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抑制N-连接寡糖加工并不妨碍甲状腺球蛋白的分泌。一项关于苦马豆素和脱氧野尻霉素的研究。

Inhibition of N-linked oligosaccharide processing does not prevent the secretion of thyroglobulin. A study with swainsonine and deoxynojirimycin.

作者信息

Franc J L, Hovsepian S, Fayet G, Bouchilloux S

出版信息

Eur J Biochem. 1986 May 15;157(1):225-32. doi: 10.1111/j.1432-1033.1986.tb09660.x.

DOI:10.1111/j.1432-1033.1986.tb09660.x
PMID:2940087
Abstract

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.

摘要

在无血清培养基中培养的猪甲状腺滤泡细胞中,研究了两种药物,苦马豆素(SW)和脱氧野尻霉素(dNM)对甲状腺球蛋白合成和分泌的影响。预期这些药物会改变甲状腺球蛋白中的N-连接聚糖。通过免疫沉淀从滤泡内容物、培养基和细胞提取物中获得用[2-³H]甘露糖或[4,5-³H]亮氨酸标记的新合成甲状腺球蛋白;前两个含有分泌型甲状腺球蛋白的部分有时一起分析。SW不抑制亮氨酸掺入,dNM仅轻微抑制。相反,dNM强烈降低甘露糖掺入(在1-3 mM时降低高达50-75%)。然而,在进行16小时甘露糖标记后,分别为2.5 microM和3 mM的SW和/或dNM并未显著改变上述部分中放射性甲状腺球蛋白的相对比例。对从这些甲状腺球蛋白制备的链霉蛋白酶糖肽进行伴刀豆球蛋白A-琼脂糖行为和Bio-Gel P-4上位置的检测。通过各种外切糖苷酶处理进一步表征由内切葡糖胺酶H释放且对凝集素具有高亲和力的寡糖,即高甘露糖型和某些杂合型。对照细胞的甲状腺球蛋白显示出与组织提取的猪甲状腺球蛋白大小和比例相当的复合型和高甘露糖型聚糖。用SW(α-甘露糖苷酶II抑制剂)处理后,复合型聚糖几乎完全被唾液酸化杂合聚糖取代。与这种几乎完全抑制相反,dNM(修剪葡糖苷酶抑制剂)仅使复合型单元标记减少30%,高甘露糖型聚糖标记增加约50%,部分被葡萄糖覆盖。最后,在不存在或存在SW和dNM的情况下,对甲状腺球蛋白分泌进行了[³H]亮氨酸脉冲追踪研究。尽管在最初几个小时可检测到分泌放缓,但该研究表明甲状腺球蛋白的长期分泌没有变化。

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