Glossary of I Exam 2
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- How are immunoglobins structurally organized?
- 2 identical light chains and 2 identical heavy chains
- How are immunoglobins organized functionally?
- into variablle regions and constant regions. Variable regions are the Ag binding sites. Constant regions define the physiologic and biologic properties of the Ig
- What types of L chains are there?
- kappa or lambda
in one Ig you can only have 2 kappa L chains or 2 lamda L chains..never a mixture
- What types of H chains are there?
- mu (IgM), epsilon (IgE), delta (IgD), 2 alpha (IgA) or 4 gamma (IgG)
- What do the H chains define?
- class and subclass of the Ig produced
- fragment, antigen binding
the variable and constant regions (Ch1) (n-termini) on each chain
- Where is the C1q binding site on an Ig?
- Where does pepsin digestion cleave an Ig?
- cleaves after disulfide bonds that hold H-chains together leaving F(ab)2
- Where does papain digestion cleave an Ig?
- before the hinge: does not cleave S-S bonds holding H chains together
- Purpose of variable regions?
- Ag binding
- What is the Fc region on an Ig?
- Fragment, constant
Effector fxn: need conserved seq to interact with effector molecules
Consists of constant regions of H chains
- What is the Ig fold or Ig domain?
- fxnl region of approx. 110 aa
Ex: variable region on 1 light chain including the S-S outpocketing
- How many variable region domains does a L chain have?
- How many constant region domains does a L chain have?
- How many variable region domains does a H chain have?
- How many constant region domains does a H chain have?
- 3-4 depending on the class of Ig (4 for IgE and IgM)
- Definition of Fab fragment.
- MONOMERIC Ag-binding fragment created by papain digestion consisting of an intact L chain and variable H domain and CH1 domain
- Definition of Fc fragment.
- constant region fragment resulting from papain digestion consisting of a DIMER of the H-chain constant region, but lacking the CH1 domain
- Definition of F(ab)2 fragment.
- DIMERIC Ag-binding fragment created by pepsin digestion consisting of intact L chain and the VH and CH1 domains; it is dimeric b/c pepsin cleaves below an interchain disulfide bridge b/n 2 H chains
- Definition of Fv fragment.
- The Vh-Vl DIMER forming the variable region of the Fab fragment
- What portion of the Ag binds to the Ig?
- antigenic determinants=epitopes
- What does each variable region have?
- 3 hypervariable regions
- what is another name for hypervariable regions?
- complementarity determining region(s) (CDR)
- How many CDRs make up an Ag binding site?
- 3 light chain CDRs and 3 Heavy chain CDRs
- What level of protein structure results in Ig structure? Why?
- secondary.. beta pleated sheets b/c the confer rigidity within a large protein molecule (form a core) providing an axis around which conformational changes take place (core where variability can occur)
- Ribbon structure of L chain C domain? of L chain V domain?
- 4 over 3 - C
5 over 4 - V
- What do hypervariable regions of an Ig determine?
- Ag specificity of the Ig molecule
- In terms of the variable domain, where are the CDRs located?
- CDR1 - beginning of domain
CDR2- middle of domain
CDR3- tail end of domain
- Do all 6 CDRs need be present in Ag binding?
- No..not if they aren't prominent in the binding
- Why types of intermolecular forces exist b/n the Ag and the Ab?
- NO covalent bonds formed, only weak, noncovalent bonds: hydrogen bonding, Van der Waals, electrostatic, and hydrophobic bonds
- How does an Ig make contact with immune cell membrane to transduce signal?
- wiggles its way in and in doing so creates lipid rafts
(conformational changes occur following binding of Ag; shift in positions when Ag sits in pocket)
- What is the structure and fxn of the hinge domain?
- hinge region does NOT have Ig domain
fxns as flexible component allowing range of motion for Ig
- How is flexibility limited in the hinge region?
- limited by # of S-S bonds
different Ig classes have different ranges of motion
- Where are carbohydrates present on an Ig?
- the constant region near the complement binding region (C1q)
- Characteristics of circulating Ig.
- characteristics of membrane bound Ig
- ex: found on BCR
bound to TRANSmembrane
- Characteristics of secretory Ig.
derived from circulatory and deposited in area where we want secretions by certain cells (breast milk)
- characteristics of cell-bound Ig.
- circulating Ig that is bound on its Fc end by a specific R on a cell (NK)
- Difference b/n secreted and membrane-bound Igs in terms of COOH terminus?
- secreted has hydrophilic aa sequence
membrane-bound has hydrophilic spacer (26aa) and hydrophobic transmembrane sequence and short cytoplasmic tail
- Which Ig's are always monomer?
- IgG, IgD, IgE
- Which Ig is pentamer when in plasma?
