ABSTRACT
Colicins are toxic proteins produced by Escherichia coli that must cross the membrane to exert their activity. The lipid insertion of their pf domain is linked to a conformational change which enables the penetration of a hydrophobic hairpin. They provide useful models to more generally study insertion of proteins, channel formation and protein translocation in and across membranes. In this paper, we study the lipid-destabilizing properties of helices H8 and H9 forming the hydrophobic hairpin of colicin E1. Modelling analysis suggests that those fragments behave like tilted peptides. The latter are characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. Fluorescence techniques using labelled liposomes clearly show that H9, and H8 to a lesser extent, destabilize lipid particles, by inducing fusion and leakage. AFM assays clearly indicate that H8 and especially H9 induce membrane fragilization. Holes in the membrane are even observed in the presence of H9. This behaviour is close to what is seen with viral fusion peptides. Those results suggest that the peptides could be involved in the toroidal pore formation of colicin E1, notably by disturbing the lipids and facilitating the insertion of the other, more hydrophilic, helices that will form the pore. Since tilted, lipid-destabilizing fragments are also common to membrane proteins and to signal sequences, we suggest that tilted peptides should have an ubiquitous role in the mechanism of insertion of proteins into membranes.
Subject(s)
Bacterial Proteins/chemistry , Membrane Lipids/chemistry , Algorithms , Computer Simulation , Hydrophobic and Hydrophilic Interactions , Microscopy, Atomic Force , Protein Structure, Secondary , Spectroscopy, Fourier Transform InfraredABSTRACT
Here, we predicted the minimal N-terminal fragment of gp41 required to induce significant membrane destabilization using IMPALA. This algorithm is dedicated to predict peptide interaction with a membrane. We based our prediction of the minimal fusion peptide on the tilted peptide theory. This theory proposes that some protein fragments having a peculiar distribution of hydrophobicity adopt a tilted orientation at a hydrophobic/hydrophilic interface. As a result of this orientation, tilted peptides should disrupt the interface. We analysed in silico the membrane-interacting properties of gp41 N-terminal peptides of different length derived from the isolate BRU and from an alignment of 710 HIV strains available on the Los Alamos National Laboratory. Molecular modelling results indicated that the 12 residue long peptide should be the minimal fusion peptide. We then assayed lipid-mixing and leakage of T-cell-like liposomes with N-terminal peptides of different length as first challenge of our predictions. Experimental results confirmed that the 12 residue long peptide is necessary and sufficient to induce membrane destabilization to the same extent as the 23 residue long fusion peptide. In silico analysis of some fusion-incompetent mutants presented in the literature further revealed that they cannot insert into a modelled membrane correctly tilted. According to this work, the tilted peptide model appears to explain at least partly the membrane destabilization properties of HIV fusion peptide.
Subject(s)
HIV Envelope Protein gp41/chemistry , HIV-1/chemistry , Membrane Lipids/chemistry , Models, Molecular , T-Lymphocytes/chemistry , Algorithms , Amino Acid Motifs , Cell Membrane , Cell Membrane Permeability , Computer Simulation , HIV Envelope Protein gp41/physiology , HIV-1/physiology , Liposomes/chemistry , Peptides/physiology , T-Lymphocytes/physiologyABSTRACT
The Chameleon peptide (Cham) is a peptide designed from two regions of the GB1 protein, one folded as an alpha-helix and the other as a beta structure. Depending on the environment, the Cham peptide adopts an alpha or a beta conformation when inserted in different locations of GB1. This environment dependence is also observed for tilted peptides. These short protein fragments, able to destabilise organised system, are mainly folded in beta structure in water and in alpha helix in a hydrophobic environment, like the lipid bilayer. In this paper, we tested whether the Cham peptide can be qualified as a tilted peptide. For this, we have compared the properties of Cham peptide (hydrophobicity, destabilising properties, conformation) to those of tilted peptides. The results suggest that Cham is a tilted peptide. Our study, together the presence of tilted fragments in transconformational proteins, suggests a relationship between tilted peptides and structural lability.
