Trafficking of lysosomal cathepsin B—green fluorescent protein to the surface of thyroid epithelial cells involves the endosomal/lysosomal compartment

Martin Linke1,*, Volker Herzog1 and Klaudia Brix1,2,{ddagger}

1 Institut für Zellbiologie and Bonner Forum Biomedizin, Universität Bonn, Ulrich-Haberland-Str. 61a, D-53121 Bonn, Germany
2 School of Engineering and Science, International University Bremen, PO Box 75 05 61, D-28725 Bremen, Germany
* Present address: Department of Human Genetics, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA

{ddagger} Author for correspondence (e-mail: k.brix{at}iu-bremen.de)

Accepted 23 September 2002


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 Summary
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 Materials and Methods
 Results
 Discussion
 References
 
Cathepsin B, a lysosomal cysteine proteinase, is involved in limited proteolysis of thyroglobulin with thyroxine liberation at the apical surface of thyroid epithelial cells. To analyze the trafficking of lysosomal enzymes to extracellular locations of thyroid epithelial cells, we have expressed a chimeric protein consisting of rat cathepsin B and green fluorescent protein. Heterologous expression in CHO cells validated the integrity of the structural motifs of the chimeric protein for targeting to endocytic compartments. Homologous expression, colocalization and transport experiments with rat thyroid epithelial cell lines FRT or FRTL-5 demonstrated the correct sorting of the chimeric protein into the lumen of the endoplasmic reticulum, and its subsequent transport via the Golgi apparatus and the trans-Golgi network to endosomes and lysosomes. In addition, the chimeras were secreted as active enzymes from FRTL-5 cells in a thyroid-stimulating-hormone-dependent manner. Immunoprecipitation experiments after pulse-chase radiolabeling showed that secreted chimeras lacked the propeptide of cathepsin B. Thus, the results suggest that cathepsin B is first transported to endosomes/lysosomes from where its matured form is retrieved before being secreted, supporting the view that endosome/lysosome-derived cathepsin B contributes to the potential of extracellular proteolysis in the thyroid.

Key words: Epithelial cells, Green fluorescent protein, Cathepsin, Lysosome, Thyroglobulin


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 Introduction
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Trafficking of lysosomal enzymes (for a review, see von Figura, 1991Go) from the trans-Golgi network (TGN) to organelles of the endocytic pathway is mediated by the mannose 6-phosphate (M6P) modification of N-linked oligosaccharides, which is recognized by 46 kDa and 300 kDa M6P-receptors (MPRs) of the TGN (Ludwig et al., 1994Go; Pohlmann et al., 1995Go; Munier-Lehmann et al., 1996Go; Sohar et al., 1998Go; Dittmer et al., 1999Go). Lysosomal enzymes are transported as proteolytically inactive precursors that, after uncoupling of receptor-ligand complexes, become matured by proteolytic processing within late endosomes or lysosomes. However, it is well known that sorting from the TGN to lysosomes is not the exclusive transport pathway of lysosomal enzymes. In various cell types, the secretion of inactive proforms of lysosomal enzymes has been observed in physiological and, more often, in pathological conditions. Indeed, a defect in M6P-based trafficking is the cause of I-cell disease in which lysosomal enzymes are secreted as proforms from patients' fibroblasts (for reviews, see Amara et al., 1992Go; Kornfeld, 1992Go; Kornfeld and Sly, 1995Go). Mistargeted and secreted lysosomal proenzymes can, however, be reinternalized by the 300 kDa MPR at the cell surface, thus providing cells with an additional mechanism to concentrate mature enzymes to lysosomes (Lobel et al., 1989Go) (for a review see, von Figura, 1991Go).

Lysosomal enzymes are also detected extracellularly in certain physiological conditions (for a review, see Andrews, 2000Go). A striking example of such conditions is the presence of mature lysosomal enzymes in the resorption lacunae of osteoclasts, where they mediate the extracellular degradation of organic bone matrix. Osteoclasts like macrophages and, in general, hematopoietic cells have the ability to redirect late endosomes or even lysosomes to the plasma membrane from where their content is secreted by exocytosis. In addition, the secretion of lysosomes might also be viewed as a ubiquitous phenomenon enabling cells to reseal their plasma membrane after rupture (Rodriguez et al., 1997Go). Hence, it became obvious that cell types not belonging to the hematopoietic lineage are also able to secrete their lysosomal enzymes, and the term `secretory lysosomes' was coined (for reviews, see Andrews, 2000Go; Blott and Griffiths, 2002Go).

We have observed, recently, that thyroid epithelial cells secrete lysosomal enzymes such as the cathepsins B, D and K (Brix et al., 1996Go; Lemansky et al., 1998Go; Tepel et al., 2000Go). Mature and proteolytically active cathepsins belonging to the papain family of cysteine proteinases, that is, cathepsins B and K, have been shown to provide thyroid epithelial cells with a mechanism of extracellular degradation of thyroglobulin (Tg) at the apical plasma membrane. Before endocytosis, Tg as the thyroid prohormone undergoes limited proteolysis, which leads to the liberation of the thyroid hormone thyroxine (T4). Consequently, it was concluded that the secretion of lysosomal enzymes from thyroid epithelial cells fulfills a physiological task in extracellular prohormone processing (Tepel et al., 2000Go; Brix et al., 2001Go). Furthermore, we have observed that the secretion of lysosomal enzymes from thyroid epithelial cells is a regulated process (Linke et al., 2002Go). Secretion of mature cathepsin B is triggered by thyroid stimulating hormone (TSH) and contributes to the extracellular release of T4. An as yet unanswered question is, however, whether the cysteine proteinases are secreted from thyroid epithelial cells as zymogens or as mature, enzymatically active enzymes.

Here, we have expressed a chimeric protein consisting of cathepsin B, a prototype of a lysosomal cysteine proteinase, and the enhanced green fluorescent protein (EGFP), a visualization tag, to analyze the transport pathways of lysosomal enzymes in rat thyroid epithelial cells, that is, FRT or FRTL-5 cells. Both cell lines differ from each other in that FRT cells better represent the epithelial phenotype of thyrocytes, whereas FRTL-5 cells rather reflect their physiological properties (Ambesi-Impiombato and Coon, 1979Go). Tg and receptors for TSH (for a review see, van de Graaf et al., 2001Go) are expressed by FRTL-5 but not by FRT cells (Garbi et al., 1987Go; Akamizu et al., 1990Go). A polarized epithelial phenotype is, however, a characteristic feature of FRT cells, whereas FRTL-5 cells grow in a much less polarized fashion and without numerous cell-cell contacts.

In both, FRT and FRTL-5, cells transport of cathepsin B-EGFP (CB-EGFP) chimeras involved the endoplasmic reticulum (ER), the Golgi apparatus, the TGN and lysosomes as deduced from colocalization and transport studies. In heterologously expressing CHO cells, CB-EGFP was also sorted into lysosomes, demonstrating that trafficking of the chimeras to lysosomes was governed by the structural motifs of its cathepsin B portion. Pulse-chase radiolabeling of CB-EGFP expressing FRTL-5 cells and immunoprecipitation studies showed the secretion of a single molecular form of the chimeric protein, consisting of matured cathepsin B linked to EGFP and of endogenous cathepsin B in a TSH-dependent fashion. The results indicate that cathepsin B is first transported to compartments of the late endocytic pathway where it matures before being secreted from thyroid epithelial cells.


