Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression

Abstract

Recently, it has been suggested that only ϳ2% of human thyroid peroxidase (hTPO 933 ) reaches the surface of stably transfected (Chinese hamster ovary) cells, most being degraded intracellularly, and this might be representative of thyroid peroxidase (TPO) behavior in thyrocytes (Fayadat, L., Siffroi-Fernandez, S., Lanet, J., and Franc, J.-L. (2000) In the past decade, cell type-dependent differences in membrane and secretory protein trafficking have been increasingly recognized. Certainly, the rate and efficiency of protein folding and export from the endoplasmic reticulum (ER) 1 varies between cell types (1). Further, in the distal secretory pathway, the expression of cDNAs encoding regulated secretory proteins has been found to result in storage in secretory granules in some regulated secretory cell types but not others (2-5). Additionally, cell surface polarity signals can be differentially interpreted by different polarized cell types (6 -10). The identification of cell type-dependent differences in protein sorting and trafficking represents a crucial first step in elucidating the underlying molecular mechanisms that account for such differences (11).Thyroid peroxidase (TPO) is a key enzyme responsible for iodination of thyroglobulin during the synthesis of thyroid hormones. Electron microscope autoradiography studies have established that iodination activity is localized primarily at the (extracellular aspect of the) apical surface of thyrocytes delimiting the thyroid follicle lumen; suggesting that under physiological circumstances, TPO is primarily an apical plasma membrane enzyme (12). TPO is neither the sole gene product responsible for the diaminobenzidine oxidation reaction (a technique that has been used to detect peroxidase activity cytochemically) nor the sole protein immunoreacting with thyroid autoimmune antisera (used to immunolocalize the "antimicrosomal antigen" (Ref. 13)). Nevertheless, TPO cytochemical activity in thyrocytes is associated with apical membrane vesicles and the outer surfaces of apical microvilli (14), while additional reaction product can be detected in the thyrocyte ER and Golgi complex. Moreover, using patients' antisera, the thyroid microsomal antigen is immunolocalized to the apical surface of thyrocytes (15), while additional immunoreaction is found in the cytoplasm (16,17). Acute thyroid-stimulating hormone stimulation of the thyroid gland causes additional TPO enzymatic activity and immunoreactivity at the apical cell surface (18 -23). These results all indicate that TPO travels via the secretory pathway to the plasma membrane.Despite this, lingering questions persist about the trafficking of TPO to the cell surface in the thyroid and in various cell culture systems (24). In primary porcine thyrocytes after 7 days in culture, only ϳ30% of endogenously expressed TPO could be chemically modified at the cell surface with a non-permeant biotinylation reagent (25). Further, it has recently been reported that only ϳ2% of recombinant wild-type hTPO (fulllength hTPO 933 ) re...

Intracellular Trafficking of Thyroid Peroxidase to the Cell Surface

“…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). Another study proposed that only 2% of cellular TPO may reside at the plasmalemma (26), but there are reasons to wonder if all of these studies might represent great underestimates of the TPO surface distribution (both because of intracellular lumina that may sequester plasmalemmal proteins during thyrocyte primary culture (25) and because of low biotinylation efficiency of surface TPO (27)). Alternatively, some investigators have examined acquisition of endo H resistance to estimate the fraction of cellular TPO molecules that have proceeded beyond the ER through the secretory pathway, and this fraction is very low in real thyroid tissue (Figs.…”

“…We and others have previously reported that recombinant TPO expression, even in clones derived from single cells, results in two cell subpopulations, each of which maintain comparable total TPO protein expression but only one of which maintains surface TPO protein expression (27). Therefore, we bathed live 293-22 cells expressing rTPO in medium containing anti-rTPO and a fluorescent-conjugated anti-rabbit secondary antibody and used flow cytometry and fluorescence-activated cell sorting (FACS) to segregate the cells into FACS-negative and FACS-positive subpopulations (Fig.…”

“…Second, sensitivity of TPO N-glycans to digestion with endoglycosidase H might be used to provide an estimate of the fraction of TPO molecules residing within or beyond the endoplasmic reticulum (18). However, as straightforward as these methods are to execute, we previously reported that cell type-dependent differences in TPO surface expression may limit the utility of recombinant TPO for physiological studies of intracellular distribution, and furthermore, we have raised the question of whether TPO molecules exiting the ER necessarily acquire endoglycosidase H resistance (27). Indeed, it is a well recognized phenomenon that the glycans on some exportable proteins fail to acquire endo H resistance upon trafficking through the Golgi en route to the plasma membrane, perhaps due to physical inaccessibility of high mannose oligosaccharides in the tertiary or quaternary structure (28).…”

“…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. Indeed, heretofore we were unable to identify polyclonal antisera against hTPO that could unequivocally identify endogenous rTPO protein (27). Therefore, 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.…”

A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

“…Newly synthesized TPO is transported from the endoplasmic reticulum to the cell surface via the Golgi complex (20,(22)(23)(24). During processing and intracellular trafficking TPO interacts with the molecular chaperones calnexin, calreticulin (25), and BiP (26).…”