SELF-PEPTIDES FROM 4 HLA-DR ALLELES SHARE HYDROPHOBIC ANCHOR RESIDUESNEAR THE NH2-TERMINAL INCLUDING PROLINE AS A STOP SIGNAL FOR TRIMMING

Naturally processed MHC class II-associated peptides proved to be hete rogeneous in size, varying from 13 to 25 amino acids. Truncation varia nts suggested sequence motifs that afford the amino termini to be shif ted for obtaining an alignment: a 9- to 11-residue core region that is bordered by primary anchor residues is surrounded by extra sequences of variable lengths and hitherto unknown functions. Herein we present bulk sequencing analyses of self-peptides from four HLA-DR alleles and HLA-DQw7 clearly showing that the length of most of the NH2-terminal preanchor sequence is limited to 1 to 3 residues. Most strikingly, pro line is the dominant residue reappearing at positions 2 and 3 in any a llele. Proline revealed to function as a stop signal for NH2-terminal trimming as well as a secondary anchor: crude cytosolic and endosomal peptide fractions could be processed by aminopeptidases in vitro, wher eupon DR1 binding peptides with increased affinity were generated. In addition, aminopeptidase treatment of DR1:self-peptide complexes impli ed that proline together with sterical constraints of the MHC molecule do protect the peptides' NH2-termini from further processing, whereas their COOH-termini were accessible to cathepsin B processing. Finally , bulk sequencing profiles contained signals from further putative anc hor residues clustering in the NH2-terminal region: tyrosine, phenylal anine, leucine, isoleucine, and valine are enriched at positions 2 to 4 in DR1, DR5, and DR6, however, at positions 4 to 6 in DR3. Isotype-s pecificity is demonstrated by DQw7 displaying glutamine and asparagine at position 2. Obviously, the degenerate occurrence of aromatic or al iphatic side chains close to the NH2-terminal guarantees for essential interactions with a hydrophobic pocket of the investigated DR molecul es. Most probably, this pocket is located in the nonpolymorphic DR alp ha-chain rationalizing previous findings of promiscuous peptide bindin g to different DR alleles.