Antigen processing

Processing refers biochemically to the unfolding of the proteins and/or the generation of peptides as a result of their partial proteolysis. Globular proteins in their native state do not bind to MHC class II

proteins: proteins are required to have conformational flexibility in order to be able to bind and mold into the combining site of an MHC molecule. This flexibility is brought about either by denaturing the protein or partially fragmenting it to small peptides. The denatured proteins or the peptides, in contrast to the native unfolded molecule, interact and complex to the class II MHC molecules.

Processing by APCs involves several successive stages: 1) the internalization of the antigen into acid intracellular vesicles; 2) its partial proteolysis; 3) its coupling to intracellular MHC II molecules; and 4) its transport to the plasma membrane.

Protein antigens internalized by APCs are taken to deep intracellular vesicles where unfolding of the protein and/or its partial catabolism to peptides then takes place. The denatured protein and/or peptides are thus available to bind to class II MHC molecules. The most recent evidence suggests that this encounter takes place preferentially, but not exclusively, with class II MHC molecules that have been recently synthesized, and that this encounter takes place in vesicles with the properties of early lysosomes.

Class II MHC molecules assemble in the endoplasmic reticulum (ER) and traverse the Golgi vesicles associated with a nonpolymorphic polypeptide, the invariant chain. Invariant chain and the ctfi dimers of class II molecules form a complex that is transported to vesicles outside the Golgi vesicles. The estimate is that a trimerized invariant chain binds to three class II molecules (i.e. three a(3 dimers), thus forming a nonameric complex. The invariant chain has signal sequences in its cytosolic portion that serves to guide the complex out of the Golgi into late vesicles. The invariant chain also has the important property of covering the combining site of the class II molecule during its travel through the ER. Thus, during their sojourn through the ER and Golgi the class II molecules have less opportunity to interact with peptides derived from catabolism of self peptides. The invariant chain has different isoforms that may be involved in different ways in the fate of the complex.

Once the invariant chain-a|3 complex leaves the Golgi, the invariant chain is catabolized in a proteolysis rich, acidic compartment; however, a segment of the invariant chain comprising residues 81-104 remains associated with the combining site. This peptide, termed CLIP, must be removed to make the dimer competent to bind foreign (or autologous) peptides. A secondary auxiliary molecule (HLA-DM in humans or H2-M in the mouse) then associates with the a(3 dimer containing the CLIP peptide. This association leads to the removal of CLIP. How this protein associates with the a|3 dimer, and the basic interaction that favors a fast off-rate of the bound peptide still requires analysis. As the CLIP peptide is removed, high-affinity binding peptides found in the vesicular 'loading' compartment then bind. Thus, there is an exchange of peptides, with CLIP leaving, and strong binding peptides occupying, the class II MHC molecule. Class II molecules with bound peptides are then transported to the plasma membrane, the site of interaction with the T cell and its receptor.

Many of the class II MHC bound peptides have a very slow off-rate and, therefore, remain bound during their entire span in the APC. In fact the half-life of the class II MHC molecules reflects their content of different affinity peptides. Some of the class II molecules bearing weak binding peptides can exchange peptides in endosomal vesicles or at the plasma membrane.

Thus, a scenario that is favored is that an internalized protein (i.e. a soluble protein or a microbe containing a large array of proteins) is taken to a deep lysosomal compartment, and it is there that peptides are generated from partial proteolysis. Nascent class II molecules meet with these peptides and an effective peptide exchange reaction takes place, as described above. Peptides that are bound, i.e. 'selected', are protected from further proteolysis. Class II molecules are, therefore, peptide-binding molecules that protect from extensive catabolism. Figure 1 illustrates the antigen-processing events depicted using the protein lysozyme.

The nature of the proteolytic event that results in the degradation of the invariant chain and of the protein antigen is still under much consideration. Most likely, cathepsins are involved in the degradation. A strong case has been made for the involvement of cathepsin S in invariant chain degradation. Concerning the protein antigen, one way of viewing the processing event is to postulate the unfolding of the protein, after which the denatured unfolded polypeptide binds to class II molecules through the segment displaying sequences that favor high binding to MHC molecules. Once bound, the portion of the unfolded peptide that spills out of the peptide-combining site is catabolized, perhaps by amino- and carboxypep-tidases, up to the limit of the combining site.

There has been much discussion about the nature of the MHC-peptide loading compartment (called MIIC). An organelle containing lysosomal proteins and class II MHC molecules, and which receives internalized protein late, has been identified by electron microscopy, and also defined by subcellular fractionation. Thus, for many protein antigens this late vesicular and lysosomal-like compartment may be the site of loading to nascent class II molecules. However, it is also clear that peptides can bind class

Figure 1 Processing of the protein hen egg white lysozyme (HEL). There are two functional compartments in APCs, depicted as the two squares: a deep compartment (left) and a recycling or endosomal compartment (right). HEL is a globular protein that requires denaturation by reduction of its four disulfide bonds (dHEL). This takes place after HEL reaches the deep compartment. The dHEL binds to the ap dimers of class II MHC, after their exit for ER-Golgi. «[3 dimers exit bound to the invariant chain (c*p-li). The invariant chain is degraded leaving CLIP peptides bound to it. The HLA-DM molecules remove CLIP making the a(3 dimers competent to bind dHEL. The dHEL bound to class II molecules is trimmed by amino- and carboxypeptidases to a peptide of 15-16 residues. The peptide is transported to plasma membrane. The dHEL not bound is catabolized to amino acids (box with dots). If APCs are given HEL already denatured, it can bind to cifi dimers in endosomes where peptide exchange takes place. Peptides can also bind directly to some peptide-free «p dimers at the cell surface.

Figure 1 Processing of the protein hen egg white lysozyme (HEL). There are two functional compartments in APCs, depicted as the two squares: a deep compartment (left) and a recycling or endosomal compartment (right). HEL is a globular protein that requires denaturation by reduction of its four disulfide bonds (dHEL). This takes place after HEL reaches the deep compartment. The dHEL binds to the ap dimers of class II MHC, after their exit for ER-Golgi. «[3 dimers exit bound to the invariant chain (c*p-li). The invariant chain is degraded leaving CLIP peptides bound to it. The HLA-DM molecules remove CLIP making the a(3 dimers competent to bind dHEL. The dHEL bound to class II molecules is trimmed by amino- and carboxypeptidases to a peptide of 15-16 residues. The peptide is transported to plasma membrane. The dHEL not bound is catabolized to amino acids (box with dots). If APCs are given HEL already denatured, it can bind to cifi dimers in endosomes where peptide exchange takes place. Peptides can also bind directly to some peptide-free «p dimers at the cell surface.

II molecules in early endosomes or, if available, at the plasma membrane. The nature of the protein, and the ease by which the protein is subjected to denaturation and partial proteolysis, clearly are factors in determining the site of loading. Autologous proteins, which are rapidly secreted from APCs, can also load into class II molecules during their short travel through APCs. This is an indication that there is some degree of proteolysis taking place during secretion and that the loading process is very effective. Lastly, peptides derived from cytosolic molecules have been found bound to class II molecules, an indication that cytoplasmic-derived peptides are also accessible to the class II MHC system. However, whenever a comparison has been made, peptides derived from cytosolic proteins are less effective than those found in vesicular proteins in reaching the class II MHC system.

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