Hence this assay can also be used to characterize subcellular fractions using CatE as an endosomal mar-ker, whereas CatD is a well-known lysosomal marker.. For measuring total aspartic p
Trang 1activity in antigen-processing organelles
Nousheen Zaidi1, Timo Herrmann1,5, Daniel Baechle2, Sabine Schleicher3, Jeannette Gogel4,
Christoph Driessen4, Wolfgang Voelter5 and Hubert Kalbacher1,5
1 Medical and Natural Sciences Research Centre, University of Tu¨bingen, Germany
2 PANATecs GmbH, Tu¨bingen, Germany
3 Children’s Hospital Department I, University of Tu¨bingen, Germany
4 Department of Medicine II, University of Tu¨bingen, Germany
5 Interfacultary Institute of Biochemistry, University of Tu¨bingen, Germany
Cathepsin E (CatE; EC 3.4.23.34) and D (CatD; EC
3.4.23.5) are the major intracellular aspartic
protein-ases They have similar enzymatic properties, e.g
susceptibility to various proteinase inhibitors such as
pepstatin A and similar substrate preferences, as
both prefer bulky hydrophobic amino acids at P1
and P1¢ positions [1] In addition, both enzymes
have approximately the same acidic pH optimum
towards various protein substrates such as hemo-globin [2,3]
However, these enzymes have different tissue distri-bution and cellular localization, suggesting that they might have more specific physiological functions CatE
is a nonlysosomal proteinase with a limited distribu-tion in certain cell types, including gastric epithelial cells [4], but is mainly present in cells of the immune
Keywords
antigen-presenting cells; cathepsin D;
cathepsin E; enzyme activity assay;
fluorescent substrate
Correspondence
H Kalbacher, Ob dem Himmelreich 7,
72074 Tu¨bingen, Germany
Fax: +49 7071 294507
Tel: +49 7071 2985212
E-mail: kalbacher@uni-tuebingen.de
Website:
http://www.kalbacher.uni-tuebingen.de
(Received 27 March 2007, revised 24 April
2007, accepted 25 April 2007)
doi:10.1111/j.1742-4658.2007.05846.x
Cathepsin E (CatE) and D (CatD) are the major aspartic proteinases in the endolysosomal pathway They have similar specificity and therefore it is difficult to distinguish between them, as known substrates are not exclu-sively specific for one or the other In this paper we present a substrate-based assay, which is highly relevant for immunological investigations because it detects both CatE and CatD in antigen-processing organelles Therefore it could be used to study the involvement of these proteinases in protein degradation and the processing of invariant chain An assay combi-ning a new monospecific CatE antibody and the substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2 [where MOCAc
is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], is presented This substrate is digested by both proteinases and therefore can be used to detect total aspartic proteinase activity in biological samples After deple-tion of CatE by immunoprecipitadeple-tion, the remaining activity is due to CatD, and the decrease in activity can be assigned to CatE The activity of CatE and CatD in cytosolic, endosomal and lysosomal fractions of B cells, dendritic cells and human keratinocytes was determined The data clearly indicate that CatE activity is mainly located in endosomal compartments, and that of CatD in lysosomal compartments Hence this assay can also be used to characterize subcellular fractions using CatE as an endosomal mar-ker, whereas CatD is a well-known lysosomal marker The highest total aspartic proteinase activity was detected in dendritic cells, and the lowest
in B cells The assay presented exhibits a lower detection limit than com-mon antibody-based methods without lacking the specificity
Abbreviations
CatD, cathepsin D; CatE, cathepsin E; EBV, Epstein–Barr virus; NAG, N-acetyl-b- D -glucosaminidase; TAPA, total aspartic proteinase activity.
