Effects of deoxymannojirimycin and castanospermine on the polarized secretion of thyroglobulin

In order to better explore a possible role of oligosaccharide structures in the polarized secretion of thyroglobulin (Tg), we labeled cultured porcine thyroid cells with L-[3,4,5-3H]leucine or D-[2-3H]mannose in the presence or absence of the following inhibitors of N-linked oligosaccharide processing: deoxymannojirimycin (dMM), an inhibitor of mannosidase I expected to give rise to high mannose units exclusively, thus abolishing the formation of lactosaminyl branches, and castanospermine (Cs), an inhibitor of glucosidases which is expected to produce glucosylated high mannose oligosaccharides. [3H]Leucine pulse-chase experiments were performed to study the rate of Tg secretion in the presence and absence of dMM (1 mM) or Cs (1.6 mM). After a 15-h chase period, dMM and Cs did not modify the relative proportions of released Tg (65% in the follicular content, 16% in the medium) and cellular Tg (19%). In contrast, Cs led to a lower rate of secretion in both secretory pathways (t1/2 increased from 42 min to 105 min in the apical pathway and from 108 min to 138 min in the basal pathway). After a long-term labeling (16 h) with [3H]leucine or [3H]mannose, drugs did not notably affect the relative proportions of labeled Tg in the different compartments (follicular content, medium, and cell). The structures of N-glycans borne by Tg, with and without drugs, were checked. After Tg immunoprecipitation, pronase-glycopeptides were fractionated on concanavalin A-Sepharose 4B. The glycopeptides tightly bound to the lectin were treated by endo-beta-N-acetylglucosaminidase H and oligosaccharides separated by HPLC. With dMM, complex-type glycans of Tg were totally replaced by high mannose-type glycans, Man8-9GlcNAc; Cs induced the accumulation of glucosylated high mannose-type structures, Glc3Man7-9GlcNAc and Glc2Man8-9GlcNAc, but the action of this inhibitor was not total. In conclusion the correct secretion of Tg does not require the presence of the sialyllactosaminyl structure; the presence of glucose residues on high mannose-type structures lowers the rate of exocytosis.