Tài liệu Báo cáo khoa học: Inhibitory properties of cystatin F and its localization in U937 promonocyte cells ppt

Inhibitory properties of cystatin F and its localization
in U937 promonocyte cells
Tomaz
ˇ
Langerholc
1
, Valentina Zavas
ˇ
nik-Bergant
1
, Boris Turk
1
, Vito Turk
1
, Magnus Abrahamson
2
and Janko Kos
3
1 Department of Biochemistry and Molecular Biology, Joz
ˇ
ef Stefan Institute, Ljubljana, Slovenia
2 Department of Clinical Chemistry, Institute of Laboratory Medicine, University of Lund, Sweden
3 Faculty of Pharmacy, Department of Pharmaceutical Biology, University of Ljubljana, Slovenia
Human papain-like cathepsins were long believed to be
responsible for terminal protein degradation in the
lysosomes. This view changed dramatically when they
were found to be involved in a number of important
cellular processes, such as antigen presentation [1],
bone resorption [2], apoptosis [3] and protein process-
ing [4], as well as several pathologies such as cancer
progression [5], inflammation [6] and neurodegenera-

tion [7]. Their high proteolytic potential, which can be
very harmful, requires the activity of papain-like cath-
epsins to be strictly regulated. Their endogenous pro-
tein inhibitors act as one of the main means of
regulation [8]. The best characterized are the cystatins,
which comprise a superfamily of evolutionarily related
proteins, each consisting of at least one domain of
100–120 amino acid residues with conserved sequence
motifs [8–11]. Type I cystatins (the stefins), stefins A
and B, are cytosolic, % 100 amino acid residue-long
proteins lacking disulfide bridges. Type II cystatins,
cystatins C, D, E ⁄ M, F, S, SA, SN are longer extra-
cellular proteins, consisting of % 120 amino acid resi-
dues and containing two disulfide bridges. Type III
cystatins, the kininogens, are large multifunctional
plasma proteins, containing three type II cystatin-like
domains.
Cystatin F was discovered recently by three inde-
pendent groups. Two of them identified it by cDNA
cloning and named the new inhibitor leukocystatin [12]
Keywords
cathepsin; cysteine protease; inhibition;
cystatin; antigen presentation
Correspondence
T. Langerholc, Department of Biochemistry
and Molecular Biology, Joz
ˇ
ef Stefan
Institute, Ljubljana, Slovenia
Fax: +386 14773984

Tel: +386 14773573
E-mail:
(Received 9 November 2004, revised 31
January 2005, accepted 2 February 2005)
doi:10.1111/j.1742-4658.2005.04594.x
Cystatin F is a recently discovered type II cystatin expressed almost exclu-
sively in immune cells. It is present intracellularly in lysosome-like vesicles,
which suggests a potential role in regulating papain-like cathepsins involved
in antigen presentation. Therefore, interactions of cystatin F with several
of its potential targets, cathepsins F, K, V, S, H, X and C, were studied
in vitro. Cystatin F tightly inhibited cathepsins F, K and V with K
i
values
ranging from 0.17 nm to 0.35 nm, whereas cathepsins S and H were inhib-
ited with 100-fold lower affinities (K
i
% 30 nm). The exopeptidases, cathep-
sins C and X were not inhibited by cystatin F. In order to investigate the
biological significance of the inhibition data, the intracellular localization
of cystatin F and its potential targets, cathepsins B, H, L, S, C and K,
were studied by confocal microscopy in U937 promonocyte cells. Although
vesicular staining was observed for all the enzymes, only cathepsins H and
X were found to be colocalized with the inhibitor. This suggests that cysta-
tin F in U937 cells may function as a regulatory inhibitor of proteolytic
activity of cathepsin H or, more likely, as a protection against cathepsins
misdirected to specific cystatin F containing endosomal ⁄ lysosomal vesicles.
The finding that cystatin F was not colocalized with cystatin C suggests
distinct functions for these two cysteine protease inhibitors in U937 cells.
Abbreviations
mAb, monoclonal antibody; pAb, polyclonal antibody; M6P, mannose-6-phosphate.

FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS 1535
and cystatin F [13]. The third group found overex-
pressed mRNA encoding cystatin F in liver metastatic
tumors and named it cystatin-like metastasis-associated
protein (CMAP) [14]. Cystatin F is an unusual type II
cystatin showing little sequence identity (29–34%) to
other members of the family. Together with cystatin
E ⁄ M it is the only known human glycosylated type II
cystatin. In addition to two disulfide bonds, common
to all type II cystatins, cystatin F contains two addi-
tional cysteines in positions 1 and 37 (cystatin C num-
bering), which were suggested to form an additional
disulfide bond [13]. Cystatin F has been shown to inhi-
bit cathepsin L (EC 3.4.22.15), papain (EC 3.4.22.2)
and legumain (EC 3.4.22.34; K
i
¼ 0.3–10 nm), but not
cathepsin B (EC 3.4.22.1), which was therefore sugges-
ted not to be a physiological target of cystatin F
[13,15].
Given that its expression is restricted to hematopoi-
etic cells [12,13] it is likely that cystatin F is involved
in processes of the immune response. Immunomodula-
tory properties have been demonstrated for another
type II cystatin, cystatin C. The process of dendritic
cell maturation leads to a reduced level and distinct
intracellular distribution of cystatin C, favoring the
activity of cathepsin S and hence efficient Ii chain clea-
vage [16]. In contrast, cystatin F mRNA levels are sig-
nificantly upregulated during dendritic cell maturation

[17]. Immunocytochemical staining of cystatin F in
human promonocyte U937 cells displays a vesicular
pattern [18]. In subcellular fractionation experiments
cystatin F coeluted with the peak of b-hexosaminidase
activity, an enzyme typically located in lysosome-like
organelles. Independently, Journet et al. [19] detected
cystatin F as a soluble protein after affinity puri-
fication of mannose-6-phosphate (M6P) containing
proteins. This means that M6P was present in the
N-linked carbohydrate moiety in cystatin F or, alter-
natively, that cystatin F was in complex with another
M6P containing protein. Nevertheless, despite secre-
tion of cystatin F from U937 cells, a high proportion
seems to reside intracellularly in lysosomes or lyso-
some-like organelles [18].
The aim of our study was to identify potential tar-
gets of cystatin F among endogenous lysosomal cys-
teine proteases. First we found that dimers of cystatin
F are inactive as inhibitors of cysteine proteases and
that the monomeric form has to be restored for the
inhibitory potential. After activation of cystatin F we
have studied the in vitro kinetics of the interaction
between cystatin F and several cathepsins, as well
as their intracellular localization in promonocyte
U937 cells, using specific antibodies and confocal
microscopy.
Results
Activation of cystatin F
Recombinant cystatin F showed one band on
SDS ⁄ PAGE (Fig. 1A) at 17 kDa under reducing con-

A
B
C
Fig. 1. (A) SDS ⁄ PAGE of cystatin F; lane 1, no reduction; lane 2,
reduction with 100 m
M dithiotreitol; ST, molecular weight stand-
ards. (B) Inhibitory activity of cystatin F against papain. Cystatin F
(500 n
M) was incubated 15 min at 37 °C in phosphate buffer
pH 6.0 with different concentrations of dithiotreitol. After dilution,
cystatin F was equilibrated with a twofold molar excess of papain.
The residual activity of papain was measured with Z-FR-AMC. Rel-
ative activity of cystatin F is shown, from 0% (uninhibited enzyme)
to 100% (the lowest activity of the enzyme). Dimerization of cysta-
tin F leads to a loss of inhibitory activity. (C) Immunoblot of cystatin
F run on nondenaturing PAGE. Cystatin F (200 n
M) was incubated
15 min at 37 °C in phosphate buffer pH 6.0 using different concen-
trations of dithiotreitol. Samples were immunoblotted using anti-
human cystatin F polyclonal antibody.
Cystatin F in U937 cells T. Langerholc et al.
1536 FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS
ditions and at 35 kDa under nonreducing conditions.
The latter corresponds to a dimer of cystatin F.
In the absence of reducing agent dithiotreitol, dimeric
cystatin F did not inhibit papain, but its relative activity
(0% for uninhibited enzyme, 100% for the highest inhi-
bition) increased substantially on incubation with 20
and 30 mm dithiotreitol (% 20 and % 100%, respect-
ively). No further changes in relative activity of cystatin

