A peptide binding protein which plays a role in antigen presentation.
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Animals↗
Biomedical subjects
E Margoliash
Publications and source records attributed to E Margoliash.
Identification of a peptide binding protein that plays a role in antigen presentation.
The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by its ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from [35S]methionine-labeled cells, appears as two discrete bands of approximately equal to 72 and 74 kDa after NaDodSO4/PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation as evidenced by the ability of rabbit antibodies raised against it to block the activation of an antigen-specific T-cell hybrid by antigen-presenting cells and pigeon cytochrome c. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation--facilitating the interaction of antigenic peptides with Ia and/or the T-cell receptor.
Animals↗
Preferred sites on cytochrome c for electron transfer with two positively charged blue copper proteins, Anabaena variabilis plastocyanin and stellacyanin.
Rate constants for the reactions of horse cytochrome c (E'0 of +260 mV) with the copper proteins Anabaena variabilis plastocyanin (E'0 of +360 mV) used as oxidant and stellacyanin (E'0 of +187 mV) used as reductant have been determined at 25 degrees C, pH 7.5 and 7.0, respectively, and an ionic strength of 0.10 M (NaCl). These rate constants were also measured with eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse cytochrome c derivatives, modified at lysine-7, -13, -25, -27, -60, -72, -86, or -87 and with the trinitrophenyl (TNP) derivative modified at lysine-13. The influence of the modifications on the bimolecular rate constants for these reactions defines the region on the protein that is involved in the electron-exchange reactions and demonstrates that the preferred site is at or near the solvent-accessible edge of the heme prosthetic group on the "front" surface of the molecule. Both reactions are strongly influenced by the lysine-72 modification to the left of the exposed heme edge and, to this extent, behave similar to the earlier studied reaction with azurin. These effects span only an order of magnitude in rate constants and are thus many times smaller than those for the physiological protein redox partners of cytochrome c. While the preferred sites of reaction on the surface of cytochrome c for small inorganic complexes appear to be dependent only on the net charge of the reactants, with the copper proteins additional factors intervene. These influences are discussed in terms of hydrophobic patches and the distribution of charges on the surface of the four copper proteins so far examined.
Binding Sites↗
Role of L3T4 and Ia in the heteroclitic response of T cells to cytochrome c.
The activation of helper T lymphocytes has been proposed to result from the sum of low-affinity interactions between the specific immune receptor, as well as nonpolymorphic receptors such as L3T4 on the T cell surface, and nominal antigen and Ia displayed in a multivalent array on the antigen-presenting cell surface. The present work takes advantage of a T cell hybridoma specific for pigeon cytochrome c in the context of I-Ek, which responds to tobacco hornworm moth cytochrome c at one hundredth the concentration of the homologous antigen, to determine if the T cell's requirement for L3T4 and Ia is directly related to its functional affinity for antigen. The results demonstrate that the T cell's activation by pigeon cytochrome c was blocked by antibodies directed to L3T4 and to I-Ek, even at antigen concentrations twofold to fourfold above those required for maximal responses. In contrast, the response to tobacco hornworm moth cytochrome c was not as affected by these antibodies under equivalent superoptimal conditions. The same phenomenon was observed for the T cell's activation by the carboxyl-terminal peptide fragments of the two cytochromes c, which do not require processing, indicating that the differences were not due to the relative efficiency of processing and/or presentation of the antigens. Although both I-Ek- and L3T4-specific antibodies blocked the T cell response to pigeon cytochrome, antibodies to I-Ak had no effect, even though I-Ak had been considered to be a ligand for L3T4. Thus, either Ia does not bind L3T4 or, if it does, I-Ek must be a sufficient ligand for L3T4 for T cells that recognize their antigen in the context of I-Ek. These studies provide more definitive evidence that the T cell's requirement for the functions of Ia and of L3T4 is dependent on the T cell's functional affinity for its antigenic determinant. This data is consistent with a model of T cell activation in which, given a high enough affinity of the T cell receptor for the processed antigen, the requirement for other components of a stimulatory complex, such as Ia and L3T4, may diminish to undetectable levels.
Animals↗
Peptides related to the antigenic determinant block T cell recognition of the native protein as processed by antigen-presenting cells.
