BCH - Immunoglobulins Flashcards
How are Immunoglobulins/Antibodies proteins made
by B lymphocytes of the adaptive immune system
How do immunoglobulins bind ligands?
with a high affinity and high degree of specificity
Immunoglobulins are heterotetramers comprised of?
2 identical light chains and 2 identical heavy chains
(2 dissimilar units)
How are immunoglobulin chains held together
through non-covalent bonding forces (hydrogen bonds, hydrophobic interactions) and covalent disulfide bonds
Each immunoglobulin chain has regions of ?
constant sequence and variable sequence
What does constant sequence mean
different from one immunoglobulin class to another
where are the regions of variable sequence found
the amino-terminal domains of each chain
what do the variable regions of immunoglobulins cooperate to form
antigen-binding sites
Why are immunoglobulins referred to as being bivalent
there are 2 antigen-binding sites per immunoglobulin molecule
Immunoglobulins assume a what shaped structure with the antigen-binding sites and where?
a Y-shaped structure ; at the tips of the two upper domains
What is the stem/base of the Y-shaped structure formed by
constant regions of the heavy chains
Do imminoglobulins exhibit elements of quaternary structure?
NO, the polypeptide chains are COVALENTLY joined to one another through DISULFIDE bonds –> quaternary structures focuses on the non-covalent interactions between the polypeptide chains and involve weaker forces (hydrogen bonds, ionic interactions, hydrophobic)
What is a paratope
the specific part of the immunoglobulin that binds epitopes
What is a ligand
any molecule that binds specifically and reversibly to a protein (or other molecule)
What is an antigen
any substance recognized by the adaptive immune system that triggers an immune response (for B lymphocytes that includes substances recognized by immunoglobulins)
What is an epitope
the specific part of an antigen recognized by an immunoglobulin
Antigens bind to immunoglobulins via ?
complementary surfaces (think of a lock and key relationship)
the regions of extreme sequence variation are known as
hypervariable regions
How many hyper variable regions are there in each variable region of a heavy chain and a light chain
Three regions
What do the hypervariable regions form
the binding regions for the epitopes in antigens recognized by antibodies
what are hyper variable regions aka
complementary determining regions
what gives immunoglobulins their remarkable ability to recognize and bind an amazing array of different molecules
the sequence variation within the complementary determining regions
Developing B lymphocytes initially express what? what are they referred to as
Membrane-bound immunoglobulins
- referred to as B cell receptors
B lymphocytes are white blood cells derived from
hematopoietic stem cells found in bone marrow
What does each B lymphocyte progenitor express
a unique immunoglobulin that is found in thousands of copies on the cell surface
How does the immunoglobulin expressed in a particular B cells differ from immunoglobulins synthesized in other B cells?
the unique amino acid sequences found within its variable domains
The binding of an antigen to a B cell receptor activates the B cell expressing this receptor, stimulating its proliferation and differentiation into?
Effector/plasma cells
What do effector/plasma cells do
produce large amounts of soluble immunoglobulins capable of recognizing the antigen and eliciting a response
what is a role of the constant regions of membrane-bound immunoglobulins
to anchor the immunoglobulin to the plasma membrane
Antigens bind to the ____ regions of immunoglobulins
variable
What are the 5 major classes of the immunoglobulins
IgA, IgD, IgE, IgG, IgM
What plays a major role in giving the different immunoglobulin classes their defining characteristics
the heavy chains
Which immunoglobulins perform the role of B cell receptors on the surface of B cells
IgM and Ig
Where do the immunoglobulins go from the ER
they transit through the Golgi and then to the cell surface
Protein trafficking - for immunoglobulins to reach the cell surface they must first be
directed to the ER
Where does the signal sequence exit? As the protein is doing what?
the ribosome tunnel as the protein is being synthesized and is recognized by the SRP
how do proteins transiting through the secretory pathway do so
by being packaged in lipid vesicles
what does the SRP do
binds the signal sequence and brings the nascent (early stage) protein and its associated ribosome to a tunnel within the ER membrane
Regarding signal peptides - for a protein to enter the secretory pathway it must contain
a signal sequence at its extreme amino terminus
What will happen if a protein only contains a signal sequence for its target information and this signal is cleaved/removed?
