)
Constant Region (Remainder)
Which isotypes are co-expressed
IgM and IgD through differential mRNA splicing of the primary RNA gene
Lamda vs Kappa vs Heavy (Signal Sequence)
- Also how many variable sections
Lambda
7-23-9-9-12-7
30 Variable
Kappa
7-12-9-9-23-7
40 Variable
7-23-9-9-12-7-7-12-9-9-23-7
65 Variable
What happens when a B-Cell’s immunoglobulin binds to an antigen
It differentiates into a antibody-secreting plasma cell
What is the most common isotype switch
Switch to IgG, which is secreted as a monomer
What determines the strength of an epitope binding (Shape)
The shape that an epitope binds to can vary
- Pocket (Tightly bound)
- Groove (Loosely bound)
- Surface (Very Loosely bound)
What is Glycosylation
- Where does it occur?
Attachment of sugars to specific amino acid residues
- Occurs in the Endoplasmic Reticulum and the Golgi Apparatus
Two types:
- N-Glycosylation (Asn)
- O-Glycosylation (Ser, Thr)
