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R Amons

Publications and source records attributed to R Amons.

At least 37 records · Page 2Linked to original sources
PubMed1995

The use of dedicated peptide libraries permits the discovery of high affinity binding peptides.

The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been obtained from a phage display peptide library (Schellekens et al., 1994). Binding studies with different length variants of this peptide identified a 12mer as a suitable lead compound for our library study. Two incomplete dedicated resin-bound synthetic peptide libraries were generated. Both consisted of 2 x 10(6) 12mers, in which positions were alternately fixed (amino acids identical to the lead sequence) and random. The libraries were screened with mAb A16 and beads with binding peptides were sequenced using Edman degradation. This resulted in a unique peptide binding motif, essentially comprising a 7mer core sequence. Comparison of the sequence of the natural epitope with the binding motif revealed that its sequence was identical to the motif except for one position. Substitution of a methionine in the natural epitope by a tyrosine or a phenylalanine at that position, as dictated by the motif, resulted in a peptide with an affinity for binding to mAb A16 about 50 times higher than that of the natural epitope. Thus, if a lead sequence is available, the use of incomplete, dedicated synthetic peptide libraries provides a fast and powerful tool for the detection of high affinity peptides.

Amino Acid Sequence↗
PubMed1995

DR4Dw4/DR53 molecules contain a peptide from the autoantigen calreticulin.

Rheumatoid arthritis (RA) occurs more frequently in HLA-DR4+ individuals than in those who do not express this MHC class II molecule. Although the role of this genetic factor in the immunopathology of this autoimmune disease is unclear, the association of RA with HLA-DR4 may indicate that DR4 molecules present autoantigen(s) to T cells. Here we report the analysis of naturally processed peptides, eluted from a mixture of HLA-DR4Dw4 (DRB1*0401) and DR53 (DRB4*0101) molecules isolated from an RA patient-derived EBV-transformed B cell line. Several (size variants of) self-peptides originating from the autologous molecules HLA-A2, HLA-Cw9, HLA-B62, HLA-DR4Dw4 and HLA-DR53, were identified. We also found a sequence that has no homology to any protein in the SwissProt protein sequence databank, and a peptide identical to an internal fragment of the autoantigen calreticulin. The association of the identified peptides with cells expressing HLA-DR4Dw4/DR53 was confirmed by peptide binding analysis. In agreement with previously described peptide binding motifs for DR4Dw4, most peptides contained an aromatic residue (Phe, Tyr, Trp) at relative position i and a small hydroxyl-containing residue (Ser, Thr) at i + 5. Our findings indicate that in RA patient-derived EBV-transformed B cells DR4Dw4/DR53 molecules present a peptide from the autoantigen calreticulin. Interestingly, autoantibodies against calreticulin have been found in various rheumatic diseases, including rheumatoid arthritis. Thus, the analysis of HLA class II-bound peptides can lead to the identification of putative T helper epitopes, which might be involved in the immunopathology of autoimmune diseases.

Amino Acid Sequence↗
PubMed1994

The subunit structure of elongation factor 1 from Artemia. Why two alpha-chains in this complex?

Elongation factor 1 (EF-1) regulates the specific interaction of aminoacyl-tRNA with the ribosome during the elongation phase of protein biosynthesis. Although individual functions of its separate chains have been well defined, to date there is hardly information about the structure and function of the whole complex. We describe here the complete subunit structure of elongation factor 1, and discuss its change during development of Artemia. Elongation factor 1 consists of a pentameric complex, composed of four different subunits alpha, beta, gamma, and delta in a molar ratio of 2:1:1:1. Although one molecule of EF-1 alpha dissociates easily from the complex EF-1 alpha 2 beta gamma delta under the influence of aminoacyl-tRNA and GTP, the second molecule of EF-1 alpha was found to remain firmly attached. Thus, in eukaryotic protein synthesis, movement of transfer RNAs to the ribosome seems under the influence of two distinct molecules of EF-1 alpha, a result possibly related to the presumed consumption of two molecules of GTP by EF-Tu during the elongation step of prokaryotic protein synthesis.

Amino Acid Sequence↗
PubMed1994

The leucine-zipper in elongation factor EF-1 delta, a guanine-nucleotide exchange protein, is conserved in Artemia and Xenopus.

Elongation factor 1, a complex involved in protein biosynthesis, contains two guanine-nucleotide-exchange proteins EF-1 beta and EF-1 delta. The sequence of EF-1 delta of Artemia was determined with the purified protein. When compared to EF-1 delta from Xenopus, a high degree of identify (80%) was found in the C-terminal domains of the proteins, which contain the guanine-nucleotide-exchange activity. The N-terminal domains share only 23% of the amino acids at identical positions, and therefore they were further analysed for less obvious types of homology. To this end, a published approach for sequence analysis, which can detect peculiar amino acid patterns in proteins was applied. In this way, a weak albeit unmistakable similarity between the two EF-1 delta proteins was demonstrated in the region of the leucine-zippers, apart from the leucine repeat itself. Apparently, they display a common structural pattern in their N-terminal domains, which so far has been observed mainly in transcription factors.

Amino Acid Sequence↗
PubMed1994

Identification of the maltose transport protein of Saccharomyces cerevisiae.

Identification of the maltose transport protein of Saccharomyces cerevisiae was attempted by searching for maltose-inducible proteins in isolated plasma membranes. Membranes from maltose-grown cells contained two proteins that were absent in glucose-grown cells. The proteins differed in size, but peptide sequence analysis indicated a high degree of homology. The amino-terminal and internal sequences of the largest protein, with an apparent molecular mass of 64 kDa, were determined. These sequences were identical to predicted amino acid sequences in the MAL61 gene product. It is concluded that this protein is the inducible maltose permease of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1994

Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing.

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogenous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.

Amino Acid Sequence↗
PubMed1994

Hb Kurdistan [alpha 47(CE5)Asp-->Tyr], a new alpha chain variant in combination with beta (0)-thalassemia.

We have characterized the structural abnormality of a new alpha chain mutant found in a Kurdish family. The clinical and hematological investigation of eight individuals have shown that the alpha variant is associated with a beta(0)-thalassemia mutation (nonsense codon 39). The tryptic peptide map and sequencing of the abnormal peptide revealed the substitution of an aspartic acid by a tyrosine residue at position 47 of the alpha chain; furthermore, selective amplification and molecular analysis of both alpha genes have assigned the new mutation to the alpha 2 gene. The variant, named Hb Kurdistan, is clinically silent but the percentage of this hemoglobin found in the only double heterozygote for beta (0)-thalassemia and alpha-Kurdistan, presumably indicates a lower affinity of the abnormal chain for the beta polypeptides.

Adult↗
PubMed1993

Isolation of an HLA-A2.1 extracted human minor histocompatibility peptide.

Purified HLA-A2.1 molecules obtained by affinity chromatography of 6 x 10(10) Epstein Barr virus (EBV)-transformed B lymphocytes were used in an attempt to isolate the human HLA-A2.1-restricted minor histocompatibility (H) peptides H-Y and HA-2. Fraction 18 of the high-performance liquid chromatography (HPLC)-separated HLA-A2.1 peptide pool was found to contain the natural HA-2 peptide. An HA-2-specific, HLA-A2.1-restricted cytotoxic T lymphocyte clone lysed HLA-A2.1+ HA-2- EBV-transformed B lymphocyte cell lines reproducibly and in a concentration-dependent fashion in the presence of fraction 18, but not in the presence of other HPLC fractions. By contrast, H-Y sensitizing activity was not found in any fraction. Amino acid sequencing of peptide fraction 18 revealed a mixture of peptides with maximal length of nine amino acids, in which the presence of Leu at positions 2 and 9 was dominant. Surprisingly, the HA-2 peptide could not be mimicked by any of the peptide mixtures synthesized according to the amino acid sequences found in fraction 18. Our failure to obtain the actual amino acid sequence of the human minor H peptide HA-2 from a peptide pool with the established pattern for binding to HLA-A2.1 may indicate that this CTL defined minor H peptide does not represent an abundant HLA-A2.1 binding peptide.

Amino Acid Sequence↗
PubMed1993

The complete primary structure of ribosomal protein L1 from Thermus thermophilus.

The primary structure of the 23S rRNA binding ribosomal protein L1 from the 50S ribosomal subunit of Thermus thermophilus ribosomes has been elucidated by direct protein sequencing of selected peptides prepared by enzymatic and chemical cleavage of the intact purified protein. The polypeptide chain contains 228 amino acids and has a calculated molecular mass of 24,694 D. A comparison with the primary structures of the corresponding proteins from Escherichia coli and Bacillus stearothermophilus reveals a sequence homology of 49% and 58%, respectively. With respect to both proteins, L1 from T. thermophilus contains particularly less Ala, Lys, Gln, and Val, whereas its content of Glu, Gly, His, Ile, and Arg is higher. In addition, two fragments obtained by limited proteolysis of the intact, unmodified protein were characterized.

Amino Acid Sequence↗
PubMed1993

Purification and primary structure of ceratotoxin A and B, two antibacterial peptides from the female reproductive accessory glands of the medfly Ceratitis capitata (Insecta:Diptera).

In the present article we report the purification and the amino acid sequence of two antibacterial peptides present in the secretion of the female reproductive accessory glands of the dipteran insect Ceratitis capitata. Both peptides consist of 29 amino acid residues, are heat stable, strongly basic and differ from each other for the substitution of two amino acids. Their primary sequence and predicted secondary structure are related to other families of peptides known to have lytic and/or antibacterial activity. We propose the name ceratotoxins (from Ceratitis) for these antibacterial peptides.

