Thyroxine liberation from N-terminus of

Thyroglobulin by Cathepsin C and PGCP

Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute,Jamova 39, 1000 Ljubljana, Slovenia

Dejan Suban, Tajana Zajc, Miha Renko, Georgy Mikhaylov, Boris Turk, Vito Turk and Iztok Dolenc

Introduction

Thyroxine is an iodine-containing hormone produced by the thyroid gland and secreted in the blood. Its main function is to increase the rate of cell metabolism (metabolism of carbohydrates, fats, proteins). It influences a great variety of effects, including growth, development, reproduction and resistance to infection. Thyroid hormones release generally requires uptake of thyroglobulin (Tg) from the colloid and a proteolytic cleavage in lysosomes. However, thyroxine (T4) can also be released to some extent by extracellular proteolysis of Tg within the colloid, at the apical surface of thyrocytes. The thyrocytes also secrete proteolitic enzymes [1, 2].

Secretion of endosomal-lysosomal enzymes from FRTL-5 cells was realized by using six components: (6H) TSH, insulin, hydrocortisone, transferrin, somatostatin, and L-glycyl-histidyl-lysine in the medium. When activity was monitored by the specific substrate Gly-Phe-4MbNA however, secretion of cathepsin C was detected in the presence of 3H and also found to be greater in the presence of 6H. No difference in cathepsin C activity was observed in lysates (Fig. 4A), except on addition of insulin to 3H medium, where cathepsin C activity was increased. Observing the influence of TSH, insulin, and/or somatostatin on the secretion of cathepsin C from FRTL-5 cells however showed that cathepsin C activity is higher when two of these three components are included in 3H

Results media (Fig. 4B). We concluded that the secretion of endosomal-

lysosomal enzymes is caused by the complex N- In the present study we show the extracellular action of glycosylation of proteins. The enzyme responsible for the

two lysosomal enzymes, cathepsin C and PGCP, on Tg, initiation of biosynthesis of hybrid and complex

and their ability to liberate a thyroid hormone, thyroxine glycosylation is N-acetylglucosaminyl transferase 1 from the position 5 of Tg. We performed in vitro (GnT1). We checked the expression of the protein in experiments to show the ability of cathepsin C and/or FRTL-5 cells in the presence of 6H and compared it to that PGCP to liberate thyroxine at conditions which simulate where 3H were used. We found an increase in GnT1

expression level when all six components were added to the natural environments in thyroid follicle lumen as is the medium of FRTL-5 cells (Fig. 5)[4]. recommended for enzyme action in vitro [3]. Dipeptides

To support data obtained from in vitro studies and cell were removed, as observed by HPLC analysis (Fig. 1A). N-culture experiments immunelabeled slices of mouse terminal sequencing (Fig. 1B) of the products resulted in thyroid gland were performed to show the presence of progressive truncation of Tg from the N-terminal part investigated enzymes, cathepsin C and PGCP, in thyroid

including the thyroxine on position 5. The reaction was epithelial cells (Fig. 6).

terminated at Arg13. The role of PGCP and cathepsin C in Tg hydrolysis was

Conclusiondemonstrated also, using FRTL-5 cells. The endogenous distribution of both enzymes in the cells was observed by

In vitro experiments proved that cathepsin C removes fluorescence microscopy, using specific antibodies. up to 12 amino acids from the N-terminus of porcine Cathepsin C and PGCP were observed to be partially thyroglobulin, including the dipeptide with the thyroxine colocalized in cells (Fig. 2A). They were enriched, on position 5.particularly in the endoplasmic reticulum and Golgi

Cell culture experiments with FRTL-5 cell line showed apparatus (Fig. 2B-C, 2D-E), organelles where N-localization of cathepsin C and PGCP and confirmed their glycosylation of proteins takes place. Cathepsin C and secretion into the medium. The secreted PGCP has a PGCP are rarely directed into lysosomes (Fig. 2F, G). They hybrid or a complex type of N-glycosylation. Secretion of were even segregated in different vesicles of secretory the active cathepsin C from FRTL-5 cells is stimulated by nature. TSH, insulin, and/or somatostatin. The released enzymes As N-glycosylation has been shown to be important for liberate thyroxin from porcine thyroglobulin added to the a variety of functions of glycoproteins, including media. trafficking, we focus on the nature of N-glycosylation of

Additionally, we show that TSH, insulin, and/or cathepsin C or PGCP in cell lysates and in media. Western somatostatin induce up-regulation of GnT1, the enzyme blot analysis was performed following enzymatic responsible for the initiation of biosynthesis of hybrid deglycosylation (Fig. 3). The intracellular cathepsin C and and complex N-glycosylation of proteins. PGCP were deglycosylated with Endo H and PNGase F.

