Synthesis and assembly of MHC-peptide complexes

Adam Benham, Abraham Tulp and Jacques Neefjes

The assembly of major histocompatibility complex (MHC) class I and class II molecules, and their asso- ciation with antigenic peptides, is analogous to a pro- duction line. Each molecule in the pathway has a spe- cific job to do before it hands over its partially processed product to the next cog in the cellular machine. This tireless molecular workforce has been quietly gener- ating functional MHC class I and class II complexes for millennia, yet it has only been in the past two dec- ades that we have begun to understand how some of the many components work. MHC class I and class II molecules use different intracellular pathways and, consequently, present different peptides (with some exceptions).

The MHC class I processing pathway MHC class I molecules present antigenic fragments

of approximately nine amino acids in length to CD8’ T cells. The majority of class I-binding peptides are de- rived from nuclear and cytosolic proteins, but peptides from surface and secreted antigens are not uncommon. Since the 8th International Congress of Immunology, in 1992, progress has been made in different aspects of the cell biology of class I-restricted antigen presentation. This discussion will follow the process from the time the antigen is exposed to the cytosol to the moment of presentation at the cell surface (Fig. 1).

A role for the proteasome? Since MHC class I molecules present peptides, intact

antigen first has to be degraded in the cytosol. The major cytosolic proteolytic complex is the proteasome, which exists as both a 20s and a 26s form. Rock and colleagues have addressed the role of the proteasome using mutant cell lines deficient in the ubiquitin path- way of degradation, as well as ‘specific’ inhibitors for proteasomes. Together, these approaches have suggested a major involvement of these proteolytic complexes in antigen generation’. However, since there may be other, as yet undefined, proteolytic cytosolic enzyme com- plexes, it is unclear whether proteasomes have an ex- clusive role in this process. Whatever the mechanism, the result of proteolysis is the generation of peptides, rather than single amino acids.

The exact substrate specificity of the proteasome, as well as the size and sequence of the final degradation products, are unknown. Two subunits of the protea- some, LMP-2 and LMP-7, have been localized to the MHC gene cluster and have been implicated in antigen degradation. Homologous deletion of the Lmp-7 gene in mice appears to affect the generation of peptidesl; however, the precise effect of LMP-2 and LMP-7 on the activity of proteasomes is still being debated.

1 Rack, KL., Gramn, C., Rot&&n, L, ez al. (1994) Cell 78, 761-771 ~2~~~,H.J., Swat, W., Lap&e, C. etul. (1994) Science 265,

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Fig. 1. Antigen presentation by major histocompatibility complex (MHCi class I molecules. Antigens are degraded rn the cytosol, and the result- ing fragments are size selected by the transporter associated with antigen processing (TAP). TAP translocates the peptides mto the endoplasmic

reticulum (ER) in a process requiring the hydrolysis of ATP. In the FIR, freshly synthesized MHC class 1 heterodimers bind peptides of correct sequence and length, and transport these to the C-ell surface.

The TAP heterodimer Following their generation in the cytosol, peptides

have to be translocated into the lumen of the endo- plasmic reticulum (ER) for association with newly syn- thesized MHC class I molecules. Numerous articles published between 1990 and 1992 unambiguously de- fined two homologous genes that are involved in pep- tide translocation. These genes encode the transporter associated with antigen processing (TAP), which is a multi-membrane-spanning heterodimer. Techniques have been developed to follow ATP-dependent peptide trans- location by TAP from the cytosol to the ER, and these have enabled the size specificity and sequence speci- ficity of TAP to be defined in rat, mouse and human cells.

TAP has the highest affinity for peptides of 8-13 amino acids in length, which is the approximate size of MHC class I-binding peptides. It can translocate shorter and longer peptides, but with considerably lower affinity3T4; a clear upper limit to peptide length has not yet been determined. TAP has a broad peptide sub- strate specificity, although it prefers peptides that do not contain proline residues at position 2 or 3. Never- theless, the latter peptides are still presented by MHC class I molecules, and are probably, translocated as longer entities that are subsequently trimmed in the ER.

Murine TAP, and rat TAP encoded by the cimb allele, prefer peptides with hydrophobic or aromatic C-terminal amino acids. Human TAP, and rat transporters en- coded by the cim” allele, are more indiscriminate. Thus, TAP limits the set of peptides that can bind to MHC class I molecules in mice and Limb-expressing rats4J. Interestingly, these MHC class I molecules generally use a hydrophobic or aromatic C-terminal anchor residue, which suggests that their specificity has co-evolved with the specificity of the murine and rat cimb transporters.

Peptide loading A peptide may follow several routes after entering

the ER: (1) it may bind to assembled MHC class I heterodimers; (2) it may be trimmed, although the rate of degradation in the ER is slow; or (3) it may be rapidly transported from the ER to the cytosol in an ATP-dependent fashion 3.4. The short half-life of pep- tides in the ER probably ensures that only high-affinity peptides bind to class I molecules. It is not yet clear whether accessory molecules such as gp94, calnexin or immunoglobulin heavy chain binding protein (BiP) are involved in the process of peptide loading.

