Jean-Pierre Szikora

Structure, Chromosomal Localization, and Expression of 12 Genes of the Mage Family

We reported previously that human geneMAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with aMAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12MAGE genes are located in the q terminal region of chromosome X. LikeMAGE-1, the 11 additionalMAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that ofMAGE-1. The coding sequences of theMAGE genes predict the same main structural features for allMAGE proteins. In contrast, the promoters and first exons of the12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of eachMAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of theMAGE family includingMAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

Plant-like traits associated with metabolism of Trypanosoma parasites.

Trypanosomatid parasites cause serious diseases among humans, livestock, and plants. They belong to the order of the Kinetoplastida and form, together with the Euglenida, the phylum Euglenozoa. Euglenoid algae possess plastids capable of photosynthesis, but plastids are unknown in trypanosomatids. Here we present molecular evidence that trypanosomatids possessed a plastid at some point in their evolutionary history. Extant trypanosomatid parasites, such as Trypanosoma and Leishmania, contain several “plant-like” genes encoding homologs of proteins found in either chloroplasts or the cytosol of plants and algae. The data suggest that kinetoplastids and euglenoids acquired plastids by endosymbiosis before their divergence and that the former lineage subsequently lost the organelle but retained numerous genes. Several of the proteins encoded by these genes are now, in the parasites, found inside highly specialized peroxisomes, called glycosomes, absent from all other eukaryotes, including euglenoids.

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma.

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented byHLA-B7 and is encoded by a new gene that we have namedRAGE1. No expression ofRAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.

Structure of the Gene of Tum- Transplantation Antigen-p35b - Presence of a Point Mutation in the Antigenic Allele

Mutagen treatment of P815 tumour cells produces tum‐ variants that are rejected by syngeneic mice because they express new transplantation antigens. These ‘tum‐’ antigens elicit a cytolytic T lymphocyte (CTL) response but no detectable antibody response. The DNA of tum‐ variant P35 was transfected into P815 cell line P1.HTR. Transfectants expressing tum‐ antigen P35B were identified on the basis of their ability to stimulate anti‐P35B CTL. This was repeated with a cosmid library and a cosmid carrying the sequence encoding antigen P35B was recovered from a transfectant expressing the antigen. Gene P35B is 6 kb long and contains 11 exons. The sequence shows no homology with the previously identified tum‐ gene P91A nor with any gene presently recorded in the data banks. The antigenic allele of gene P35B differs from the normal allele by a point mutation located in exon 5. This mutation, which replaces a Ser by an Asn residue, was shown by site‐directed mutagenesis to be responsible for the expression of the antigen. A synthetic decapeptide covering the sequence surrounding the tum‐ mutation rendered P815 cells sensitive to lysis by anti‐P35B CTL. Surprisingly, the homologous peptide corresponding to the normal sequence of the gene had the same effect, indicating that this tum‐ mutation does not exert its effect by generating the aggretope or the epitope of the antigenic peptide. As observed previously with gene P91A, we found that fragments of gene P35B containing only exons 4 and 5, which were cloned in non‐expression vectors, transferred efficiently the expression of the antigen.

INVOLVEMENT OF TWO ETS BINDING SITES IN THE TRANSCRIPTIONAL ACTIVATION OF THE MAGE1 GENE

The MAGE1 gene codes for an antigen recognized on melanoma cell line MZ2-MEL by autologous cytolytic T lymphocytes. It is expressed in a number of tumors of different histological origins, but not in normal tissues except in testis. The MAGE1 promoter region was analyzed by performing transient transfections in MZ2-MEL cells with luciferase reporter plasmids. A fragment extending from nucleotide −792 to +118 exhibited high transcriptional activity. By deletional analysis of this fragment, we identified five activating regions designated C, A, B′, B, and D. The activity of region A depends on the presence of region B′ and vice versa. Two inverted Ets motifs contained in regions B′ and B were found to drive 90% of the activity of the MAGE1 promoter in MZ2-MEL cells. Electrophoretic mobility shift assays performed with a nuclear extract from MZ2-MEL cells and with competitor oligonucleotides containing an Ets consensus site showed that nuclear proteins bind to the Ets motif of regions B′ and B. Similar experiments suggested that region A binds transcription factors of the Sp1 family. The MAGE1 promoter was found to exert transcriptional activity in tumor cells where the MAGE1 gene is not expressed, suggesting that other mechanisms, such as demethylation, may contribute to the tumor-specific expression of the gene.

Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei

Label-free semi-quantitative differential three-dimensional liquid chromatography coupled to mass spectrometry (3D-LC-MS/MS) was used to compare the glycosomal and mitochondrial proteomes of the bloodstream- and insect-form of Trypanosoma brucei. The abundance of glycosomal marker proteins identified in the two life-cycle stages corresponded well with the relative importance of biochemical pathways present in the glycosomes of the two stages and the peptide spectral count ratios of selected enzymes were in good agreement with published data about their enzymatic specific activities. This approach proved extremely useful for the generation of large scale proteomics data for the comparison of different life-cycle stages. Several proteins involved in oxidative stress protection, sugar-nucleotide synthesis, purine salvage, nucleotide-monophosphate formation and purine-nucleotide cycle were identified as glycosomal proteins.

The disulphide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner

In Escherichia coli, DsbA introduces disulphide bonds into secreted proteins. DsbA is recycled by DsbB, which generates disulphides from quinone reduction. DsbA is not known to have any proofreading activity and can form incorrect disulphides in proteins with multiple cysteines. These incorrect disulphides are thought to be corrected by a protein disulphide isomerase, DsbC, which is kept in the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway is considered to be isolated from the DsbA/DsbB pathway. We show that the DsbC and DsbA pathways are more intimately connected than previously thought. dsbA‐dsbC‐ mutants have a number of phenotypes not exhibited by either dsbA‐, dsbC‐ or dsbA‐dsbD‐ mutations: they exhibit an increased permeability of the outer membrane, are resistant to the lambdoid phage Φ80, and are unable to assemble the maltoporin LamB. Using differential two‐dimensional liquid chromatographic tandem mass spectrometry/mass spectrometry analysis, we estimated the abundance of about 130 secreted proteins in various dsb‐ strains. dsbA‐dsbC‐ mutants exhibit unique changes at the protein level that are not exhibited by dsbA‐dsbD‐ mutants. Our data indicate that DsbC can assist DsbA in a DsbD‐independent manner to oxidatively fold envelope proteins. The view that DsbCs function is limited to the disulphide isomerization pathway should therefore be reinterpreted.

Efficient expression of tum− antigen P91A by transfected subgenic fragments

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum− antigens) recognized by cytolytic T cells. The gene encoding tum− antigen P91A comprises 12 exons and a mutation located in exon 4 is responsible for the production of a new antigenic peptide. Transfection experiments showed that the expression of the antigen could be transferred not only by the entire gene but also by gene segments comprising only the mutated exon and parts of the surrounding introns. This was observed with subgenic regions that were not cloned in expression vectors. Antigen expression did not require the integration of the transfected gene segment into a resident P91A gene by homologous recombination. It also occurred when the subgenic segment was transfected without the usual selective gene, which comprises an eucaryotic promoter, and also without plasmid sequences, which are known to contain weak promoters. When a stop codon was introduced at the beginning of exon 4, the expression of the antigen was maintained and evidence was obtained that an ATG codon located in this region served as initiation site for the translation of the antigenic peptide. But we have not obtained evidence indicating that antigenic peptides are direct translation products rather than degradation products of entire proteins.

Tum- mutation P35B generates the MHC-binding site of a new antigenic peptide.

Immunogenic tumor cell variant P35 was obtained by mutagen treatment of mouse mastocytoma P815. It express a potent new antigen recognized by syngeneic cytolytic T lymphocytes (CTL). This antigen is the result of a point mutation in a gene that is expressed by most healthy cells. A decapeptide encoded by the region spanning the mutation sensitized P815 cells to the relevant CTL, whereas the homologous decapeptide corresponding to the normal sequence did not. Only the mutant decapeptide was capable of enhancing the expression of the Dd-presenting molecule at the cell surface, indicating that the mutation generates a motif which enables the antigenic peptide to bind to Dd.