F were observed at dithiotreitol concentrations above
30 mm (Fig. 1B). In addition, the effect of increasing the
concentration of reductant on cystatin F was followed
by electrophoresis under native conditions, where the
transition between 20 and 30 mm dithiotreitol was
accompanied by a shift to a smaller molecular mass
(Fig. 1C). These results suggest that dimerization of
cystatin F is linked to disulfide bond formation, which is
responsible for the loss of inhibitory activity of the pro-
tein. We also noticed that monomerization of cystatin F
was enhanced in acidic environment, especially below
pH 5 (T. Langerholc, unpublished data).
Cystatin F was remarkably stable under reducing
conditions, as no loss of inhibitory activity was
observed even after prolonged incubation at dithiotrei-
tol concentrations as high as 100 mm, and the K
i
value
for the inhibition of papain (K
i
¼ 1.4 nm) was similar
to that reported for inhibition at low dithiotreitol con-
centration (K
i
¼ 1.1 nm) [13].
Based on these results, 100 mm dithiotreitol was
used in the cystatin F activation buffer in order to
ensure total conversion of cystatin F to the active
monomeric state prior to kinetic studies. It should be
noted that, after dilution of cystatin F solution to the

final dithiotreitol concentration of 2.5 mm, which was
used in all subsequent inhibition studies, no dimer for-
mation was observed.
Inhibition of potential target enzymes
in U937 cells
In preliminary experiments, nanomolar to submicro-
molar concentrations of cystatin F were found to be
sufficient to completely abolish the activity of cathep-
sins F (EC 3.4.22.41), K (EC 3.4.22.38), L, V (EC
3.4.22.43), S (EC 3.4.22.27) and H (EC 3.4.22.16).
However, the true exopeptidases cathepsins C
(EC 3.4.14.1) and X (EC 3.4.22 ) were not inhibited
at all, even at the highest concentration of the inhib-
itor (200 nm for cathepsin C and 600 nm for cathep-
sin X, respectively). Therefore, detailed kinetic studies
were performed only with cathepsins F, K, L, V, S
and H.
A linear dependence of the pseudo first order rate
constant k on inhibitor concentration was observed for
all the enzyme–inhibitor pairs investigated, providing
no evidence for a binding model more complex than
the assumed one. The k
ass
and k
diss
values obtained by
linear regression analysis (Table 1) were used for calcu-
lating the K
i
values. The final K

i
values, which were
corrected for substrate competition, are listed in
Table 1. Cystatin F was observed to be a tight binding
inhibitor of cathepsins F, K, L, V, with K
i
values ran-
ging from 0.17 to 0.35 nm. Surprisingly cathepsin S,
despite being an endopeptidase, was inhibited by cysta-
tin F substantially more weakly, with K
i
¼ 33 nm,
comparable to the inhibition of the aminopeptidase
cathepsin H (K
i
¼ 30 nm ). In comparison with other
cystatins, cystatin F is a rather slow binding inhibitor
of the cathepsins, characterized by k
ass
values in the
range of 10
6
)10
7
m
)1
, and high k
diss
values in the
range of 10

)3
to 10
)4
for the tightly inhibited cathep-
sins F, K and V. The reason for the considerably
Table 1. Interaction of cystatin F with cysteine proteases. The experimental conditions and methods are described in the Experimental pro-
cedures section. SD, standard deviation. Data from literature are shown for comparison.
Enzyme
K
i
±SD
[n
M]
10
4
· k
diss
[s
)1
]
10
)6
· k
ass
[M
)1
Æs
)1
] Substrate
Cathepsin F 0.17 ± 0.05 20 ± 4 12 ± 1 Z-FR-AMC

Cathepsin K 0.35 ± 0.15 11 ± 3 3.2 ± 0.6 Z-FR-AMC
Cathepsin V 0.30 ± 0.15 4.8 ± 1.4 1.6 ± 0.3 Z-FR-AMC
Cathepsin S 33 ± 13 3.7 ± 0.7 0.011 ± 0.002 Z-FR-AMC
Cathepsin H 36 ± 15 0.57 ± 0.2 0.0016 ± 0.00024 H-R-AMC
Cathepsin C > 100 H-SY-bNA
Cathepsin X > 100 Dnp-GFFW
Papain 1.4 ± 0.4 3.5 ± 0.6 0.25 ± 0.03 Z-FR-AMC
Cathepsin L
a
0.31 Z-FR-AMC
Legumain
b
10 Z-AAN-AMC
Cathepsin B
a
>1000 Z-FR-AMC
Papain
a
1.1 Z-FR-AMC
a
[13].
b
[15].
T. Langerholc et al. Cystatin F in U937 cells
FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS 1537
lower inhibition constants for cathepsins S and H was
due mainly to the low k
ass
values.
Colocalization of cystatin F and potential target

