Lecture 5 - Antigen Presentation II Flashcards
In general, which cells can capture Ag from the environment and present it?
Professional antigen presenting cells
Describe the overall process of Ag presentation in the context of MHC class II
- APC endocytoses Ag
- Endosomal proteases (cathepsins) degrade the Ag
- Ag peptides are contained in endosomal compartments
- MHC II molecules are synthesised in the ER
- Trafficking of vesicles w/ MHC II from ER to endocytic compartment
- MHC II molecules bind the Ag peptides
- MHC II-peptide complexes are transported to the plasma membrane
What is the ‘goal’ of MHC II presentation?
To display in real-time on the plasma membrane a sample of all the proteins contained in endosomal compartments of the cell
When does Ag presentation on MHC II occur?
Constitutively:
In the absence of infection, only peptides derived from ‘normal self’ proteins are presented
Upon infection or cellular transformation ‘altered self’ proteins are also presented in conjunction with ‘normal self’ protein derived peptides
What degrades proteins in endocytic compartments?
Endosomal proteases called cathepsins
Describe the mechanisms of endocytosis that occur
What does each result in the formation of?
What is each process dependent on?
Describe the specificity of each mechanism
Which cells are capable of each mechanism of endocytosis?
Which mechanisms are most important in MHC II presentation?
- Macropinocytosis
• ‘Drinking’ a large volume
• Actin dependent (to bend the plasma membrane)
• Non-specific
• Results in the formation of a macropinosome
• DCs - Phagocytosis
• ‘Eating’ a large particle (bacterium, protozoans, cells)
• Actin dependent
• Triggered by a surface receptor (specific)
• Results in formation of a phagosome
• DCs, Macrophages - RME
• Capture of a soluble molecule
• Clathrin-dependent
• Specific: triggered by a surface receptor
• Results in the formation of a micropinosome
• DCs, Macrophages, B cells
also:
4. Pinocytosis
• 'Drinking' a small volume
• Clathrin-dependent
• Non-specific ("fluid phase")
• Results in the formation of a Micropinosome
- Autophagocytosis (?)
• Cytosolic proteins are transferred to endosomes
• Mechanism unclear
• Results in the formation of an autophagosome
Most important:
• Macropinocytosis
• Phagocytosis
• RME
Which mechanisms of endocytosis can the following cells perform:
• DCs
• Macrophages
• B cells?
DCs:
• Macropinocytosis
• RME
• Phagocytosis
Macrophages:
• RME
• Phagocytosis
B cells:
• RME
Which receptors do each of the following cells use to perform RME:
• DCs
• Macrophages
• B cells?
DCs:
• Mannose receptors
• FcRs
etc.
Macrophages:
• Mannose receptors
• FcRs
etc.
B cells:
• Only surface Ig
Describe the different aspects of the endocytic pathway
Describe the biochemical changes that occur along the endocytic pathway
- Early endosome
•The immediate product of endocytosis
•Contents largely materials from extracellular environment - Late endosome
•Contents are more ‘cellular’, and less ‘extracellular’ - Lysosome
•Final destination of materials in endosomes
•Degradation of materials into smaller components that may be recycled and used again by the cell
As the endosome ‘matures’, contents become increasingly:
• Acidic (pH decreases)
• Reducing (sulphide bonds destroyed)
• Richer in proteases
Describe the process of degradation of endocytosed protein
– Denaturation –
- Acidification:
• H+-pump ATPases lower the pH of endosomal compartments - Reduction of disulphide bonds
• GILT cleaves S-S bonds
• GILT: gamma interferon lysosomal thiol reductase
Cleavage of disulphide bonds plus low pH results in the denaturation of proteins → more accessible to proteolysis
– Proteolysis –
By endosomal proteases, known as Cathepsins (Cts + letter; e.g. Cts D)
Describe the features of cathepsins
• Abundant in all cells (not just APCs) → indicates general role in turn-over of endogenous membrane proteins and endocytosed extracellular proteins
Different active sites:
• Cysteine (Cts B, L, S)
• Aspartic acid (Cts D, E)
• Serine (Cts G)
Variable optimum pH
Rather nonspecific and redundant
• Different cathepsins will do the same thing (i.e. cleave at the same location)
Can be secreted for extracellular proteolysis
Describe the route that MHC II molecules take before they are loaded with peptide
- Synthesised in the ER
- Ii (invariant chain) binds to peptide binding cleft of MHC II in ER (to prevent binding of other ER components)
- Specific Ii sequence on cytoplasmic tail tags MHC II for transport to the endocytic route (not to the plasma membrane like MHC I)
- Zipper region of Ii is cleaved by an unknown mechanism, releasing three αβ dimers, each bound to Iip10
- Cathepsin S cleaves Iip10 at remove transmembrane tail (only CLIP still bound to αβ dimer)
- Chaperone HLA-DM interacts with αβ-CLIP, resulting in an ‘open conformation’ of the αβ dimer
- CLIP substituted by an antigenic peptide
8a. Preformed peptide binds to MHC II binding cleft
8b. Epitope capture:
• Longer polypeptides bind to the MHC II binding cleft, then are further trimmed down to size
- Complexes trafficked to the cell surface
- Remain on cell surface until the peptide dissociates from the MHC II binding cleft
Describe the peptide binding cleft of MHC II molecules
Open on both sides (“hot dog”)
Can bind peptides of any size
(Unlike MHC I, which binds peptides 9 aa in length)
Describe the structure of Ii
Transmembrane: embedded in the ER membrane, tail on the inside of the ER
Two regions:
- “Zipper” region
• Promotes formation of a trimer (three Ii) - CLIP region
• Occupies the peptide binding cleft of the αβ dimer
• CLIP is promiscuous: fits the peptide binding cleft of all MHC II allotropes
What are CIIV and MIIC?
