title: Self-peptides from four HLA-DR alleles share hydrophobic anchor residues near the NH2-terminal including proline as a stop signal for trimming.
abstract: Naturally processed MHC class II-associated peptides proved to be heterogeneous in size, varying from 13 to 25 amino acids. Truncation variants suggested sequence motifs that afford the amino termini to be shifted 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, proline is the dominant residue reappearing at positions 2 and 3 in any allele. 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, whereupon DR1 binding peptides with increased affinity were generated. In addition, aminopeptidase treatment of DR1: self-peptide complexes implied 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 anchor residues clustering in the NH2-terminal region:tyrosine, phenylalanine, 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-specificity is demonstrated by DQw7 displaying glutamine and asparagine at position 2. Obviously, the degenerate occurrence of aromatic or aliphatic side chains close to the NH2-terminal guarantees for essential interactions with a hydrophobic pocket of the investigated DR molecules. Most probably, this pocket is located in the nonpolymorphic DR alpha-chain rationalizing previous findings of promiscuous peptide binding to different DR alleles.
authors.affiliation: Center of Medical and Natural Sciences, University of Tübingen, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Kropshofer
authors.forename: H
authors.initials: H
authors.lastname: Max
authors.forename: T
authors.initials: T
authors.lastname: Halder
authors.forename: M
authors.initials: M
authors.lastname: Kalbus
authors.forename: C A
authors.initials: CA
authors.lastname: Muller
authors.forename: H
authors.initials: H
authors.lastname: Kalbacher
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D002401:Cathepsin B / Q000494:pharmacology; D002461:Cell Line, Transformed; D005802:Genes, MHC Class II; D006684:HLA-DR Antigens / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007931:Leucyl Aminopeptidase / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011392:Proline / Q000032:analysis
chemical_list: D006684:HLA-DR Antigens; D010446:Peptide Fragments; D011392:Proline; D007931:Leucyl Aminopeptidase; D002401:Cathepsin B