enzymes in U937 cells
Using confocal immunofluorescence microscopy, vesi-
cular staining of cystatin F was observed. Colocalization
of cystatin F with the lysosomal proteins LAMP-2
(Fig. 2A) and CD68 (Fig. 2B) revealed at least partial
endosomal ⁄ lysosomal localization of cystatin F.
Cathepsins are considered as typical endosomal ⁄
lysosomal enzymes, characterized by a slightly acidic
pH optimum (reviewed in [4]). Cathepsins B, C, H, K,
L, S and X were all found to be expressed in U937
cells. Their amounts varied considerably, as judged by
a semiquantitative approach based on the level of the
fluorescence signal observed and the concentration of
primary antibodies used. However, when subcellular
localization of cystatin F was compared with that of
the cathepsins, cystatin F was found to be colocalized
with cathepsins X and H (Fig. 2C,D), but not with
cathepsins L (Fig. 3A), B, C and K (not shown). The
results for cathepsin S were less clear and showed
partial colocalization of the two proteins (Fig. 3B). As
both primary antibodies for cathepsin F and cystatin
F were of rabbit origin, a different approach was used.
In this approach cathepsin F was tested for possible
colocalization with cathepsin H, but no colocalization
between the two proteases was observed (not shown).
The fact that cathepsin H colocalized with cystatin F,
as described above, suggested that cathepsin F was not
colocalized with cystatin F. Cystatin F was not colo-
calized with cystatin C (Fig. 3C), a typical secreted
type II cystatin.

Discussion
Cystatin F has been known for some years, but its
activity and functional properties have not been com-
pletely determined. However, initial studies revealed an
inhibitory profile that was not typical of other type II
cystatins [13]. Cystatin F, isolated from a baculovirus
expression system, can form disulfide-bonded dimers,
as shown for the inhibitor expressed in Escherichia coli
[12]. This type of dimerization mechanism is different
from general domain-swapping in the cystatin family
[20]. Although both additional cysteines in cystatin F
at positions 1 and 37 (cystatin C numbering) can form
a disulfide bond [13], the cysteine at position 1 has
been suggested to be involved in dimerization of cysta-
tin F [12], similar to cysteine 3 in stefin B [21]. Higher
dithiotreitol concentrations than previously reported
[12] were needed to restore monomers and inhibi-
tory activity under nondenaturing conditions. Loss of
inhibitory potential of dimerized cystatin F can be
explained by blocking of the N-terminal part, disabling
protease access, or by a conformational change result-
ing from disruption of an intramolecular disulfide
bond between cysteines 1 and 37. As dimers have been
observed in U937 cells under physiological conditions
[22], dimerization of cystatin F could be a process
regulating its inhibitory properties.
Screening of proteases for their inhibition showed
that cystatin F is different from other cystatins, both
in terms of specificity and strength of binding to the
target enzyme. Cystatins are generally rather non-

selective inhibitors. An interesting feature of cystatin
F is the 100-fold stronger inhibition of cathepsins F,
L, and V than of cathepsin S. Cathepsin S is closely
related to other endopeptidases of the papain-like
enzyme family, and its crystal structure contains no
pronounced features which would discriminate it from
the related enzymes [23]. Most of our knowledge
about type II cystatins is based on mutagenesis stud-
ies of human cystatin C, where inhibition of cathep-
sin S depends strongly on the Gln55–Gly59 segment
in the first hairpin loop of cystatin C [24]. In con-
trast, substitutions in this wedge-shaped region have
been shown to be of little importance for the inhibi-
tion of cathepsin L [25]. The region Gln55–Gly59 in
cystatin F is the same as in cystatin C, except for an
unfavorable substitution of the nonpolar Ala58 by
Lys. Molecular modeling on the stefin B–papain com-
plex indeed shows steric clashes between Lys58 of
cystatin F and the bulky Tyr18 of cathepsin S. In
contrast, Tyr18 is replaced by the smaller Asn or
Asp in all other known lysosomal cysteine proteases,
indicating easier accommodation of bulky side chains
like that of Lys. This structural feature may in part
contribute to weaker inhibition of cathepsin S by
cystatin F.
The side chain of Val10 in cystatin C, which enters
the S
2
pocket of the cysteine protease, is generally
important for making a strong contribution to the