- Which Ig is a dimer 50% of the time that it is in plasma?
- Which Ig is most prominent in adaptive 2ndary response/most abundant Ab?
- Where is IgG found?
- free in plasma
- Which Ig crosses the placenta?
- IgG-provides passive immunization to fetus
- Where is IgM found?
- free in plasma (PENTAMER)
surface of B cell (MONOMER when transmembrane)
- What is the first class of Abs released by B cells during primary response?
- Where is IgD found?
- ONLY found embedded on surface of B cell (transmembrane)
does NOT circulate
- What is the fxn of IgD?
- important in B cell activation
- Where is IgA found?
- found in plasma as monomer
found in secretions (tears, milk, saliva) as dimer
- What is the fxn of IgA?
- protects mucosal surfaces; prevents attachment of pathogens to epithelial cells
- Which Ig is an example of cell bound?
- IgE: secreted by plasma cells in skin and tissues linig GI tract and respiratory tract and found on mast cells and basophils
- What is the fxn of IgE?
- triggers release of histamine from mast cell or basophil that contributes to inflammation and some allergic responses
- Which Ig's have 4 C domains?
- IgM and IgE
- Purpose of glycosylation on Ig's?
- increase hydrophilicity
- Where on an Ig is glycosylation found?
- ONLY on C domains b/c the V domains' job is to bind Ag
around C1q (complement) binding site
- How many forms of IgG are there?
- 4--they differ in hinge regions
- Which class of IgG is the most flexible?
- What holds the IgA and IgM dimeric and pentameric forms together?
- S-S bonds and 1 J chain
- How many H-chain isotypes are there of IgA?
- 2: alpha 1 and alpha 2
- Usual molecular form of IgM?
- pentamer or hexamer
- Usual molecular form of IgA?
- monomer, dimer, tetramer
- What is the molecular wt. of IgM?
- >950 kDa
- What is the molecular wt. of IgG1-4?
- 150 kDa
- What is the molecular wt. of IgA1,2?
- monomer: 160
- What is the molecular wt. of IgE?
- 190 kDa
- What is the molecular wt. of IgD?
- 180 kDa
- Which Ig is primarily for 1' antibody responses?
- Which Ig is primarily for 2' antibody responses?
- IgG and IgA
- Which Ig is secretory?
- IgA1 and IgA2
- Which Ig is for antiparasite responses and allergy?
- Which Ig's activate the classical complement pathway?
- IgM, IgG1, IgG3
- Which Ig's bind to macrophage Fc R's?
- IgG1, IgG3 and IgE
- Which Ig's induce mast cell degranulation?
- How many Ag binding sites are available on an IgM?
- 10 Ag binding sites
- List the Ig's in decreasing order in terms of % circulating.
- IgG, IgA, IgM, IgD, IgE
- What is the difference b/n the pentameric and hexameric forms of IgM?
- pentameric: J chain, found in secretions
hexameric: NO J chain, not found in secretions
- Which Ig increases in cases of autoimmune diseases?
- What is the most prominent form of IgG?
- What is the least abundant form of IgG?
- What is the Bramble R?
- FcR that carries IgG across endothelium into extracellular spaces (blood to tissue): endocytoses IgG in endothelial cell and moves it to tissue where the Bramble is then recycled
- What is the R for transplacental transfer?
- What happens to the mechanism for which IgG leaves blood to enter tissue during times of trauma?
- no Bramble R needed b/c leaky and Ab's will leave
- What is the fxn of Poly-Ig receptor?
- binds dimeric IgA by binding to J chain then transcytosis of IgA-Poly Ig complex through epithelial cell into lumen
- What happens to the poly-Ig when IgA is secreted into the lumen?
- IgA carries part of the Poly-Ig with it to protect the IgA from digestive enzymes
- Where is the secretory component of the poly Ig attached to IgA?
- to the C domain of each IgA monomer--stabilizes union and protects dimer from degradation
- The FcR on basophils and mast cells binds to what region on the IgE?
- CH3 domain
- Where are isotypic determinants found and what is there fxn?
- found on C domains of H and L chains
determine isotype/class (IgG1 from IgM)
- What are allotypic determinants?
- finite/discrete areas w/n C regions defining difference in class b/n SAME species
-subtle aa differences defining me from you
- What are idiotypic determinants?
- Found in V regions
not constant among classes
unique to individuals derived for given Ag (ex: my reaction to strep is different from Ry's rxn to strep)
- What determinants do identical twins share?
- isotypic and allotypic. Different idiotypes
- What are the predominant characteristics of Ig Superfamily?
- the structural unit is the Ig domain (110 aa,50-70aa outpocketed)--characterizes all molecules, homologous cell-cell recognition
- What is the smallest Ig domain?
no transmembrane segment therefore no cytoplasmic tails
- Does having an Ig domain mean that molecule has immune fxn?
