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.

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 Mrs of 65,000 and 59,000, respectively. These are approximately equal to 2,000 greater than the Mrs 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 two patients with Sandhoff disease did not direct the translation of immunoprecipitable beta chain; RNA of fibroblast cultures from four patients with Tay-Sachs disease (three of Ashkenazi Jewish descent and one of non-Jewish descent) did not direct the translation of immunoprecipitable alpha chain. In contrast, 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--lysosomes and the exterior of the cell.