Effects of thyroglobulin spontaneus mutations localized within the region I on its intracelular distribution and secretion

CITTERIO, CINTIA E; SIFFO, SOFÍA; MOYA, CHRISTIAN MARTÍN; RIVOLTA CARINA M; REY OSVALDO; ARVAN, PETER ; TARGOVNIK , HÉCTOR M

Mar del PLata

Congreso; LXI Reunión Anual de la Sociedad Argentinta de Investigación Clínica; 2016

Sociedad Argentina de Investigación Clínica

Thyroglobulin (TG), the matrix for thyroid hormone synthesis, is a large glycoprotein (330 kDa monomer) containing 4 structural and functional regions. TG region I is composed of multiple type-1 Cys reach repeats plus linker and hinge segment and its folding is the rate-limiting step of TG maturation. Defective TG synthesis cause congenital hypothyroidism; the incidence is 1:100,000 births.The aim was to characterize the effects of rat TG spontaneous mutations in the region I and their sequential reversion to the wild type sequence, on TG intracellular distribution and secretion. The analysis of TG triple mutant p.L571P, p.Q676R and p.Q907R (mutTG) in a previously validated expression system of eukaryotic cell monolayers followed by Western Blot with anti-TG antibody, showed that mutTG was expressed but not secreted to the culture media. Moreover, immunofluorescence images showed massive intracellular accumulation of mutTG. The double mutant p.L571P/p.Q907R or p.L571P/p.Q676R, obtained by reversion of a single mutation p.Q676R or p.Q907R in mutTG, respectively, still did not allow the secretion of TG. This could indicate that the aforementioned TG mutants do not complete their post-translational modifications, therefore are not secreted and thyroid hormonogenesis would be impaired by the lack of TG arrival to the follicular lumen. However, the reversion of the single mutation p.L571P in the double mutant p.Q676R/p.Q907R allowed the secretion of TG. This result was confirmed in the TG triple reverted clone (obtained by sequential reversion of single mutations). Interestingly, p.L517P falls in the linker of TG, which has been previously reported to belong to the critical segment of TG (interval between linker and hinge segments of region I) involved in the final acquisition of secretory competence of TG. Overall, this work is a contribution to the understanding of the implications of multiple mutations on the TG intracellular transport and secretion.