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Y Paterson

Publications and source records attributed to Y Paterson.

At least 73 records · Page 4Linked to original sources
PubMed1989

Substitutions engineered by chemical synthesis at three conserved sites in mitochondrial cytochrome c. Thermodynamic and functional consequences.

Analogues of the 39-residue CNBr fragment of horse cytochrome c (66-104) have been prepared by total chemical synthesis. Conformationally assisted ligation of these peptides with the native cytochrome c fragment 1-65 (homoserine lactone form) occurred in high yield. Semisynthetic protein molecules of the expected molecular weight were obtained that had folded structures similar to the native molecule as shown by spectral properties and by cross-reactivity with a panel of monoclonal antibodies sensitive to the three-dimensional integrity of cytochrome c. Point mutations were introduced into the horse sequence at three strongly conserved sites: Tyr67, Thr78, and Ala83. The contributions of these 3 residues to the stability of the heme crevice were estimated by titration of the 695 nm absorption due to coordination of ferric iron by the sixth ligand methionine sulfur. The roles of these residues in catalysis of electron transfer and in establishing the value of the redox potential of cytochrome c were also investigated. The hydroxyl group of Tyr67 modulates the spectral properties of the heme and has a profound influence on its redox properties, but hydrogen bonding involving this phenolic hydroxyl does not stabilize the heme crevice. In contrast, we find that Thr78 is strongly stabilizing and that asparagine is not an adequate substitute for this residue because of the greater entropic cost of burying its side chain. The low biological activity of analogues modified at this position, despite normal redox potentials, imply a role for Thr78 in the electron transfer mechanism. The replacement of Ala83 by proline induces a similar phenomenon. An involvement of this residue in the catalysis of electron transfer provides an explanation of the low reactivity of plant mitochondrial cytochromes c in mammalian redox systems.

Amino Acid Sequence↗
PubMed1989

Mapping of four discontiguous antigenic determinants on horse cytochrome c.

The epitopes (antigenic determinants) recognized by four different monoclonal antibodies on horse cytochrome c have been partially characterized by differential acetylation of lysine residues of free and antibody-bound cytochrome c. The degree of acetylation in the bound and free antigen molecule was assessed by a double-labeling procedure with [3H]acetic anhydride and [14C]acetic anhydride. Out of the 19 lysine residues of cytochrome c only very few were less reactive in the antigen-antibody complex, i.e. presumably located at the epitope for the antibody under study. The protection varied from 1.5-fold to over 20-fold lower reactivity in antibody-bound cytochrome c. The present results are complemented by previous data obtained by cross-reactivity analysis with cytochromes c from different species, with chemically modified cytochrome c derivatives, and by inhibition of proteolysis of cytochrome c in the presence of the antibodies. From the combined data we conclude that each of the four epitopes depends on the precise spatial folding of the antigen and contains residues which are brought together by the folding of the polypeptide chain. This work exemplifies that mapping of conformation-dependent epitopes can be achieved by applying a combination of mapping procedures of which each by itself provides partial information.

Acetylation↗
PubMed1989

The autoantigenic response to rabbit cytochrome c.

In this report we describe the production and characterization of autoantibody responses to rabbit cytochrome c (cyt c) in rabbits immunized with either the native monomeric or polymerized form of rabbit cyt c. Fine-specificity analyses of the response indicated that the majority of the response was to the evolutionarily conserved amino-terminal region of the molecule. The relative affinity of the autoantibody interaction with rabbit cyt c was assessed by a solid-phase assay and was found to be lower than that observed for rabbit anti-horse cyt c antibody populations. These findings are consistent with the prediction that low-affinity self-reactive B cells may escape tolerance induction.

Amino Acid Sequence↗
PubMed1989

An analysis of the physical properties of peptides that influence the pigeon cytochrome c specific T lymphocyte response.

The ability to modify T cell responses was analyzed using synthetic peptide analogues of the T cell determinant for pigeon cytochrome c. Although the B10.A T cell proliferative response is directed to residues 95-104, residues to the amino-terminal side of this determinant influence antigen-specific T cell recognition. The proposed role of this non-determinant leader sequence has been to stabilize the core determinant in a helical conformation. Previous studies from our laboratory, however, using non-native leader sequences that were designed to examine the changes to T cell recognition invoked when the determinant was made more or less helical, amphipathic, or lipid binding in character than the native determinant. The structure of each analogue in aqueous, non-polar (TFE) and lipid environments was determined by circular dichroism. The ability of each antigen analogue to bind to phospholipid membranes and to stimulate two different pigeon cytochrome c T cell hybridomas, 2B4 and 22.D11, was also investigated. Our findings suggest that neither helicity or amphipathicity are necessary features of T cell recognition but that electrostatic interactions involving either the lipid membrane or the I-Ek molecule may influence T cell stimulation.