A mouse T cell hybrid specific for pigeon cytochrome c in the context of I-Ek responds by secreting interleukin 2 when co-cultured with the native antigen and the B cell lymphoma, LK-35.2, or naive splenic B cells as antigen-presenting cells (APC). Cytochromes c and their corresponding C-terminal fragments which are not capable of stimulating the TPc9.1 cells, including the autologous mouse cytochrome c, block the T cells' response to pigeon cytochrome c. In contrast, nonstimulatory N-terminal peptides of cytochrome c, which share no homology with the antigenic peptide, do not block. Blocking is observed when the nonstimulatory cytochromes c or peptides are present in culture with the live APC and nonsaturating concentrations of pigeon cytochrome c. With tobacco hornworm moth cytochrome c as antigen, a protein for which the T cell has a higher functional affinity, the response of TPc9.1 cannot be blocked by the nonstimulatory cytochromes c or by peptides, even when limiting concentrations of the tobacco hornworm moth cytochrome c are used. When paraformaldehyde-fixed APC are employed, no native cytochrome c can stimulate the T cells, including the tobacco hornworm moth protein which with the live APC is effective at 50 to 100-fold lower concentrations than pigeon cytochrome c. However, with fixed APC the T cells are stimulated by the C-terminal fragments containing residues 81-104 of the pigeon protein or residues 81-103 of the tobacco hornworm moth protein as readily and with the same relative efficiencies as the native protein, presented by live APC. The nonstimulatory peptides, but not the native cytochromes c, block T cell activation by pigeon cytochrome c pulsed-fixed APC, indicating that the nonstimulatory peptides compete with the stimulatory pigeon cytochrome c peptides produced by the APC. This competition appears to be due to nonstimulatory peptides which associate at the APC surface and not to those acting from solution because the APC which have been incubated with pigeon cytochrome c and nonstimulatory peptides and washed free of excess antigen and peptides are not stimulatory to the T cell hybrid. It was concluded that the activation of a pigeon cytochrome c-specific T cell, which recognizes a peptide fragment of the native protein on the surface of an APC, can be blocked by an excess of nonstimulatory homologous peptides when these are also associated on the surface of the APC.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Anti-immunoglobulin augments the B-cell antigen-presentation function independently of internalization of receptor-antigen complex.
All mouse splenic B cells, including small resting B cells, process and present the native globular protein antigens, pigeon and tobacco hornworm moth cytochromes c, to a cytochrome c-specific T-cell hybrid in a major histocompatibility complex-restricted fashion, in the micromolar to nanomolar antigen-concentration range. As is the case for macrophages, treatment with paraformaldehyde or the lysosomotropic agents chloroquine and ammonium chloride blocked processing of the native pigeon protein but did not affect the presentation of a carboxyl-terminal peptide fragment of pigeon cytochrome c (residues 81-104) which contained the T-cell antigenic determinant. However, in contrast to macrophages, whose antigen-processing and -presentation functions are insensitive to radiation, radiation blocked the processing of the native protein but not the presentation of the peptide fragment. The processing and presentation function of the B cells was augmented by F(ab')2 of rabbit anti-mouse Ig antibodies, in that 1/10th to 1/30th as many cells and 1/10th as much antigen were required to maximally activate the T-cell hybrid. This augmentation did not appear to be due to either crosslinking of the Ig receptors or to B-cell activation, as monovalent Fab fragments were nearly as effective as the bivalent reagent, and the concentrations of F(ab')2 anti-Ig used did not induce measurable proliferative responses. Furthermore, enhancement can occur in the absence of cytochrome c binding and internalization, since B cells that were fixed with paraformaldehyde after treatment with F(ab')2 anti-Ig were more effective in presenting the carboxyl-terminal peptide than were untreated fixed cells. The same phenomenon followed the binding of an irrelevant antigen (carboxydinitrophenylated bovine serum albumin) by antigen-binding B cells, resulting in enhanced processing and/or presentation of native pigeon cytochrome c. Thus, nonspecific enhancement of antigen processing and presentation can be obtained by either antigen or anti-Ig binding to the B-cell antigen receptor, both treatments presumably delivering the same signal without requiring internalization of the specifically bound antigen for subsequent processing.
Animals↗
Developmental expression of nuclear genes that encode mitochondrial proteins: insect cytochromes c.