However, only PNGase F was effective in deglycosylation REFERENCES

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hormone liberation in the thyroid epithelial cells. Endocrinology 137:1963-1974.forms of PGCP and cathepsin C were modified mainly by [2] Dunn JT. (1996) Thyroglobulin: chemistry and biosynthesis. In the Thyroid. A Fundamental and Clinical Text,

mannose-type glycans, whereas the secreted PGCP was eds. Braverman LE, and Utiger RD. Philadelphia: Lippincott-Raven, p. 85-95.[3] Jordans S., Jenko-Kokalj S., Kuhl N.M., Tedelind S., Sendt W., Bromme D., Turk D., Brix K. (2009) modified by hybrid or complex type of oligosaccharide Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and

redox conditions. BMC Biochemistry 10: 23.chains. The Western blot for cathepsin C showed only [4] Suban D., Zajc T., Renko M., Turk B., Turk V. and Dolenc I. (2012) Cathepsin C and plasma glutamate carboxypeptidase secreted from Fischer rat thyroid cells liberate thyroxin from the N-terminus of the precursor form. thyroglobulin. Biochimie 94:719-726.

Fig. 2. Localization of PGCP and cathepsin C in FRTL-5 cells. The enzymes are partially colocalized (A). Co-localization is seen in endoplasmic reticulum (B, C) and in Golgi (D, E).Delivery to lysosomes was rare (F, G), because both enzymes were secreted. Bars represent the micrometre (µm) scale.

Fig. 1. Gradual cleavage of the N-terminal part of porcine thyroglobulin. (A) HPLC analysis of thyroglobulin (dashed line), its hydrolysis with cathepsin C (dotted line) or its hydrolysis with cathepsin C and PGCP (solid line). (B) Newly formed terminals after cleavage with cathepsin C are marked by arrows, as determined by protein sequence analysis. Thyroxin on position 5 is marked as an inverse Y. The reaction is stopped at Arg-Pro-, marked by inverse RP.

Fig. 4. Control of active cathepsin C by components added to the media, monitored by the hydrolysis of the specific substrate Gly-Phe-4MβNA. All samples include hydrocortisone, transferrin and L-glycyl-histidyl-lysine (3H). Addition of thyrotropin (TSH), insulin (I), and/or somatostatin (S) is marked. (A) Intracellular activity of cathepsin C under the control of added components. (B) Secretion of active cathepsin C under the control of added components.

Fig. 5. Expression of N-acetylglucosaminyl transferase 1 (GnT1), the starting enzyme in complex N-glycosylation, under the presence of three (3H) or six components (6H) supplemented to the media. TSH, insulin and/or somatostatin induced upregulation of N-acetylglucosaminyl transferase 1 expression, while the level of β-actin remained constant.

Fig. 6. Distribution of cathepsin C (A) and PGCP (B) in mouse thyroid tissue. Cryo-sections of mouse thyroid tissue were stained for DNA with DAPI (blue) and immunolabeled with antibodies directed against cathepsin C or PGCP (green). Cell nuclei were localized by DNA-DAPI signals. Cathepsin C and PGCP were detected in vesicles and near apical plasma membrane of thyrocytes. Bars represent 10 μm.

Fig. 3. Deglycosylation and Western blot of cathepsin C and PGCP in FRTL-5 cells and their secretion from the cells. Endo H deglycosylation of cathepsin C and PGCP enzymes from cell lysates (A, C) indicates the presence of high-mannose N-glycosylation. The secreted cathepsin C (B) showed only the proenzyme form of the protein. The secreted PGCP (D) hybrid or complex N-glycosylation, as determined by endoglycosidase PNGase F.