IMMUNOLOGY - Roit A. - Mir 2000

Chapter 9. Antigen Recognition

FORMATION AND STRUCTURE OF THE ANTIGENIC PEPTIDE-MHC MOLECULE COMPLEX

The groove in the MHC molecule where antigenic peptide binding takes place features various pockets and clefts, along with protruding and recessed surface areas. Their fine topology partly depends on The amino acid residues lining this groove and thus varies among MHC molecules of different haplotypes. The parameters of peptide-MHC binding depend on The Nature of its side chains and the mutual complementarity of the contacting areas of both association partners. A certain proportion of the peptide's side chains does not participate in the interaction with the MHC molecule and is designed for contact with the TCR (see Chapter 7).

The formation of complexes from antigenic Peptides and MHC molecules occurs in specialized intracellular Organelles, with Class I and class II molecules interacting in different Regions of the Cell.

Peptides are held within the designated binding groove of the MHC molecule by specific anchor amino acid residues

Peptide fragments resulting from ANTIGEN Processing AND subsequently displayed by APCs in association with MHC molecules can be obtained as purified preparations and sequenced. These include not only peptides from cell-engulfed foreign Antigens (such as Viral Particles), but also the Organism's own peptides cleaved within the APC or endocytosed by it from extracellular fluid. Analysis (via purification and sequencing) of these self-peptides eluted from complexes with class I MHC molecules showed that they consist of nine amino acid residues; furthermore, The Structure of several such peptides was precisely determined, and characteristic amino acid residues were identified—one at the C-terminus and another closer to the N-terminus. These characteristic Structural motifs distinguish peptides bound by class I molecules of different haplotypes (Fig. 9.22).

Fig. 9.22. Allospecific structural motifs in peptide molecules eluted from complexes with class I MHC molecules of the H-2K^b or H-2K^d haplotype. MHC molecules were isolated by immunoprecipitation. The associated peptides were purified and sequenced. Anchor amino acid residues occurring most frequently at specific positions are defined as dominant, while those occurring only relatively frequently are defined as functional. Positions without a specified amino acid have an equal probability of featuring several different residues. The single-letter code is used to designate Amino Acids. Top views of the peptide-binding grooves (top view) of two class I MHC molecules of different haplotypes are shown above. The positions of anchor residues within the peptide bound to each haplotype's MHC molecule are highlighted in color.

The Significance of these conserved residues became apparent through the Analysis of the three-dimensional structure of certain class I MHC molecules, which provided a fairly clear picture of the antigenic peptide's arrangement within the peptide-binding groove. It was established that the edges of this groove are closed at its ends, and the peptide—in the form of an extended (non-α-helical) chain of nine amino acid residues—rests its N- and C-termini against both ends. Some of the antigenic peptide's side chains sink into pockets formed by the variable region of the class I MHC heavy chain, and numerous Hydrogen Bonds are established along the entire length between the amino acid residues of this molecule and the peptide. For instance, Tyrosine residues frequently found at the N-terminus of the peptide interact in this manner with a conserved Lysine residue of the class I MHC molecule, stably holding the antigenic peptide in its peptide-binding groove. Meanwhile, the central part of the peptide projects from the groove, presenting its epitopes to T-cell receptors. These findings are consistent with the fact that characteristic structural motifs bound by class I MHC molecules are found at the ends of antigenic peptides.

Analysis of the three-dimensional structure of class II MHC molecules revealed a peptide-binding groove with a series of pockets, very similar to (though sometimes differently positioned than) that of class I molecules. Its main difference from the peptide-binding groove of class I molecules is its open ends. Due to this non-closure of the edges, the ends of the bound peptide protrude from the groove, and it contains more than 15 amino acid residues, i.e., exceeding the length of peptides eluted from complexes with class I MHC molecules. It was possible to identify conserved anchor amino acid residues within peptides eluted from the complex with class II MHC molecules. This was more difficult to accomplish than in the case of peptides bound by class I molecules, because in class II molecules the peptide ends protrude beyond the groove, and the distribution of the complex-forming bonds is more uniform (Fig. 9.23). The Functions of the terminal PARTS OF THE peptide-binding groove in class I and class II MHC molecules differ significantly: in the former case, the binding of the N- and C-termini of the peptide at the ends holds it within the groove, whereas in the latter case, the open ends allow the peptide to protrude past its boundaries.

