Mechanisms of peptide loading into MHC
Peptides are anchored in the peptide-binding cleft of MHC molecules by fitting specific amino acid residues into "pockets" at the bottom of this cavity. The sources of peptides and their binding geometry depend on the molecule class.
- MHC-I binding: The peptide is secured by its ends in two pockets. The first pocket binds the C-terminal residue, while the second most commonly corresponds to position 9.
- MHC-II binding: The peptide is anchored in several (typically four) pockets located at various sites along the bottom of the cleft (typical positions: 1, 4, 6, and 9).
Interestingly, the loading process is not selective for foreign molecules. Up to 90% of the peptide pool in MHC consists of fragments of the cell's own proteins. During infection, the proportion of foreign peptides is no more than 0.1% (about 100 out of 100,000 MHC molecules on a cell). Total presentation of self-proteins is used by T cells as a baseline to detect pathological changes.
MHC-I pathway: processing of intracellular proteins
MHC-I molecules are designed to present peptides of intracellular origin. The process of their preparation begins in the cytoplasm.
- Proteasomal degradation: Cytoplasmic proteins are tagged with polyubiquitin and degraded in proteasomes (20S complexes with a mass of ~1500 kDa). Cleavage is mediated by the $\beta$-subunits of the inner ring ($\beta_1$, $\beta_2$, $\beta_5$).
- Effects of IFNγ: Under the influence of interferon-gamma, new subunits (LMP2, MECL1, LMP-7) are synthesized, which form the immunoproteasome. It more efficiently produces peptides with hydrophobic and basic C-termini, which are ideally suited for MHC-I.
- Transport to the ER: Peptides are transported from the cytoplasm into the endoplasmic reticulum (ER) via specific transporters TAP1 and TAP2.
- Assembly and stabilization: The MHC-I $\alpha$-chain and $\beta_2$-microglobulin are synthesized in the ER. While the molecule is empty, its conformation is supported by chaperones (calnexin and calreticulin). As soon as the peptide is embedded in the cleft, the chaperones dissociate, and the stabilized complex is transported to the cell membrane via the Golgi apparatus.
MHC-II pathway: presentation of exogenous antigens
MHC-II molecules present peptides of extracellular origin. Antigen-presenting cells (APCs) uptake them via endocytosis (phagocytosis, macropinocytosis, clathrin- or caveolae-mediated endocytosis).
- Synthesis and transport of MHC-II: The $\alpha$- and $\beta$-chains of the molecule are synthesized in the ER. To prevent premature binding of self-proteins to the antigen-binding cleft, it is blocked by the invariant Ii chain. The complex is packaged into vesicles and directed to the MIIC compartment.
- Endosomal processing: Internalized exogenous proteins enter endosomes, where the environment is gradually acidified. The acidic environment activates proteases—cathepsins L and S.
- Peptide loading: Cathepsins cleave foreign proteins into peptides and degrade the MHC-II Ii chain, leaving only a small fragment—CLIP—in its cleft. The acidified endosome fuses with the MIIC vesicle. A specialized molecule, HLA-DM, catalyzes the displacement of CLIP, making room for the insertion of the generated exogenous peptide. Afterward, the complex is transported to the cell surface.
Cross-presentation
The classical model assumes that intracellular proteins are loaded onto MHC-I and extracellular ones onto MHC-II. However, mounting a cytotoxic response against extracellular pathogens requires presentation in the context of MHC-I. This process is ensured by mechanisms of cross-presentation.
The main pathway is retrograde transport, in which exogenous proteins exit the endosomes into the cytosol, are degraded in cytosolic proteasomes, and their peptides are then transported into the ER for loading onto MHC-I molecules. There are also mechanisms of direct protein penetration into the cytosol and cleavage directly within endosomes.
Antigen recognition by the T-cell receptor
Antigenic peptides for T cells do not exist in a free state; the T-cell receptor (TCR) recognizes them only in a membrane-bound form together with MHC. The epitope for the TCR is always linear (rather than conformational, as for B cells).
This mechanism is described by the concept of "altered self": the antigen-recognizing region (V domains) reads both the peptide itself and the adjacent conformationally altered regions of the self-MHC molecule.
- The most variable region of the receptor, CDR3, contacts the peptide.
- The CDR1 and CDR2 regions recognize the adjacent areas of MHC.
Coreceptors are involved in recognition: CD4 binds to the $\beta_2$ domain of the MHC-II molecule, while CD8 has an affinity for the $\alpha_3$ domain of the MHC-I molecule.
Briefly: answers to topical questions
Which proteins are part of the peptide-loading complex (PLC) for MHC-I molecules?
The sources do not provide a complete list of all components of the peptide-loading complex (PLC) for MHC-I molecules. The presented materials mention only individual molecules that ensure the stability of the empty molecule and peptide transport: the chaperones calnexin and calreticulin, as well as transport systems TAP (encoded by the TAP1 and TAP2 genes), which deliver peptides from the cytoplasm to the endoplasmic reticulum.
What are the main intracellular pathways of cross-presentation of exogenous antigens?
Proteins from the extracellular environment can enter the MHC-I processing-presentation pathway through the following cross-presentation mechanisms:
- Retrograde transport (the main described pathway) — exogenous proteins enter endosomes, penetrate into the cytosol, are degraded in proteasomes, and the resulting peptides are then transported into the endoplasmic reticulum.
- Direct cytosolic entry — extracellular proteins penetrate directly into the cytosol, bypassing the endosomal stage.
- Endosomal cleavage — the degradation of engulfed proteins into peptides occurs directly within endosomes with the participation of associated proteasomes, after which the peptides are immediately incorporated into MHC-I molecules.
Which cytokines, besides interferon-gamma, are capable of inducing the synthesis of immunoproteasome subunits?
The provided materials contain information only about a single inducer of immunoproteasome subunit synthesis—interferon-gamma (IFNγ). Information regarding other cytokines capable of triggering this process is absent from the source.
To quickly remember who "removes the block" in the MHC-II pathway, use this mnemonic: HLA-DM acts like a key that "removes the CLIP" (displaces the residual CLIP peptide) to make room for the antigen.
Class and exam questions
Are T cells capable of recognizing carbohydrate antigens?
Currently, there is no evidence of the ability of carbohydrates (oligosaccharides) to be incorporated into MHC molecules. It is believed that they are not directly presented to T cells.
How does the presentation of lipid epitopes occur?
Lipids and glycolipids are presented by non-classical molecules of the CD1 family (e.g., CD1d for NKT cells). Their loading occurs in the ER with the participation of carrier proteins, and recycling of the complex through endosomes is mandatory for antigen renewal on the membrane.
What happens in the event of a genetic defect in the HLA-DM molecule?
A lack of HLA-DM activity will lead to the failure of the CLIP fragment to be displaced from the MHC-II cleft. As a result, MHC-II molecules carrying only the self-peptide CLIP will reach the cell surface, making the presentation of exogenous antigens impossible.
What else to study on this topic
- Structural differences between the proteasome and immunoproteasome (effect of IFNγ)
- Types of endocytosis in antigen-presenting cells and the role of the DEC-205 receptor
- CD1 molecules: recycling and presentation of lipid antigens
- Structure of the antigen-recognizing regions of the TCR (CDR1, CDR2, CDR3)
- Alternative pathways of cross-presentation of antigens
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