Animals↗
PubMed1988

The heme moiety of cytochrome c is an autoreactive Ir gene-restricted T cell epitope.

In these studies, we have shown that the heme moiety of cyt c is a dominant T cell epitope that induces a large proliferative response in lymph node T cells derived from SJL and B10.A mice when presented on either unfixed or fixed syngeneic APCs. Not only is this vigorous response observed for cyt c-primed T cell populations but also for populations obtained from naive SJL or B10.A mice. The reactivity to the heme moiety falls under strict MHC restriction, in that it is present only in murine strains bearing either the I-Ak or I-As molecule and can be blocked by antibodies specific for these class II molecules. Therefore, these findings require that the current models describing the nature of T cell epitopes be extended to include nonpeptide molecules. Furthermore, as the heme moiety is ubiquitous throughout the organism, although sequestered within proteins, the existence of heme-reactive T cell populations in unprimed animals provides another example of the existence of self-reactive T cell clones.

Animals↗
PubMed1988

The activation of pigeon cytochrome c-specific T cell hybridomas by antigenic peptides is influenced by non-native sequences at the amino terminus of the determinant.

We recently demonstrated that the sequence 95-104 contains all the residues necessary for direct recognition of the I-Ek restricted pigeon cytochrome c determinant but that residues located in sequences to the amino-terminal side of residue 95 improve the ability of peptides containing the sequence 95-104 to stimulate Ag-specific T cell clones. In this study we use synthetic peptides with amino-terminal leader sequences containing residues that differ with respect to their conformational stabilizing effects, charge, and hydrophilicity to examine the mechanism by which they modulate T cell recognition. Our findings indicate that the role of these residues in T cell stimulation is not related to their ability to stabilize alpha-helical secondary structure, nor do they appear to be processed differently. The leader sequences do not differentially influence the ability of the peptides to be presented by APC displaying Ia molecules of related haplotype, i.e., E alpha kE beta k, E alpha kE beta b, and E alpha kE beta s, to T cells which recognize the pigeon cytochrome c determinant on such presenting cells. Because antigenic potency correlates with the inclusion of hydrophobic residues and positively charged residues in the leader sequences, we discuss our findings with reference to the possibility that they non-specifically enhance the interaction of the antigenic peptides with the APC membrane.

Amino Acid Sequence↗
PubMed1988

Monoclonal antibodies as probes of conformational changes in protein-engineered cytochrome c.

Determination of the nature of the antigen-antibody complex has always been the ultimate goal of three-dimensional epitope mapping studies. Various strategies for epitope mapping have been employed which include comparative binding studies with peptide fragments of antigens, binding studies with evolutionarily related proteins, chemical modifications of epitopes, and protection of epitopes from chemical modification or proteolysis by antibody shielding. In this study we report the use of protein engineering to modify residues in horse cytochrome c that are in or near the epitopes of four monoclonal antibodies specific for this protein. The results demonstrate not only that site-specific changes in the antigen binding site dramatically affect antibody binding, but, more importantly, that some of the site-specific changes cause local and long-range perturbations in structure that are detected by monoclonal antibody binding at other surfaces of the antigen. These findings emphasize the role of native conformation in the stabilization of the interaction between protein antigens and high affinity monoclonal antibodies. Furthermore, the results demonstrate that monoclonal antibodies are more sensitive probes of changes in conformation brought about by protein engineering than low resolution spectroscopic methods such as circular dichroism, where similar spectra are observed for all the analogues. These findings suggest a role for monoclonal antibodies in detecting conformational changes invoked by nonconservative amino acid substitutions or substitutions of evolutionarily conserved residues in protein-engineered or recombinant proteins.

Animals↗
PubMed1988

Processing of a minimal antigenic peptide alters its interaction with MHC molecules.