Subject(s)
Lipid Bilayers/chemistry , Lipid Metabolism , Lipids/chemistry , Peptides/chemistry , Proteins/chemistry , Hydrophobic and Hydrophilic Interactions , Liposomes/chemistry , Molecular Conformation , Time FactorsABSTRACT
BACKGROUND: Interleukin-12 (IL-12) has been identified as a key inducer of a type 1 T-helper cell cytokine pattern, which is thought to contribute to the development of atherosclerosis. We sought to study the role of IL-12 in atherosclerosis by inhibition of IL-12 using a newly developed vaccination technique that fully blocks the action of IL-12. METHODS AND RESULTS: LDL receptor-deficient (LDLr(-/-)) mice were vaccinated against IL-12 by 5 intramuscular injections of IL-12-PADRE complex in combination with adjuvant oil-in-water emulsion (low dose)/MPL/QS21 every 2 weeks. Two weeks thereafter, atherogenesis was initiated in the carotid artery by perivascular placement of silicone elastomer collars. IL-12 vaccination resulted in the induction of anti-IL-12 antibodies that functionally blocked the action of IL-12 as determined in an IL-12 bioassay. Blockade of IL-12 by vaccination of LDLr(-/-) mice resulted in significantly reduced (68.5%; P<0.01) atherogenesis compared with control mice without a change in serum cholesterol levels. IL-12 vaccination also resulted in a significant decrease in intima/media ratios (66.7%; P<0.01) and in the degree of stenosis (57.8%; P<0.01). On IL-12 vaccination, smooth muscle cell and collagen content in the neointima increased 2.8-fold (P<0.01) and 4.2-fold (P<0.01), respectively. CONCLUSIONS: Functional blockade of endogenous IL-12 by vaccination resulted in a significant 68.5% reduction in atherogenesis in LDLr(-/-) mice. Vaccination against IL-12 also improved plaque stability, from which we conclude that the blockade of IL-12 by vaccination may be considered a promising new strategy in the treatment of atherosclerosis.
Subject(s)
Carotid Artery Diseases/immunology , Interleukin-12/antagonists & inhibitors , Vaccines/therapeutic use , Animals , Autoantibodies/therapeutic use , Biological Availability , Carotid Artery Diseases/surgery , Disease Models, Animal , Epitopes/immunology , Epitopes/therapeutic use , Humans , Interferon-gamma/blood , Interleukin-12/blood , MiceABSTRACT
"Cancer germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. We report the identification of a new MAGE-1-encoded peptide that is recognized by a cytolytic T-lymphocyte (CTL) clone on human leukocyte antigen (HLA)-B*5701. The sequence of the peptide, corresponding to position 102-112 of the MAGE-1 protein sequence, is ITKKVADLVGF. When tumor cells expressing MAGE-1 were transfected with HLA-B*5701, they were lyzed by the CTL clone, indicating that the peptide is processed in tumor cells and can, therefore, be used as a target for anti-tumoral vaccination.
Subject(s)
Antigens, Neoplasm/immunology , HLA-B Antigens/immunology , Neoplasm Proteins/immunology , Peptides/immunology , T-Lymphocytes, Cytotoxic/immunology , Antigens, Neoplasm/genetics , Base Sequence , Cell Line, Tumor , Cell Transformation, Viral , Herpesvirus 4, Human , Humans , Melanoma-Specific Antigens , Neoplasm Proteins/geneticsABSTRACT
The lipid-destabilizing properties of the N-terminal domain of the GP2 of Ebola virus were investigated. Our results suggest that the domain of Ebola virus needed for fusion is shorter than that previously reported. The fusogenic properties of this domain are related to its oblique orientation at the lipid/water interface owing to an asymmetric distribution of the hydrophobic residues when helical.
Subject(s)
Ebolavirus/pathogenicity , Membrane Fusion , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Amino Acids/chemistry , Animals , Humans , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers , Liposomes/chemistry , Membranes/chemistry , Models, Molecular , Peptides/chemistryABSTRACT
'Cancer-germline' genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens, which have been used in therapeutic vaccination trials of cancer patients. MAGE-A4 is expressed in more than 50% of carcinomas of esophagus, head and neck, lung, and bladder. We report here the identification of a new MAGE-A4 encoded peptide, which is recognized by a cytolytic T lymphocyte (CTL) clone on HLA-B*3701. The sequence of the peptide is SESLKMIF. It corresponds to the MAGE-A4156-163 protein sequence. When tumor cells expressing MAGE-A4 were transfected with HLA-B*3701, they were recognized by the CTL clone, demonstrating that the peptide ought to be processed in tumor cells and could therefore serve as a target for therapeutic antitumoral vaccination.