Amino Acid Sequence↗
PubMed1992

Identification of the sites in the eukaryotic elongation factor 1 alpha involved in the binding of elongation factor 1 beta and aminoacyl-tRNA.

In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1 beta and aminoacyl tRNA to the alpha-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemia. For this purpose the polypeptide chain of EF-1 alpha, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-1 alpha and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129-461 and 69-461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1-36, that remained tightly bound. The ability of the mixture of the 37+43-kDa fragments, including this amino-terminal peptide 1-36, to bind GDP or to facilitate aminoacyl tRNA binding to salt-washed ribosomes was severely reduced, compared to intact EF-1 alpha. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1 beta. A 30-kDa fragment, comprising the residues 1-287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1 beta was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1 beta is induced through binding of this factor to the carboxy-terminal part of EF-1 alpha. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-1 alpha, especially in the region of its presumed effector loop is induced.

Amino Acid Sequence↗
PubMed1991

Phosphorylation regulates the activity of the eEF-2-specific Ca(2+)- and calmodulin-dependent protein kinase III.

The activity of the eukaryotic elongation factor 2 (eEF-2)-specific Ca(2+)- and calmodulin-dependent protein kinase III (CaM PK III) is regulated by phosphorylation. The kinase can be inactivated by treatment with alkaline phosphatase and subsequently reactivated by endogenous protein kinase. This kinase can be substituted for by the catalytic subunit of cAMP-dependent protein kinase but not by casein kinase II. The purified kinase preparation contains only one protein as judged by gel electrophoresis. This protein has a molecular mass of approximately 90 kDa and an isoelectric point of 5.2. Reactivation of the eEF-2 kinase is associated with the phosphorylation of this protein. The amino acid sequence obtained from the 90-kDa protein reveals substantial homology with that of murine heat shock protein 86 (HSP 86) a member of the HSP 90-family. Conventional preparations of HSP 90 contain an inactive eEF-2 kinase that could be activated after dephosphorylation and phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.

Alkaline Phosphatase↗
PubMed1991

Mapping the functional domains of the eukaryotic elongation factor 1 beta gamma.

The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha.

Antibodies, Monoclonal↗
PubMed1990

The primary structure of artemin from Artemia cysts.

The primary structure of artemin, a major protein isolated from Artemia cysts, has been determined by direct Edman degradation of the purified protein. The amino-terminal acetylated protein has 229 amino acid residues and a high content of histidine and cysteine/cystine. A search in the GenBank Data Base at Los Alamos, using the FASTA program [Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448] revealed a limited but unmistakable similarity to ferritin from vertebrates.

Amino Acid Sequence↗
PubMed1990

Elongation factor 1 beta of artemia: localization of functional sites and homology to elongation factor 1 delta.

Elongation factor (EF)-1 beta, a 26 kDa protein, is the eukaryotic equivalent of bacterial EF-Ts, the nucleotide exchange factor in protein synthesis. EF-1 beta catalyzes the exchange of guanine nucleotides bound to EF-1 alpha; the latter protein is the eukaryotic equivalent of bacterial EF-Tu. Limited proteolytic cleavage studies on EF-1 beta lead to the following picture: the protein is composed of two domains, an aminoterminal and a carboxyterminal domain, connected to each other by a stretch of hydrophilic, charged amino acids situated in the middle of the molecule. The carboxyterminal domain supplies the catalytic site for the nucleotide exchange reaction, whereas the aminoterminal domain interacts with EF-1 gamma, the third component of elongation factor 1. The regulatory, serine phosphate residue, Ser-89, localized in the hydrophilic stretch of EF-1 beta, does not appear to be necessary for the basic exchange reaction. The fourth component of the high molecular weight elongation factor complex (EF-1H), named EF-1 delta or 28 K protein, is homologous to EF-1 beta and contains regions very similar to the carboxyterminal part. EF-1 delta was found to be active in the nucleotide exchange reaction.

Amino Acid Sequence↗
PubMed1990

Elongation factor EF-1 alpha from Artemia is specifically labeled at Lys-63 by the guanine nucleotide analog pyridoxal-5'-diphospho-5'-guanosine.

We have synthesized the guanine nucleotide analog pyridoxal-5'-diphospho-5'-guanosine. This compound specifically modifies a single lysine residue in elongation factor 1 alpha from Artemia, indicating that this residue is in close contact with the reactive part of the guanine nucleotide analog. This result is discussed in terms of the structure of the nucleotide-binding domain of the factor.

Affinity Labels↗
PubMed1990

Hb J-Anatolia [alpha 61(E10)Lys----Thr]: structural characterization and gene localization of a new alpha chain variant.

We report the characterization of a new hemoglobin variant having a single amino acid substitution (Lys----Thr) at position 61 of the alpha chain. In addition to the structural analysis, we also describe the strategy used for the identification of the base substitution and the localization of the defect at the gene level using polymerase chain reaction and hybridization with allele-specific oligonucleotides.

Alleles↗