    Materials and Methods
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 Materials and Methods
 Results
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 References
 
Cryosections from rat thyroid glands
Sprague Dawley rats were bled by opening of the Aorta descendens. Prewarmed PBS supplemented with 10 IU per ml heparin (Braun, Melsungen, Germany), followed by 3% formaldehyde in phosphate-buffered saline (PBS) was perfused via the Vena porta. Thyroid glands were dissected and postfixed with 8% formaldehyde in PBS, infiltrated with 2.3 M sucrose as a cryoprotectant and frozen in liquid propane. Sections of 1 µm were prepared with a cryotome (Ultracut E, FC4D, Reichert-Jung, Wien, Austria) and were mounted on poly-L-lysine-coated microscope slides. Blocking was performed with BSA and immunolabeling was with rabbit anti-rat cathepsin B antibodies (Upstate Biotechnology, distributed by Biozol, Eching, Germany) and DTAF-conjugated secondary antibodies (Dianova, Hamburg, Germany).

Cell culture
CHO-K1 and FRTL-5 cells were obtained from ATCC (Manassas, USA) or ECACC (Salisbury, UK). FRT cells were kindly provided by Lucio Nitsch (Naples, Italy). Cells were grown in Ham's-F12 (CC Pro, Neustadt/Weinstrasse, Germany) supplemented with 10% fetal calf serum (FCS) (GibcoTM Invitrogen GmbH, Karlsruhe, Germany) for CHO-K1 cells, F12 Coon's (Sigma, Deisenhofen, Germany) supplemented with 5% FCS for FRT or F12 Coon's (Sigma) supplemented with 5% calf serum (CS) (GibcoTM Invitrogen GmbH), 0.1 U/ml TSH, 0.166 mg/ml insulin, 0.326 µg/ml hydrocortisol, 2 µg/ml glycyl-histidyl-lysine, 1 µg/ml somatostatin and 0.5 mg/ml transferrin (all from Sigma) for FRTL-5 cells. All cell culture media contained 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 µg/ml amphotericin B (GibcoTM Invitrogen GmbH). Cells were incubated at 37°C in a 5% CO2 atmosphere (Heraeus Instruments GmbH, Osterode, Germany). For microscopic inspection, cells were incubated with the above media, but buffered with 20 mM HEPES instead of sodium bicarbonate and adjusted to pH 7.4. For transport studies, FRT cells were kept at 20°C in HEPES-buffered medium before mounting on microscope slides and in vivo microscopy at the permissive temperature of 37°C. Before TSH stimulation, FRTL-5 cells were kept for two days under `five hormone' conditions (5H), that is, complete medium without TSH. Stimulation was with `five hormone medium' supplemented with 50 µU/ml TSH.

Vector construction
Messenger RNA (mRNA) from cultured FRTL-5 cells was isolated using an mRNA isolation kit (Perkin Elmer, Langen, Germany). 1 µg of mRNA was used for reverse transcription (RT) with AMV reverse transcriptase (Invitrogen, Groningen, Netherlands). For polymerase chain reactions (PCR), specific primer pairs were designed on the basis of the published sequence of cathepsin B from Sprague Dawley rats (AC X82396) (Guenette et al., 1994Go). The primers (underlined) introducing restriction sites ({downarrow}) for EcoRI and BamHI to allow the insertion of the complete coding sequence of FRTL-5 derived cathepsin B from nucleotides 49 to 1068 into the multiple cloning site of the plasmid pEGFP-N1 (Clontech, Heidelberg, Germany) were:

PCR was performed under the following conditions: 0.1 µg cDNA, 0.3 mM each of dNTPs (AGS, Heidelberg, Germany), 0.3 µM of each primer (MWG Biotech GmbH, Ebersberg, Germany), 2.5 U Pwo DNA polymerase (Thermo Hybaid, Ashford, UK) and 10 cycles of 15 seconds at 94°C, 30 seconds at 62°C, 45 seconds at 72°C, followed by 20 cycles of 15 seconds at 94°C, 30 seconds at 62°C, 45 seconds + 20 seconds per cycle at 72°C and followed by a final extension period for 7 minutes at 72°C. 3.9 pmol of the amplification product and 1.3 pmol of the plasmid pEGFP-N1 (Clontech) were digested with BamHI (NEB, Frankfurt, Germany) for 1 hour at 37°C, followed by ethanol precipitation and restriction with EcoRI (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) for 1 hour at 37°C. After ethanol precipitation, the restricted plasmid was dephosphorylated with alkaline phosphatase (MBI, St. Leon-Rot, Germany), and the restricted cDNA was ligated into the plasmid using T4-DNA-Ligase (MBI) for 120 minutes at 16°C, resulting in the vector pCathB-EGFP. Competent E. coli JM 109 were transformed with pEGFP-N1 or pCathB-EGFP, and kanamycin resistant clones were used for isolation of the vector DNAs (JETstar 2.0) (Genomed GmbH, Bad Oeynhausen, Germany). The complete sequence of the fusion construct encoded by pCathB-EGFP was sequenced (Sequiserve, Vaterstetten, Germany) in both directions using the primer pair:

Nucleotide and amino-acid sequences were analyzed with Omiga 1.1.3 (Oxford Molecular Group, Inc., Campbell, CA, USA) using Clustal W 1.60 algorithm for alignments (Pearson and Lipman, 1988Go) and PROSITE to search for structural motifs (Bairoch and Bucher, 1994Go; Falquet et al., 2002Go). The entire sequence of the vector pCathB-EGFP is available from GenBank under accession number AF490378.

Transfection
Vector DNA was isolated, precipitated sequentially with isopropanol, 100% and 70% ethanol, and dissolved in 10 mM Tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8). One day before transfection, 300,000 cells per well were seeded on cover glasses in six-well plates (Fisher Scientific, Schwerte, Germany) and incubated over night at 37°C and 5% CO2. Transfection experiments were carried out using either 50 µl effectene (Qiagen, Hilden, Germany) or 1.5 µl Fugene 6 (Roche Diagnostics, Mannheim, Germany) transfection reagent per µg vector DNA. The transfection procedures were performed according to the suppliers' instructions. For selection, transfected cells were passaged in ratios of 1 to 12 three days after transfection and thereafter incubated in culture medium supplemented with 1 mg/ml G418 (Calbiochem, Schwalbach, Germany).

