Identification and characterization of the tumor-specific P1A gene product.
In murine mastocytoma P815, gene P1A directs the expression of antigens P815A and B which are the target of a T cell-mediated rejection response in syngeneic animals. This gene is expressed at a high level in various tumors, but is silent in normal tissues except testis and placenta; its activation is thus possibly related to malignant transformation. An anti-synthetic peptide rabbit antiserum reacted by immunoblotting with a cellular protein migrating near 40 kDa on SDS-PAGE. The immunoreactive protein was detected only in lysates from cells which express antigen P815A: P1.HTR mastocytoma cells and, after transfection with cosmids carrying the P1A gene, the antigen-loss variant P0.HTR cells and DAP-3 H-2Ld fibroblasts. The identity of this protein as the P1A gene product was confirmed by cell-free transcription-translation of the P1A cDNA, the product of which also migrated near 40 kDa in SDS-PAGE and was captured by protein A-Sepharose in the presence of the antiserum. Subcellular fractionation by differential and isopycnic centrifugation indicated that the P1A protein is associated with cytoplasmic membranes demonstrating a broad distribution with respect to size and density. Immunofluorescence microscopy also revealed a cytoplasmic signal, particularly intense in small vesicles, which coincides with that produced by an anti-mouse type I collagen guinea pig antiserum except near the cell periphery where the P1A signal is weaker. We conclude that the P1A protein is bound to membranes of the secretory pathway, at a concentration which goes increasing from the endoplasmic reticulum to secretion vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Amar-Costesec
A
A
International Institute of Cellular and Molecular Pathology, Université Catholique de Louvain, Brussels, Belgium.
Godelaine
D
D
Van den Eynde
B
B
Beaufay
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D012515:Mast-Cell Sarcoma / Q000276:immunology*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; C076546:tumor rejection antigen P815A, mouse