- NO..you can have an Ig domain and have no immune fxn
Ex: Platelet derived growth factor
- Pre-antigen exposure takes place where? post?
- pre: BM
post: secondary organs
- What state is the B cell in when it is released from the BM?
- mature, but naive b/c it hasn't encountered any Ags
- What is unique to the lymphoid lineage as the end result of antibody diversity?
- mature B cell w/ DNA that is different than the stem cell that generated it
- What are the 3 gene complexes that encode Igs?
- one for kappa chains
one for lamda chains
one for all heavy chains
- What is the variable region domain encoded by?
- 2 (VJ in light chain) to 3 (VDJ in heavy chain) distinct segments
- What exon segments are involved in L-chain formation?
- L: leader segment
J: joiner segment
C: constant segment
- What exon segments are involved in H-chain formation?
- L: leader segment
V: variable segment
D: diversity segment
J: joining segement
C: constant segment
- What is the first constant exon on H chain?
- Cmu and ending in C epsilon
- Are the 3 gene complexes on the same chromosome?
- No, each on different chromosome
- Why are there more Heavy chain combinations thant light chain?
- b/c of 9 different C regions and 3 aa D region
- What parts of the Light chain are constructed at the DNA level?
- J added to V
C brought into proximity but NOT added
- What parts of the heavy chain are constructed at the DNA level?
- D added to J
V added to DJ: VDJ rearranged DNA
- What parts of the heavy and light chains are constructed at the RNA level?
- C exon added to VJ in L
C exon added to VDJ in H
splicing of introns
- What does RSS stand for?
- recombination signal sequence
- What is the purpose of RSS?
- to regulate joins such that a V gene segment joins to a D or J and not to another V. DNA rearrangements are in fact guided by conserved noncoding DNA sequences. These sequences consist of a conserved block of seven nucleotidesthe heptamer which is always contiguous with the coding sequence, followed by a nonconserved region known as the spacer, which is either 12 or 23 nucleotides long. This is followed by a second conserved block of nine nucleotides,the nonamer
- a 2 turn RSS has what bp spacer?
- 23 bp
- a 1 turn RSS has what bp spacer?
- 12 bp
- What is the purpose of the 12 or 23 bp spacer in an RSS?
- consists of conserved bps/length so as to not disrupt the DNA helix
- What is the 12/23 rule?
- only a gene segment flanked by a RSS with a 12-base pair (bp) spacer can be joined to one flanked by a 23 bp spacer RSS
- Why can't VH gene segments be joined to JH gene segments directly?
- B/C both VH and JH gene segments are flanked by 23 bp spacers, they need a Dh gene to be in the center b/c it has 12bp on either side
- What are Rag 1/Rag 2?
- nuclear phosphoproteins involved in the V(D)J recombinase
They recognize RSSs and form RSS synapse
- Explain VDJ recombination.
- RAG complex recognizes RSSs
RAG complexes bind to each other, bringing together segments to be joined (synapse)
ssDNA nick to form signal joint (2 RSSs coming together) and hairpin structures on ends of each coding segment
2 bps from 1 coding segment "slide" down hairpin creating overhang
hairpin is cleaved
DNA Pol fills in gaps of overhang w/ correct bases (P-Nucleotide addition)
Free DNA is trimmed by exonucleases
TdT adds N-nucleotides to open end of chain
Free DNA ends are aligned, polymerized, and ligated
- In P-nucleotide addition is there a template?
- yes, template created by overhang
- How can added or deleted nucleotides disrupt the reading fram?
- Such frameshifts will lead to a nonfunctional protein, and DNA rearrangements leading to such disruptions, known as nonproductive rearrangements.
End is STOP codons
- Explain TdT N-nucleotide addition.
- attach bases to open end of chain (nucleotides are added at random to the ends of the single-stranded segments )
pol fills in by basepairing
- In N-nulceotide addition is there a template?
- What are P-nucleotides?
- stretches of nucleotides that originate from the complementary strand are known as P-nucleotides
- What are N-nucleotides?
- nontemplated, or N, nucleotides added by TdT at random to ssDNA following P-nucleotide addition
- Is TdT present in rearranging H chains and L chains?
- ONLY H-chains
- What regions determine the Ag specificity of the Ig molecule?
- hypervariable regions
- Which hypervariable region can exhibit diversity?
- only CDR3 b/c it is not encoded in V exon like CDR1 and 2 are
CDR3 is formed by jxn of D &J
- P and N nucleotide additions occur when encoding what hypervariable region?
- How many Igs can 1 B cell produce?
- only 1 Ig/B cell
- What is allelic exclusion?
