SUBCELLULAR-LOCALIZATION OF METALLOTHIONEIN IIA IN HUMAN BLADDER-TUMOR CELLS USING A NOVEL EPITOPE-SPECIFIC ANTISERUM

Human metallothioneins (hMTs) are a family of highly homologous protei ns thought to be critical in cellular protection against toxins. The c omparative function of individual isoforms is, however, obscure. Antib odies to individual MT isoforms might help clarify this issue but thei r generation has been challenging. We now describe a strategy that has successfully produced an epitope-specific antiserum to a major human isoform, hMT IIA. The immunogen, a conjugated tridecyl amino acid synt hetic peptide unique to residues 8-20 in hMT IIA (AAGDSCTCAGSCK), yiel ded an antiserum (B2) that reacted specifically with hMT IIA in a conc entration- and titer-dependent manner and showed no reactivity with hu man or rabbit liver MT I or horse MTs. This antiserum recognized rabbi t liver MT II and, surprisingly, also reacted weakly with chicken MT. MT amino acid sequence comparisons and peptide blocking studies sugges ted aspartate-11 and threonine-14 are important antigenic determinants for B2. Using confocal immunofluorescence microscopy and B2 antiserum , we observed nuclear localization of hMT IIA with human bladder T24 t umor cells in exponential growth but more cytoplasmic localization at confluence. These results suggest the subcellular location of hMT IIA is a function of cell density in T24 bladder carcinoma cells. The gene ral approach of epitope-specific antibodies may be useful for the gene ration of antibodies to other MT isoforms and for studying the role of individual MT isoforms in biology and toxicology. (C) 1994 Academic P ress, Inc.