the resulting protein will be localized initially to the lumen of the Er and in lipid vesicles for transit through the secretory pathway to the cell surface
When the lipid vesicles fuse with the plasma membrane, the protein is released from the cell and secreted into the extracellular milieu (environment)
REMOVING/CLEAVING does NOT prevent the protein from proceeding through the secretory pathway
Secretory vesicles are essential for transporting ?
the protein from the Golgi apparatus to the plasma membrane, where the protein is then secreted into the extracellular milieu (environment)
What immunoglobulin proteins are located on the cell surface and what do they contain
IgM and IgD, they contain targeting information in addition to the signal sequence
STOP-TRANSFER SQUENCES
What are stop transfer sequences?
hydrophobic stretches of amino acids that stop the polypeptide chain from being translocated across the ER membrane
Explain IgM and IgD and their stop-transfer sequences
they disengage from the translocation channel allowing the protein to cross into the ER membrane with the result that the protein is now embedded within the ER membrane
Prior to fusion of transport vesicles, the carboxyl terminus of an immunoglobulin is oriented toward the
cytosol
As the IgM/IgD transit through the secretory pathway, it will remain ?
within the membrane of the lipid vesicles
Prior to fusion of transport vesicles, the amino terminus of an immunoglobulin is oriented toward the
Lumen of the ER
When the lipid vesicles fuse with the plasma membrane the protein is retained on the _______ ? in an orientation ______ of that in which it was first inserted into the ER membrane
plasma membrane ; opposite
When localized to the plasma membrane, which terminus of the protein in the figure above will be found on the outside of the cell?
the Amino terminus
Remember, when the vesicles fuse with the plasma membrane the protein is retained on the plasma membrane in an orientation OPPOSITE of that in which it was first inserted into the ER membrane
This can be simplified by: membrane vesicles invert their orientation upon fusion with the plasma membrane
Membrane vesicles ______ their orientation upon fusion with the plasma membrane
invert!!
a protein oriented with its amino terminus toward the LUMEN of the ER will have its amino terminus oriented to the OUTSIDE of the cell upon fusion of transport vesicles with the plasma membrane
each immuboglobulin chain is synthesized _____ and contains a
independently ; signal peptide directing it to the ER
heavy chains may contain ______ , meaning ?
both signal peptides and stop-transfer sequences
meaning they can become embedded in the ER membrane with their amino-termini localized to the luminal side of the membrane and their carboxyl-termini localized to the cytoplasmic surface
Light chains only contain
signal peptides and pass through to the lumen of the ER
Within the ER, light chains assemble with heavy chains to do what?
form the characteristic heterotetrameric structure
Secreted forms of IgM generally have
lower affinities for their antigens than IgG antibodies
B1 lymphocytes are a subclass of B cells, what do they synthesize/produce
secreted form of IgM –> “natural IgM antibodies”
Which immunoglobulin is the first class of immunoglobulins expressed on immature B cells in the bone marrow
IgM
As vesicles containing the assembled immunoglobulins fuse with the plasma membrane, what happens to certain immunoglobulins ?
they remain bound to the plasma membrane via the stop-transfer sequence, which serves as a membrane anchoring domain
Which 2 classes of immunoglobulins are found embedded in the plasma membranes of the B cells in which they are made
IgM and IgD
As B cells transit from the bone marrow into the peripheral blood they begin to express ____ and are called ?
IgD immunoglobulins ; mature naive B cells
Secreted forms of IgM assemble into multimers of _____ IgM units with an associated joining (J) chain
5
the lower affinities of secreted forms of IgM generally are compensated for by
higher avidities (overall strength) due to the multiple antigen binding sites present in multimers
Which domain must be left out of IgM for it to be secreted from cells rather than being localized to the plasma membrane?
stop transfer sequence
What is different about natural IgM antibodies
They are spontaneously produced without prior immunization
They are produced by unique “unmutated” variable gene segments and are directed initially against self and non-self antigens
They’re important when discussing the ABO blood system
Stop transfer sequences play a role in
anchoring proteins to the plasma membrane
We know that forms of IgM lacking stop transfer sequences do not disengage from the transfer channel and embed in the ER membrane during synthesis, so what happens instead?
Instead, an IgM lacking a stop transfer sequence completely transits the ER membrane to the lumen of the ER.
