Reinout Amons

Peptide binding characteristics of the coeliac disease-associated DQ(α1*0501, β1*0201) molecule

Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of theHLA-DQ2 (α1*0501, β1*0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several sizevariants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClα46–63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClα46–63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides.Substitutions at P1 and P4 were most critical, suggesting a more prominent role as anchor residues. Structural features of the DQ2 molecule that may relate to the binding motif and to gluten sensitivity are discussed.

Unique peptide binding characteristics of the disease-associated DQ(α1*0501, β1*0201) vs the non-disease-associated DQ(α1*0201, β1*0202) molecule

Abstractu2003To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(α1*0501, β1*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(α1*0201, β1*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I α chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. At p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(α1*0201, β1*0202) molecule. The most striking difference between the two DQ molecules is the presence of an additional anchor residue at p3 for the DQ(α1*0201, β1*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(α1*0501, β1*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(α1*0501, β1*0201) and DQ(α1*0201, β1*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(α1*0501, β1*0201) molecule should be helpful in the identification of CD-inducing epitopes.

The primary structure of elongation factor EF-1 alpha from the brine shrimp Artemia.

cDNA as well as amino acid sequencing has revealed the complete primary structure of elongation factor EF‐1 alpha from the brine shrimp Artemia. A comparison with the published sequences of bacterial EF‐Tu, mitochondrial EF‐Tu and chloroplastic EF‐Tu shows that distinct areas of these polypeptide chains are conserved in evolution. The evolutionary distance between prokaryotic and eukaryotic types of EF‐Tu is larger than among bacterial and organellar EF‐ Tus . A number of regions present in both EF‐Tu and EF‐G from Escherichia coli are also found in EF‐1 alpha from Artemia.

Natural peptides isolated from Gly86/Val86-containing variants of HLA-DR1,-DR 11, -DR13, and -DR52

Peptide binding motifs for human major histocompatibility complex (MHC) class II (HLA) molecules of the DR subtype invariably predict a hydrophobic anchor residue near the N-terminus of the peptide (Rammensee et al. 1995). The crystal structure of HLA-DR1 (DR β1*0101) complexed with the influenza haemagglutinin (HA) 307–319 peptide has revealed that the side chain of the major hydrophobic anchor residue, tyrosine at position 309, is bound in a deep and conserved hydrophobic pocket (Stern et al. 1994). This pocket is lined by DR β residue 86, which can be either glycine (Gly) or valine (Val). Various studies have demonstrated that this Gly 86/ Val86 dimorphism at DRβ86 can affect allorecognition (Lang et al. 1988; Johnson et al. 1991; de Koster et al. 1992; Demotz et al. 1993) and antigen presentation (Zeliszewski et al. 1990; Krieger et al. 1991; Busch et al. 1991; Ong et al. 1991). Moreover, this dimorphism has been shown to influence the stability of HLA-DR molecules in the detergent sodium dodecyl sulphate (SDS; Verreck et al. 1993). Together, these results suggest that different sets of peptides may be selected by Gly 86/Val86-DR variants. Indirect evidence for such differential peptide selection has been obtained by peptide binding studies (Krieger et al. 1991; Busch et al. 1991; Marshal et al. 1994) and the characterization of peptides associated to non-related Val 86or Gly86-containing DR allelic products (Vogt et al. 1994). In order to directly analyze the influence of DR β86 on peptide selection we performed a systematic analysis of the peptide content of Gly 86/Val86 variants of the subspecificities DR1, DR11, DR13, and DR52 by pool sequencing as well as by the determination of individual peptide seImmunogenetics (1996) 43: 392–397 uf6d9 Springer-Verlag 1996

Sequence homology between EF-1α, the α-chain of elongation factor 1 from Artemia salina and elongation factor EF-TU from Escherichia coli

The occurrence, in the eukaryotic factor, of several ϵ‐trimethyllysine residues, is remarkable.

Protein-ribonucleic acid interactions in ribosomes

Abstract 1. 1. The unfolding of ribosomes from Escherichia coli renders ribosomal RNA (rRNA) susceptible to both specific and nonspecific nuclease action. 2. 2. The nuclease digestion produces a precipitate which contains most of the total ribosomal protein and 15% of the total RNA. 3. 3. RNA fragments isolated from the precipitate are heterodisperse, as judged by gel electrophoresis, and have a 28 (±7) nucleotide-residue chain length. 4. 4. The RNA fragments, obtained with nonspecific nucleases have a higher G and C content than intact rRNA. 5. 5. The concept, that the RNA fragments derive from nonhelical RNA regions, which in situ preferentially bind ribosomal proteins, is discussed.