Fig. 9.23. Purified preparations of HLA-DR of two haplotypes (DRB1*0401 and DRB1*1101) were incubated with a peptide library cloned in the M13 bacteriophage. After numerous Selection steps, peptides efficiently bound by class II MHC molecules were identified and sequenced. Amino acid residues within these peptides occurring at the same positions with a frequency greater than 20% were defined as dominant anchors. Other residues occurring only relatively frequently were termed functional anchors. (According to Hammer et al., 1993). Note that the class II MHC peptide-binding groove accommodates a longer peptide compared to that bound in the groove of class I MHC molecules.

Class I MHC molecules form complexes with endogenously synthesized peptides

It has been repeatedly demonstrated that T Cells restricted in antigen recognition by class I MHC molecules (e.g., Tc cells) recognize endogenous antigens synthesized by the target cell, whereas T cells restricted by class II MHC molecules (specifically, Th cells) recognize exogenous antigens. By arbitrarily changing the localization of a protein, one can elicit an Immune Response restricted by either class I or class II MHC molecules. For instance, Influenza virus hemagglutinin (HA), a glycoprotein associated with host cell membranes, elicits only a weak Tc response. However, if the sequence encoding the N-terminal signal peptide is excised from the HA Gene (cDNA) and this modified gene is then introduced into The Cell using an appropriate vector, a cytoplasmic form of HA will be synthesized, eliciting a very strong anti-HA Tc response. Similarly, introducing Ovalbumin into the Cytoplasm of a target cell using the osmotic Shock method induces the appearance of Tc cells, whereas conventional immunization with this exogenous antigen elicits exclusively a Th response.

Proteasomes are cellular organelles that degrade cytoplasmic Proteins. The formation of antigenic peptide-class I MHC molecule complexes occurs in the rough Endoplasmic reticulum (ER), but these (endogenous) peptides themselves are generated from cytosolic proteins. The First stage of the process involves organelles termed proteasomes. These are complexes of 12-15 different subunits that provide the primary proteolytic activity of the Cytosol. Proteasomes contain, for example, a set of Endopeptidases that cleave denatured or ubiquitin-tagged proteins into peptides ranging from 5 to 15 amino acid residues in length. (Ubiquitin is a polypeptide attached to proteins as a tag that destines them for degradation.) Two proteasome subunits are encoded by the MHC-resident genes LMP2 and LMP7. Gamma-interferon induces the expression of these genes, and their products displace constitutive (non-inducible) proteasome components. A wealth of evidence, including results from targeted gene modification and proteasome inhibitor experiments, indicates the involvement of LMP2 and LMP7 in the processing of all peptides that form complexes with class I MHC molecules.

Peptides generated in the cytosol by proteasome action gain entry into the ER lumen via heterodimeric transport proteins from the "ABC" superfamily, encoded by two genes—TAP1 and TAP2. These genes are mapped near the LMP2 and LMP7 genes (Fig. 9.24). It has been established that microsomes isolated from cells are capable of transporting antigenic peptides into the ER, whereas microsomes from cells deficient in the TAP1 and TAP2 genes lack this ability. The most efficient transport occurs when peptides contain 8–15 amino acid residues. Peptides of precisely this length are preferentially bound to class I MHC molecules, although they may undergo additional processing prior to complexation.

Fig. 9.24. The genes for transporter associated with antigen processing (TAP) and proteasome components (LMP) are located within the H-2 complex in the mouse and the HLA complex in humans.

As recently established, during endogenous antigen processing, interaction partners can associate both physically and functionally. For instance, newly synthesized complexes of class I MHC molecules and β₂-microglobulin associate with TAP in the ER and dissociate following Transport from the ER to the cis-side of the Golgi complex. The compartmentalization of various processing products presumably promotes the most efficient accumulation of antigenic peptides within the antigen-presenting cell.

Antigenic peptides transported into the ER form complexes with class I MHC molecules (Fig. 9.25). The formation of such complexes is a complex process involving chaperone proteins (i.e., companions, assistants) such as calnexin. Chaperones initiate and organize the assembly of a stable complex—transportable to the cell surface—consisting of the class I heavy chain, β₂-microglobulin, and the antigenic peptide. In the absence of the antigenic peptide, such complexes are unstable; therefore, only fully functionally active complexes can be presented to T cells.

Fig. 9.25. Proposed pathway of antigenic peptide-MHC molecule complex formation. Cytoplasmic antigens are processed by proteasomes, two subunits of which are encoded by the LMP2 and LMP7 genes from the MHC complex. Peptide transport is carried out by two transport proteins (from the "ABC" superfamily) encoded by the TAP1 and TAP2 genes, which also belong to the MHC. Antigenic peptides form a complex with class I MHC heavy chains and β₂-microglobulin in The endoplasmic reticulum (ER). Molecular chaperones, such as calnexin, attach to not-yet-fully-assembled antigenic peptide-class I MHC molecule complexes. Following this, the assembled complexes are transported to the cell surface (Fig. 9.26).