The lumenal IgM transits in vesicles from the ER through the Golgi to the plasma membrane where fusion of the vesicle with the plasma mebrane releases the soluble lumenal contents into the extracellular environment
What is the most abundant immunoglobulin found in the serum
IgG
How many subclasses of IgG containing slightly different heavy chains are there
4: γ1, γ2, γ3, and γ4
IgGs are secreted immunoglobulins so they lack
stop-transfer sequences in their heavy chains
Once naïve B cells are activated and become effector B cells (plasma cells), IgG are ?
produced in large amounts
What is the primary role of plasma cells
the synthesis of secreted immunoglobulin molecules
The majority of therapeutic antibodies are of what class
IgG
Immunoglobulins can be cleaved into Fc and Fab fragments by ?
digestion with the proteolytic enzyme papain
Pathogens are recognized by immunoglobulins through sequences in their
variable regions (Fab fragments)
Pathogens can be targeted for destruction by macrophages and neutrophils through sequences in their
Fc fragments
Tail regions on IgE molecules bind to _________ promoting _______ attracting _______?
Fc receptors on mast cells
cytokine and histamine release
other components of the immune system
What immunoglobulin is found in the saliva and the surface of mucous membranes, including those of the oral cavity
IgA
What antibody is the first line of defense against inhaled and ingested pathogens
IgA
Why are IgA unusual
they are synthesized by mature plasma cells lying beneath epithelial cell surfaces
IgA dimers synthesized by these B cells are recognized by ____________, and then are ________ , and are released from the
receptors on the basolateral surface of epithelial cells (pIgR)
then they are trafficked/transcytosed through the endothelial cells
released from the apical suface
Release of IgA from the epithelial cells involves ?
proteolytic cleavage of the IgA receptor
What are are found in the Ig domain proximal to the amino termini of light and heavy chains
Variable regions of immunoglobulins
A portion of the IgA receptor remains associated with the
secreted IgA and is referred to as the secretory component
Variable domains are regions where amino acid sequences
differ between one immunoglobulin of a given class and another
Immunoglobulins are made up of repeating structural motifs called ______
Light chains contains ______ of these structural motifs, whereas heavy chains contain _______
Ig domains
2, 4
Only a small number of residues within the hypervariable regions contribute to
the antigen-binding site
In what element of secondary structure are the hypervariable loop regions of immunoglobulins found
random structure
their size and structural contains would not allow for the high degree of variability needed for reverse turns
Hypervariable sequences are found in loops at the ends of
the variable domains where they form antigen-binding sites
Naïve B cells form approximately 10^12 different immunoglobulin sequence variants referred to as the
primary Ig repertoire
The primary repertoire of IgM and IgD molecules produce immunoglobulins capable of
recognizing a tremendous number of different types of antigens, but with relatively low affinities
What is an example of class switching
B lymphocytes switching from IgM and IgD to IgG
As B lymphocytes switch from IgM and IgD to IgG (class switching) during activation, affinities for antigens increase through _________ , this is known as ______ ?
continued amino acid sequence variation in variable regions
secondary Ig repertoire
Effector helper T lymphocytes play an important role _____ , particularly during
in B cell activation
during class switching
NA Rearrangements During B Cell Development Produce
Immunoglobulin Diversity
Immunoglobulin light and heavy chains are encoded ______ on different _____
separately ; chromosomes
Example using light chains as regarding how pieces of a gene come together to create immunoglobulins of diverse sequence
Each expressed light chain is derived from three distinct segments of DNA: V (variable), J (joining), and C (constant)
The κ light chain locus has approximately 35 segments to choose from in creating the V domain, 5 J region segments, and 1 constant region
Thus, there are approximately 175 potential sequence permutations of κ light chains
The heavy chain locus contains ___ V (variable) segments, ___ D (diversity) segments, and ___ J (joining) segments for ____ different sequence permutations