affinity for cathepsins B, H, L and S [24] and is
replaced by an unfavorable proline in cystatin F, thus
partially explaining the overall lowered affinity of cyst-
atin F for these enzymes. Proline in the S
2
site is a fea-
ture of human stefin A and cystatins F, S and SN, all
of which are significantly less potent inhibitors of cath-
epsin B than cystatin C [9,26]. Unlike Val10, Leu9
which occupies the S
3
pocket in cystatin C is the most
discriminating residue for binding to cathepsins B, H,
L, S [24]. No L9K mutants of cystatin C have been
prepared yet to study the effect of incorporating the
Cystatin F in U937 cells T. Langerholc et al.
1538 FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS
Fig. 2. Immunolabeling of cystatin F in U937 cells, where colocalization was found. Specific monoclonal (mAb) and polyclonal (pAb) antibod-
ies were applied. In all pictures, cystatin F was labeled with primary rabbit anti-(cystatin F) pAb and goat anti-rabbit Alexa Fluorä 488-labeled
secondary antibody (Ab) (green). Red color originates from labeling with: (A) mouse anti-(LAMP-2) mAb and goat anti-mouse Alexa Fluorä
546-labeled secondary Ab; (B) mouse anti-CD68 mAb and goat anti-mouse Alexa Fluorä 546-labeled secondary Ab; (C) mouse anti-(cathepsin
X) 1F12 mAb and goat anti-mouse Alexa Fluorä 546-labeled secondary Ab; (D) sheep anti-(cathepsin H) pAb and donkey anti-sheep Alexa
Fluorä 546-labeled secondary Ab. Before merging the images, signals for red and green fluorescence were adjusted to comparable levels.
The sites of colocalization are shown in yellow.
T. Langerholc et al. Cystatin F in U937 cells
FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS 1539
bulky, charged lysine, which is unique for cystatin F at
this position.
Cystatins are considered to be typical representatives
of the so-called emergency inhibitors. They are present

in large excess over the potential target concentration
and primarily act on escaped proteases by trapping
them rapidly into stable complexes and preventing any
additional proteolysis [8]. Secreted cystatin F, however,
does not fit this classification. Its median concentration
of around 60 pm [27] in pleural fluids is five times
lower than the K
i
value for cathepsin L (K
i
¼ 310 pm).
Cystatin F is a rather slow binding inhibitor, its associ-
ation rate constants for the most strongly inhibited
cathepsins F, K and V being 10–100 times lower than
those for cystatin C with cathepsin L [28]. It is prob-
able that the role of cystatin F in extracellular fluids is
not to neutralize excessive protease activity, at least
not that of known lysosomal cysteine proteases.
Cystatin F is a secreted type II cystatin, although it
is present intracellularly in a much higher proportion
of the total protein than is observed for a typical type
II cystatin C (25% vs. 3–4%, respectively) [18]. The
Fig. 3. Immunolabeling of cystatin F in U937 cells, where weak or no colocalization was found. Specific monoclonal (mAb) and polyclonal
(pAb) antibodies were applied. In all pictures, cystatin F was labeled with primary rabbit anti-(cystatin F) pAb and goat anti-rabbit Alexa
Fluorä 488-labeled secondary antibody (Ab) (green). Red color originates from labeling with: (A) mouse anti-(cathepsin L) N135 mAb and goat
anti-mouse Alexa Fluorä 546-labeled secondary Ab; (B) mouse anti-(cathepsin S) 1E3 mAb and goat anti-mouse Alexa Fluorä 546-labeled
secondary Ab; (C) mouse anti-(cystatin C) 1A2 mAb and goat anti-mouse Alexa Fluor ä 546-labeled secondary Ab. Before merging the ima-
ges, signals for red and green fluorescence were adjusted to comparable levels. The sites of colocalization are shown in yellow. Weak colo-
calization of cystatin F can be observed with cathepsin S, but none with cathepsin L and cystatin C.
Cystatin F in U937 cells T. Langerholc et al.

1540 FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS
fact that cystatin F does not colocalize with cystatin C
in U937 cells suggests different intracellular functions
for these cystatins. Although cystatin C has been sug-
gested to control antigen presentation by regulating
the activity of cathepsin S [16], this view has recently
been challenged [29]. Cystatin F is present predomin-
antly in cells of the immune system, and it would
therefore be expected to be a better candidate to con-
trol activity of cathepsins. Although it is partially
secreted from promonocyte U937 cells, a large propor-
tion resides in the lysosome-like vesicles [18,19]. The
colocalization of cystatin F with LAMP-2 and CD68
shown here confirms these observations, in contrast to
a recent report that cystatin F is not targeted to endo-
somes and lysosomes [22]. A role for cystatin F, invol-
ving a function other than its inhibition of cysteine
proteases, cannot be excluded, as shown for chicken
cystatin [30,31] and cystatin C [32].
If the same criteria that are valid for emergency type
inhibitors were met in lysosomes, proteases would be
inactivated and there would be no proteolyis. Hence
the concept that inhibitors can modulate protease
activity, and not only abolish it [33]. These modulatory
inhibitors are often colocalized with their targets. Cyst-
atin F could be a candidate for modulating the activity
of cathepsin H; in vitro inhibition is tight enough to
impair its activity at concentrations, which can be
found inside the lysosomes [34]. Additionally, such an
inhibitor would have a protective role against misdirec-