Trang 2system, such as macrophages [5], lymphocytes [5],
microglia [6] and dendritic cells [7] It is reported to be
localized in different cellular compartments, such as
plasma membranes [8], endosomal structures [6],
endo-plasmic reticulum and Golgi apparatus [6,9,10] In
contrast, CatD is a typical lysosomal enzyme widely
distributed in almost all mammalian cells [5,9,11,12]
Studies with CatE-deficient and CatD-deficient mice
have provided additional evidence of the association
of these enzymes with different physiological effects
CatD-deficient mice develop massive intestinal necrosis
[13], thromboembolia [13], lymphopenia [13], and
neur-onal ceroid lipofuscinosis [14] CatE-deficient mice are
found to develop atopic dermatitis-like skin lesions
[15] It was reported recently that CatE-deficient mice
show increased susceptibility to bacterial infection
associated with decreased expression of multiple cell
surface Toll-like receptors [16] According to a very
recent study [17], CatE deficiency induces a novel form
of lysosomal storage disorder in which there is an
accumulation of lysosomal membrane
sialoglycopro-teins and an increase in lysosomal pH in macrophages
CatD has also been suggested to play a role in
deter-mining the metastatic potential of several types of
can-cer; high levels of CatD have been found in prostate
[18], breast [19] and ovarian cancer [20] CatE is
expressed in pancreatic ductal adenocarcinoma [21],
and its presence in pancreatic juice is reported to be a
diagnostic marker for this cancer [22] Increased
con-centrations of CatE in neurons and glial cells of aged
rats are suggested to be related to neuronal
degener-ation and re-activdegener-ation of glial cells during the normal
aging process of the brain [23]
CatE and CatD both play an important role in the
MHC class II pathway CatD is reported to be
involved in processing MHC II-associated invariant
chain [24] in antigen processing and presentation
[25,26] CatE is also reported to be involved in antigen
processing by B cells [27,28] microglia [29] and murine
dendritic cells [7]
Several studies have determined the subcellular
localization of CatE and CatD in different cell types,
but there are few reports on the activity of these
enzymes in organelles relevant to antigen-processing
[5,30] Previous reports have described highly selective
substrates for aspartic proteinases, but none of the
substrates described is exclusively specific for CatE or
CatD [30–32] In most of the studies, additional
meth-ods or inhibitors are used to measure the specific
activ-ity of CatE or CatD For example, to specifically
determine CatD activity, a CatD digest and pull-down
assay has been described [30] Other studies have
util-ized a specific inhibitor of CatE, the Ascaris pepsin
inhibitor, which inhibits pepsins and CatE [33], but does not affect other types of aspartic proteinases including CatD [31,34] This inhibitor was originally isolated from the round worm Ascaris lumbricoides [35] However, it is not commercially available
In the present study, CatE and CatD activities were determined in subcellular fractions (lysosomal, endo-somal and cytosolic) of antigen-presenting cells For measuring total aspartic proteinase activity (TAPA) in biological samples, the previously described peptide substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 [where MOCAc is (7-meth-oxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl] [31] was used, which is digested by both CatE and CatD
It is an intramolecularly quenched fluorogenic peptide derivative in which the fluorescent signal of the fluoro-phore MOCAc is quenched by the chromophoric resi-due Dnp After cleavage of the peptide, the quenching efficiency is decreased, resulting in an increase in fluorescence The activity determined in subcellular fractions was completely inhibited by pepstatin A Therefore, this activity can be only attributed to aspar-tic proteinases and represents TAPA For the specific determination of CatE and CatD activity, CatE was specifically depleted by immunoprecipitation The remaining activity is due to CatD, and the decrease in activity is assigned to CatE This approach allows the specific and highly sensitive measurement of both CatE and CatD activities in biological samples
Results and Discussion Expression of CatE mRNA in different cell lines