To investigate tissue-specific developmental regulation of mitochondrial biogenesis, we studied the expression of the Manduca sexta (tobacco hornworm moth) thoracic muscle cytochrome c gene during adult eclosion and used this information to obtain a cDNA clone for this gene, which in turn was used to isolate the corresponding Drosophila melanogaster gene. Over the 3 days prior to adult Manduca emergence, mitochondrial inner membranes become progressively more electron dense and lamellar, and, while there is no accumulation of apocytochrome c, the amount of the holoprotein increases 40-fold per insect thorax. As determined by in vitro translation and blot hybridization analysis, the major thoracic muscle cytochrome c gene is primarily regulated at the transcriptional level, with cytochrome c mRNA increasing from less than 0.01% to 0.04% of total poly(A)+ RNA and declining to an undetectable level by day 2 after eclosion. Furthermore, the ratio of cytochrome c to the other cytochromes remains the same at all times, indicating that these components of the respiratory chain follow coordinated developmental programs. By using polysome immunoadsorption, a poly(A)+ RNA population of greater than or equal to 95% cytochrome c mRNA was isolated from thoracic muscle tissue and was used to construct a cDNA library, which was screened by hybrid selection/translation. We report the sequence of one of those clones, pMSc750, and its use to isolate the major thoracic muscle cytochrome c gene of Drosophila.
Animals↗
Assembled topographic antigenic determinants of pigeon cytochrome c.
The specificity of the IgG fraction of the sera of several rabbits hyperimmunized with glutaraldehyde-polymerized pigeon cytochrome c was examined by fluorescence-quenching titration and a sensitive competitive plate-binding radioimmunoassay developed for the analysis of small amounts of antiserum. Four pigeon cytochrome c-specific Fab fragments were found to bind simultaneously to the immunogen. Competition assays, using an extensive set of naturally occurring, chemically prepared hybrid and enzymically modified cytochromes c, implicated in antibody binding all seven amino acid residues at which the immunogen differs from the homologous rabbit cytochrome c. Thus, rabbits produce small amounts of three antibody populations directed against the regions of serine 15, alanine 44, and glycine 89, respectively, on pigeon cytochrome c, and a large amount of the population which binds to an assembled topographic determinant composed of isoleucine 3, glutamine 100, alanine 103, and lysine 104. The latter four residues are from the amino-terminal and carboxyl-terminal alpha-helices, and cluster where the two helices cross each other on the back surface of the molecule. Antibodies against native pigeon cytochrome c reacted very poorly with the several cyanogen bromide-cleaved fragments of the molecule, consisting of residues 1 to 65, 1 to 80, 66 to 104, and 81 to 104.
Amino Acid Sequence↗
The B10.A mouse B cell response to pigeon cytochrome c is directed against the same area of the protein that is recognized by B10.A T cells in association with the Ek beta:Ek alpha Ia molecule.
An analysis of the fine specificities of the primary and hyperimmune antibody responses of B10.A mice to pigeon cytochrome c showed that both were qualitatively very similar. Small amounts of antibody appeared to be directed against the regions of serine 15 and/or glutamic acid 44. The remaining antibodies (greater than 70%) bound to the same complex topographic determinant (including residues 3, 103, and 104) on the back surface of pigeon cytochrome c which had been found to dominate the rabbit antibody response to this protein, and to be involved in Ia-restricted T cell stimulation. The mouse antibodies reacted very poorly with fragmented forms of the immunogen or with tobacco hornworm moth cytochrome c, even though both of these antigens had been shown previously to strongly stimulate pigeon cytochrome c-primed T cells. The specificities of the primary IgG responses of seven other mouse strains were found to be very similar, but not identical, to that of B10.A mice. The cytochrome c-specific antibodies in the hyperimmune serum were shown to bind to determinants involving residues that vary between pigeon and mouse cytochromes c. Comparison of the binding of the antibodies to the immunogen and to the corresponding host protein enabled the calculation of the proportion of the overall binding energy contributed by the variant residues. This was as low as 19 to 35% for the primary response, rose to 25 to 46% for the hyperimmune mouse antibodies, and reached 40 to 63% for hyperimmune rabbit antibodies. The remaining energy of interaction (37 to 81%) was necessarily contributed by the surface of the protein surrounding the variant residues, which is the same for the immunogen and the host protein. These results illustrate the relatively subtle differences in binding affinities which can distinguish self from non-self recognition by antibody molecules.
