Humoral immune responses to cathepsin D and glucose-regulated protein 78 in ovarian cancer patients.
Many cancer patients develop tumor-reactive immune responses against antigens that are either expressed on the surface of tumor cells or released from them into the peripheral circulation. In this study, tumor-reactive immunoglobulins, present in the sera of ovarian cancer patients, were used to identify commonly recognized tumor-associated antigens on ovarian tumor cells. Western immunoblot analysis of cellular proteins, obtained from UL-1 ovarian tumor cell line, demonstrated several commonly recognized immunoreactive proteins. Two of these proteins (Mr 32,000 and 71,000) were selected for further investigation. Cellular proteins isolated from normal human ovarian epithelia, in a similar fashion, failed to exhibit corresponding immunoreactivity to these proteins. As an additional control, sera from normal (nontumor-bearing) individuals failed to identify these proteins on Western immunoblots. Furthermore, the absorption of the ovarian cancer patients' sera with normal ovarian epithelial tissue did not remove the reactivity of these two proteins. The Mr 32,000 and 71,000 proteins were subsequently purified by reverse-phase high-performance liquid chromatography, separated by SDS-PAGE, transferred to the polyvinylidene difluoride membrane, and digested with trypsin. These resulting tryptic fragments were separated by microbore reverse-phase high-performance liquid chromatography, and selected fragments were sequenced by mass spectrometry. This sequence analysis identified the Mr 32,000 protein as cathepsin D and the Mr 71,000 as glucose-regulated protein 78 (member of the heat shock protein family). The identities of cathepsin D and glucose-regulated protein 78 were confirmed by Western blot analysis. Additionally, the presence of cathepsin D was demonstrated in association with immune complexes in vivo. Currently, the common antigenic epitopes of these proteins are being defined.
Chinni
S R
SR
Departments of Obstetrics and Gynecology and Biochemistry, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA.
Falchetto
R
R
Gercel-Taylor
C
C
Shabanowitz
J
J
Hunt
D F
DF
Taylor
D D
DD
D002288:Adenocarcinoma, Mucinous / Q000097:blood / Q000276:immunology* / Q000473:pathology; D000231:Adenocarcinoma, Papillary / Q000097:blood / Q000276:immunology* / Q000473:pathology; D000368:Aged; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000097:blood; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000097:blood; D000951:Antigens, Neoplasm / Q000276:immunology*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000276:immunology* / Q000302:isolation & purification; D002402:Cathepsin D / Q000276:immunology* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D018292:Cystadenoma, Papillary / Q000097:blood / Q000276:immunology* / Q000473:pathology; D000091342:Endoplasmic Reticulum Chaperone BiP; D000939:Epitopes / Q000276:immunology; D005260:Female; D006360:Heat-Shock Proteins; D006801:Humans; D013058:Mass Spectrometry; D008875:Middle Aged; D018832:Molecular Chaperones / Q000276:immunology* / Q000302:isolation & purification; D008970:Molecular Weight; D010051:Ovarian Neoplasms / Q000097:blood / Q000276:immunology* / Q000473:pathology; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis; D014407:Tumor Cells, Cultured
D000912:Antibodies, Neoplasm; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D000939:Epitopes; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D002402:Cathepsin D