Fig 1: Perturbation of N-linked glycosylation distinctly impacts A2234D Tg.A, schematic detailing the N-glycosylation and lectin-mediated folding pathway. Glycosylation is carried out by two distinct OST complexes containing STT3A or STT3B as catalytic subunits. Glucosidases then trim terminal glucose residues, lectin chaperones (CANX, CALR), and PDIA3 assist in folding, followed by further glucose trimming. Subsequently, glucosyltransferases (UGGT1, UGGT2) serve as quality control sensors to re-glucosylate improperly folded proteins for iterative chaperoning cycles. B–E, interaction changes of Tg mutants compared with WT with individual N-glycosylation quality control factors that were identified as high-confidence interactors of Tg. Error bars show SEM. Student’s parametric t test with adjusted p values (Storey q values) was used to determine significant changes. *q < 0.05, **q < 0.005, ***q < 0.0005. B, lectin chaperones and lectin-associated protein disulfide isomerase PDIA3. C, glucosyltransferases sensing misfolded proteins. D, glucosidases involved in glycan trimming. E, catalytic subunits of the OST complex STT3A responsible for cotranslational glycosylation and STT3B responsible for posttranslational glycosylation. F, comparison of WT Tg secretion in parental, STT3A, or STT3B KO HEK293T cells. WT Tg was transiently transfected into the respective cells, and newly synthesized proteins were metabolically labeled for 30 min with 35S and then chased with unlabeled media. 35S-labeled protein was quantified in the media, and lysate after 4 h % Tg secreted was calculated as Tgmedia,4h/(Tglysate,0h+Tgmedia,0h). Error bars represent SEM of three to four biological replicates. STT3A or STT3B KO do not significantly alter WT secretion. Student’s parametric t test was used to determine significant changes in Tg secretion. Representative autoradiograms are shown in supplemental Figure S5G. G, comparison of A2234D Tg degradation in parental, STT3A, or STT3B KO HEK293T cells. A2234D Tg was transiently transfected into the respective cells and subjected to the same 35S-pulse labeling scheme as in F. % Tg degraded was calculated as 1-(Tglysate,4h/Tglysate,0h). Error bars represent SEM of three to four biological replicates. Representative autoradiograms are shown in supplemental Figure S5H. Student’s parametric t test was used to determine significant (*p < 0.05) changes in Tg degradation. OST, oligosaccharyl transferase complex; Tg, thyroglobulin; UGGT1, UDP-glucose glycoprotein glucosyltansferase 1;UGGT2, UDP-glucose glycoprotein glucosyltansferase 2.