Subject(s)
Antigen Presentation , Antigens, Neoplasm/immunology , HLA-B Antigens/immunology , Neoplasm Proteins , Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , Antigen Presentation/immunology , Cells, Cultured , Dendritic Cells/immunology , HLA-B37 Antigen , HumansABSTRACT
Recombinant cofactor-independent phosphoglycerate mutase from Trypanosoma brucei was inactivated by EDTA, and reactivated by Co(2+) much more than by Mn(2+) or Fe(2+). It displayed a minor phosphoglycerate phosphatase activity, which was stimulated by Mn(2+) more than by Co(2+). Upon incubation with [(32)P]phosphoglycerate, radioactivity was incorporated into the enzyme, most particularly in the presence of Mn(2+) or Fe(2+). The phosphorylated residue was identified by tandem mass spectrometry as Ser74, a residue homologous to the phosphorylated serine in alkaline phosphatase. However, the rates of formation and of disappearance of this phosphoenzyme were quite low compared to the mutase reaction. This and other properties indicated that the observed phosphoenzyme is an intermediate in the minor phosphatase activity rather than in the phosphomutase reaction.
Subject(s)
Phosphoglycerate Mutase/metabolism , Trypanosoma brucei brucei/enzymology , 2,3-Diphosphoglycerate/metabolism , Animals , Cations, Divalent/metabolism , Phosphorylation , Phosphoserine/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Trypanosoma brucei brucei/geneticsABSTRACT
The heterotrimeric complex of the human major histocompatibity complex (MHC) molecule HLA-A*0201, beta2-microglobulin and the decameric peptide GVYDGREHTV derived from the melanoma antigen (MAGE-A4 protein has been determined by X-ray crystallography at 1.4 A resolution. MAGE-A4 belongs to a family of genes that are specifically expressed in a variety of tumours. MAGE-A4-derived peptides are presented by MHC molecules at the cell surface to cytotoxic T-lymphocytes. As the HLA-A*0201:MAGE-A4 complex occurs only on tumour cells, it is considered to be an appropriate target for immunotherapy. The structure presented here reveals potential epitopes specific to the complex and indicates which peptide residues could be recognised by T-cell receptors. In addition, as the structure could be refined anisotropically, it was possible to describe the movements of the bound peptide in more detail.
Subject(s)
Antigens, Neoplasm/chemistry , Antigens, Neoplasm/metabolism , HLA-A Antigens/chemistry , HLA-A Antigens/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Amino Acid Sequence , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Binding Sites , Circular Dichroism , Crystallography, X-Ray , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , HLA-A Antigens/immunology , Humans , Immunotherapy , Ligands , Models, Molecular , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Peptide Fragments/immunology , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Protein Binding , Protein Conformation , Protein Denaturation , Receptors, Antigen, T-Cell/immunology , Temperature , Thermodynamics , beta 2-Microglobulin/chemistry , beta 2-Microglobulin/metabolismABSTRACT
We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.
Subject(s)
Antigens, Neoplasm/genetics , Lymphocytes, Tumor-Infiltrating/immunology , Macrophage Colony-Stimulating Factor/genetics , Open Reading Frames , Peptides/genetics , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Antigens, Neoplasm/immunology , Base Sequence , Carcinoma, Renal Cell/immunology , Cytotoxicity, Immunologic , HLA-B35 Antigen , Humans , Kidney Neoplasms , Macrophage Colony-Stimulating Factor/immunology , Molecular Sequence Data , Peptides/immunology , Protein BiosynthesisABSTRACT
Intact human erythrocytes catalyse the conversion of fructose into fructose 3-phosphate with an apparent K(m) of 30 mM [Petersen, Kappler, Szwergold and Brown (1992) Biochem. J. 284, 363-366]. The physiological significance of this process is still unknown. In the present study we report that the formation of fructose 3-phosphate from 50 mM fructose in intact erythrocytes is inhibited by 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine, with an apparent K(i) of 100 microM. (31)P NMR analysis of cell extracts incubated with DMF indicated the presence of an additional phosphorylated compound, which was partially purified and shown to be DMF 3-phosphate by tandem MS. Radiolabelled DMF was phosphorylated by intact erythrocytes with an apparent K(m) ( approximately 100 microM) approx. 300-fold lower than the value reported for fructose phosphorylation on its third carbon. These results indicate that the physiological function of the enzyme that is able to convert fructose into fructose 3-phosphate in intact erythrocytes is probably to phosphorylate fructosamines. This suggests that fructosamines, which are produced non-enzymically from glucose and amino compounds, may be metabolized in human erythrocytes.