ted or prematurely activated cathepsins F, K, L, but
less against cathepsin S, the cysteine protease with the
most important role in invariant chain processing [35].
As the lysosomal cathepsins B, C, F, K, L, S show
minimal or zero colocalization with cystatin F, the lat-
ter might be present in a lysosomal subpopulation,
colocalized with cathepsins H and X. Lysosome-like
organelles are not homogenous, but rather a dynamic
and complex class of vesicles, in which lysosomal cath-
epsins are distributed in a nonrandom manner. Murine
J774 macrophages concentrate cathepsin H in early
endosomes and cathepsin S in late endosomes [36], in
contrast to human B lymphoblastoid cells, where cath-
epsin S is active in all endocytic compartments, while
cathepsins B and X are prominent in early and late
endosomes [37]. Our results suggest that in U937 cells
cathepsins H and X concentrate in different vesicles
from those containing cathepsins B, C, F, K and L,
and that cystatin F is not distributed throughout the
whole endosomal ⁄ lysosomal pathway.
In conclusion, the endosomal ⁄ lysosomal localization
of cystatin F, its restricted and readily regulated expres-
sion [18], selective and not too potent inhibition of
cathepsins are all in favor of the active role of cystatin
F in processes of antigen presentation. Further work to
localize cystatin F in cells other than the model pro-
monocyte U937 cell line is necessary to shed more light
on the biological function of this unusual inhibitor.
Experimental procedures
Materials

trans-Epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-
64) was obtained from the Peptide Research Institute
(Osaka, Japan). Fluorogenic substrates benzyloxycarbonyl-
FR-7-amido-4-methylcoumarin (Z-FR-AMC), R-AMC and
SY-b-naphthylamide (SY-bNA) were purchased from
Bachem (Bubendorf, Switzerland). The specific cathepsin X
substrate 2,4-dinitrophenyl (Dnp)-GFFW-OH [38] was a
gift of L. Juliano (University of Sao Paolo, Brazil). Stock
solutions of substrates were made in dimethysulfoxide
(Merck, Darmstadt, Germany).
Enzymes and inhibitors
Cystatin F was produced in a baculovirus expression system
and purified to homogeneity as described [13]. Papain (2·
crystallized; Sigma, St. Louis, MO, USA) was further puri-
fied by affinity chromatography as described [39]. Human
cathepsins were expressed in E. coli (cathepsin K [40]), in
Pichia pastoris (cathepsin F (M. Fonovic
ˇ
, Jozˇ ef Stefan Insti-
tute, Ljubljana, Slovenia, unpublished data), cathepsin S
(M. Mihelic
ˇ
, Jozˇ ef Stefan Institute, Ljubljana, Slovenia, un-
published data) and cathepsin V [41]) or isolated from spleen
(cathepsin C [42]) or liver (cathepsin X [38]). Cathepsin H
was isolated from porcine spleen [43]. All enzymes were 10%
(cathepsin K) to 100% active (papain) as determined by act-
ive site titration with E-64 or chicken egg white cystatin [44].
Activation of cystatin F
Twenty microliters of 0.1 m phosphate buffer, pH 6.0, con-