Amino Acid Sequence↗
Kinetics and energetics of intramolecular electron transfer in yeast cytochrome c peroxidase.
The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yonetani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D. C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618]. The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Cytochrome-c Peroxidase↗
Characterization of the interaction of cytochrome c and mitochondrial ubiquinol-cytochrome c reductase.
Characterization of the steady state kinetics of reduction of horse ferricytochrome c by purified beef ubiquinol-cytochrome c reductase, employing 2,3-dimethoxy-5-methyl-6-decylbenzoquinol as reductant, has shown that: 1) the dependence of the reaction on quinol and on ferricytochrome c concentration is consistent with a ping-pong mechanism; 2) the pH optimum of the reaction is near 8.0; 3) the effect of ionic strength on the apparent Km and the TNmax of the reaction for the native cytochrome c is small, and at higher cytochrome c concentrations substrate inhibition is observed; 4) the effect of ionic strength on the kinetic parameters for the reaction of 4-carboxy-2,6-dinitrophenyllysine 27 horse cytochrome c is much larger than for the native protein; and 5) competitive product inhibition is also observed with a Ki consistent with the binding affinity of ferrocytochrome c for Complex III, as determined by gel filtration. In addition, direct binding measurements demonstrated that ferricytochrome c binds more tightly than the reduced protein to Complex III under low ionic strength conditions and that under these conditions more than one molecule of cytochrome c is bound per molecule of Complex III. Exchange of Complex III into a nonionic detergent decreases this excess nonspecific binding. Measurement of the rates of dissociation of the oxidized and reduced 1:1 complexes of cytochrome c and Complex III by stopped flow was consistent with the disparity of binding affinities, the dissociation rate constant for ferrocytochrome c being about 5-fold higher than that for the ferric protein. A model which accounts for the properties of this system is described, assuming that cytochrome c bound to noncatalytic sites on the respiratory complex decreases the catalytic site binding constant for the substrate.
Animals↗
Synergistic effects of antigen and soluble T-cell factors in B-lymphocyte activation.
Supernatants from phorbol 12-myristate 13-acetate-activated cultures of the mouse EL4 thymoma, or of several mouse T-cell hybridomas stimulated either by their specific antigen or by concanavalin A, induced primary splenic B cells to proliferate and differentiate to antibody-secreting cells. This effect was not due to interleukin 2 and did not require the presence of macrophages. The antibody response was polyclonal, including antibodies specific for 2,4-dinitrophenyl and pigeon cytochrome c, present in amounts of 1% or less of the total immunoglobulin produced. The addition of either of these antigens increased the amount of the corresponding specific antibody. At very high concentrations of dinitrophenyl-hemocyanin the specific response could be depressed. These observations were taken to demonstrate that soluble T-cell factors are sufficient to activate a portion of naive B cells to antibody secretion and that under these conditions in vitro the presence of antigen merely enhances the specific response.
2,4-Dinitrophenol↗
Single catalytic site model for the oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase.
A single catalytic site model is proposed to account for the multiphasic kinetics of oxidation of ferrocytochrome c by cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). This model involves nonproductive binding of substrate to sites near the catalytic site on cytochrome c oxidase for cytochrome c, decreasing the binding constant for cytochrome c at the catalytic site. This substrate inhibition results in an increase in the first-order rate constant for the dissociation of the ferricytochrome c-cytochrome c oxidase complex, the rate-limiting step in the steady-state turnover of electrons between cytochrome c and cytochrome c oxidase in the spectrophotometric assay, yielding increases in the initial rate as well as the Michaelis constant--namely, multiple kinetic phases.
Animals↗
The antigenic structure of proteins: a reappraisal.
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Amino Acid Sequence↗
Role of phospholipid in the low affinity reactions between cytochrome c and cytochrome oxidase.
The steady-state oxidation of ferrocytochrome c by cytochrome oxidase monitored spectrophotometrically showed that: (1) the kinetics were strictly biphasic with purified enzyme, while mitochondrial membrane-bound enzyme exhibited multiphasic kinetics with extended low affinity phases; (2) the TNmax for the highest affinity phase was as slow as 5-10 electron X s-1 for both preparations, while for the low affinity phases it was about 45 electron X s-1 for the purified enzyme and 150 electron X s-1 for the mitochondrial membrane-bound enzyme; (3) reconstitution of purified enzyme into acidic phospholipid vesicles partially repleted the extended low affinity phases, while reconstitution into uncharged vesicles had no effect.