Subject(s)
Erythrocytes/enzymology , Fructosamine/analogs & derivatives , Fructosamine/metabolism , Fructose/analogs & derivatives , Fructose/metabolism , Morpholines/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Erythrocytes/drug effects , Erythrocytes/metabolism , Fructosamine/chemical synthesis , Fructosamine/pharmacology , Fructose/chemical synthesis , Fructose/pharmacology , Fructosephosphates/metabolism , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Morpholines/chemical synthesis , Morpholines/pharmacology , Phosphorylation/drug effects , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Substrate SpecificityABSTRACT
Fructosamines are thought to play an important role in the development of diabetic complications. Little is known about reactions that could metabolize these compounds in mammalian tissues, except for recent indications that they can be converted to fructosamine 3-phosphates. The purpose of the present work was to identify and characterize the enzyme responsible for this conversion. Erythrocyte extracts were found to catalyze the ATP-dependent phosphorylation of 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine. The enzyme responsible for this conversion was purified approximately 2,500-fold by chromatography on Blue Sepharose, Q Sepharose, and Sephacryl S-200 and shown to copurify with a 35,000-M(r) protein. Partial sequences of tryptic peptides were derived from the protein by nanoelectrospray-ionization mass spectrometry, which allowed for the identification of the corresponding human and mouse cDNAs. Both cDNAs encode proteins of 309 amino acids, showing 89% identity with each other and homologous to proteins of unknown function predicted from the sequences of several bacterial genomes. Both proteins were expressed in Escherichia coli and purified. They were shown to catalyze the phosphorylation of DMF, fructoselysine, fructoseglycine, and fructose in order of decreasing affinity. They also phosphorylated glycated lysozyme, though not unmodified lysozyme. Nuclear magnetic resonance analysis of phosphorylated DMF and phosphorylated fructoseglycine showed that the phosphate was bound to the third carbon of the 1-deoxyfructose moiety. The physiological function of fructosamine-3-kinase may be to initiate a process leading to the deglycation of fructoselysine and of glycated proteins.
Subject(s)
Cloning, Molecular , Fructose/analogs & derivatives , Gene Expression , Phosphotransferases (Alcohol Group Acceptor)/blood , Phosphotransferases (Alcohol Group Acceptor)/genetics , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Chromatography , DNA, Complementary/chemistry , Electrophoresis, Polyacrylamide Gel , Erythrocytes/enzymology , Fructose/metabolism , Humans , Magnetic Resonance Spectroscopy , Mice , Molecular Sequence Data , Morpholines/metabolism , Phosphorylation , Sequence Alignment , TransfectionABSTRACT
We obtained a lytic CD4 T-cell clone that recognized an antigen presented by HLA-DRB1*1101 on the tumor cells of a melanoma patient who enjoyed an unusually favorable clinical evolution. The antigen appeared to be shared between several melanoma cell lines. To identify the encoding gene, we used a new method, based on the cotransfection into human embryonal kidney cell line 293 of a cDNA library from the tumor together with a cDNA clone encoding the class II transactivator, which induces the expression of HLA class II molecules. The product of the gene coding for the antigenic peptide is EphA3, a member of the Eph family of tyrosine kinase receptors, which mediate the repulsion of neural cells by cells carrying the ligand Ephrins on their surface. EphA3 is expressed at a high level in the retina and fetal brain, at a lower level in several normal tissues, and not at all in hematopoietic cells, the only cells that constitutively express HLA class II molecules. It is overexpressed in several types of tumors, including melanoma, lung carcinoma, and sarcoma. On the basis of this pattern of expression, EphA3 may be a source of tumor-specific antigens recognized on tumor cells that express HLA class II molecules. Anti-EphA3 T cells may have participated in a tumor rejection response in the patient, because the cells of metastases collected several years later than the metastasis used to characterize the antigen had lost expression of HLA-DR or EphA3, therefore escaping recognition by these lymphocytes.