- the rearrangement of complete heavy and light chain sequences
- How many somatic recombinations occur at the DNA level in light chains? heavy chains?
- 1 in light chains: V and J joined together
2 in heavy chains: V,D, and J joined together
- What happens at the RNA level in Heavy chains?
- o First thing done at the RNA level is to assemble all of the constant regions that we are going to need. (CH1, CH2, and CH3) and CH4 if it is M or E.
- How do we prevent inappropriate linkage b/n exons?
- Regulation is done by recombination signal sequences in conjunction with the 12/23 rule.
- How do we alter the DNA helix?
- 12 bp= 1 turn in helix
23 bp=2 turn in helix
If you are talking about inserting and recombining a coiled DNA helix, we have to be restrained somehow by the shape of that coil. If start randomly inserting DNA in, it will lead to kinks. Keeps the molecule in tertiary conformational comfort.
12 is going to align with 23 b/c of the shape of the helix
- What are Rag1 and Rag2?
- phosphoproteins in the nucleus and provide the basis for the recognition sequence of the 12/23 rule
- What is junctional flexibility?
- •Trimming of free DNA by exonuclease
o Exonucleases come in and trim the ends. They do this by nicking off the exisiting gene or after the polymerase has added extra. Causes flexibility by removing bases
- VDJ exons and VJ exons make up what component of the Ig molecule?
- hypervariable region
- Do the junctional rearrangements affect all CDR's?
- NO only CDR3
CDR3 results being encoded by the end of the very end of the V region, D region and J region. CDR3 is a joining of the product of these 3 exons
- Why can't we have N-nucleotide addition in Light chains?
- b/c there is no TdT, so we can’t have N nucleotide addition and all we can have is P nucleotide addition
- What keeps the cell from undergoing continuous rearrangement?
- A process known as allelic exclusion.
- What is the basis of allelic exclusion?
- We ensure that once we have rearranged the gene and put it together (joined exons) that after that it cannot be altered.
All other possibilities are excluded by suppressing other alleles
- Which chains are put together first?
- Heavy chain rearrangement happens first sequentially: B cell always starts by putting together a heavy chain.
- When does light chain rearrangement occur?
- when the cell has committed itself to a heavy chain.
- Explain the process of heavy chain rearrangement.
- In the first DNA recombination event, D and J are combined. If they are successfully combined, all other attemps to combine D and J are suppressed. Once D and J are together, then we want to add a V chain. Have two options. One from the mom and one from the dad. Once one of the Vs is successfully joined with D and J then all other V chains are blocked from joining.
If we have a D and J and we try to hook up to a V but something is wrong in the Rag1 Rag 2 complex and it doesn’t hook up properly, then you can try again with a V from another chromosome.
If keep marching down and trying non productive connections then the cell will just die via apoptosis.
Once we have selected D, J and V, then we can start to assemble the light chain.
- At what stage of B cell maturation does the B cell have both heavy and light chains rearranged?
- when it is an immature B cell
- What is the first Ig that the B cell can express?
- IgM, b/c in the process of development it hooks up to the first constant region, mu, which it presents as a monomer (transmembrane form). It is secreted as a pentamer
- where is the immature B cell located?
- in the BM
- Why could a B cell be killed while in the BM when still immature?
- If when the cell is in the bone marrow it grabs on to stromal cells in the bone marrow because it recognizes the protein, then it will be killed, because it is recognizing self.
If IgM binds to self it is eliminated via apoptosis
- When will an IgD present on the surface of a B cell?
- • Once B cell has undergone selection (doesn’t bind to self) it will undergo alternative mRNA splicing and you will see IgD
This indicates a mature, but naive B cell
- How is the IgD on the mature B cell different from the IgM?
- In this case the IgD molecule expressed by the cell which has the delta constant region on its tail, has the exact same variable region as IgM that is produced by the cell
- What is the purpose of an IgD on the surface of B cell?
- identifier of molecules that have undergone mRNA splicing and selection. Will never really see IgD in circulation
- What leads to either an RNA encoding an IgM or an IgD heavy chain.
- •This is done at the RNA level by recognition of polyadenylation sites.
•The primary transcription site in the B cell, for instance, contains both the gene for the IgM and IgD in close proximity. They are separated by polyadenylation.
•If the transcript is cut in the region of the pA1 you get transcription of IgM.
•If you cut the transcript at pA2 instead then get expression of IgD
If we cut at pA2, all mu exons are removed and only delta exons are expressed
- Production of secreted or membrane bound Ig forms depends on what?
