Abstract
For thyroid hormone synthesis, thyroid peroxidase (TPO) molecules must be transported from the endoplasmic reticulum via the Golgi complex to be delivered at the cell surface to catalyze iodination of secreted thyroglobulin. Like other glycoproteins, TPO molecules in transit to the cell surface have the potential to acquire endoglycosidase H resistance as a consequence of Golgi-based modification of their N-linked carbohydrates, and measurement of the intracellular distribution of TPO has often relied on this assumption. To examine TPO surface distribution in thyrocyte cell lines, we prepared new antibodies against rat TPO. Antibody reactivity was first established upon expression of recombinant rat (r) TPO in 293 cells, which were heterogeneous for surface expression as determined by flow cytometry. By cell fractionation, surface rTPO fractionated distinctly from internal pools of TPO (that co-fractionate with calnexin), yet surface TPO molecules remained endoglycosidase H (endo H)-sensitive. Although the FRTL5 (and PC Cl3) rat thyrocyte cell line also exhibits almost no endo H-resistant TPO, much of the endogenous rTPO is localized to the cell surface by immunofluorescence. Similar results were obtained by fractionation of FRTL5 cell membranes on sucrose gradients. We conclude that in FRTL5 cells, a large fraction of rTPO is delivered to the plasma membrane yet does not acquire Golgi-type processing of its N-glycans. Rat and mouse thyroid tissue TPO also shows little or no endo H resistance, although cell fractionation still needs to be optimized for these tissues.
Highlights
In the biosynthesis of thyroid hormones, iodination of thyroglobulin occurs primarily at the apical surface of thyrocytes, delimiting the thyroid follicle lumen
As a surrogate for thyrocytes, recombinant thyroid peroxidase (TPO) has been studied in nonthyroid cells where experimental access to the plasma membrane is straightforward; from such studies it has been suggested that the fraction of TPO molecules at the cell surface may be very limited [26]
In primary porcine thyrocytes after 7 days in culture, ϳ30% of endogenously expressed TPO could be biotinylated at the cell surface [40], and a similar number has been reported for recombinant hTPO [41]
Summary
The FRTL5 (and PC Cl3) rat thyrocyte cell line exhibits almost no endo H-resistant TPO, much of the endogenous rTPO is localized to the cell surface by immunofluorescence. It is clear that the same glycoprotein may achieve complex glycosylation in one cell type yet not receive complex glycosylation in another cell type [31] For all of these reasons we have been interested in developing a system with which to pursue the distribution of endogenous TPO at the cell surface using thyrocyte cell lines that express thyroid differentiated function [32]. The best studied thyrocyte cell line is the rat-derived, FRTL5 cell model In these cells, TPO mRNA levels have been reported in numerous studies, but no examination of the endogenously expressed TPO protein has yet been performed. We set out to prepare an antiserum against rTPO to begin to characterize behavior of the protein in transfected cells, in thyrocyte cell lines, and in thyroid tissue
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