Animals↗
Preferred sites for electron transfer between cytochrome c and iron and cobalt complexes.
The kinetics of oxidation of eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse ferrocytochromes c, modified at lysine 7, 13, 25, 27, 60, 72, 86, or 87, and of one trinitrophenyl horse ferrocytochrome c, modified at lysine 13, by the 3- and 3+ inorganic complexes hexacyanoferrate(III) (Fe(CN)6(3-) ) and tris(1,10-phenanthroline)cobalt(III) (Co(phen)3(3+) ) have been characterized. The influence of the modified residues on the bimolecular rate constants for these reactions define the protein molecular surface involved. The site of electron exchange for both oxidants appears to be the solvent accessible edge of the heme prosthetic group or a closely related structure on the "front" surface of the molecule. The reaction with Fe(CN)6(3-) is most strongly influenced by modification of lysine 72, a residue to the left of the exposed heme edge. (CDNP lysine 72 cytochrome c yields a 3.6-fold decrease in the bimolecular rate constant, as compared to that for the native protein.) However, it is the region around lysine 27, to the right of the heme edge, that is most influential in the reaction with Co(phen)3(3+). (CDNP-lysine 27 cytochrome c exhibits a 7.3-fold increase in the rate constant, as compared to that for the native protein.) The kinetics of reaction of the CDNP-lysine 13, 60, 72, and 87 modified cytochromes c with Fe(CN)5(4-aminopyridine)2- as oxidant and Fe(CN)5(4-aminopyridine)3- and Fe(CN)5-(imidazole)3- as reductants have also been determined and further illustrate the influence of electrostatics on the kinetics of such protein-small molecule electron exchanges.
Chemical Phenomena↗
Interaction of cytochrome c with the blue copper proteins, plastocyanin and azurin.
Bimolecular rate constants have been determined for the reactions of native horse cytochrome c, eight 4-carboxy-2,6-dinitrophenyl (CDNP-) cytochromes c singly modified at lysines 7, 13, 25, 27, 60, 72, 86, or 87 and one 2,3,6-trinitrophenyl cytochrome c singly modified at lysine 13, with the blue copper proteins, plastocyanin (from parsley leaves) and azurin (from Pseudomonas aeruginosa). Plastocyanin, a protein having a negative charge of about -7, yields a bimolecular rate constant with native ferrocytochrome c of 1.5 x 10(6) M-1 S-1, which decreases with the modified cytochromes c to a minimum of 7.5 x 10(5) M-1 S-1 for the CDNP-lysine 13 derivative. Conversely azurin, a protein with an overall negative charge of only about -1 to -2, exhibits bimolecular rate constants with native ferrocytochrome c of 6.6 x 10(3) M-1 S-1 at pH 6.1 and 4.0 x 10(3) M-1 S-1 at pH 8.6, which increase upon modification of the cytochrome c to a maximum of 4.1 x 10(4) M-1 S-1 at pH 6.1 and 2.7 x 10(4) M-1 S-1 at pH 8.6, for the CDNP-cytochrome c modified at lysine 72. This behavior indicates that: 1) the reaction of cytochrome c occurs at a negatively charged site on plastocyanin, whereas azurin behaves as a positively charged reactant, the electrostatics governing to a large extent the relative reactivities of the modified cytochromes c; 2) in both cases the interaction domain on cytochrome c is located on the "front" surface of the protein and encompasses the solvent accessible edge of the heme prosthetic group, as is the case for all the reactions of cytochrome c with its mitochondrial protein redox partners, as well as for small inorganic redox complexes; and 3) the bimolecular rate constants for plastocyanin and azurin are orders of magnitude slower and the effects of lysine modifications far smaller than for the reactions with physiological systems, indicating that: (a) the electric fields generated by the reactants do not align them, prior to electron transfer, as effectively as for the physiological reaction partners of cytochrome c; and (b) there is an absence of a precise molecular fit between cytochrome c and the nonphysiological redox partners.
Azurin↗
The reaction of primate cytochromes c with cytochrome c oxidase. Analysis of the polarographic assay.
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Animals↗