Inhibition of N-linked oligosaccharide processing does not prevent the secretion of thyroglobulin. A study with swainsonine and deoxynojirimycin.
The effects of two drugs, swainsonine (SW) and deoxynojirimycin (dNM), on synthesis and export of thyroglobulin were studied in folliculized porcine thyroid cells cultured in a serum-free medium. These drugs were expected to alter N-linked glycans in thyroglobulin. Newly synthesized thyroglobulin labeled with [2-3H]mannose or [4,5-3H]leucine was obtained by immunoprecipitation from the follicular contents, culture media and cell extracts; the first two compartments, containing secreted thyroglobulin, were sometimes analyzed together. Leucine incorporation was not inhibited by SW and only slightly by dNM. In contrast dNM strongly decreased mannose incorporation (by up to 50-75% at 1-3 mM). However after 16-h mannose labelings, SW and/or dNM at 2.5 microM and 3 mM respectively did not significantly modify the relative proportions of radioactive thyroglobulin in the above-mentioned compartments. Pronase glycopeptides prepared from these thyroglobulins were examined with respect to behaviour on concanavalin-A-Sepharose and position on Bio-Gel P-4. Oligosaccharides released by endoglucosaminidase H and with high affinity for the lectin, i.e. high-mannose and certain hybrids, were further characterized by various exoglycosidase treatments. Thyroglobulin from control cells displayed complex and high-mannose glycans comparable in size and proportion to those attributed to tissue-extracted porcine thyroglobulin. After treatment with SW (an inhibitor of alpha-mannosidase II), complex glycans were almost totally replaced by sialylated hybrid glycans. In contrast to this nearly total suppression, dNM (an inhibitor of the trimming glucosidases) caused only a 30% decrease in labeling of complex units and an about 50% increase in high-mannose glycans, covered to some degree by glucose. Finally a [3H]leucine pulse-chase study was performed on thyroglobulin secretion in the absence or presence of both SW and dNM. Though a slowdown was detectable in the first few hours, this study revealed no change in the long-term export of thyroglobulin.
D017485:1-Deoxynojirimycin; D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000378:metabolism; D006596:Hexosaminidases / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis*; D011402:Pronase / Q000378:metabolism; D017026:Swainsonine; D013552:Swine; D013954:Thyroglobulin / Q000096:biosynthesis*; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism; D043323:alpha-Mannosidase
D000470:Alkaloids; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; D013954:Thyroglobulin; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D008361:Mannosidases; D043323:alpha-Mannosidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D011402:Pronase; D007930:Leucine; D005944:Glucosamine; D008358:Mannose; D017026:Swainsonine