- alternative mRNA splicing
Each heavy-chain C gene has two exons (membrane-coding) that encode the transmembrane region and cytoplasmic tail of the transmembrane form, and a secretion-coding (SC) sequence that encodes the carboxy terminus of the secreted form
if the primary transcript is cleaved and polyadenylated at a site DOWNSTREAM of these exons, the sequence encoding the carboxy terminus of the secreted form is removed by splicing and the CELL-SURFACE form of immunoglobulin is produced. Alternatively, if the primary transcript is cleaved at the pA site located before the last two exons, only the secreted molecule can be produced.
Secreted forms will have hydrophilic ends
Transmembrane forms will have hydrophobic ends
- Have B cells with both IgM and IgD on surface undergone isotype switching?
Later in the immune response, however, the same assembled V region may be expressed in IgG, IgA, or IgE antibodies. This change is known as isotype switching. It is stimulated in the course of an immune response by external signals such as cytokines released by T cells or mitogenic signals delivered by pathogens
- The events that occur BEFORE B cells encouter Ag provide for what?
- primary Ag receptor repertoire
all events that lead to genetic diversity BEFORE Ag exposure
- Explain main events in Generation of Diversity AFTER B cells encounter antigen
- •Mature B cells are expressing IgM with a so so affinity. It is going to become a B cell with exquisite specificity.
•Going to achieve this specificity by molding product of B cell with antigen: known as clonal selection.
•Going to change Ig from transmembrane to secreted form
•Going to generate changes by somatic hypermutation. No more rag, no more P or N nucleotide addition or junctional flexibility. We have already selected out CDR3.
•Going to generate higher affinity Ig
- Compare Pre Ag G.O.D. and Post Ag G.O.D.
- pre: junctional flexibility (with framshifts and STOP codons)affecting ONLY CDR3 that occurs during recombination
post: somatic hypermutation affecting ALL CDR's (no framshifts, but can have STOP codons) requires proximity to promoter
- Where does the randomness of somatic hypermutation occur?
- point mutations only in the variable regions
don't want to mess with effector fxn of C regions
- Where does SHM occur?
- germinal center of 2ndary lympoid follicle
- What effect does SHM have on affinity?
- •Can have a positive, no or negative effect on IgM affinity
- When in B cell maturation can SHM occur?
- • Can only occur with a mature B cell with an IgM presented on its surface that has migrated to secondary lymphoid tissue
- Why does clonal selection occur?
- •With sequential antigen administration we are refining the immune response with vaccination over time.
Clonal selection is the process of trying to adjust fit to Ag by inserting different aa to repulse Ag or tighten affinity of have NO affect at all
- How does frequency of SHM depend on distance?
- frequency decreases with distance from the rearranged
V(D)J gene b/c mutations DO NOT extend into the portion of the gene encoding the constant region
- What does isotype switching depend on?
- Dependent upon secretion of cytokines from T cells makes them change effector function and dependent on microenvironment
- What does isotype switching do to the Ig?
- translation of H chain constant region and LOSS of intervening DNA
-DOES NOT affect specificity or affinity of variable region
-increases the diversity of Ig function
- What occurs during isotype switching?
- Switch regions under the influence of cytokines, which direct, come together and DNA is looped out. Now constant region for IgM and D is removed and V D J region is untouched and is aligned with the C region of IgG3. So the cell now produces IgG3
IgG3 can go on to produce other classes but can never go back to IgM or IgD. The DNA for that is lost.
Once DNA is excised, cell is commited to any Ig downstream of the one it has switched to
- Which heavy chain gene doesn't have a preceding switch region?
- Cdelta b/c its S region is the same as IgM so you don't need it
- What is the purpose of V(D)J recombination vs. isotype switching?
- recom: Ag diversity
switch:change of isotype of Ig
- When is reading frame affected?
- only in V(D)J recombination, NOT possible in class switch
- What happens when the functional mRNA is exported from the nucleus?
- mRNA for the light chain and heavy chain are each transcribed on different poly ribosomes.
•They are lead into the rER by the leader sequence; series of amino acids on terminal region of V region that is taken into rER and orients them.
•Then the leader sequence is cleaved off in the lumen of the rER, it has served its purpose.
•The heavy and the light chains pass through the cisterna moving through the rER and that is where we get disulfide bonds that connect H and L chains. Make the H2L2 structure.
•Then H2L2 goes to golgi where it gets glycosylated
•Then they get incorporated into secretory vesicles.
•If you have transcribed one with transmembrane domain, it becomes embedded in part of the secretory vesicle membrane, goes to the surface, and fuses with the membrane and is expressed as a membrane Ig. If you have transcribed a secreted form, then it is in the secretory vesicle but is released from the cell.
- What are the molecules that are absolutely necessary for the presentation of antigen to T cells?
- major histocompatability complex proteins• T cells cannot see antigens unless they are presented by MHC or MHC like molecules
- What happens to B cells once they enter 2ndary lymphoid organs?