Immunolabeling and colocalization experiments
Cells were grown on cover glasses, rinsed with PBS and fixed with 8% formaldehyde in 200 mM HEPES (pH 7.4) for 30 minutes at 37°C. After washing with HEPES buffer, cells were permeabilized with 0.2% Triton X-100 (Merck, Darmstadt, Germany) in HEPES buffer for 5 minutes at room temperature. In some experiments, permeabilization with Triton was omitted. For blockage of nonspecific binding sites, cells were incubated with 3% BSA in calcium- and magnesium-free (CMF-)PBS. Primary antibodies were diluted in CMF-PBS containing 0.1% BSA, and cells were incubated at 4°C overnight with either rabbit anti-rat cathepsin B (Biozol), rabbit anti-rat protein disulfide isomerase (PDI) (Stressgen, distributed by Biomol, Hamburg, Germany), monoclonal mouse anti-rat mannosidase II (Babco, Richmond, CA, USA) or rabbit anti-rat lysosomal membrane glycoprotein 96 (lgp96) antibodies [AMC2; kindly provided by Ana Maria Cuervo, Boston, MA, USA (Cuervo and Dice, 1996Go)]. After washing, cells were incubated for 90 minutes at 37°C with secondary antibodies, that is, TRITC-labeled goat anti-rabbit or Cy3-labeled goat anti-mouse antibodies (Dianova). Cells were mounted in mowiol (Hoechst AG, Frankfurt, Germany) containing 50 mg/ml 1,4-diazabicyclo(2,2,2)octane to avoid photobleaching.

Labeling of endocytic compartments with Lyso Tracker
Transfected cells were grown on cover glasses, rinsed with PBS and incubated at 20°C for 60 minutes before loading with 1 nM Lyso Tracker Red DND-99 (Molecular Probes, Leiden, Netherlands) in medium without serum for 30 minutes at 37°C. Thereafter, cells were chased in complete culture medium for 120 minutes at 37°C, washed with PBS, fixed with 3% formaldehyde in PBS for 30 minutes at 37°C and mounted (see above).

Microscopy and documentation
Cells or cryosections were mounted on microscope slides and viewed with confocal laser scanning microscopes (LSM) (TCS 4D, Leica, Bensheim, Germany, or LSM 510, Zeiss, Oberkochen, Germany). Micrographs were stored in TIFF format and color coded with Image Pro Plus 3.0.01.00 software (Media Cybernetics, L.P., Silver Springs, MD).

Subcellular fractionation, SDS-PAGE and immunoblotting
Subcellular fractionation was performed as previously described (Brix et al., 1996Go). In brief, non-transfected or G418-selected CB-EGFP expressing cells were grown in 75 cm2 culture flasks until confluence, rinsed with ice-cold PBS, then harvested using rubber policemen. Cell suspensions were pelleted by centrifugation (5 minutes, 900 g) at 4°C. The pellets were resuspended in 100 mM Soerensen phosphate buffer (KH2PO4 and Na2HPO4, pH 7.2), supplemented with 0.25 M sucrose and 5 mM ethylenediaminetetraacetic acid (EDTA) and homogenized on ice using a Dounce homogenizer (Kontes Co., Vineland, NJ). Cellular debris and nuclei were removed from cell homogenates by centrifugation (5 minutes, 900 g, 4°C). Lysosomes were enriched by centrifugation at 10,000 g for 10 minutes at 4°C. The resulting pellet was used as the lysosomal fraction. The supernatant was layered onto cushions of 0.32 M and 1.2 M sucrose in the same buffer (see above), and centrifuged at 100,000 g for 2 hours at 4°C. The resulting band at the interphase between 0.32 M and 1.2 M sucrose was removed and resuspended in PBS. Plasma membrane vesicles were collected as the pellet of the following centrifugation at 100,000 g for 1 hour at 4°C.

Cell homogenates or lysosomal fractions were lysed on ice with 0.2% Triton X-100 in PBS supplemented with protease inhibitors (0.2 µg/ml aprotinin, 10 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), 2 mM EDTA, 1 µM pepstatin) for 30 minutes, cleared by centrifugation and boiled in sample buffer consisting of 10 mM Tris-Cl (pH 7.6), 0.5% (w/v) sodium dodecyl sulphate (SDS), 25 mM dithiothreitol (DTT), 10% (w/v) glycerol, 25 µg/ml bromophenol blue. Proteins were separated by SDS-polyacrylamide gelelectrophoresis (PAGE) (Laemmli, 1970Go) and blotted onto nitrocellulose. For the detection of procathepsin B and cathepsin B, rabbit anti-rat cathepsin B propeptide (Linke et al., 2002Go), rabbit anti-rat cathepsin B antibodies (Biozol) and horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG (Dianova) were applied. The procathepsin-B-specific antiserum was kindly provided by Lukas Mach, Vienna, Austria and John S. Mort, Montreal, Canada, and it was raised against a synthetic peptide comprising the first 56 amino acids of the rat cathepsin B propeptide (Fox et al., 1992Go) essentially as described previously (Rowan et al., 1992Go). For the detection of EGFP (Clontech) and CB-EGFP chimeric proteins, mouse anti-green fluorescent protein (GFP) antibodies (Roche Diagnostics) and HRP-coupled goat anti-mouse IgG (Dianova) were used. Immunoreactions were visualized by enhanced chemiluminescence on Hyperfilm-MP (Amersham Pharmacia Biotech) and scanned using a transmitted light scanner device (Hewlett-Packard, Palo Alto, CA).

Thyroid hormone liberation assay and reversed phase chromatography
Plasma membrane fractions from FRT cells were resuspended in PBS (pH 7.2) and incubated with Tg for 30 minutes at 37°C. In controls, plasma membrane preparations were preincubated with 1 mM E64 for 5 minutes to irreversibly inhibit cysteine protease activities. Thyroid hormones, iodotyrosines and iodothyronines liberated by Tg proteolysis were then enriched by absorptive chromatography on sephadex LH-20 (Sweeting and Eales, 1992Go) and analyzed by reversed phase chromatography on µRPC C2/C18 PC 3.2/3 columns using a SMARTTM system (Amersham Pharmacia Biotech). Flow rate was 100 µl/minute, sample loading and washing was with 0.1% trifluoroacetic acid (TFA) and elution was with 50% isopropanol, 50% acetonitrile and 0.1% TFA. The absorption of the eluent was monitored at 214 nm. For the identification of the eluting peaks, triiodothyronine (T3) or T4 were run as standards on the same column.

Radiolabeling and immunoprecipitation
Radiolabeling of non-transfected or G418-selected CB-EGFP expressing cells was for 1 hour at 37°C in methionine- and cysteine-free DMEM (Biowhittaker, Verviers, Belgium) supplemented with [35S]-methionine and [35S]-cysteine (Redivue Pro-Mix, 28 and 12 µCi/ml, respectively) (Amersham Pharmacia Biotech). After the indicated pulse periods, or after chase periods of up to 24 hours in non-radioactive culture medium at 37°C, culture media of radiolabeled cells was collected and cleared by centrifugation for 10 minutes at 1,000 g and 4°C. For immunoprecipitation, rabbit anti-GFP antibodies (Clontech), rabbit anti-rat cathepsin B (Biozol) or rabbit anti-rat cathepsin B propeptide antibodies were pre-adsorbed to protein-A-coupled magnetic micro beads (Miltenyi Biotec, Bergisch Gladbach, Germany) overnight at 4°C. Immunobeads were then incubated overnight at 4°C with culture media of radiolabeled cells. Magnetic immobilization of immunobeads on µ-columns, washing and elution of immunoprecipitated proteins with heated SDS-sample buffer was performed according to the manufacturer's protocol (Miltenyi Biotec). After SDS-PAGE and western blotting, radiolabeled proteins were visualized by a phosphor storage imager (Fuji BAS1000, Düsseldorf, Germany) or by exposure onto Hyperfilm-MP (Amersham Pharmacia Biotech).