The finding that MHC class I molecules are associ- ated with TAP suggests that the processes of peptide

Fig. 2. Intracellular transport and manipulation of the mamr histo- compatibility complex (MHC) class U-invariant chain (It) complex. In the endoplasmic reticulum, a nonamer of three MHC class II molecules and three Ii molecules are assembled (I). This complex is transported through the Golgi into the endosomal pathway, where Ii is degraded (2). However, a fragment of li, named class U-associated li peptide (CLIP), remains associated to class 11 molecules and prevents binding of peptides. CLIP removal is catalysed by the class II-like structure

HLA-DM (3), and class 11 molecules are loaded with peptide (4).

translocation and loading are coupled6,‘. Further analy- sis indicates the existence of two pools of MHC class 1 molecules: one is associated with TAP and the other is free. The existence of these two pools presumably en- sures efficient binding to peptides in a compartment where peptides have a short lifespan. Peptide binding disrupts the MHC class I-TAP complex and class I mol- ecules are subsequently transported to the cell surface to present their cargo to CDS+ T cells.

The MHC class II processing pathway It is well known that MHC class II molecules pre-

sent peptides from antigens degraded by the endosomal/ lysosomal pathway to CD4+ T cells. MHC class II molecules are assembled in the ER and associate with the invariant chain (Ii). This association is required for efficient release of MHC class II molecules from the ER. The signals that target the class II-Ii complex to the endocytic pathway are located in the cytoplasmic tail of Ii. Upon arrival in endosomes, Ii is degraded, probably exposing the MHC class II peptide-binding groove and allowing peptide loading. Furthermore, dissociation of Ii fragments from MHC class II mol- ecules is essential for class II release from the endocytic pathway and transport to the cell surface (Fig. 2).

Getting peptides into the groove Peptide loading of MHC class II molecules has been

investigated in cells with specific deletions in the class II region. These cells failed to present antigen and their MHC class II molecules only contained a fragment of Ii, named class II-associated Ii peptide (CLIP) (Refs 8,9). The class II-like structure HLA-DM was absent in

these cells, and was subsequently defined as being re- quired for efficient CLIP release and loading of MHC class II moleculesi(‘.’ ’ (although not all class II mol- ecules are equally dependent on HLA-DM for loading). HLA-DM appears to be a chaperone that helps in the last step of MHC class II folding, which results in the loading of peptide.

In 1992, it was already established that loading oc- curred in endosomal structures. Electron microscopical analysis of different cell lines had shown that MHC class II molecules accumulated in a lysosomal-like struc- ture known as MIIC (for MHC class II compartment)r2. This was often observed to have a multilaminar ap- pearance that seemed to be induced by the expression of MHC class II molecules. Although class II molecules were sometimes found in earlier compartments, the con- sensus was that peptide loading occurred in the MIIC. This seemed likely because: (1) Ii fragments could be found in the MIIC; and (2) HLA-DM was localized in these structures13. However, recent subcellular fraction- ation studies complicated this issue by showing that MHC class II loading could occur in a ‘novel’ medial endosome, a ‘novel’ recycling endosome, an endosomal MIIC-like structure (reviewed in Ref. 14) or even in multiple compartments’“. Whatever the case, the intra- cellular location of HLA-DM may be decisive in unam- biguously establishing the site of loading of MHC class II molecules.

Transport to the cell surface The signal that releases MHC class II molecules

from endosomes, and the mechanism of class II trans- port to the cell surface, remains unclear. Although the

immunology Today 36 1 Vol. 16 No. 7 199.F

half-life of MHC class II molecules is similar to that of presentable peptide (suggesting that a class II molecule only binds once in its lifetime to peptide), it cannot be excluded that a fraction of MHC class II molecules associates with peptide during recycling.

Exceptions In nature, nothing is absolute and there are excep-

tions to the general scheme outlined above. For exam- ple, it has been suggested that exogenous proteins can serve as antigens in the MHC class I pathway, at least in macrophages . l6 MHC class I molecules are perfectly capable of presenting peptides generated in the ER, as has been illustrated by analysis of peptides eluted from MHC class I molecules expressed in a cell line with deficient TAP expression . “xl8 These peptides were de- rived from signal sequences that are removed in the ER during translation of proteins on ER-bound ribosomes.

As for MHC class I molecules, class II molecules are assembled in the ER. Since peptides are continuously pumped into the ER lumen, MHC class II molecules could also be loaded there, but this is generally pre- vented by association with Ii. Loading of MHC class II molecules in the ER of mice lacking Ii is improved but still inefficienti9. Analysis of peptides eluted from MHC class II molecules has revealed that a relatively large number are derived from cytosolic protein@‘. How these enter the class II processing pathway is unclear and is a subject for future research.

Future work One major question that still exists concerns the

specificity of antigen degradation. Clearly, the proteins involved (and their specificity) need to be defined. If degradation is not stochastic, then determining the specificity of peptide binding to MHC molecules may allow us to improve the predictions of correct present- able peptides from a linear protein sequence.

Adam Benham, Abraham Tulp and Jacques Neefies are at the Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

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