Synthesis of beta-hexosaminidase in cell-free translation and in intact fibroblasts: an insoluble precursor alpha chain in a rare form of Tay-Sachs disease.

Abstract

RNA was isolated from human term placenta or cultured fibroblasts and translated in a rabbit reticulocyte system in the presence of [35S]methionine; the translation products were immunoprecipitated with antisera made against .beta.-hexosaminidase or its isolated .alpha. and .beta. chains and analyzed by polyacrylamide gel electrophoresis. The largest translated .alpha. and .beta. chain polypeptides had MW of 65,000 and 59,000, respectively. These are .apprx. 2,000 greater than the MW of precursor chains synthesized by intact fibroblasts and deglycosylated with endo-.beta.-N-acetylglucosaminidase H suggesting the presence of a signal sequence. RNA of fibroblast cultures from 2 patients with Sandhoff disease did not direct the translation of immunoprecipitable .beta. chain; RNA of fibroblast cultures from 4 patients with Tay-Sachs disease (3 of Ashkenazi Jewish descent and 1 of non-Jewish descent) did not direct the translation of immunoprecipitable .alpha. chain. A normal amount of .alpha. chain was made in the presence of RNA from the fibroblast culture of another non-Jewish Tay-Sachs patient (GM 1110). Intact fibroblasts from this patient also synthesized the .alpha. chain as shown by labeling with [3H]leucine; however, strong detergent was required for extraction. The .alpha. chain could be labeled with [3H]mannose but not with [32P]-phosphate; it was neither secreted nor accumulated in the proteolytically processed form, and it disappeared within a day of synthesis. A plausible though not unique explanation is that the insoluble .alpha. chain is not transported from the endoplasmic reticulum (the site of glycosylation) to the Golgi apparatus (the site of phosphorylation) nor to further points of destination.sbd.lysosomes and the exterior of the cell.