Identification of the Peptide Binding Motif for HLA-B44, One of the Most Common HLA-B Alleles in the Caucasian Population

Abstract

Most peptides that bind to a particular MHC class I molecule share amino acid residues that are thought to physically "anchor" the peptide to polymorphic pockets within the class I binding site. Sequence analysis of endogenous peptides bound to HLA-B44 revealed two potential dominant anchor residues: Glu at P2 and Tyr, or occasionally Phe, at P9. In vitro assembly assays employing synthetic peptides and recombinant HLA-B44 produced by Escherichia coli revealed that an acidic amino acid at P2 was necessary for promoting stable peptide binding to HLA-B44. Surprisingly, although Tyr was almost exclusively found at P9 of the endogenous peptide sequences, a wide variety of amino acid residues such as Leu, Ala, Arg, Lys, His, and Phe could be tolerated at this position. Using this information, we identified antigenic peptides from the influenza virus components nonstructural protein 1 and nucleoprotein that are presented by HLA-B44 to antiinfluenza type A cytotoxic T lymphocytes. In addition, cytotoxic T lymphocytes induced by these antigenic peptides were shown to be capable of recognizing endogenously processed peptides from influenza-infected cells, indicating a potential use for these peptides in vaccine development. Finally, molecular models were created to investigate the possible ways in which the anchor residues might function to stabilize the binding of peptides to HLA-B44, and these models indicate that the acidic residue at P2 most likely interacts primarily with Lys 45 of the HLA-B44 heavy chain and makes additional contacts with Ser 67 and Tyr 9.

HLA Photoaffinity Labeling Reveals Overlapping Binding of Homologous Melanoma-associated Gene Peptides by HLA-A1, HLA-A29, and HLA-B44

“…4C, the MAGE decapeptides were considerably more efficient competitors than the corresponding MAGE nonapeptides (63 versus 22% inhibition). This is in accordance with the peptide-binding motif for HLA-B44, which is glutamic acid in position 2 and tyrosine or phenylalanine at the C terminus (25,26). Similar results were obtained when C1R.B*4402 cells were used instead of C1R.B*4403 cells (data not shown), indicating that the two main subtypes of HLA-B44 bind these MAGE peptides with similar efficiency.…”

“…These differences are most likely explained by secondary anchor residues. For example sequencing of peptides eluted from HLA-B44 showed a preference for an aliphatic hydrophobic residue in P3 and often also in P6 (25,26); this is consistent with the observation that the MAGE-1 and the two MAGE-4 decapeptides, which lack hydrophobic residues in these positions, bound less avidly to HLA-B44 (Figs. 3 and 4C).…”

A Single Polymorphic Residue Within the Peptide-Binding Cleft of MHC Class I Molecules Determines Spectrum of Tapasin Dependence

“…To determine the influence of residue 114 on peptide loading as a function of tapasin, we examined peptide loading into MHC class I molecules by using the reporter peptide. We incubated intact microsomes derived from B4402 and B44D114H transfectants with or without tapasin and AEIDKVTGY peptide, a high-affinity ligand for B4402 (19), in the presence of the competitors at different concentrations. The amount of tapasin-dependent peptide loading was determined by measuring the level of incorporation of labeled peptide into W6/32-reactive class I complexes.…”

HLA-B44 Polymorphisms at Position 116 of the Heavy Chain Influence TAP Complex Binding via an Effect on Peptide Occupancy

“…Baculoviruses encoding HLA-B*44 were used to express HLA-B*4402 or HLA-B*4405 in insect cells, and the abilities of different exogenous peptides to bind HLA-B*4402 and HLA-B*4405 were compared using thermostability assays. Two HLA-B*44-specific peptides, SEIDTVAKY (41) and EEFGRAFSF (42) were synthesized, and additionally, two nonapeptide libraries were synthesized, one of which was completely random at each position while the other was synthesized with a HLA-B*44 glutamic acid anchor at position 2. Metabolically labeled insect cells expressing the two class I heterodimers were lysed in the presence or absence of excess exogenous peptide, and lysates were heated to 37°C for 12 min or maintained at 4°C, followed by immunoprecipitation analyses with the W6/32 Ab.…”