Class II MHC molecules form complexes with exogenous antigenic peptides in endosomes

Class II MHC α- and β-chains are found in the ER as complexes with a polypeptide termed the invariant chain (Ii); this protein is encoded by a non-MHC gene. The αβ-Ii complex is transported through the Golgi apparatus to an endosome or lysosome, where Ii is released in an acidic environment. Based on experimental data, it can be hypothesized that the dissociation of Ii from the αβ-complex allows the antigenic peptide to take its place. The class II MHC molecule-antigenic peptide complex resides in the endosome/lysosome for 1–3 hours before moving to the cell surface.

How do exogenous antigen peptides encounter class II molecules within the appropriate cellular organelles? To answer this question, we need to trace the intracellular trafficking pathways of MHC molecules. Following their assembly in the ER, both classes of MHC molecules pass through the Golgi apparatus—class I molecules associated with endogenously derived antigenic peptides, and class II molecules bound to the invariant chain (Ii). MHC molecules of each class diverge at the trans-Golgi network. Subsequently, on their way to The Plasma Membrane, class II molecules enter specialized endosomes or Lysosomes that differ from conventional ones, apparently being uniquely dedicated to the storage and transport of these molecules (Fig. 9.26).

Fig. 9.26. Newly synthesized class I molecules associate with an antigenic peptide (1). Class II molecules bind Ii chains in the ER lumen (2). The Ii protein prevents class II molecules from prematurely associating with antigenic peptides and contains signals that allow these complexes to exit the ER. Class I and class II molecules separate after passing through the Golgi apparatus (3). Class I molecules are routed directly to the cell surface (4). Class II molecules enter an acidic compartment—MHC class II compartments (MIIC)—where they form complexes with exogenous antigen peptides after the invariant CLIP peptide vacates the peptide-binding groove (5).

A major breakthrough in recent research has been the identification of the cellular organelles (vesicles) where MHC class II molecules associate with antigenic peptides. These vesicles, designated as MIIC, exhibit a complex membranous structure (resembling an onion bulb under Electron Microscopy) and possess The properties of both endosomes and lysosomes.

As it turned out, a key role in the formation of complexes with antigenic peptides is played by the HLA-DM molecule, which is structurally homologous to class II molecules. This molecule consists of an α chain and a β chain encoded by the DMA and DMB genes (located in the class II region of the HLA complex). In mutant cell lines lacking these genes, class II molecules are unstable, and the cells themselves are unable to process and present protein antigens. When class II molecules were isolated from such mutant cells and the bound peptides analyzed, the associated peptides proved to be predominantly an invariant polypeptide known as CLIP (class II-associated invariant peptide). Research has established that HLA-DM catalyzes the dissociation of CLIP from the class II molecule complex in an acidic environment in vitro, thereby enabling the binding of exogenous peptides.

In all likelihood, exogenous antigens enter antigen-presenting cells via receptor-mediated or fluid-phase endocytosis. Enzymatic Cleavage of endocytosed proteins occurs within endosomes or lysosomes, and the resulting peptides bind to class II molecules, facilitated by HLA-DM acting as a catalyst. The newly formed complexes are then transported to the cell surface.

Questions for Thought

■ What does the term epitope mean? What are the dimensions of an epitope? Can epitopes overlap? In which regions of a protein antigen molecule are epitopes most likely to be found? What types of amino acid residues typically make up epitopes?

■ If Monoclonal Antibodies to an antigen of known structure are available, how can one determine the specific region of the antigen recognized by these antibodies?

■ Is antibody Specificity a precisely measurable characteristic for a given sample, or does it depend on the physiological and experimental conditions under which the antibody activity is assessed?

■ Do monoclonal antibodies possess greater antigen specificity compared to a polyclonal antiserum?

■ Whether an antigenic peptide is presented in a complex with MHC class I or class II molecules depends not on its structure, but on the type of intracellular organelles where it encounters the respective MHC molecule. What are the advantages of such an antigen presentation mechanism for the functioning of The Immune System?

■ How can an understanding of the mechanisms of antigen presentation aid in the design of peptide-based Vaccines?

■ What impact might Mutations or modifications of TAP or LMP genes have on the antigen presentation process?

Introduction/47.html">Further Reading

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