Subject(s)
Antigen Presentation/immunology , Antigens, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , HLA-DR Antigens/immunology , Nuclear Proteins , Receptor Protein-Tyrosine Kinases/immunology , Amino Acid Sequence , Base Sequence , Clone Cells , DNA, Complementary/genetics , Epitopes, T-Lymphocyte/immunology , Gene Expression , HLA-DR Serological Subtypes , Humans , Melanoma/immunology , Molecular Sequence Data , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Receptor, EphA3 , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators/genetics , Trans-Activators/immunology , Transfection , Tumor Cells, CulturedABSTRACT
A CTL clone that recognizes autologous tumor cells was previously isolated from the blood of a head-and-neck cancer patient. The Ag was identified as peptide FPSDSWCYF presented by autologous HLA-B*3503 molecules. This peptide was encoded by a mutated CASP-8 gene, which is implicated in the triggering of apoptosis. Here, we show that this CTL clone, which expresses a single TCR, also recognizes two unrelated peptides on allogeneic HLA-B*3501 molecules. One peptide, HIPDVITY, is encoded by squalene synthase, and the other one, QFADVIVLF, is encoded by 2-hydroxyphytanoyl-CoA lyase. Both genes are expressed ubiquitously. These antigenic peptides are processed and presented by HLA-B*3501 cells. The two HLA-B35 alleles are closely related. Our results might reinforce the notion that the recognition of allogeneic HLA molecules depends on the presence in their groove of a limited number of peptides processed from ubiquitous proteins.
Subject(s)
Antigen Presentation , Caspases/metabolism , HLA-B35 Antigen/metabolism , Oligopeptides/metabolism , T-Lymphocytes, Cytotoxic/enzymology , T-Lymphocytes, Cytotoxic/metabolism , Aged , Alleles , Amino Acid Sequence , Animals , Antigen Presentation/genetics , B-Lymphocytes/metabolism , Base Sequence , COS Cells , Caspase 8 , Caspase 9 , Caspases/immunology , Cell Line , Clone Cells , DNA, Complementary/analysis , Farnesyl-Diphosphate Farnesyltransferase/genetics , Female , HLA-B35 Antigen/biosynthesis , HLA-B35 Antigen/genetics , Humans , Lymphocyte Activation , Molecular Sequence Data , Oligopeptides/genetics , Oligopeptides/immunology , Protein Binding/immunology , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes, Cytotoxic/immunology , Transfection , Tumor Cells, CulturedABSTRACT
Phosphoserine phosphatase belongs to a new class of phosphotransferases forming an acylphosphate during catalysis and sharing three motifs with P-type ATPases and haloacid dehalogenases. The phosphorylated residue was identified as the first aspartate in the first motif (DXDXT) by mass spectrometry analysis of peptides derived from the phosphorylated enzyme treated with NaBH(4) or alkaline [(18)O]H(2)O. Incubation of native phosphoserine phosphatase with phosphoserine in [(18)O]H(2)O did not result in (18)O incorporation in residue Asp-20, indicating that the phosphoaspartate is hydrolyzed, as in P-type ATPases, by attack of the phosphorus atom. Mutagenesis studies bearing on conserved residues indicated that four conservative changes either did not affect (S109T) or caused a moderate decrease in activity (G178A, D179E, and D183E). Other mutations inactivated the enzyme by >80% (S109A and G180A) or even by >/=99% (D179N, D183N, K158A, and K158R). Mutations G178A and D179N decreased the affinity for phosphoserine, suggesting that these residues participate in the binding of the substrate. Mutations of Asp-179 decreased the affinity for Mg(2+), indicating that this residue interacts with the cation. Thus, investigated residues appear to play an important role in the reaction mechanism of phosphoserine phosphatase, as is known for equivalent residues in P-type ATPases and haloacid dehalogenases.
Subject(s)
Adenosine Triphosphatases/metabolism , Phosphoric Monoester Hydrolases/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Amino Acid Substitution , Aspartic Acid/metabolism , Borohydrides/pharmacology , Catalysis , Humans , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Mass Spectrometry/methods , Mutagenesis, Site-Directed , Oxidation-Reduction/drug effects , Oxygen Isotopes , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Trypsin/metabolism , Water/metabolismABSTRACT
MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.