Cathepsin B activity assays
Conditioned media of non-transfected or CB-EGFP expressing CHO, FRT or FRTL-5 cells were used for cathepsin B activity assays, as were anti-GFP immunoprecipitates from conditioned media of TSH-stimulated CB-EGFP-expressing FRTL-5 cells. The activity of cathepsin B was determined at pH 6.0 by a colorimetric assay (Linke et al., 2002Go) using N-benzyloxycarbonyl-arginyl-arginine-p-nitroanilide (Z-Arg-Arg-pNA; Bachem Biochemica GmbH, Heidelberg, Germany) as the cathepsin-B-specific substrate.

Statistic evaluations
All statistics were done using standard computer software and levels of significance were determined by one-way ANOVA (Origin, MicroCal Software, Northampton, MA, USA).


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Localization of cathepsin B in rat thyroid epithelial cells
In immunolabeled cryosections from rat thyroids, cathepsin B was detected in vesicles of various sizes resembling endosomes and lysosomes (Fig. 1A, arrows) and in association with the apical plasma membrane of thyroid epithelial cells (Fig. 1A, arrowheads). Immunolabeling of formaldehyde-fixed and Triton-X-100-permeabilized FRT cells with cathepsin-B-specific antibodies revealed the presence of the protease in numerous vesicles, that is, lysosomes (Fig. 1C, arrows), and in cisternal structures surrounding the nucleus (Fig. 1C, stars). When FRT cells were immunolabeled directly after fixation but without Triton X-100 permeabilization the antibodies against cathepsin B showed an intense labeling at the borders between neighboring cells (Fig. 1B, arrowheads), that is, cell-surface-associated cathepsin B was identified.



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Fig. 1. Localization and possible function of cathepsin B in rat thyroid epithelial cells. A confocal fluorescence micrograph of a segment of a rat thyroid follicle (A), and a conventional fluorescence micrograph of formaldehyde-fixed (B) and Triton-X-100-permeabilized (C) FRT cells after immunolabeling with rabbit anti-rat cathepsin B antibodies. Cathepsin B was recognized within vesicles resembling endosomes or lysosomes (arrows) or in association with the plasma membrane (arrowheads) of rat thyroid epithelial cells in situ (A) and in vitro (B,C). In vitro degradation of Tg with plasma-membrane-associated proteases of FRT cells without (blue curve) or after inhibition of cysteine proteases by E64 (red curve) and identification of liberated thyroid hormones (eluting positions marked by green arrows) by reversed phase chromatography (D). Note that preincubation with E64 completely abolished liberation of T3 and T4 by proteases associated with plasma membrane preparations (D, cf. red with blue curve), indicating the contribution of cell-surface-associated cysteine proteases in Tg processing for thyroid hormone liberation. N, nuclei; stars, Golgi cisternae. Bars, 10 µm (A), 50 µm (B,C).

 

To analyze whether cell-surface-associated cathepsin B might be involved in thyroid hormone liberation by the processing of Tg, plasma membrane fractions from FRT cells were incubated with Tg at neutral pH. By reverse phase chromatography, the liberation of the thyroid hormones T3 and T4 was detectable (Fig. 1D, blue curve). The liberation of thyroid hormones was not observed when E64, that is, a cysteine proteinase inhibitor, was added to plasma membrane fractions prior to incubation with Tg (Fig. 1D, red curve), demonstrating that proteolysis of Tg was mediated by cell-surface-associated cysteine proteinases.

Taken together, these results indicated the extracellular presence of mature and proteolytically active cathepsin B at the surface of FRT cells. The occurrence of mature cathepsin B at extracellular locations can only be explained by its secretion and subsequent association with the plasma membrane of thyroid epithelial cells and prompted us to further analyze the transport pathways of cathepsin B in rat thyrocytes, that is, FRT or FRTL-5 cells.

Features of the vector pCathB-EGFP
The cDNA of rat cathepsin B was amplified by RT-PCR using mRNA from FRTL-5 cells, that is, Fischer rat thyroid epithelial cells, and primers introducing restriction sites for EcoRI and BamHI allowed insertion of the cathepsin-B-coding sequence into the multiple cloning site of pEGFP-N1. The resulting vector was named pCathB-EGFP and, as confirmed by sequencing, coded for a chimeric protein (Fig. 2) consisting of the 17 amino-acid signal peptide (light grey, S), the 62 amino-acid propeptide (grey, pro), the 254 amino-acid mature portion of rat cathepsin B (blue), fused to the 239 amino-acid EGFP (green) by a six amino-acid spacer peptide (pink) derived from the residual multiple cloning site of pEGFP-N1. Hence, the chimeric protein CB-EGFP contained all targeting signals and structural motifs of the lysosomal cysteine proteinase cathepsin B that are known to be required for correct transport and proper maturation of the enzyme. In many cell types including rat thyroid epithelial cells, the mature enzyme is present as the so-called single chain form, but it also occurs as the two-chain form of the enzyme, in which the amino acids at positions 127-128 (Fig. 2, white box) are proteolytically removed, whereas the light (LC) and heavy chains (HC) of the enzyme remain linked by a disulfide bridge. The chimeric protein CB-EGFP lacked the last six amino acids at the C-terminus of rat cathepsin B, the so-called C-terminal extension. Because recombinant rat cathepsin B devoid of this region exerts full enzymatic activity (Hasnain et al., 1992Go), the C-terminal extension was replaced by the six amino-acid spacer peptide provided by the vector to completely ensure covalent attachment of EGFP to the heavy chain of cathepsin B by the spacer peptide (Fig. 2, pink), regardless of whether the cathepsin B portion of the chimeric protein is processed to the single- or to the two-chain form of the enzyme. In addition, the two known potential N-glycosylation sites of rat cathepsin B, that is, Asn-38 and Asn-192, were present in the chimeric protein CB-EGFP (Fig. 2, orange). When compared with the published sequence of cathepsin B from Sprague Dawley rats (Guenette et al., 1994Go), the chimeric protein CB-EGFP differed in two amino acids within the N-terminal propeptide of its cathepsin B portion, that is, substitutions Ser-23 to Phe-23, and Asn-67 to Lys-67. Because the structural motifs of the deduced amino-acid sequences of cathepsin B from Sprague Dawley rats and from FRTL-5 cells were the same, as predicted by PROSITE, it is unlikely that the substitutions in the propeptide of FRTL-5 cathepsin B alter the specific functions of this protein. Furthermore, the nucleotide sequence encoding the cathepsin B portion of the chimeric protein CB-EGFP was in complete agreement with the published sequence of rat procathepsin B cDNA (Chan et al., 1986Go).



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Fig. 2. Schematic drawing of the chimeric protein CB-EGFP. The cathepsin B portion of the chimeric protein consists of the signal peptide (S, light grey), the propeptide (pro, dark grey) and the light (LC, blue) and heavy chains (HC, blue) of the protease, which are covalently linked to EGFP (green) by a spacer peptide (pink). Exchanges of two amino acids within the propeptide of cathepsin B from FRTL-5 cells are indicated in grey, the positions of two potential N-glycosylation sites in orange and the active site residues of the cathepsin B portion of the chimeric protein are shown in blue. The complete coding sequence of the vector pCathB-EGFP is available from GenBank under accession number AF490378.