** If you wanted to include the fact that there are 120 sequence permutations of λ light chains, what does this change?
40 V, 23 D, 6 J for
40 x 23 x 6 = 5520
** by recombination alone there are 295 x 5520 > 1.5 million different combinations of heavy and light chains
Not to be answered as a question –> this is the full breakdown of using light chains as an example of how pieces of a gene come together to create immunoglobulins of diverse sequence
Each expressed light chain is derived from three distinct segments of DNA: V (variable), J (joining), and C (constant)
The κ light chain locus has approximately 35 segments to choose from in creating the V domain, 5 J region segments, and 1 constant region
Thus, there are approximately 175 potential sequence permutations of κ light chains
The heavy chain locus contains 40 V (variable) segments, 23 D (diversity) segments, and 6 J (joining) segments for 5520 different sequence permutations
By including the fact that there are 120 (30 V x 4 J) sequence permutations of λ light chains
By recombination alone there are (120 + 175 =) 295 x 5520 > 1.5 million different combinations of heavy and light chains
The joining of V, (D), and J regions occurs via ______ and is catalyzed by an enzyme known as the ______ recombinase
recombination ; V(D)J
The V(D)J enzyme randomly selects a ______ V site and links it to one of the
downstream, J segments (for light chains)
What happens to any upstream V regions
excluded by transcription
Any downstream J segments arising via the recombination process are
removed by RNA splicing
What happens when V and J segments in making a light chain (reading frames) are joined
mistakes occur leading to a further increase in sequence diversity
When V and J segments join in making a light chain, mistakes are made –> what is one of these mistakes involving amino acids?
Some of these mistakes simply change the amino acid that is coded for and that leads to immunoglobulin diversity
When V and J segments join in making a light chain, mistakes are made –> what is one of these mistakes involving functional proteins?
these mistakes prevent a functional protein from being expressed
If this occurs, the B cell that made such a mistake dies in the bone marrow
This process is known as clonal deletion
What is clonal deletion
V and J segments join = mistake –> functional protein is not expressed –> B cell that makes the mistake dies in the bone marrow
When and why is expression of the V(D)J recombinase shut off
Once a B cell begins producing a functional immunoglobulin formed by the joining of heavy and light chains
to assure that only a single immunoglobulin is made by any individual B cell
When and why would the V(D)J recombinase remain active?
If the immunoglobulin produced recognizes a self antigen in the bone marrow
it remains active to give the B cell a chance to make an immunoglobulin that does not recognize a self antigen
What happens if B cells that recognizes a self antigen in the bone marrow escape the process of V(D)J remaining active?
it will produce self-antigens and can cause autoimmune diseases
Somatic hyper-mutation further enhances the affinity of
immunoglobulins for antigens
Once B cells are activated by antigen and begin making a unique immunoglobulin, cells producing this immunoglobulin enter into a
enter into a hyper-mutable state
Explain the hyper-mutable state from B cells making a unique immunoglobulin
This state occurs to actively transcribed genes, and since the immunoglobulin expressed in a particular B cell is the major protein being produced, its gene is subjected to an ENHANCED rate of mutation
These mutations tend to be directed to the VARIABLE part of the immunoglobulin genes
This hyper-mutable state occurs though the induction of ____ and what does this change create?
an activation-induced deaminase enzyme that converts cytosines to uracils on single-stranded DNA in the act of being transcribed
This change creates G-U mismatches, and repair of these mismatches can produce various types of mutations
B cell clones with higher affinities for antigens are preferentially
activated leading to their survival and enhanced proliferation allowing them to dominate as the amount of antigen declines
B cell clones with increased affinity are selected from
the initial population of B cell clones providing enhanced protection against pathogens
What is final change that occurs to immunoglobulin genes upon antigen activation
the switching between different immunoglobulin classes
Class switching is promoted by the
same activation-induced deaminase enzyme involved in hypermutation of the immunoglobulin variable region
What is the major change that occurs as a consequence of class switching
exchanging one heavy chain constant region for another and the subsequent production of secreted rather than membrane bound immunoglobulins
Deamination events cause
double-stranded breaks in switch sequences between heavy chain constant regions
Recombination between Cμ and other C regions
allows class switching to occur
Recombination is mediated by a
recombinase distinct from V(D)J recombinase
Class switching is irreversible as the circular DNA released by the recombinase process is
degraded
In this way (class switching) a B cell goes from making IgM and IgD immunoglobulins to making
IgG, IgA, and IgE
High affinity forms of secreted immunoglobulins are the types of immunoglobulins used
therapeutically