To determine the expression of CatE at the mRNA level in different cell lines, RT-PCR was performed using RNA extracted from DCs (monocyte-derived human dendritic cells), WT100 [Epstein–Barr virus (EBV)-transformed B-cell line] and HaCaT (immortal-ized human keratinocyte cell line) PCR products from the cell lines were analyzed by gel electrophoresis and found to contain a band of the expected size (241 bp) (Fig 1) As these cell lines were found to be positive for CatE mRNA, they were used to determine the enzymatic activity of CatE and CatD Previous studies have also shown that murine dendritic cells [7] as well
as another EBV-transformed B-cell line (Fc7) are pos-itive for CatE mRNA [27]
Determination of antibody specificity The monospecific antibody for CatE was raised against the antigenic peptide SRFQPSQSSTYSQPG (CatE
Trang 3118–132) This peptide was selected from the CatE
sequence using laser gene software (dnastar,
Madi-son, WI, USA) for antigenicity and surface
probabil-ity blast tool analysis showed that the selected
peptide sequence does not exhibit significant homology
with sequences in CatD or any other known protein,
therefore it is specifically present in CatE Figure 2A
shows sequence alignment of CatE and CatD
The antiserum obtained was further purified by
affinity chromatography on CH-activated Sepharose
containing the peptide SRFQPSQSSTYSQPG
immobi-lized via stable peptide bonds
To determine the specificity and cross-reactivity of
the resulting CatE antibody, indirect ELISA,
competit-ive inhibition ELISA (CI-ELISA) and western blot
analysis were performed
The results of indirect ELISA (Fig 2B) showed that
the antibody specifically recognized CatE and the
anti-genic peptide SRFQPSQSSTYSQPG used to generate
the antibody, and gave a complete negative reaction
towards CatD
CI-ELISA was performed to further enhance the
specificity of the antibody The antibody was
preincu-bated with different concentrations of CatE and CatD,
before a standard ELISA was performed to detect the
antigenic peptide SRFQPSQSSTYSQPG
Preincuba-tion of CatE with the antibody showed a
dose-depend-ent inhibition of antibody binding (IC50¼ 48.6 ng;
Fig 2C) Increasing concentrations of CatD did not
affect antibody binding This experiment shows that
CatE specifically binds to the monospecific antibody in
a free system
Western blot analysis also confirmed that the
mono-specific antibody mono-specifically recognizes CatE and not
CatD (data not shown)
Characterization of subcellular fractions
To control the quality of subcellular fractions, N-acetyl-b-d-glucosaminidase (NAG; EC 3.2.1.52) activity was determined, as it is a wide-spread and well-established marker for endosomal⁄ lysosomal compartments [36] Table 1 shows the activity of NAG in subcellular fractions of different cell lines
As expected, all cell lines showed highest NAG activity in lysosomal fractions with lower activity in endosomal fractions Cytosolic fractions had very low NAG activity
Western blot analysis of subcellular fractions from different cell lines used for CatE and CatD determination
For immunochemical determination of subcellular localization of CatE and CatD, western blot analysis was performed No CatE was recovered from any sub-cellular fraction of WT100 Endosomal fractions of DCs and HaCaT contained a significantly larger amount of CatE than the respective lysosomal frac-tions, but no CatE was found in the cytosolic fractions
of any of the cell lines (Fig 3) As expected, higher amounts of CatD were detectable in lysosomal frac-tions No CatD was detected by western blotting in the cytosolic fraction of any of the three cell types (Fig 3)
Specific inhibition of CatE by immunoprecipitation
To determine the specificity of our immobilized CatE antibody in depleting CatE from the samples, we tes-ted it with CatE and CatD CatE (recombinant) was completely immunoprecipitated by the antibody against CatE (Fig 4A), whereas it had almost no effect on CatD activity (Fig 4B) This approach for depleting proteinase activity from complex biological samples is flexible and can be used for other proteinases
as well
Activity of Cat E and CatD in subcellular fractions
of different cell types The activity of CatE and CatD was determined in subcellular fractions of different cell types using a combination of the peptide substrate, aspartic prote-inase inhibitor (pepstatin A) and depletion of CatE
by immunoprecipitation Activities were determined
by linear regression using a minimum of five measurement points as described in Experimental
CatE (241bp)
M Negative Control DCs HaCaT WT100
Fig 1 CatE expression at mRNA level in different cell lines Total
RNA was extracted from HaCaT, WT100 and DCs Equal amounts
of total RNA (2 lg) from each sample were used for RT-PCR After
reverse transcription, specific primers for human CatE were used
to amplify CatE cDNA.