 

Heterologous expression of CB-EGFP in CHO cells
To characterize the expression pattern of the chimeric protein beyond the rat system, CHO cells were transiently transfected with pCathB-EGFP. Immunolabeling with antibodies specific for rat cathepsin B showed that the green fluorescence of the chimeras' EGFP portion and the red fluorescence of its immunolabeled rat cathepsin B portion colocalized within numerous vesicles concentrated in the perinuclear region (Fig. 3A, yellow signals). The antibodies did not cross-react with endogenous cathepsin B of non-expressing CHO cells (Fig. 3A), demonstrating specificity of immunolabeling for rat cathepsin B. The CB-EGFP containing vesicles were identified as endocytic compartments, that is, lysosomes, because they were abundant within the perinuclear region and because they were reached by the fluid phase marker Lyso Tracker Red after pulse-chase incubation (Fig. 3B). The results indicated that the cathepsin B portion of the chimeric protein contained all structural motifs required for targeting to endocytic compartments.



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Fig. 3. Heterologous expression of CB-EGFP and its lysosomal localization in CHO cells. Single channel fluorescence (lower panels, middle and right), merged (A,B) and phase contrast micrographs (lower panels, left) of CHO cells transiently expressing CB-EGFP (green in A,B) were taken with a confocal LSM after immunolabeling with antibodies specific for heterologously expressed rat cathepsin B (A, red) and after pulse-chase loading of lysosomes with Lyso Tracker (B, red). Yellow signals are indicative of colocalization. N, nuclei. Bars, 20 µm.

 

Transport of the chimeric protein CB-EGFP in FRT or FRTL-5 cells
Rat thyroid epithelial cell lines were transiently transfected with pEGFP-N1 or pCathB-EGFP using BES-calcium-phosphate-, DEAE-dextran- or liposome-mediated transfection protocols. In general, transfection with pEGFP-N1 resulted in higher transfection efficiencies compared with pCathB-EGFP, which was most probably due to the size differences of the vectors of approximately 1 kbp. Best results with transfection efficiencies of up to 20% were achieved with liposome-mediated transfection procedures.

To analyze the sorting and transport of the chimeric protein, colocalization studies were performed with FRT or FRTL-5 cells transiently expressing EGFP or CB-EGFP. After immunolabeling of EGFP expressing cells with antibodies against PDI, that is, an ER-resident protein, no colocalization with cytosolic or nuclear EGFP was observed (not shown). By contrast, CB-EGFP-expressing FRT or FRTL-5 cells showed green fluorescence within a reticular network, which was identified as the lumen of the ER by its colocalization with PDI (Fig. 4A and Fig. 5A, arrowheads). The chimeric protein was also present in PDI-negative vesicles (Fig. 4A and Fig. 5A, arrows) or in crescent-shaped cisternal structures at the nuclei (stars). Immunolabeling of the Golgi apparatus with antibodies against mannosidase II revealed colocalization of the glycosidase and the chimeric protein CB-EGFP within the cisternal structures at the nuclei (Fig. 4B and Fig. 5B, stars), that is, within the Golgi apparatus of FRT and FRTL-5 cells. CB-EGFP-containing vesicles of FRT or FRTL-5 cells were identified as compartments of the endocytic pathway, that is, lysosomes, by the colocalization with Lyso Tracker Red after 30 minutes of endocytosis followed by a 120 minute chase period (Fig. 4C and Fig. 5C, arrows). Expression of CB-EGFP in FRT or FRTL-5 cells and subsequent immunolabeling of endogenous cathepsin B was carried out to test for authentic localization of the chimeras. Both, endogenous cathepsin B and expressed CB-EGFP colocalized within numerous vesicles (Fig. 4D and Fig. 5D, arrows). The distribution of these vesicles was comparable to that of cathepsin-B-containing vesicles of non-transfected FRT or FRTL-5 cells (Fig. 4D and Fig. 5D, insets).



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Fig. 4. Trafficking of CB-EGFP in FRT cells. Single channel fluorescence (lower panels, middle and right, and inset in D), merged (A-D) and phase contrast micrographs (lower panels, left) of FRT cells transiently expressing CB-EGFP were taken with a confocal LSM after immunolabeling with antibodies against the ER-resident protein PDI (A, red), the Golgi mannosidase II (B, red), the endogenous lysosomal cysteine protease cathepsin B (D, red) and after pulse-chase loading of lysosomes with Lyso Tracker (C, red). For reference, the inset in D shows non-transfected FRT cells after immunolabeling of cathepsin B. Yellow signals are indicative of colocalization, arrowheads point to ER-cisternae, stars mark the positions of the Golgi and arrows indicate lysosomes. N, nuclei. Bars, 40 µm.

 


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Fig. 5. Trafficking of CB-EGFP in FRTL-5 cells. Single channel fluorescence (lower panels, middle and right, and inset in D), merged (A-D) and phase contrast micrographs (lower panels, left) of FRTL-5 cells transiently expressing CB-EGFP were taken with a confocal LSM after immunolabeling with antibodies against the ER-resident protein PDI (A, red), the Golgi mannosidase II (B, red), the endogenous lysosomal cysteine protease cathepsin B (D, red) and after pulse-chase loading of lysosomes with Lyso Tracker (C, red). For reference, the inset in D shows non-transfected FRTL-5 cells after immunolabeling of cathepsin B. Yellow signals are indicative of colocalization. Arrowheads point to ER-cisternae; stars mark the positions of the Golgi and arrows indicate lysosomes. N, nuclei. Bars, 40 µm.

 

CB-EGFP-expressing FRT cells were used to directly demonstrate the trafficking of the chimeric protein in living cells. Steady-state expression showed the presence of the chimeric protein within cisternae of the ER (Fig. 6A, arrowheads), the Golgi-apparatus (stars) and within numerous vesicles (arrows). Upon incubation at 20°C, CB-EGFP accumulated within the TGN (Fig. 6B), from where the chimeric protein was trafficked to vesicles at the permissive temperature of 37°C (Fig. 6C). Such vesicles were identified as lysosomes by their colocalization with lgp96 (Fig. 6D).



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Fig. 6. Transport of CB-EGFP in living FRT cells. Single channel fluorescence (A,B,D'',D'''), merged (C,D) and phase contrast micrographs (D') of FRT cells transiently expressing CB-EGFP taken in the conventional (A) or the confocal mode (B-D''') at 37°C (A,C), 20°C (B) or after fixation and immunolabeling with antibodies against rat lgp96 (D). During steady state, CB-EGFP was detectable within the lumen of the ER, the Golgi apparatus and within vesicles (A). Note, the accumulation of CB-EGFP within the TGN after incubation of the cells at 20°C (B), its subsequent transport to vesicles after shifting to the transport-permissive temperature of 37°C (C) and its colocalization with the lysosomal marker lgp96 (D). Micrographs of different focal planes are merged in C, and colored in blue, green and red as indicated. Yellow signals in D are indicative of colocalization of green fluorescent CB-EGFP (D''') with lgp96 (D''). Arrowheads point to ER-cisternae, stars mark the positions of the Golgi and the TGN and arrows indicate lysosomes. N, nuclei. Bars, 50 µm (A,C), 20 µm (B,D,D').