CIIV:
• Early endosome that contains MHC II molecules
MIIC:
• Late endosome that contains MHC II molecules
What determines how long a MHC II:peptide complex remains at the cell surface?
Half life at cell surface is directly proportional to the affinity of the interaction between the MHC II:peptide binding site and the peptide
What is responsible for the removal of CLIP from the MHC II molecule?
Humans: HLA-DM
Mice: H2-DM
Describe how only high affinity MHC II:peptide complexes make it to the surface
HLA-DM / H2-DM subjects the MHCII:peptide complexes to editing
Only complexes carrying peptides that confer a minimum stability leave the endosomesIf the peptide does not bind strongly enough, it is removed again by HLA-DM/H2-DM and a new peptide will bind
This process continues until a complex with at least the minimum stability is formed
What is the role of MARCH1?
A membrane-associated ubiquitin ligase
Ubiquitinates MHC II:peptide complexes at the cell surfaces
Once this happens, the MHC II is taken back up, delivered to lysosomes and degraded
This faster turnover of MHC II:peptide complexes from the cell surface occurs when the APC has not encountered a pathogen
The process stops once the APC encounters pathogens
Why is it important to present contents of endosomes on the surface of cells?
Some microbes infect cells, but reside in the endosome
e.g. M. tuberculosis
Topologically, the endosome is equivalent to…
the outside of the cell
Compare the 'mechanisms' of the following: • Macropinocytosis • Micropinocytosis • RME • Phagocytosis
Macropinocytosis: actin-dependent
Micropinocytosis: clathrin specific
RME: Clathrin dependent
Phagocytosis: actin-dependent
What role does actin play in some mechanisms of endocytosis?
Strength of the actin cytoskeleton required to push the pull the plasma membrane into a macropinosome / phagosome
What role does Clathrin play in some mechanisms of endocytosis?
Forms a ‘cage’ around the vesicle’
Differentiate between RME and phagocytosis
Phagocytosis
• Invagination of a large portion of membrane
• Eating of a large structure, such as a microbe or a human cell
• Actin dependent
RME:
• Invagination of a small portion of the membrane
• Clathrin dependent
NB Both processes are specific (triggered by surface receptors)
How does the endosome become acidic?
H+ pump (ATP dependent), H+ pumped into endosome to lower pH of endosomal compartments
What is GILT?
γ-interferon lysosomal thiol
This is an enzyme increasingly present in the endosomes
This enzyme cleaves disulphide bonds present in the proteins in the endosomal compartments
Why is it important to break disulphide bonds in proteins as they are being degraded?
The proteins become denatured (linear) and more accessible to proteases
Describe the specificity of proteases
Largely non-specific
Can not predict where proteases cleave
They merely cleave at regions where they can fit
(i.e. unlike restriction enzymes)
Are cathepsins Ag presentation specific, or ubiquitous?
Ubiquitous
Characterise the abundance of the various antigenic peptides along the endocytic route
The abundance / availability various along the endocytic route
As proteins travel along this route, they are exposed to more and more proteases.
Different peptides will be generated at different points along this pathway
This generators a large repertoire of peptides for presentation by the cell.
This is desirable, as it means there is a greater chance that an immune response will be primed
Which protease removes Iip10 from the αβ dimer?What is the result of this?
Cathepsin S
The result is CLIP bound to the binding cleft of the αβ dimer
Describe the structure of HLA-DM
Looks like a HLA class II moleculeDoes not have an open peptide binding cleft
What are the two models for peptide binding to the binding cleft of MHC II molecules?
- Binding of preformed peptides
• Occurs later on in the endocytic pathway (LE, MIIC) - “Epitope capture”
• Ensures protection of peptides from being completely degraded before they can bind to the MHC II molecule
• Occurs early on in the endocytic route (i.e. EE, CIIV)
Probably both of these mechanisms are occurring at different points
What is special about MHC II molecules in terms of the lysosome?
They are one of the few proteins that are resistant to degradation in the lysosome, and that can escape back to the plasma membrane from the lysosome
In summary, MHC I presents material from the …, while MHC II presents material from the …
MHC I: cytosol
MHC II: endosomes
What is the effect of IFN-gamma on the MHC II pathway of Ag presentation?
IFN-gamma induces:
• GILT expression
• HLA-DM expression
List the functions of the Ii
- Prevent peptides from binding the MHC II molecules in the ER
- Targeting of vesicles containing MHC II molecules to CIIV and MIIC