- Ag-dependent proliferation and differentiation into plasma and memory cells
- What is expressed on the Pro-B surface?
Calnexin holds these markers together
- In terms of H and L chains, which chains if any are rearranged in the Pro-B stage?
- Only the DJ exons of the H chain are rearranged
L chain configuration is still that of germ line
- What is expressed on surface of Pre-B cell?
- In terms of H and L chains, which chains if any are rearranged in the Pre-B stage?
- The complete V(D)J of H chain is rearranged
L chain configuration is still that of germ line
Surrogate L chain present
- In terms of H and L chains, which chains if any are rearranged in the Immature B?
- H chain rearranged, L chain rearranged, Ab present (IgM)
- What percent of mature B cells encounter Ag?
- Only 10% . If they don’t encounter Ag they die of lonliness..huge amt of cell loss
- Which chromosome is MHC on?
- The major histocompatibility complex is located on chromosome 6 in humans and chromosome 17 in the mouse
- Is the beta2-microglobin located on the same chromosome as MHC class I?
- The gene for b2-microglobulin, although it encodes part of the MHC class I molecule, is located on a different chromosome
- What are the three main class MHC I genes?
- HLA-A, -B, and -C in humans
- What genes are present in MHC class II?
- genes for the a and b chains of the antigen-presenting MHC class II molecules HLA-DR, -DP, and -DQ
- Interferon gamma regulates what MHC class genes?
- MHC II
- What is the fxn of the HLA-DM genes?
- catalyze peptide binding to MHC class II molecules
- What are the MHC class I and II genes called in humans?
- In humans these genes are called HumanLeukocyteAntigen or HLA genes
- The particular combination of MHC alleles found on a single chromosome is known as what?
- MHC haplotype
there is only a small chance that the corresponding MHC locus on both the homologous chromosomes of an individual will have the same allele; most individuals will be heterozygous at MHC loci
- What does it mean that expression of MHC alleles is codominant?
- the protein products of both the alleles at a locus being expressed in the cell, and both gene products being able to present antigens to T cells
- The a chain of the class I molecule has three extracellular domains, what are they?
- a1, a2 and a3, encoded by separate exons
- What is the advantage of two different alleles at any given MHC locus (heterozygotes)?
- have a better chance of resisting infection than those with identical alleles at the locus, as they have a greater capacity to present peptides from a wide range of microbes and parasites
- Explain the structure of class I MHC proteins.
- Class I MHC proteins consist of a transmembrane a chain, which is encoded by a class I MHC gene, and a small extracellular protein called b2-microglobulin. The b2-microglobulin does not span the membrane and is encoded by a gene that does not lie in the MHC gene cluster. The a chain is folded into three extracellular globular domains (a1, a2, a3), and the a3 domain and the b2-microglobulin, which are closest to the membrane, are both similar to an Ig domain. The two N-terminal domains of the a chain, which are farthest from the membrane, contain the polymorphic (variable) amino acids that are recognized by T cells in transplantation reactions. These domains bind a peptide and present it to cytotoxic T cells.
- Explain the structure of class II MHC proteins.
- Like class I MHC proteins, class II MHC proteins are heterodimers with two conserved Ig-like domains close to the membrane and two polymorphic (variable) N-terminal domains farthest from the membrane. In these proteins, however, both chains (a and b) are encoded by genes within the MHC, and both span the membrane. The two polymorphic domains bind a peptide and present it to helper T cells
- What is the primary fxn of the MHC gene complex-encoded proteins?
- the presentation of antigenic peptides to T cells, thereby controlling the maturation of the ADAPTIVE immune response, both humoral and cellular
- What is the multigenic gene complex in humans? mice?
- humans: HLA gene complex
mice: H-2 gene complex
- The MHC class I genes are similar to what genes in the mouse?
- H2-K, -D, and -L in the mouse
H-2 locus controls reactivity
ability to respond depends on H-2 locus and allele on locus
- How do T cells recognize Ag?
- via their TCR when Ag is bound to MHC protiens on the APC
- What cells express MHC class I?
- • MHC class1 are expressed on every single cell of the body except for erythrocytes and platelets. Help define us as self.
ONLY on nucleated cells
- What cells express MHC class II?
- • MHC class 2 are confined to those cells important to the immune response and are expressed on antigen presenting cells (APC) which are macrophage, dendritic cells and B cells. They can be upregulated by cytokines
- What protein structurs do MHC molecules express?
- complex tertiary and quarternary structures
- Why are MHC molecules considered to be part of the Ig Superfamily?
- b/c they have an Ig fold/domain
- What is the major fxn of MHC I?