 

Thus, the results indicated that trafficking of the chimeric protein CB-EGFP and of the endogenous cathepsin B occurred along the same pathway. After entry into the lumen of the ER, cathepsin B and CB-EGFP are transported to the Golgi and the TGN, from where both proteins were sorted into identical compartments, that is, lysosomes.

Integrity of the chimeric protein CB-EGFP
Lysates of lysosomal fractions of non-transfected or CB-EGFP-expressing CHO, FRT or FRTL-5 cells were analyzed by immunoblotting. Antibodies against rat cathepsin B recognized the proform (pro), the single chain form (SC) and the heavy chain (HC) of the two chain form of cathepsin B in lysosomes of non-transfected FRT or FRTL-5 cells (Fig. 7A, lanes 5 and 7), as well as the lysosomal fractions of CB-EGFP-expressing CHO, FRT or FRTL-5 cells (Fig. 7A, lanes 4, 6, 8). A major portion of cathepsin B was present as the single chain form of ~30 kDa rather than as the two chain form of the enzyme (Fig. 7A, lanes 4-8, compare SC with HC). In lysosomal fractions of CB-EGFP-expressing cells, a protein band with an apparent molecular mass of ~60 kDa was additionally recognized by the antibodies against cathepsin B (Fig. 7A, lanes 4, 6, 8, CB-EGFP). This protein band represented the chimeric protein, because it was absent from non-transfected controls and because it was also detectable in lysosomal fractions of CB-EGFP-expressing cells when blots were probed with antibodies against GFP (Fig. 7B, lanes 11, 13, 15, CB-EGFP). Recombinant EGFP was detectable at ~30 kDa (Fig. 7B, lane 9, EGFP), and no immunoreactions were observed in the lysosomal fractions of non-transfected controls (Fig. 7B, lanes 10, 12, 14), proving the specificity of the GFP antibodies.



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Fig. 7. Lysosomal CB-EGFP and its secretion from transfected cells. Lysates of lysosomal fractions of non-transfected (lanes 3, 5, 7, 10, 12, 14) or CB-EGFP-expressing CHO, FRT or FRTL-5 cells after selection with G418 (lanes 4, 6, 8, 11, 13, 15) were normalized to contain equal amounts of protein and separated on 12.5% SDS gels. After blotting, proteins were immunolabeled with antibodies against rat cathepsin B (A) or GFP (B). Recombinant human procathepsin B (lane 1), bovine spleen cathepsin B (lane 2) and EGFP (lane 9) were used as standards. C shows an autoradiograph of SDS-PAGE-separated anti-GFP immunoprecipitates from culture media collected after the indicated time intervals from radiolabeled non-transfected (lane 18) or CB-EGFP-expressing FRT cells (lanes 16 and 17). Molecular mass markers are given in the left margin. The positions of the intact chimeric protein (CB-EGFP) and its degradation fragment (F1) as well as those of procathepsin B (pro), single chain (SC) and heavy chain of two-chain cathepsin B (HC) are indicated in the right margins. The proteolytic activity of cathepsin B within conditioned media of transfected (+) or non-transfected CHO, FRT or FRTL-5 cells was determined at pH 6.0 by using a colorimetric assay (D). Cathepsin B activities in D are given as mean±standard deviation; levels of significance are indicated as ** for P<0.01, n=3.

 

In FRT and FRTL-5 cells, endogenous pro-, single- and two-chain cathepsin B forms were far more abundant than the CB-EGFP form and present in similar amounts to non-transfected controls (Fig. 7A, compare lanes 6 and 8 with lanes 5 and 7), demonstrating that the chimeric protein was not overexpressed in thyroid epithelial cell lines. In CHO cells, however, chimeric CB-EGFP was expressed in higher amounts compared with FRT or FRTL-5 cells (Fig. 7A, compare lane 4 with lanes 6 and 8; Fig. 7B, compare lane 11 with lanes 13 and 15). In addition, degradation products of the chimeric protein were detected in lysosomes of CHO cells, because both antibodies against cathepsin B and GFP recognized several protein bands with higher electrophoretic mobility than the intact CB-EGFP (Fig. 7A, lane 4; Fig. 7B, lane 11). In lysosomes of FRT and FRTL-5 cells, antibodies against GFP reacted mainly with intact CB-EGFP and faint amounts of a protein fragment running slightly above the EGFP standard were additionally immunostained (Fig. 7B, lanes 13 and 15, F1). A protein fragment of similar molecular mass was also detectable in anti-GFP immunoprecipitates from the 48 hour secretion media of pulse-radiolabeled CB-EGFP-expressing FRT cells (Fig. 7C, lane 17, F1). The extracellular appearance of high amounts of intact CB-EGFP and of faint amounts of the F1 fragment argues for an exchange of the lysosomal content of FRT cells with the extracellular environment, that is, for the secretion of lysosomal proteins.

Secretion of CB-EGFP from FRT cells was a fast process; it was already detectable within 1 hour of the pulse (Fig. 7C, lane 16, CB-EGFP). CB-EGFP was not detected within the secretion media of non-transfected FRT cells (Fig. 7C, lane 18). Furthermore, CB-EGFP was stable in both the lysosomes and the extracellular medium of FRT cells, because the F1 fragment was almost absent from lysosomes isolated at steady state (Fig. 7B, lane 13) and because first signs of proteolytic degradation of pulse-labeled CB-EGFP secreted from FRT cells were observed not before 48 hours (Fig. 7C, compare lane 17 with lane 16). Most importantly, the apparent molecular mass of secreted CB-EGFP was identical to that of the lysosomal CB-EGFP (compare Fig. 7C, lane 16, with Fig. 7B, lane 13), suggesting that FRT cells secreted lysosomally matured CB-EGFP.

To gain information on whether secreted mature CB-EGFP exerts proteolytic activity, conditioned media of non-transfected or CB-EGFP-expressing CHO, FRT or FRTL-5 cells were assayed for their ability to cleave the cathepsin-B-specific substrate Z-Arg-Arg-pNA at pH 6.0. Proteolytic activity of cathepsin B was detectable within the secretion media of non-transfected and CB-EGFP-expressing cells (Fig. 7D). Cathepsin B activity was higher in CB-EGFP expressing CHO cells when compared with non-transfected controls (Fig. 7D, compare CHO + with CHO), which was most probably due to the overexpression of CB-EGFP in CHO cells (compare with Fig. 7A). The conditioned media of CB-EGFP-expressing FRT cells contained slightly lower amounts of proteolytically active cathepsin B than the non-transfected controls (Fig. 7D, compare FRT + with FRT), whereas the levels of cathepsin B activity within conditioned media of non-transfected FRTL-5 cells were comparable to those of their CB-EGFP expressing counter-parts (Fig. 7D, compare FRTL-5 + with FRTL-5). These results indicated that extracellular cathepsin B activity was not altered by the expression of CB-EGFP in FRTL-5 cells, suggesting that transfection with pCathB-EGFP did not affect cathepsin B export in this cell line.