taining 500 nm cystatin F and 0–100 mm dithiotreitol (Bio
Vectra, Charlottetown, Canada), was incubated for 15 min
at 37 °C. After dilution to 900 lL, 50 lL of activated
papain was added and the mixture was incubated for an
additional 30 min at 37 °C to equilibrate. Dithiotreitol
concentration in the final solution was 2.5 mm. The final
concentration of papain was twice that of cystatin F (20 nm
vs. 10 nm, respectively). Residual activity of papain was
determined by measurements of Z-FR-AMC hydrolysis.
Electrophoresis and immunoblotting
Samples were separated by SDS ⁄ PAGE under denaturing
conditions using 20% polyacrylamide gels and the Phast-
System apparatus (Pharmacia Biotechnology, Uppsala,
T. Langerholc et al. Cystatin F in U937 cells
FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS 1541
Sweden). Non-denaturing electrophoresis was performed
using the P8DS Penguin
TM
apparatus (Owl Separation
Systems, Portsmouth, NH, USA) in continuous setting,
using 13% polyacrylamide gels. Samples of cystatin F were
incubated for 15 min at 37 °C in phosphate buffer, pH 6.0,
containing 0–100 mm dithiotreitol, before application to the
gel. Electrophoresis was run in 50 mm acetate buffer,
pH 5.3, at 10 mA constant current. After electrophoresis,
proteins were transferred to PVDF membranes (Millipore,
Billerica, MA, USA) by passive diffusion. Non-specific
binding was blocked with 0.4% Tween in NaCl ⁄ P
i
, pH 7.2.

After this and all subsequent steps membranes were washed
with NaCl⁄ P
i
, pH 7.2, containing 0.05% Tween. Mem-
branes were incubated with primary anti-(human cystatin
F) polyclonal antibody [13], followed by goat anti-(rabbit
IgG) secondary antibody (Jackson Immunoresearch Labor-
atory, West Grove, PA, USA). Bands were detected using
0.05% 3,3¢-Diaminobenzidine (Sigma-Aldrich, Steinheim,
Germany) and 0.01% H
2
O
2
in 0.05 m Tris ⁄ HCl buffer,
pH 7.5.
Kinetic measurements
All measurements were performed at 37 °C under pseudo-
first order conditions with at least 10-fold molar excess of
the inhibitor. The following assay buffers were used: 0.1 m
phosphate buffer, pH 5.5 (for cathepsin F), pH 6.0
(papain), pH 6.5 (cathepsin S) or pH 6.8 (cathepsins C and
H), or 0.1 m acetate buffer, pH 5.5, for cathepsins K, V
and X. All buffers contained 2.5 mm EDTA and 0.1%
(w ⁄ v) polyethyleneglycol. In addition, the assay buffer for
cathepsin C contained 0.02 m NaCl. Activating buffers for
all the enzymes consisted of 5 mm dithiotreitol in assay
buffer. The fluorogenic substrates used to measure the
activity of each cathepsin are listed in Table 1. In all experi-
ments the dimethylsulfoxide concentration was less than
2% and the final dithiotreitol concentration was 2.5 mm.

Prior to measurements, cystatin F was incubated for
15 min at 37 °Cin20lL of assay buffer (see above) contain-
ing 100 mm dithiotreitol, followed by the addition of fluoro-
genic substrate to 950 lL. The reaction was then started by
the addition of 50 lL enzyme in activating buffer. The
enzyme concentration varied from 0.03 nm (cathepsin V) to
30 nm (cathepsin X). The release of product was monitored
continuously in a C-61 spectrofluorimeter (Photon Techno-
logy International, Lawrenceville, NJ, USA). The excitation
and emission wavelengths for the AMC substrates were set
to 370 and 460 nm, respectively. Measurement of SY-bNA
was performed at an excitation wavelength of 335 nm and an
emission wavelength of 415 nm, while tryptophan liberation
from Dnp-GFFW was detected at excitation and emission
wavelengths of 280 and 360 nm, respectively.
All the progress curves showed an exponential approach
to a final linear rate and the experimental data was fitted to
the following integrated rate equation [45] by nonlinear
regression analysis using grafit 3.0 software (Erithacus
Software Ltd, Horley, Surrey, UK)
½P¼v
s
Á t þðv
z
À v
s
ÞÁð1 À e
Àk:t
Þ=k ð1Þ
P is the product concentration, v

z
and v
s
are the initial and
steady-state velocities, respectively, and k is the pseudo-first
order rate constant describing the presteady state of the
reaction. Based on previous results (reviewed in [8]) we
assumed a competitive mechanism of inhibition without a
pre-equilibrium step for the interaction between cystatin F
and its potential target proteases. In this mechanism k is
given by the following equation [45]:
k ¼ k
ass
Á½I
0
=ð1 þ½S
0
=K
m
Þþk
diss
ð2Þ
where I
o
is the inhibitor concentration, K
m
is the Michaelis–
Menten constant for the enzyme-substrate pair, S
o
is the

substrate concentration and k
ass
and k
diss
are the associ-
ation and dissociation rate constants, respectively. k was
determined for four to six inhibitor concentrations. Linear
regression analysis of the plot of k (obtained at different I
o
)
vs. I
o
gave k
ass
and k
diss
, and K
i
was calculated as K
i
¼
k
diss
⁄ k
ass
.
Cell culture
The human promonocyte cell line U937 (ATCC no. CRL-
2367) was cultured in RPMI 1660 medium (Life Tech-
nologies, Paisley, UK) with 10% (v ⁄ v) fetal bovine serum