- present peptides to T cells which express CD 8. express altered self proteins that are endogenously processed within the cell
- Each MHC I locus encodes how many different chains?
- only 1 alpha chain/locus
many allele choices
- Each MHC II locus encodes how many different chains?
- 2 chains: alpha and beta/locus
many allele choices
- What is embedded in the center of the MHC class II complex? What is its fxn?
- o Embedded in the center is the DM/DO locus. It is not an MCH2 molecule in terms of presentation but instead encodes those molecules that are important in the process of presenting antigen in these molecules
- What is the major fxn of MHC II?
- oThey serve to present antigenic peptides to CD4 or helper cells and are primarily inducing an adaptive immune response
- What is located in the MHC class III?
- oNot really an MHC molecule at all
oThe S molecule encodes molecules that are important in the immune response but have nothing to do with antigen presenting.
oIe. Compliment proteins, TNF alpha, TNF beta
oThese are encoded on the MHC complex but not a MHC molecule
- What is linkage disequilibrium?
- when you have the suppression of crossing over within an area.
The combination of alleles or sequence found on a chromosome is known as a haplotype and they are co dominant.
•Most are heterozygous. Very rare to find homozygous alleles.
•Maintained because there are low levels of recombination within the MCH gene complex.
- Why don't we want crossing over to occur within the MHC?
- to select for HLA haplotypes that confer resistance to disease or survival.
- What is a haplotype?
- the combo of HLA alleles found on a given chromosome 6;each individual expresses both alleles for each HLA gene
- In one mating, what types of combos of haplotypes can occur?
- four possible combos of haploytypes can be found in the offspring: 1 chance in 4 that an individual will share the same haplotypes with a sibling
- At a single gene locus, how many alleles can an individual express? Therefore, how many different MHC molcules can human typically express?
- no more than 2 alleles/locus
human typically express 6 different MHC I and 6 different MHC II on his/her cells
- Which MHC class I gene has the most alleles?
- HLA-B +600 alleles
- How many loci/gene in MHC Class II?
- 2 loci :alpha and beta / gene with the exception of HLA-DR which has multiple beta loci (9) each with multiple alleles
- What is the predominant class 2 molecule for tissue rejection and antigen presentation ?
- Which Beta loci of DR are the most heterogenous?
- Explain the complexity of DR.
- •If you are DRB1 you have the B1 block
•If you are DRB3 you inherit that block to make the B 1 beta chain and you also inherit a gene called B3 which allows you to make a different beta chain.
•If you are B4 or B5 you have the option of making a B1 or B4, or B1 or B5 beta chain respectively.
- What is the purpose of pseudogenes?
- These exons or loci have arisen evolutionarily through gene duplication and through that process you get mutations. A pseudogene either has a stop codon or has a deleterious frameshift.
•Theses are loci within the complex that have arisen but are dysfunctional (they are damaged or don’t work), however they have the capacity, through mutation, to be functional again. So they cannot be ignored. Occasionally they will undergo a productive mutation and encode a beta chain.
- Which genes in HLA-DR are pseudogenes?
- HLA-DRB2, B6, B7 B8 and B9
- If you are homozygous, how many isoforms of MHC class I will you have? MHC 11?
- 3 isoforms (HLA-A1, HLA-A1, HLA-B2, HLA-B2, HLA-C4, HLA-C4)
minimum 3 isoforms for MHC II
- If you are heterozygous, how many isoforms of MHC class I will you have? MHC 11?
- there are 3 loci therefore 6 isoforms can occur for both MHC I and II
- How many haplotypes do we inherit from our parents? How can a new haplotype (R) arise?
- 1 haplotype/parent
new haplotype can arise from recombination of maternal haplotypes: when rare crossing over does occur it provides for continuing source of heterogenity allowing new haplotypes to develop
- Describe MHC class I gene organization.
- •There are 6 exons primarily. 2 that make the cytoplasmic region. 1 that makes the transmembrane domain and three functional regions called alpha 1, 2 and 3. These exons are transcribed together.
•Leads to mRNA with a leader sequence followed by alpha 1,2 and 3 with transmembrane and cytoplasmic tail.
•Amino terminus is labeled alpha 1. Alpha 3 is proximal to the membrane
•All these proteins have quaternary structure
•There is Beta 2 MG which is the simpliest member of the IG superfamily. Not covalently linked to the alpha chain. Supported by non covalent interactions.
- Which domains of the MHC I are true Ig domains?
- •Domains alpha 1 and alpha 2 is not a true immunoglobulin domain. Only Alpha 3 and B2M are.
- What is the fxn of beta2MG in MHC class I?
- provide same support that alpha3 does to prevent alpha 3 from sagging
- Describe MHC class II gene organization.