Regulated secretion of the chimeric protein CB-EGFP from FRTL-5 cells
To further analyze the secretion of the chimeric protein and to study its possible regulation, CB-EGFP-expressing FRTL-5 cells were radiolabeled for 1 hour and chased for up to 24 hours in culture medium without or supplemented with 50 µU/ml TSH. The secretion media were then used for immunoprecipitation experiments with antibodies against rat cathepsin B or against the propeptide of rat cathepsin B. The antibodies against the propeptide of rat cathepsin B reacted with both the proforms of endogenous cathepsin B and of expressed CB-EGFP (Fig. 8A, proCB-EGFP, pro) but not with the mature forms of either of the proteases, proving their specificity for procathepsin B.



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Fig. 8. Stimulated secretion of lysosomally matured CB-EGFP from FRTL-5 cells. Lysates of non-transfected (A, lane 1) or CB-EGFP-expressing FRTL-5 cells (A, lane 2) were normalized to provide equal amounts of protein and separated on 12.5% SDS gels for subsequent blotting and immunolabeling with antibodies against the propeptide of rat cathepsin B. Autoradiography of 12.5% SDS gels of secretion media from CB-EGFP-expressing, G418 selected FRTL-5 cells after 1 hour of pulse radiolabeling (B, lanes 3 and 4) and chasing for the indicated time intervals in media without TSH (B, 5H, lanes 5-10) or with 50 µU/ml TSH (B, 5H + TSH, lanes 11-16). Immunoprecipitation was with antibodies against rat cathepsin B (CB, odd numbered lanes in B) or against the propeptide of rat cathepsin B (PP, even numbered lanes in B). Molecular mass markers are given in the margins. The positions of the proform (proCB-EGFP) and the mature chimeric protein (CB-EGFP), as well as of procathepsin B (pro) and single chain cathepsin B (SC) are indicated in the margin between A and B. In C, the proteolytic activity of CB-EGFP secreted from continuously TSH-stimulated FRTL-5 cells after immunoprecipitation with anti-GFP antibodies is given as mean±standard deviation. Note that antibodies against the propeptide of cathepsin B failed to immunoprecipitate CB-EGFP from the secretion media (B) and that secreted CB-EGFP was proteolytically active (C).

 

In the immunoprecipitates with antibodies against cathepsin B, endogenous single chain cathepsin B and expressed CB-EGFP were primarily detected, whereas only faint amounts of procathepsin B were immunoprecipitable from the secretion media of FRTL-5 cells (Fig. 8B, CB). Accordingly, antibodies against the propeptide of cathepsin B immunoprecipitated the proform of the endogenous protease at a level similar to those immunoprecipitated with antibodies against cathepsin B (Fig. 8B, compare PP with CB). Most importantly, antibodies against the propeptide failed to precipitate CB-EGFP, demonstrating that the chimeric protein was secreted in a form lacking the propeptide, that is, matured into its single-chain form. The highest amounts of the mature forms of the endogenous and the expressed protease were present in the secretion media of non-stimulated cells after 4 hours of chase (Fig. 8B, lane 7), whereas incubation with TSH led to a maximum secretion within 2 hours of the chase (lane 11), indicating that TSH stimulation of FRTL-5 cells upregulated the secretion of cathepsin B and of the chimeric protein within a few hours. Consequently, secretion media of continuously TSH-stimulated, CB-EGFP-expressing FRTL-5 cells were used for immunoprecipitation with GFP-specific antibodies to isolate the secreted chimeric protein. These immunoprecipitates were then used for cathepsin B activity assays, demonstrating that the secreted chimeric protein CB-EGFP was proteolytically active (Fig. 8C). The results strongly suggested that lysosomally matured forms of endogenous cathepsin B and of expressed CB-EGFP were the principal cathepsin B forms secreted by FRTL-5 cells (see Fig. 9).



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Fig. 9. Transport pathways of lysosomal enzymes in rat thyroid epithelial cells. Schematic drawing summarizing the results on lysosomal enzyme trafficking in FRT or FRTL-5 cells. The occurrence of mature cathepsin B at the surface of thyroid epithelial cells might be explained by extracellular processing of secreted procathepsin B (light green) to the mature enzyme (dark green, left portion, crossed arrows). This report shows that intralysosomal processing was the prerequisite for the retrograde transport of mature cathepsin B from lysosomes to the apical plasma membrane and its subsequent secretion into the extracellular space (dark green, right portion, bold arrow). Extracellularly occurring CB-EGFP was resistant to proteolytic degradation for long time intervals of up to two days, suggesting the stability of secreted lysosomal enzymes, thus explaining their function in the extracellular proteolysis of Tg at the surface of thyroid epithelial cells.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cathepsin B, a lysosomal cysteine protease, is present in endocytic compartments and at the plasma membrane of thyroid epithelial cells (Brix et al., 1996Go) (this study). The biological significance of the extracellular occurrence of cathepsin B is explained by its involvement in extracellular processing of Tg as shown by the liberation of the thyroid hormones T3 and T4 from their precursor molecule (Brix et al., 1996Go; Linke et al., 2002Go) (this study). The occurrence of a mature lysosomal enzyme at the plasma membrane of thyroid epithelial cells assumes its secretion and subsequent reassociation with the cell surface. To further support this assumption, EGFP was used as a tag to study the transport pathways of the lysosomal cysteine protease cathepsin B in rat thyroid epithelial cells. Colocalization and transport studies revealed the entry of the chimeric CB-EGFP into the lumen of the ER, its subsequent transport through the Golgi and the TGN to lysosomes. Fluorescence of CB-EGFP was stable in the acidic milieu of the compartments of the endocytic pathway. Furthermore, the cathepsin B portion of the chimeric protein was proteolytically processed to the mature single chain form of cathepsin B before its secretion, because only one variant of the expected molecular mass was detectable in the supernatants of FRT and FRTL-5 cells. This variant lacked the cathepsin B propeptide, co-migrated with the lysosomal form of the chimeric protein and exhibited proteolytic activity. In addition, the extracellular occurrence of lysosomally matured CB-EGFP was upregulated to a maximum extent after 2 hours of TSH stimulation of FRTL-5 cells. The same time course of TSH stimulated secretion of lysosomal proteins was recently also observed for the secretion of endogenous cathepsin B from non-transfected FRTL-5 or primary porcine thyroid epithelial cells, and it was accompanied by a decrease in the amount of lysosomal single chain cathepsin B (Linke et al., 2002Go). We conclude that endogenous and GFP-tagged lysosomal enzymes are matured within late endosomes or lysosomes before being retrieved into transport vesicles that shuttle the proteolytically active enzymes to the plasma membrane of thyroid epithelial cells (see Fig. 9).

GFP as a tag for studying protein transport
In the past years, GFP has become a commonly used tag for transport studies of various proteins (for reviews, see Cubitt et al., 1995Go; Tsien, 1998Go). Suitable mutations have been established to improve the properties of GFP for its use in mammalian expression systems and in intravital fluorescence microscopy (Tsien, 1998Go). Fusions of GFP and its variants with a large variety of cytosolic, secretory or organelle proteins have allowed studies on the expression, localization, transport and regulation of the GFP-tagged proteins (Chalfie et al., 1994Go; Cubitt et al., 1995Go). Most of the published work pointed out that the chimeric proteins mimicked the properties and dynamics of the endogenous proteins (Cubitt et al., 1995Go). The enormous stability of GFP against denaturing conditions and most proteases as well as its stability over a broad pH range has made this protein a widely used tag for in vivo studies of protein transport (Cubitt et al., 1995Go; Tsien, 1998Go; Lippincott-Schwartz et al., 2001Go).