Before their recognition by T lymphocytes, protein antigens generally require processing by antigen-presenting cells. In a poorly understood series of events, the protein antigen is internalized, transformed and re-expressed on the surface of the antigen-presenting cell in association with gene products of the major histocompatibility complex (MHC). Small peptides derived from the native protein can be recognized in the absence of antigen processing, suggesting that processing involves proteolytic degradation. These peptides are thought to mimic the naturally produced peptide fragment. We describe here a synthetic peptide antigen of this type which does not require processing but which is nevertheless further processed by splenic antigen-presenting cells. Interestingly, this processing event specifically alters the interaction of the peptide with the class II MHC (Ia) molecule, markedly affecting both its potency as an antigen in vitro and its immunogenicity in vivo (IR gene control).

Animals↗
PubMed1987

Site-directed chemical modification of horse cytochrome c results in changes in antigenicity due to local and long-range conformational perturbations.

Comparative binding studies with peptide fragments of the whole antigen, or with evolutionarily related intact proteins with varying degrees of sequence homology, have been used extensively to map antigenic sites on proteins to the resolution of single amino acid residues. These methods are limited, however, since high affinity antibodies will often not react with peptides and evolutionarily related proteins are available for only a few antigens. In this study we use site-directed chemical modification of horse cytochrome c to identify residues involved in the binding sites of four monoclonal antibodies specific for this protein. Thus, we have N-formylated the single tryptophan found in horse cytochrome c at position 59 and N-carbethoxylated one of the histidyl residues, which was determined to be at position 26 by the analysis of proteolytic cleavage fragments of the modified protein using liquid secondary ion-mass spectrometry on triple quadropole or tandem quadropole Fourier transform instruments. We discuss the impact of these modifications on the antigenicity of horse cytochrome c with regard to the conformational perturbations introduced by such modifications and with reference to our previous studies on the binding sites of these antibodies using other methodologies (Jemmerson, R., and Paterson, Y. (1986) BioTechniques 4, 18-31).

Animals↗
PubMed1987

The use of hydrophobic, alpha-helix-defined peptides in delineating the T cell determinant for pigeon cytochrome c.

The B10.A T cell proliferative response to pigeon cytochrome c is largely directed to a single site in the molecule located at the carboxyl terminus within the amino acid sequence of residues 81 to 104. This study uses the pigeon cytochrome c-specific T cell clone A.E7 and synthetic peptide analogs to clarify the role of certain residues within this sequence in T cell recognition. By using the helically constrained amino acid, alpha-aminoisobutyric acid, alternated with alanine in an amino-terminal leader sequence, we generated a series of molecules of similar length and alpha-helical conformation but which contain increasing lengths of the native sequence. By comparing the stimulatory ability of this series of peptides, we have clearly identified that the isoleucyl residue at position 95 in pigeon cytochrome c is essential for T cell recognition. This series, when compared with a series containing the same native sequences but without the leader sequence, also showed that the presence of the leader sequence has a general effect on enhancement of T cell recognition. An analysis of the conformational preferences of the peptides using circular dichroism indicated that all of the peptides with leader sequences have a strong preference for the alpha-helical conformation in nonpolar solvents. However, the introduction of helix-breaking residues into these peptides, with a concomitant measured reduction in alpha-helix, did not affect their recognition by clone A.E7. This implies that factors other than conformational stabilization are responsible for the full potency of these peptides. Binding studies to phospholipid vesicles indicated that residues in the leader sequence and in the amino terminus of segment 81-104 beyond residue 95 were important in increasing the ability of the antigens to bind to membranes. These results suggest that the capacity to bind to membranes may be a significant factor in the dose response of T cells to exogenously presented peptides.

Animals↗
PubMed1987

A new T helper cell specificity within the pigeon cytochrome c determinant 95-104.

A T cell hybridoma with unusual antigen specificity has been isolated from B10.BR mice immunized with pigeon cytochrome c. Using synthetic peptide analogues of the pigeon cytochrome c T cell determinant 81-104 we have determined that this hybridoma is specific for residues within the sequence 95-104. However, the mode by which this T cell hybridoma recognizes this determinant in the context of the I-Ek molecule differs from all previously described T cell clones in that it is not stimulated by the moth cytochrome c analogue 81-103.

Amino Acid Sequence↗
PubMed1986

Mapping epitopes on a protein antigen by the proteolysis of antigen-antibody complexes.

A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

Animals↗
PubMed1985

Delineation and conformational analysis of two synthetic peptide models of antigenic sites on rodent cytochrome c.