Subject(s)
Dendritic Cells/immunology , Epitopes, T-Lymphocyte/metabolism , Lymphocyte Activation , Neoplasm Proteins/metabolism , T-Lymphocytes, Cytotoxic/immunology , Transfection/immunology , Amino Acid Sequence , Animals , Antigen Presentation/genetics , Antigens, Neoplasm , Avipoxvirus/genetics , Avipoxvirus/immunology , Cells, Cultured , Clone Cells , Coculture Techniques , Cytotoxicity, Immunologic , Dendritic Cells/metabolism , Dendritic Cells/virology , Epitopes, T-Lymphocyte/immunology , HLA Antigens/immunology , HLA Antigens/metabolism , HLA-A Antigens/immunology , HLA-A Antigens/metabolism , HLA-C Antigens/immunology , HLA-C Antigens/metabolism , Humans , Lymphocyte Activation/genetics , Lymphocyte Culture Test, Mixed , Melanoma-Specific Antigens , Mice , Molecular Sequence Data , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Peptides/immunology , Peptides/metabolism , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
Previously undescribed medium-chain acylcarnitines were identified in a urine sample from a patient with medium-chain acyl-CoA dehydrogenase deficiency. These are the 4-methylvaleryl, 4- and 5-methylhexanoyl, 6-methylheptanoyl-, 6-methyloctanoyl-, 4,5-dimethylhexanoyl- and 4,7-decadienoylcarnitines. Their chemical structures were obtained by gas chromatographymass spectrometry analysis of their fatty acid moieties as picolinyl esters.
Subject(s)
Acyl-CoA Dehydrogenases/deficiency , Carnitine/analogs & derivatives , Acyl-CoA Dehydrogenase , Carnitine/urine , Child, Preschool , Female , Glucuronates/analysis , Glycine/analysis , HumansABSTRACT
MAGE-type genes are expressed by many tumors of different histological types and not by normal cells, except for male germline cells, which do not express major histocompatibility complex (MHC) molecules. Therefore, the antigens encoded by MAGE-type genes are strictly tumor specific and common to many tumors. We describe here the identification of the first MAGE-encoded epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules to CD4(+) T lymphocytes. Monocyte-derived dendritic cells were loaded with a MAGE-3 recombinant protein and used to stimulate autologous CD4(+) T cells. We isolated CD4(+) T cell clones that recognized two different MAGE-3 epitopes, MAGE-3114-127 and MAGE-3121-134, both presented by the HLA-DR13 molecule, which is expressed in 20% of Caucasians. The second epitope is also encoded by MAGE-1, -2, and -6. Our procedure should be applicable to other proteins for the identification of new tumor-specific antigens presented by HLA class II molecules. The knowledge of such antigens will be useful for evaluation of the immune response of cancer patients immunized with proteins or with recombinant viruses carrying entire genes coding for tumor antigens. The use of antigenic peptides presented by class II in addition to peptides presented by class I may also improve the efficacy of therapeutic antitumor vaccination.
Subject(s)
Antigen Presentation , Antigens, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , HLA-DR Antigens/immunology , Melanoma/immunology , Neoplasm Proteins/immunology , Amino Acid Sequence , Clone Cells , Coculture Techniques , Dendritic Cells/immunology , Epitopes/immunology , HLA-DR Serological Subtypes , Humans , Male , Molecular Sequence Data , Peptides/immunologySubject(s)
Adenosine Triphosphatases/chemistry , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases/chemistry , Adenosine Triphosphatases/metabolism , Phosphoric Monoester Hydrolases/chemistry , Phosphotransferases/metabolism , Phosphotransferases (Phosphomutases)/chemistry , Phosphotransferases (Phosphomutases)/metabolismABSTRACT
When incubated with their substrates, human phosphomannomutase and L-3-phosphoserine phosphatase are known to form phosphoenzymes with chemical characteristics of an acyl-phosphate. The phosphorylated residue in phosphomannomutase has now been identified by mass spectrometry after reduction of the phosphoenzyme with tritiated borohydride and trypsin digestion. It is the first aspartate in a conserved DVDGT motif. Replacement of either aspartate of this motif by asparagine or glutamate resulted in complete inactivation of the enzyme. The same mutations performed in the DXDST motif of L-3-phosphoserine phosphatase also resulted in complete inactivation of the enzyme, except for the replacement of the second aspartate by glutamate, which reduced the activity by only about 40%. This suggests that the first aspartate of the motif is also the phosphorylated residue in L-3-phosphoserine phosphatase. Data banks contained seven other phosphomutases or phosphatases sharing a similar, totally conserved DXDX(T/V) motif at their amino terminus. One of these (beta-phosphoglucomutase) is shown to form a phosphoenzyme with the characteristics of an acyl-phosphate. In conclusion, phosphomannomutase and L-3-phosphoserine phosphatase belong to a new phosphotransferase family with an amino-terminal DXDX(T/V) motif that serves as an intermediate phosphoryl acceptor.