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
Author for correspondence (e-mail:
k.brix{at}iu-bremen.de)
Accepted 23 September 2002
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Summary |
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Key words: Epithelial cells, Green fluorescent protein, Cathepsin, Lysosome, Thyroglobulin
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Introduction |
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Lysosomal enzymes are also detected extracellularly in certain
physiological conditions (for a review, see
Andrews, 2000). 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.,
1997
). 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, 2000
;
Blott and Griffiths, 2002
).
We have observed, recently, that thyroid epithelial cells secrete lysosomal
enzymes such as the cathepsins B, D and K
(Brix et al., 1996;
Lemansky et al., 1998
;
Tepel et al., 2000
). 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., 2000
;
Brix et al., 2001
).
Furthermore, we have observed that the secretion of lysosomal enzymes from
thyroid epithelial cells is a regulated process
(Linke et al., 2002
).
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,
1979). Tg and receptors for TSH (for a review see,
van de Graaf et al., 2001
) are
expressed by FRTL-5 but not by FRT cells
(Garbi et al., 1987
;
Akamizu et al., 1990
). 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.
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Materials and Methods |
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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., 1994). The primers (underlined) introducing restriction
sites (
) 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,
1988) and PROSITE to search for structural motifs
(Bairoch and Bucher, 1994
;
Falquet et al., 2002
). 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, 1996)].
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., 1996). 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,
1970) and blotted onto nitrocellulose. For the detection of
procathepsin B and cathepsin B, rabbit anti-rat cathepsin B propeptide
(Linke et al., 2002
), 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., 1992
)
essentially as described previously (Rowan
et al., 1992
). 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, 1992) 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.,
2002) 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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Results |
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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., 1992), 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., 1994
), 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., 1986
).
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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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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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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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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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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.
|
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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Discussion |
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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., 1995;
Tsien, 1998
). Suitable
mutations have been established to improve the properties of GFP for its use
in mammalian expression systems and in intravital fluorescence microscopy
(Tsien, 1998
). 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., 1994
;
Cubitt et al., 1995
). Most of
the published work pointed out that the chimeric proteins mimicked the
properties and dynamics of the endogenous proteins
(Cubitt et al., 1995
). 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., 1995
;
Tsien, 1998
;
Lippincott-Schwartz et al.,
2001
).
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., 1999).
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., 1997
). 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., 1998
).
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., 1999
). 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., 1999
). 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., 1998
). 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., 1993). 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.,
1993), 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.,
2000).
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.,
2000) 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., 2000
).
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., 1999), 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., 1990
). 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., 1996;
Tepel et al., 2000
;
Brix et al., 2001
;
Linke et al., 2002
). 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).
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Acknowledgments |
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