So far, GFP has been used mostly to tag secretory or cytosolic proteins. Secreted, plasma membrane or lysosomal proteases have been tagged with GFP far less frequently. In trypanosome protozoa, GFP-tagging of the cathepsin L-like protease cruzain demonstrated the necessity of the prodomain for targeting of the chimeras to endocytic compartments (Huete-Perez et al., 1999Go). During bile-salt-induced apoptosis of hepatocytes, a cathepsin-B-GFP construct was used to visualize the entry of cathepsin B into nuclei after its release from lysosomes into the cytosol by an as yet unexplained mechanism (Roberts et al., 1997Go). The rat tissue plasminogen activator, a secretory serine protease, has been tagged with GFP, and the transport and secretion of the chimeric protein was analyzed in transiently expressing PC12 cells exhibiting a neuronal phenotype (Lochner et al., 1998Go). Furthermore, the N-terminal cytoplasmic domain together with the hydrophobic membrane-anchoring domain of rabbit enkephalinase, that is, neutral endopeptidase 24.11, targeted GFP to the surface of transfected cells (Simonova et al., 1999Go). In the latter example, the catalytic domain of the enzyme was not part of the chimeric fusion, whereas the entire protease sequence was GFP-tagged for the study of transport and secretion of tissue plasminogen activator. Similarly, the entire sequence of lysosomal apyrase-like protein of 70 kDa (LALP70) was fused to EGFP, resulting in the targeting of the chimeric protein to autophagosomes or lysosomes (Biederbick et al., 1999Go). This construct differs from ours in that LALP70 is a type IIIb transmembrane protein and in that EGFP was fused to the cytosolic domain of LALP70. The cytosolic membrane-associated phospholipase D1 fused to EGFP was also shown to colocalize with lysosomes (Brown et al., 1998Go). However, the entire construct consisting of phospholipase D1 and EGFP was cytosolic. In clear contrast, our CB-EGFP chimeras are soluble lysosomal proteins exposing the EGFP portion to the lumen of lysosomes.

The plant enzyme papain is one of the few proteases known to digest GFP, and consequently papain digestion was employed to study the chromophor of wild-type GFP from the Pacific Northwest jellyfish Aequorea victoria (Cody et al., 1993Go). Because cathepsin B is a lysosomal cysteine protease of the papain family, we considered possible degradation of the EGFP portion of the chimeric protein CB-EGFP within endocytic compartments. Our results demonstrate, however, that CB-EGFP remains stable over several days and even under optimal conditions for cathepsin B activity, that is, within lysosomes. This conclusion is drawn from the observation that no degradation products of CB-EGFP were detectable with anti-GFP antibodies within fractions of lysosomes isolated from CB-EGFP-expressing FRT cells under steady-state conditions. Some minor amounts of degradation of CB-EGFP occurred, however, within the lysosomes of FRTL-5 cells, and degradation of lysosomal CB-EGFP was also observed in CHO cells. Because the latter expressed high amounts of CB-EGFP, the stability of the chimeric protein within lysosomes seems to be dependent on its expression level. Most importantly, CB-EGFP secreted from thyroid epithelial cells after lysosomal maturation appeared to be stable in the extracellular environment for up to 2 days.

Although it has been reported that the fluorescence of GFP fades away at acidic pH conditions (Cody et al., 1993Go), we have shown here that the green fluorescence of CB-EGFP is detectable within all compartments of the endocytic pathway, thus, ruling out quenching or fading artefacts of CB-EGFP during transport in thyroid epithelial cells.

Physiological importance of lysosomal enzyme secretion from epithelial cells
We have shown here that in rat thyroid epithelial cells CB-EGFP is colocalized with endogenous cathepsin B. Thus, the chimeras and the endogenous protease were transported through the same compartments. Furthermore, the chimeric protein was secreted from thyrocytes in a TSH-stimulated fashion. Because the time courses of TSH-stimulated secretion of both the endogenous protease and the expressed CB-EGFP were comparable, these findings indicate that the chimeric protein reflected the entire transport pathway of the endogenous cathepsin B. However, contrary to our findings, a construct of human cathepsin B and EGFP appeared to be located in a reticular compartment of human breast carcinoma cells or mouse embryonic fibroblasts deficient in cathepsin B, and this chimeric protein was not secreted from either of the cell lines used (Moin et al., 2000Go).

It is as yet unknown whether these differences in intracellular trafficking are characteristic for the different cell types, that is, carcinoma cells or fibroblasts (Moin et al., 2000Go) versus thyroid epithelial cells (this study), or whether species-specific differences of the cathepsin B portion of the chimeras dictate their transport pathways. The latter seems less reasonable because cathepsin B is highly conserved, for example, human and rat cathepsins B share 78% identity in their sequences. Furthermore, human cathepsin B-EGFP seems to be transported in a comparable fashion in both human breast carcinoma cells and mouse embryonic fibroblasts (Moin et al., 2000Go).

We favor the hypothesis that various cell types have developed distinct transport pathways for lysosomal enzymes in order to fulfill their specific functions, for the following reasons. In addition to the preferential expression of altered isoforms of lysosomal enzymes in invasive breast carcinoma cells compared with their normal counterparts (Moin et al., 1999Go), carcinoma cells might utilize alternative pathways of lysosomal enzyme trafficking to enable secretion of the enzymes at the basolateral plasma membrane. These mechanisms of extracellular matrix degradation might facilitate the invasion of the tumor cells (for a review, see Sloane et al., 1990Go). By contrast, normal thyroid epithelial cells use extracellularly occurring lysosomal cysteine proteases, such as cathepsin B, for the proteolysis of Tg before its endocytosis, that is, at the apical cell surface. Thus, the direction of transport of lysosomal enzymes in normal epithelial cells varies from that in carcinoma cells. Similarly, the extracellular function of the secreted enzymes differs under normal conditions compared with pathological conditions.

Thyroid epithelial cells are a proven cellular model system where the secretion of lysosomal enzymes at the apical plasma membrane fulfills an essential task in thyroid physiological function, that is, prohormone processing (Brix et al., 1996Go; Tepel et al., 2000Go; Brix et al., 2001Go; Linke et al., 2002Go). The expression of the chimeric protein CB-EGFP has now shown that the maturation within endosomes/lysosomes is a prerequisite for the secretion of mature lysosomal enzymes from thyroid epithelial cells (see Fig. 9).


    Acknowledgments
 
The authors are grateful to Lucio Nitsch (Naples, Italy) for kindly providing FRT cells, to Bonnie F. Sloane (Detroit, USA) for recombinant human procathepsin B, to Ana Maria Cuervo (Boston, USA) for the antibodies against rat lgp96, and to Lukas Mach (Vienna, Austria) and John S. Mort (Montreal, Canada) for the antibodies against the rat cathepsin B propeptide. We also thank Lukas Mach for discussions and his comments on the manuscript. This study was supported by the Bonner Forum Biomedizin and by grants from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 284, projects B1 (V.H.) and B9 (K.B.).


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 Introduction
 Materials and Methods
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 Discussion
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