THE PROTEOLYTIC FRAGMENTS GENERATED BY VERTEBRATE PROTEASOMES - STRUCTURAL RELATIONSHIPS TO MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I BINDING PEPTIDES

Proteasomes are involved in the proteolytic generation of major histoc ompatibility complex (MHC) class I epitopes but their exact role has n ot been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequenc es encompassing either an immunodominant or a marginally immunogenic e pitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most d ual cleavage fragments derived from 22-mer peptides were 7-10 amino ac ids long, with octa- and nonamers predominating. Digestion of 41/44-me r peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by furth er degradation of the latter as well as of larger single cleavage frag ments. The final size distribution slightly broader than that of fragm ents derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in m ultiple overlapping proteolytic fragments including a limited number o f abundant peptides. The immunodominant epitope was generated abundant ly whereas only small amounts of the marginally immunogenic epitope we re detected. The frequency distributions of amino acids flanking prote asomal cleavage sites are correlated to that reported for correspondin g positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may t hus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.