- 2 chains: alpha and beta
alpha: a1 exon, Ig domain a2exon, transmembrane and cytoplasmic tail exon
beta: b1 exon, b2 Ig domain,transmembrane and cytoplasmic tail exon
- Which domains of MHC I have the binding site for Ag?
- •Antigen peptides are held and presented to TCR in the pocket formed by alpha 1 domain and alpha 2 domain
- What 2ndary structure are Ig domains?
- beta pleated sheets
- What 2ndary structure encompasses the area of MHC for Ag presenting?
- oUpper part of molecule where the antigen is presented is a combination of alpha and beta pleated sheets
B sheet floor, and 2 side walls formed by alpha helices
- What structural characteristics are similar b/n MHC I and II?
- highly similar quarternary structure
- What is the major site of hypervariability in MHC I?
- both alpha 1 and alpha 2 domains contribute to specificity
"hot spots" that differ b/n individuals in terms of aa present
- What is the major site of hypervariability in MHC II?
- major site of variability is Beta 1 , alpha 1 not involved as much, more involved w/ stability of binding site
- What determines the shape of the peptide binding groove?
- changes in 2ndary and tertiary conformation b/n alleles..changes in aa sequence "hot spots"
- What is the mass of the MHC I molcule?
- he entire MHC 1 molecule is approximately 45 KD and the B2M is approximately 12 KD giving it a total of 57-60 KD.
- What is the mass of the MHC II molecule?
- In the case of class 2, the alpha chain is approximately 33 KD (it smaller because it only has 2 domains made by the alpha 1 and the alpha 2). The beta chain is slightly shorter at approximately 28 KD. The class 2 molecule has a total mass of 60-61 KD.
- How are the transmembrane segments of MHC I and II similar? cytoplasmic segments?
- The transmembrane regions of these chains are all similar. These transmembrane regions are highly conserved and are approximately 25 amino acids in length and are hydrophobic because they are inserted in the lipid bilayer.
The cytoplasmic tails of these molecules are very short and they are hydrophilic and are approximately 30 amino acids in length.
- Why is there is a much higher variability frequency in alpha one and two as opposed to alpha 3?
- b/c alpha 3 is not involved in Ag presentation
- How big of a peptide can MHC I accomodate? MHC II?
- MHC I: 8-10 amino acid sequences long
MHC II:11-15 aa long
- Explain the meaning of "motif" in peptide anchor sites.
- These are amino acids that the peptides like to present
•An allele can present a wide range of peptides as long as it has those anchor residues in those positions. Each alleles gives you the capacity to present several peptides but has to accommodate a certain motif.
If nothing fits the pattern the MHC molecule will not present that as antigenic.
Ex: HLA-A2 and HLA-A3 have different motifs, bind different Ag
- What are 3 features of peptide binding to MHC molecules?
- 1. each MHC displays 1 peptide at a time
2. low affinity, broad specificity so different peptides can bind to the same MHC molecule
3. MHC molecules bind only peptides
- Do MHC molecules bind lipids, carbs, or nucleic acids?
- No! T cells do respond to both glycoproteins and glycolipids but molecules that present glycolipids are presented by CD1, not MHC
- What is CD1?
- MHC-like Ag presenting protein that associates with B2m but is HYDROPHOBIC allowing the presentation of hydrophobic lipid Ags: glycolipids
- is CD1 encoded on chromosome 6?
- no, chromosome 1
- How is CD1 structurally different from MHC I
- deeper pockets
more pockets (4)
pockets capped by alpha 1 and 2 so water is excluded and lipids can be presented (hydrophobic binding site as comparted to hydrophilic binding sites of MHC I and II)
- Does MHC I present endogenous Ag or exogenous Ag?
- MHC I: endo to CD8
MHC II: exo to CD4
- What do professional APC's express and what are pro-APC?
- express MHC class II as well as MHC class I
B cells (activated)
- Can both B and T cells see free Ag?
- no, only B can see free Ag
T need Ag to be presented on MHC
- Will unactivated B and macrophages have MHC II?
- no, must be activated
- What is the important step in the induction of the adaptive immune response?
- the processing of foreign antigen via MHC II by APC's
- What is Ag processing?
- o degradation of proteins into antigenic peptides
o antigens (proteins, lipids or glycolipids) are degraded to smaller sizes that can now be presented in the context of MHC
- what is Ag presenting?
- o expression of the peptide on a cell’s surface in association with MHC molecules or CD1
- What is the purpose of MHC I?
- to present self or tag self
- When do dendritic cells express MHC II?
- • Dentritic cells constitutively, (all the time), express MHC 2 molecules and molecules that are called co stimulatory:
- What do the costimulatory molecules, CD80/CD86 on the APC bind to?
- CD28 on T cell
required for complete induction of an immune response
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