Trang 4procedures The activity in all subcellular fractions
of these different cell types was completely inhibited
when the samples were preincubated with
pepsta-tin A (TAPA)
For differential measurements of CatE and CatD
activity, samples were subjected to
immunoprecipita-tion of CatE The decrease in activity after
immuno-precipitation is attributed to CatE, and the
remaining activity is assigned to CatD As expected,
the highest CatD activity was determined in the
lyso-somal fractions of all three cell types tested [30] In contrast, CatE activity was mainly detected in endo-somal fractions, as indicated in Table 2 and Fig 5
A low level of CatD activity was determined in endosomal fractions of all three cell types In HaCaT and DCs, a low level of CatE activity was found in lysosomal fractions In the EBV-trans-formed B-cell line (WT100) an almost equal level of CatE and CatD activity was found in the lysosomal fraction, probably because of overlapping subcellular
Fig 2 (A) Sequence alignment of CatE and
CatD The alignment was performed using
a conventional BLAST search engine Only
the small region of CatE containing the
sequence SRFQPSQSSTYSQPG (antigenic
peptide, CatE 118–132, which was used for
generating monospecific antibody) was
included during the BLAST operation
(sequence can be seen underlined in the
figure) This peptide was selected from the
CatE sequence using laser gene software
( DNASTAR , Madison, WI, USA) for
antige-nicity and surface probability BLAST tool
ana-lysis showed that the selected peptide
sequence does not exhibit significant
homol-ogy with sequences in CatD or any other
known protein, therefore it is specifically
present in CatE (B) Determination of
specif-icity of monospecific antibody (raised
against SRFQPSQSSTYSQPG) by indirect
ELISA The purified monospecific antibody
specifically recognized CatE (10 ng) and the
antigenic peptide (SRFQPSQSSTYSQPG),
and gave a complete negative reaction
towards the same amount of CatD (10 ng).
Values are mean ± SD, n ¼ 3 (Insertion:
10 ng CatE and CatD and 1 ng antigenic
peptide were incubated on an ELISA plate.
CatE and CatD antibodies were used for the
detection at dilutions of 1 : 10000 and
1 : 5000.) (C) Competitive inhibition of
anti-body (raised against SRFQPSQSSTYSQPG)
binding to SRFQPSQSSTYSQPG-coated
plates by CatE Immunoplates were
coated with antigenic peptide
(SRFQPSQSSTYSQPG; 0.1 lg ⁄ well).
Monospesific antibodies were preincubated
with different concentrations of CatE or
CatD, before standard ELISA ELISA was
performed as described in Experimental
pro-cedures The increasing concentration of
CatE caused inhibition of antibody binding
giving the IC50value of 48.6 ng The same
concentrations of CatD had no effect
on antibody binding Data points are
mean ± SD, n ¼ 2.
Trang 5fractions Cytosolic fractions of all three cell types
showed very low CatE activity, and no CatD
activ-ity Moreover, the overall activity in the subcellular
fractions of the three cell types tested varied
substan-tially, as did CatE and CatD activity DCs showed
the highest, and WT100 cells, the lowest overall
activity
As shown in Table 2, endosomal fractions of
HaCaT showed 5.5-fold higher CatE activity than
the corresponding fractions of WT100, whereas
endo-somal fractions of DCs showed 19 times higher CatE
activity than the endosomal fractions of WT100
Table 2 also shows that the lysosomal fraction of
HaCaT had 7.2 times higher CatD activity than the
corresponding fraction of WT100, and this subcellular
fraction from DCs had 16.6 times higher CatD
activ-ity than that from WT100
Analysis of peptide fragments obtained by digestion of the fluorogenic substrate with subcellular fractions, CatE or CatD, using RP-HPLC and MALDI-MS
To further confirm that the activity measured in the subcellular fractions by the fluorescence assay was only due to aspartic proteinases, the peptide substrate was digested by CatE, CatD or subcellular fractions (as described in Experimental procedures) The peptide fragments thus generated were separated by RP-HPLC using fluorescence detection (kex¼ 350, kem ¼ 450) and identified by MALDI-MS (Table 3) This method allowed detection of only N-terminal fragments con-taining the fluorophore MOCAc
Figure 6A shows the chromatogram of the
undigest-ed peptide substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2 as a negative control The fluorescence signal is quenched as a result
of resonance energy transfer between the fluorophore
Table 1 NAG activity (fluorescence per min per lg protein) in
sub-cellular fractions of different cell lines Activities were determined
by linear regression analysis taking at least seven measurement
points Values are mean ± SD (n ¼ 3).
