Principles that govern the excision of an antigenic peptide from a protein resulting in antigen presentation by MHC class I molecules are studied. It is intended to improve poorly immunogenic epitopes and thus contribute to the development of vaccines.
Biological virus and tumor models served to study the basic principles of antigen processing. Protein processing was tested after molecular construction of model proteins that differed with regard to amino acid sequences located within or in the vicinity of the antigenic peptide sequence to be presented by MHC class I proteins. The fate of the model proteins was tested in animals, within cells, or in vitro in presence of proteasome preparations. The analysis of degraded peptides by tandem mass spectrometry and sequencing revealed the fine specificity of the proteasome. Biological assays involving peptide specific cytotoxic T cell lines as probes revealed the efficacy of antigen presentation. The cooperative efforts led to the main and new conclusion that next to those peptide properties that define the peptide binding to a given MHC class I allele, also other information contained in the peptide sequence must contribute to antigen presentation. This sequence information is used by the proteolytic potential of the proteasome in the cytosol.
Sequence alteration can result an an unwanted proteasomal cleavage that results in the interruption of an antigenic sequence and consequently, in a lack of antigen presentation. This situation was found in a natural tumor model of antigen presentation. Sequence alteration can also result in the generation of cleavage sites outside the MHC presented peptide sequence that are used by the proteasome with high or low efficacy. In an attempt to correct poor processing, amino acid sequences were exchanged with the result that antigen processing in vitro and in vivo could be improved at will. The caveat that remains is, however, that only very few model proteins have been tested and there is no algorithm so far that could predict the situation for a new and unknown protein. Further work did shed some light on the function of proteasomal subcomponents. These components vary with the activation of cells and modify cleavage specificity and cleavage activity.
Peptides generated by the proteasome must be transported into the endoplasmatic reticulum (ER) by transporters associated with antigen processing (TAP). TAP alleles define the transport of peptides by recognizing and binding C terminal side chains. The residues in Tap2 which are largely responsible for the differential transport of peptides code in two clusters of 2 and 3 residues, respectively, at the two ends of a putatively cytosolic loop, including residues implicated in the control of substrate transport by the related ABC transporters. TAP molecules with MHC class I alleles without preference for those MHC molecules that can be loaded by the respective TAP. MHC class I molecules appear at the plasma membrane independent of Tap loading, provided that any form of appropriate ER peptide loading occurs.
Attempts to introduce the new information into more effective virus vector or ISCOM vaccines were so far not successful.
MAJOR SCIENTIFIC BREAKTHROUGHS:
Establishment of a firm linkage between the proteasome and the Antigen processing pathway for MHC class I molecule loading. Identification of principles for modulation of antigenicity of proteins.
Funding SchemeCSC - Cost-sharing contracts
2300 RC Leiden
RG6 2AJ Reading