Two regions of rodent cytochrome c, one within the first four residues of the molecule, which is N-acetylated, and one at a beta bend around residue 44, are known to be immunogenic and antigenic in rabbits. Using sequential peptide synthesis, we have determined the residues required for linear synthetic peptides within these sequences to bind to antibody raised in rabbits to intact rat cytochrome c. The residues that were important in binding the N-terminal peptides were N-acetylglycine at position 1 and valine at position 3. The smallest peptide sequence around residue 44 that would bind to antibodies was Gln-Ala-Ala-Gly-Phe. A theoretical conformational analysis of these peptides showed that the amino-terminal tetrapeptide adopts a wide statistical ensemble of conformational states and that the addition of residues beyond 41 and 45 in the other sequence does not appear to stabilize longer peptides in the native beta-bend conformation. Thus, the antigenicity conferred by Phe-46 and Gln-42 in this peptide is most likely due to the direct interaction of the side chains of these residues with the antibody binding site. The demonstration here that native conformation is not essential for antigenic peptides to bind to antibodies raised against the whole protein indicates that the association energy between antigen and antibody can be sufficient to induce conformation in conformationally flexible peptides. This supports the concept that anti-protein and anti-peptide antibodies may invoke conformational changes in cross-reactive protein antigens and may explain why longer peptides, which may adopt stable nonnative secondary structure, often do not bind to antibodies raised to the whole molecule.

Amino Acid Sequence↗
PubMed1985

Analysis of an evolutionarily conserved antigenic site on mammalian cytochrome c using synthetic peptides.

Two synthetic peptides inclusive of the NH2-terminal N-acetyl-Gly-Asp-Val-Glu tetrapeptide of mammalian cytochrome c (cyt c) were used in this study to address the question of whether mammals can respond immunologically to an evolutionarily conserved region of a protein. These peptides were assessed for their capacity (i) to act as immunogens for the production of anti-self cyt c antisera and (ii) to bind rabbit anti-rodent cyt c antibody. The findings from these studies indicate the existence of an immunogenic determinant in an evolutionarily conserved region of cyt c that contains residues 1-4. This determinant can induce anti-self cyt c antibodies whether presented as a peptide on a carrier protein or in the context of the intact molecule as polymerized mammalian cyt c.

Amino Acid Sequence↗
PubMed1985

Monoclonal antibodies to horse cytochrome c expressing four distinct idiotypes distribute among two sites on the native protein.

Antibody-secreting hybridoma cell lines produced from BALB/c mice that had been immunized with horse cytochrome c (cyt c) conjugated to hemocyanin yielded six hybridoma subclones that produced four monoclonal antibodies (mAb) with different patterns of cross-reactivity with a panel of evolutionarily variant cyt c. The recognition sites for three of these mAb lay in the same region of the intact molecule, because two of the mAb were sensitive to the amino acid residue present at sequence position 44, with one requiring threonine at position 47. The fourth mAb bound in another region of the molecule at a site that involves either residue 60 or residue 89. Synthetic peptides that included these residues did not react with these mAb, indicating that these sites may require interactions from noncontiguous regions of the molecule to bind antibody. The association constants for the interaction of the mAb with horse cyt c were very similar and of the order of 10(10) M-1. Specificity studies with anti-idiotypic sera and competition assays between mAb for binding to horse cyt c confirmed that the six positive hybridoma subclones produced from this fusion produced mAb that had one of these four distinct specificities. The idiotypes of these four mAb were serologically distinct, and were derived from Vh genes of the J558 family.

Animals↗
PubMed1982

Solution conformations of oligomers of alpha-aminoisobutyric acid.

The N-acetyl(Aib)nN'-methylamides (with n = 1, 2 and 3) and the N-acetyl-(Aib)3 methyl ester have been synthesized using an oxazolone procedure. An experimental conformational analysis of this series of oligomers has been carried out in water, DMSO-d6 and CDCl3 using n.m.r. techniques, and in chloroform using i.r. spectroscopy. Deuterium exchange rates of amide protons in DMSO-d6 and the rates of these proton chemical shifts with temperature in water, DMSO-d6 and CDCl3 indicate that the oligomeric N'-methylamides adopt conformations that have no hydrogen bonds when n = 1, one hydrogen bond when n = 2, and two hydrogen bonds when n = 3, and that Ac(Aib)3OMe has a conformation with one hydrogen bond. An analysis of the N-H stretching region of the i.r. spectra of these compounds in CHCl3 also suggests the existence of these conformational states. These data imply that the peptides adopt the 3(10)-helical and not the alpha-helical conformation in solution. This conclusion supports the hypothesis that the Aib residue has asymmetric geometry at the C alpha atom in solution, similar to that reported in the literature for the crystalline state.

Aminoisobutyric Acids↗