Fig 3 CatE and CatD expression at protein level in relevant
anti-gen-processing organelles of different cell lines Equal amounts of
total protein (50 lg) from each sample were applied for SDS ⁄ PAGE
followed by western blot analysis Representative immunoblots
with the monospecific CatE antibody and reprobe of the same blot
with the CatD antibody are shown C, Cytosolic fraction; L,
lyso-somal fraction; E, endolyso-somal fraction.
Fig 4 Effect of immunoprecipitation of CatE and pepstatin A treat-ment on (A) CatE and (B) CatD activities (A) (j) Hydrolysis of the fluorogenic peptide substrate (1 l M ) by 10 ng CatE in 50 m M sodium acetate buffer (pH 4) at 37 C (m) Incubation with pepsta-tin A for 15 min at 37 C before hydrolysis reaction inhibited the activity of CatE completely (d) immunoprecipitation of CatE before hydrolysis reaction also completely inhibited the activity of CatE (B) (j) Hydrolysis of the fluorogenic peptide substrate (1 l M ) by
10 ng CatD in 50 m M sodium acetate buffer (pH 4) at 37 C (m) Incubation with pepstatin A for 15 min at 37 C before hydrolysis reaction inhibited the activity of CatD completely (d) immunopre-cipitation of CatE before hydrolysis has no effect on CatD activity, hence immunoprecipitation was specific for CatE only.
Trang 6and the quencher group Figure 6B shows the results
of digestion of the substrate with CatE, leading to only
one cleavage product, because only the Phe-Phe bond
is susceptible to cleavage by CatE or CatD [31] The
peak with a retention time of 25.54 min corresponds
to the fragment, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe, as
analyzed by MALDI-MS (Table 3) Figure 6C shows
digestion of the substrate with CatD, giving a profile
similar to that of CatE, i.e only one peak is visible
with the same retention time However, when digested
with the lysosomal fraction of HaCaT (Fig 6E), an
additional peak with a retention time of 22.87 min was
observed Digestion of substrate with the endosomal
fraction of HaCaT (Fig 6F) gave a similar RP-HPLC
profile to the lysosomal fraction
Digestion of the substrate with lysosomal and
endo-somal fractions (Fig 6H,I) was completely inhibited
by pepstatin A, confirming that the activity observed
in our assay was solely due to aspartic proteinases
The additional peak observed after digestion of the
substrate with these fractions (Fig 6E,F) was a
C-ter-minal-truncated peptide
(MOCAc-Gly-Lys-Pro-Ile-Leu), as analyzed by MALDI-MS This carboxypeptidase
activity can only occur after aspartic proteinases have
created cleavage products, as the undigested substrate
contains a protective d-Arg residue at the C-terminus
Substrate digestion by the lysosomal fraction
(Fig 6K) after immunoprecipitation of CatE had almost
no effect on the RP-HPLC profile This indicates that
the activity observed in the lysosomal fraction was
mainly due to CatD Digestion by the endosomal
frac-tion (Fig 6L) was inhibited after immunoprecipitafrac-tion
of CatE, indicating that the activity in this fraction was
primarily CatE activity No cleavage was indicated in the
cytosolic fraction, hence no CatE or CatD activity was
observed by RP-HPLC This agrees with the results from
the fluorescence assay, in which only very low activity
was determined in the cytosolic fraction Digestion of
substrate with subcellular fractions of DCs and WT100 gave similar RP-HPLC profiles (data not shown)
In conclusion, the combination of methods described here facilitates the specific and parallel measurement of CatE and CatD activity in antigen-processing organ-elles The data clearly show that our approach for detecting CatE and CatD is more sensitive than immu-nodetection by western blot analysis It allows detec-tion of CatE activity in subcellular fracdetec-tions of WT100, as compared to western blot analysis by which
no CatE was detectable in any WT100 fraction It was also possible to discriminate between CatD activity in endosomal and lysosomal fractions, whereas the distri-bution of CatD in lysosomal and endosomal fractions was not significantly distinguishable when detected by western blot
Theses experimental conditions are also more
speci-fic than previous assays, because specispeci-ficity of detec-tion was not only based on the peptide sequence but was markedly increased by the use of a monospecific antibody used to deplete CatE This type of assay is flexible and can be used to discriminate activity of other proteinases with similar enzymatic properties This approach distinguishes between the activities of the enzymatically similar proteinases, CatE and CatD, and can therefore be used to investigate the involvement
of these enzymes in antigen processing and presentation
Experimental procedures Enzymes and chemicals
CatD (bovine kidney) was purchased from Calbiochem
CatE was purchased from R&D systems (Wiesbaden,
50 mm sodium citrate buffer, pH 6.5, containing 150 mm
Table 2 CatE and CatD activity (pmol MOCAc liberated per min per 20 lg total protein) in subcellular fractions of different cell lines Activit-ies were determined by linear regression analysis taking at least five measurement points Values are mean ± SD (DCs, n ¼ 2; HaCaT and WT100, n ¼ 3; where n is the number of individual experiments performed) ND, not detectable.
Trang 7NaCl at)20 C Pepstatin A (Calbiochem) was dissolved in
methanol Activated CH Sepharose 4B was purchased from
Amersham Biosciences (Munich, Germany) The substrate
Germany)
Generation and immobilization of a monospecific
CatE antibody
The antigenic peptide SRFQPSQSSTYSQPG (CatE 118–
132) was selected from the protein sequence using the laser
controlled for specificity to CatD It was synthesized as a single peptide and as a multiple antigen peptide,
Syro II (MultiSynTech, Witten, Germany) The peptides were purified using RP-HPLC and the identity was con-firmed using ESI-MS Peptide purities were determined by analytical RP-HPLC and were >90% The single peptide was coupled to key hole limpet hemocycanin using the glu-tardialdehyde method The antiserum was obtained after repeated immunization of a rabbit with a 1 : 1 mixture of the peptide–key hole limpet hemocycanin conjugate and the multiple antigen peptide This antiserum was further puri-fied by affinity chromatography on a CH-activated Seph-arose 4B column (Amersham Biosciences) containing the peptide immobilized via a stable peptide bond Peptide immobilization was performed as described by the manu-facturer The antiserum was applied to the column at
Technologies, Paisley, UK) Elution was performed with 10
membrane The resulting antibody was retested by ELISA and showed the expected specificity to the peptide epitopes and the CatE protein, but a completely negative reaction to CatD The purified monospecific antibody was immobilized
on CH-activated Sepharose as described by the manufac-turer After coupling for 3 h at room temperature, the gel
addi-tional 2 h at room temperature To block any remaining active sites, the material was further incubated with 5%
ELISA The wells of microtiter plates (Nunc Brand Products, Maxi-Sorb surface, Wiesbaden, Germany) were coated with CatE (10 ng), CatD (10 ng) or the peptide SRFQPSQSSTYSQPG
overnight The plates were washed three times with 200 lL
Tween 20, pH 7.0, containing 0.5% BSA) or commercial CatD antibody After a wash, the plates were incubated with horseradish peroxidase-conjugated goat anti-rabbit Ig
Endosomes
Lysosomes
Cytosol
Endosomes
Lysosomes
Cytosol
Endosomes
Lysosomes
Cytosol
pmol MOCAc liberated/min/20µg total protein
pmol MOCAc liberated/min/20µg total protein
pmol MOCAc liberated/min/20µg total protein
TAPA CatE activity CaD activity
TAPA CatE activity CaD activity
TAPA CatE activity CaD activity
A
B
C
Fig 5 Distribution of TAPA, CatE and CatD activity in subcellular
fractions of the cell lines (A) HaCaT, (B) WT100 and (C) DCs Equal
amounts of total protein (20 lg) were used for the determination of
CatE and CatD activities, determined by linear regression analysis
using a minimum of five measurement points Values are
mean ± SD (DCs, n ¼ 2; HaCaT and WT100, n ¼ 3; where n is the
number of individual experiments).
Trang 8(100 mm sodium citrate buffer, pH 4.5) was added per well,
and the colour development analyzed at a wavelength of
405 nm
For competitive inhibition ELISA, antiserum was
prein-cubated with different concentrations of CatE or CatD
(40 min, room temperature) and then used as primary
anti-body for standard ELISA to detect the antigenic peptide
Cell culture
The EBV-transformed human B-cell line, WT100, and the
immortalized human keratinocyte cell line, HaCaT, were
cultured in RPMI 1640 medium (Gibco Life
Technol-ogies) supplemented with 10% (v⁄ v) heat-inactivated fetal
(Nunc)
Peripheral blood mononuclear cells were isolated by
den-sity gradient centrifugation of heparinized blood obtained
from buffy coats Isolated peripheral blood mononuclear
Cellstar tissue culture flasks (Greiner Bio-One GmbH,
Fric-kenhausen, Germany) in RPMI 1640 under the same
cul-ture conditions as for WT100 and HaCaT After 1.5 h of
adherent cells were cultured in complete culture medium
colony-stimu-lating factor (Leukomax; Sandoz, Basel, Switzerland) and
interleukin 4 (R&D systems) for 6 days as described
previ-ously [38] This resulted in a cell population consisting of
70% DCs (data not shown), as determined by flow
cytometry (BD FACSCalibur, Heidelberg, Germany)
Determination of CatE mRNA expression levels
using RT-PCR
RNA was extracted from DCs, WT100 and HaCaT cells
using the TRIazol reagent as described by the manufacturer
(Invitrogen, Karlsruhe, Germany) Reverse transcription of
2 lg total RNA was initialized by 200 U Superscript II reverse transcriptase (Invitrogen), 4 lL synthesis buffer (fivefold concentrated; Invitrogen), 2.5 lL Random Primers (10 mm; Promega, Mannheim, Germany), 1 lL dithiothrei-tol (100 mm; Invitrogen), 1 lL dNTP mix (10 mm; Prome-ga) and 0.5 lL rRNAsin (PromeProme-ga) in a final volume of
20 lL After incubation at room temperature for 10 min,
amplification was carried out, adding 5 lL generated
(10 mm; Promega) in molecular-grade water and 1.1%
Single PCR amplicons were analysed using agarose gel electrophoresis
Subcellular fractionation and western blot analysis
Cell fractionation was performed as previously described by
homo-genized using a cell cracker (HGM Laboratory Equipment, Heidelberg, Germany) Then debris was separated by cen-trifugation at 8000 g for 10 min with a Minifuge RF 2150 (Heraeus, Osterode, Germany) Mitochondria and the endolysosomal fractions were separated by ultracentrifuga-tion at 100 000 g for 5 min (Beckman TL100 ultracentri-fuge, Palo Alto, CA, USA) Finally, lysosomes were separated from endosomes by hypotonic lysis with double-distilled water ( 2.5-fold of the pellet volume for keratino-cytes and DCs, and fivefold of the pellet volume for B cells) and centrifugation at 100 000 g for 5 min with a Beckman TL100 ultracentrifuge Lysosomal material was released into the supernatant, and endosomes remained in the pellet Total protein content was determined as described
by Bradford [40]
Table 3 Peptides after digestion of fluorogenic peptide substrate by CatE, CatD and subcellular fractions of HaCaT identified by MALDI-MS Retention times allude to those in Fig 6.
Retention time (min)
Expected mass
Trang 9Subcellular fractions were separated by SDS⁄ PAGE
(50 lg total protein per lane) on a 12% separating gel
and transferred to a poly(vinylidene difluoride) membrane
(Amersham Biosciences, Freiburg, Germany) Membranes
Germany) Rabbit antibody to human CatD
(Calbio-chem) was diluted 1 : 5000, and rabbit antibody to
human CatE was diluted 1 : 2000 Western blots were
developed according to the ECL protocol of Amersham
Biosciences
Detection of NAG activity NAG activity was measured as described by Schmid et al [36] Briefly, 1 lg protein from each fraction was added to
100 lL 0.1 m citrate buffer, pH 5, containing 0.8 mm
Deisenhofen Germany) and 0.1% Triton X-100
Fluor, Crailsheim, Germany) NAG activity was deter-mined by linear regression using a minimum of seven meas-urement points
A Undigested substrate B Substrate + CatE C Substrate + CatD
25.54
5 10 15 20 25 30 35
25.19
5 10 15 20 25 30 35
5
10
15
20
25
30
35
5
10
15
20
25
30
35
5
10
15
20
25
30
35
5 10 15 20 25 30 35
22.87 25.90
5 10 15 20 25 30 35
22.38 25.44
5 10 15 20 25 30 35
22.91 25.92
5 10 15 20 25 30 35
5 10 15 20 25 30 35
5
10
15
20
25
30
35
D Substrate + CF E Substrate + LF F Substrate + EF
G Substrate + CF + PepA H Substrate + LF + PepA I Substrate + EF + PepA
J Substrate + CF (IP) K Substrate + LF (IP) L Substrate + EF (IP)
5 10 15 20 25 30 35
Fig 6 RP-HPLC profiles of peptide fragments obtained after digestion of the substrate with CatE, CatD or subcellular fractions of HaCaT Fluorogenic peptide substrate (10 l M ) was incubated at 37 C in digestion buffer (50 m M sodium acetate buffer, pH 4.0) containing CatE (10 ng), CatD (10 ng) or a subcellular fraction (20 lg) (A) Undigested fluorogenic substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)- D -Arg-NH2 Substrate digested with (B) CatE, (C) CatD, (D) cytosolic fraction (CF), (E) lysosomal fraction (LF), (F) endosomal fraction (EF), (G) cytosolic fraction after pepstatin A treatment, (H) lysosomal fraction after pepstatin A treatment, (I) endosomal fraction after pepsta-tin A treatment, (J) cytosolic fraction after immunoprecipitation of CatE, (K) lysosomal fraction after immunoprecipitation of CatE, and (L) endosomal fraction after immunoprecipitation of CatE.
Trang 10Parallel detection of CatE and CatD activity
TAPA and specific catalytic activities of CatE and CatD
were determined fluorimetrically by hydrolysis of the
CatD or subcellular fraction (20 lg total protein) were
added to 80 lL digestion buffer (50 mm sodium acetate
buffer, pH 4.0), and the reaction was started by the
addi-tion of 1 lL substrate soluaddi-tion (stock soluaddi-tion 2 mm in
dimethyl sulfoxide) Fluorescent product formation was
recorded using a fluorescence reader (Tecan Spectra Fluor)
were determined by linear regression analysis using a
mini-mum of five measurement points All the experiments were
performed in triplicate yielding TAPA, i.e CatE and CatD
activity Aspartic proteinase activity could be completely
inhibited using 1 lL 1 mm pepstatin A solution in
meth-anol (1 lL methmeth-anol showed no inhibitory effect)
For the specific determination of CatE activity, samples
were subjected to immunoprecipitation of CatE before the
above assay Then 20 lg total protein from each subcellular
fraction was incubated with 20 lL monospecific CatE
fluorescence intensity is exclusively caused by CatD The
difference between total aspartic proteinase and CatD
activ-ity can be assigned to CatE activactiv-ity
Analytical RP-HPLC
The fluorogenic peptide substrate (1 mm in dimethyl
(50 mm sodium acetate buffer, pH 4.0) containing the
appro-priate amount of CatE, CatD or a subcellular fraction (with
or without pepstatin A treatment or after
immunoprecipita-tion of CatE) The reacimmunoprecipita-tion was terminated by addiimmunoprecipita-tion
of 25 lL stop solution [5% (v⁄ v) acetonitrile, 1% (v ⁄ v)
tri-fluoroacetic acid] in water Then 5 lL of the reaction mixture
was separated by analytical RP-HPLC using a C8 column
Germany) with the following solvent systems: (A) 0.055%
was performed using a linear gradient from 5% to 80%
sol-vent B within 35 min Fluorescence detection was carried out
collected and analysed by MALDI-MS
MALDI-MS
First, 0.5 lL each RP-HPLC fraction was mixed with
trifluoroacetic acid] and applied to a gold target for MALDI-MS using a MALDI-TOF system (Reflex IV, serial number 26159.00007; Bruker Daltonics, Bremen, Ger-many) Signals were generated by accumulating 120–210 laser shots Raw data were analyzed using the software Flex Analysis 2.4 (Bruker Daltonics)
Acknowledgements
We gratefully acknowledge Andreas Dittmar and Flo-rian Kramer for their technical assistance This work was supported by Deutsche Forschungsgemeinschaft (SFB 685), Higher Education Commission Pakistan, and German Academic Exchange Service (DAAD), Germany
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