(Hyclone, Logan, USA), in a humidified atmosphere con-
taining 5% (v ⁄ v) CO
2
at 37 °C. For immunolabeling
experiments, 4 · 10
5
cellsÆmL
)1
were grown in fresh culture
medium for 24 h.
Immunofluorescence
For colocalization studies, cystatin F, paired successively
with the cathepsins, cystatin C, LAMP-2 and CD68, was
double immunolabeled. Cells (10
5
) were cytocentrifuged
onto poly(l-lysine) coated slides. Cells were fixed in NaCl ⁄ P
i
,
pH 7.4, containing 4% (v ⁄ v) paraformaldehyde for 30 min
and permeabilized for an additional 10 min in 0.1% (v ⁄ v)
Triton X-100. Non-specific staining was blocked with 3%
BSA (Sigma-Aldrich) and 10% normal serum (Sigma,
St. Louis, MO, USA). Either polyclonal or monoclonal high
affinity primary antibodies were used, and were all tested
for cross-reactivity to other cathepsins or cystatins. The poly-
clonal antibodies were rabbit anti-(human cystatin F) [13],
rabbit anti-(human cathepsin F) (H-110, Santa Cruz Bio-
technology, Santa Cruz, CA, USA) and sheep anti-(human
cathepsin H). The mouse monoclonal antibodies 2A2–1F5,

6G1–1G8, N135 and 1E3 were used against human recom-
binant cathepsins B, K, L and S, respectively. Mouse mono-
clonal antibodies 2A8–3C1–1F9, 1F12 and 1A2 were against
human native cathepsins C, X and cystatin C, respectively.
Cystatin F in U937 cells T. Langerholc et al.
1542 FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS
All monoclonal antibodies were prepared in our laboratory
except mouse anti-(human LAMP-2) (clone H4B4; BD
Pharmingen, San Diego, CA, USA) and mouse anti-(human
CD68) (clone KP1; Dako, Glostrup, Denmark). Primary
antibodies against cystatin F and the protein examined
(cathepsin or cystatin C) were added for 1 h at 37 °C. After
the washing step with NaCl ⁄ P
i
, species specific Alexa
Fluor
TM
-labeled secondary antibodies (Molecular Probes,
Eugene, OR, USA) were added for 1 h at 37 °C. After the
final washing step with NaCl ⁄ P
i
, coverslips were mounted
on the glass slides using ProLongÒ Antifade kit (Molecular
Probes). Control experiments in the absence of primary
antibodies were run in parallel using the same procedure.
The specificity of the antibodies was controlled with prein-
cubation with the antigen as reported [46,47]. Fluorescence
microscopy was performed using a Zeiss LSM 510 confocal
microscope. Alexa Fluor
TM

488 and Alexa Fluor
TM
546
were excited with an argon (488 nm) and He ⁄ Ne (543 nm)
laser, respectively, and emission was filtered using a narrow
band LP 505–530 nm (green fluorescence) and LP 560 nm
(red fluorescence) filter, respectively.
Molecular modeling
Cystatin F was modeled on chicken cystatin (1CEW) with
the program modeller [48]. Models of cystatin F-cathepsin
S and cystatin F-cathepsin L were made with the program
main [49] by fitting modeled cystatin F, human cathepsin S
(1GLO) and human cathepsin L (1ICF) to the structure of
stefin B–papain complex (1STF).
Acknowledgements
The authors thank Marko Fonovic
ˇ
, Mojca Trstenjak-
Prebanda, Sas
ˇ
a Jenko Kokalj, Olga Vasiljeva, Dieter
Bro
¨
mme and Ivica Klemenc
ˇ
ic
ˇ
for kindly providing us
with cathepsins F, K, H, S, V and X. We thank Sas
ˇ

a
Jenko Kokalj for molecular modeling experiments. We
acknowledge Professor Roger Pain for critical reading
of the manuscript. Confocal images were taken at the
Carl Zeiss Reference Center for Confocal Microscopy
(LN-MCP, Institute of Pathophysiology, School of
Medicine) in Ljubljana. This work was supported by
the Ministry of Science and Sport of the Republic of
Slovenia.
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FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS 1545