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: tomaz.langerholc@ijs.si (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). 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