RESUMEN
The serological analysis of antigens by recombinant expression cloning (SEREX) has identified a multitude of new tumor antigens in many different tumor entities. These antigens can be grouped into different classes according to their specificities, with cancer/testis antigens appearing to be the most attractive candidates for vaccine development. The observation that CD8 and CD4 T-cell responses against cancer/testis antigens such as NY-ESO-1 correlate with the presence of specific antibodies demonstrates the importance of serological monitoring patients participating in vaccine trials. However, all serological assays available (Western blot, phage display and ELISA) are hampered by the fact that the protein cannot be analyzed in its natural conformation. We have thus developed a yeast display system where the antigen is expressed on the yeast surface (RAYS), allowing for a more natural folding of the protein. To validate this approach we displayed the A33 colorectal cancer antigen on the yeast cell surface and demonstrated specific binding by an A33 monoclonal antibody recognizing a conformation-dependent epitope on the A33 antigen. We then compared RAYS with the more commonly used ELISA and Western blot serological monitoring methods by analyzing 50 sera from cancer patients with known NY-ESO-1 antibody status and 10 sera from patients with unknown SSX2 antibody status in a blind fashion. RAYS appears at least equivalent to both ELISA and Western blotting for the monitoring of antibodies against NY-ESO-1 as regards specificity and sensitivity, while antibodies against SSX2 were detected more frequently by RAYS than by ELISA or phage display.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/genética , Proteínas de la Membrana , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antineoplásicos/biosíntesis , Anticuerpos Antineoplásicos/sangre , Anticuerpos Antineoplásicos/metabolismo , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Humanos , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/sangre , Biosíntesis de Proteínas , Proteínas/genética , Proteínas/inmunología , Proteínas Recombinantes/inmunología , Proteínas Represoras/biosíntesis , Proteínas Represoras/sangre , Saccharomyces cerevisiae/metabolismo , Neoplasias Testiculares/sangre , Neoplasias Testiculares/inmunología , Testículo/química , Transfección , Células Tumorales CultivadasRESUMEN
A yeast cell surface display technology was used for the isolation and characterization of tumor antigens recognised by autologous or allogeneic breast cancer serum. More than 100 clones recognized by patient serum were isolated using high-through-put fluorescence activated cell sorting. Combined serological and sequence analysis confirmed that a number of proteins known to be overexpressed in breast cancer tissue could be detected. A recently identified small breast epithelial mucin almost exclusively expressed in mammary gland tissue was isolated as a mutated protein variant. Subsequent serological analysis using the yeast expression system for the wild-type and mutant form showed a strong recognition by patient sera, whereas no significant recognition was observed for the respective prokaryotically expressed proteins. The small breast epithelial mucin is present to a large extent in a membrane bound format and might be used for tumor targeting strategies.
Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Superficie/inmunología , Neoplasias de la Mama/genética , ADN Complementario/genética , Mucinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/inmunología , Secuencia de Aminoácidos , Antígenos de Neoplasias/sangre , Antígenos de Neoplasias/inmunología , Secuencia de Bases , Neoplasias de la Mama/sangre , Neoplasias de la Mama/inmunología , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Biblioteca de Genes , Humanos , Glándulas Mamarias Humanas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mucinas/genética , Mucinas/inmunología , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Pruebas SerológicasRESUMEN
We examined the association of acetyl-CoA:alpha-glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4 degrees C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6 MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifugation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts.