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Substrate specificity of human matriptase-2
Biochimie 97 (2014) 121e127
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Research paper
Substrate specificity of human matriptase-2 M. Wysocka a, N. Gruba a, A. Miecznikowska a, J. Popow-Stellmaszyk a, M. Gütschow b, M. Stirnberg b, N. Furtmann b, c, J. Bajorath c, A. Lesner a, *, K. Rolka a a
Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk, Poland Pharmaceutical Institute, Rheinische Friedrich-Wilhelms-Universität, An der Immenburg 4, 53121 Bonn, Germany c Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität, Dahlmannstr. 2, 53113 Bonn, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2013 Accepted 1 October 2013 Available online 22 October 2013
Human matriptase-2 is an enzyme that belongs to the family of type II transmembrane serine proteases. So far there is a limited knowledge regarding its specificity and protein substrate(s). One of the identified natural substrates is hemojuvelin, a protein involved in the control of iron homeostasis. In this work, we describe the synthesis and evaluation of internal quenched substrates using a combinatorial approach. The iterative deconvolution of two libraries to define the specificity of matriptase-2 yielded to the identification of the substrate ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 with a kcat/Km value of 4.5 105 M1 s1, i.e. the highest specificity constant reported so far for matriptase-2. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Matriptase-2 Combinatorial chemistry Fluorogenic substrates Internally quenched substrates
1. Introduction Matriptase-2 (MT-2, also referred to as TMPRSS6) belongs to the class of type II transmembrane serine proteases (TTSPs) [1] located at the plasma membrane with a C-terminal protease domain directed to the extracellular space and a stem region with domains of different types, between the transmembrane domain near the N-terminus and the protease domain [2]. Since mutations in MT-2 have been linked to iron-refractory iron deficiency anemia (IRIDA), MT-2 was identified as a regulatory protease controlling iron homeostasis [3,4]. MT-2 was shown to cleave the BMP (bone morphogenetic protein) co-receptor hemojuvelin [5] and thereby to downregulate the expression of hepcidin, the systemic iron regulatory peptide hormone. The cleavage site within hemojuvelin targeted by MT-2 was determined [6]. So far, hemojuvelin represents the only known putative substrate of MT-2 beside the protease itself. In a transfected cell system, MT-2 is cleaved at two shedding sites within the stem region to yield a released form and at the activation site at the junction of the protease domain [7]. From these four reported processing sites an attempt was made to draw a conclusion about the substrate specificity of MT-2 [8]. Recently, the Kunitz-type serine protease inhibitor HAI-2 (hepatocyte growth factor activator inhibitor type 2) has been shown to inhibit the proteolytic activity of MT-2 [9]. Since Kunitz-type
* Corresponding author. Tel.: þ48 585235095; fax: þ48 585235472. E-mail addresses: [email protected], [email protected] (A. Lesner). 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.10.001
inhibitors bind to their target proteases in a substrate-like manner [10], HAI-2 can also be considered to bear a processing site attacked by MT-2. For instance, Kunitz domain I exhibited two critical Arg residues which might be recognized by MT-2. As a typical trypsin-like serine protease, MT-2 bears a conserved Asp located at the bottom of the S1 pocket of the active site determining the primary substrate specificity to cleave behind positively charged residues like Arg and Lys [11]. Recently, a set of FRET peptides was used to define the substrate specificity of matriptase, a TTSP closely related to MT-2. Other members of transmembrane serine proteases, such as MT-2, hepsin, and DESC1, a human airway trypsin like serine protease were also investigated in this study [12]. There is, however, a strong need to elucidate the substrate specificity of MT-2 more in detail, in particular with respect to the identification of further endogenous substrates and to the structural optimization of peptide-derived inhibitors for MT-2. Such compounds are expected to be of great value as pharmacological tools and, even more, as potential therapeutic agents for the treatment of iron overload diseases and b-thalassemia [8,13,14]. 2. Materials and methods 2.1. Peptide synthesis The peptide libraries and individual peptides were synthesized manually by the solid-phase method applying Fmoc chemistry,
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as described previously [15]. TentaGel S RAM (substitution 0.25 meq/g) (RAPP Polymere, Germany) was used as a solid support. The a-amino groups of the amino acids were Fmoc-protected and these amino acids were attached to the resin using N,N’-diisopropylcarbodiimide (DIPCI) and N-hydroxybenzotriazole (HOBt). The synthesis of the ACC-based library was initiated by deprotection of the amino groups of the resin with 20% piperidine in the mixture of DMF/N-methyl-2-pyrrolidone (NMP) (1:1, v/v) and coupling of Fmoc-7-aminocoumarin-4-acetic acid (Fmoc-ACC) using the N,N,N0 ,N0 -tetramethyl-O-(benzotriazol-1-yl)uranium tetrafluoroborate (TBTU) and HOBt [16]. Briefly, two equiv of Fmoc-ACC and two equiv of TBTU/HOBt were dissolved in 5 ml DMF and the solution was added to the resin. After 30 s, four equiv of N,N-diisopropylethylamine (DIPEA) were added and the mixture was stirred for 5 h. The solution was filtered off and the resin was washed with DMF. The procedure was repeated three times. Next, the whole resin was incubated with five-fold molar excess of N-acetylimidazole in DMF to cap the remaining amino groups. After deprotection with 20% piperidine in a mixture of DMF/NMP (1:1, v/v), the peptide chain was elongated as follows. A mixture of the N-protected amino acid derivative, DIPCI and HOBt (molar ratio, 1:1:1) was dissolved in DMF:NMP solution (1:1, v/v) and added to the resin. A three-fold excess over the resin active sites was used. The library of the internally quenched ABZ/Tyr(3-NO2) peptides was similarly synthesized as described [15], also on TentaGel S RAM resin, starting with of Fmoc-protected 3-nitrotyrosine. After completing the synthesis, the peptides were cleaved from the resin using a TFA/phenol/triisopropylsilane/H2O mixture (88:5:2:5, v/v) [17]. Purity of the peptides was checked on RP-HPLC Pro Star system (Varian, Australia) equipped with a Kromasil 100 C8 column (8 250 mm) (Knauer, Germany) and a UVeVIS detector. A linear gradient from 20 to 80% B within 40 min was applied (A: 0.1% TFA; B: 80% acetonitrile in A). The peptides were monitored at 226 nm. Mass spectra of the synthesized peptides were recorded using a Biflex III MALDI TOF mass spectrometer (Bruker, Germany) and a-cyano-4-hydroxycinnamic acid as a matrix.
fluorescent method with peptides solution at concentration: of 1 mg/ml (1.6 107 M) (ACC-based library) and 3 mg/ml (2.76 107 M) for the internally quenched library, respectively. Stock solutions of the peptides were prepared in DMSO. All measurements were performed using the Omega plate reader (BMG, Germany) with an excitation wavelength of 360 nm and emission wavelength 450 nm for ACC-based library, while for the internally quenched library wavelengths of 320 nm and 450 nm were used. Enzymatic hydrolysis of the peptide was performed in 50 mM Trise HCl (pH 8.0) buffer with 150 mM NaCl at 37 C and followed over 30 min. 2.4. Determination of the matriptase-2 concentration using a ‘burst’ substrate The concentration of MT-2 was determined by spectrophotometric titration with 4-nitrophenyl-40 -guanidinobenzoate (NPGB) [21]. Briefly, 10 ml of an NPGB solution (3.36 mg/ml) was added into 1500 ml of veronal buffer (0.1 M with 20 mM CaCl2, pH 8.3) followed by adding 20 ml of the conditioned medium of transfected HEK cells, or purified enzyme solution, respectively. 2.5. Determination of KM and kcat Assay conditions for the determination of the Michaelis constants (KM) and catalytic constants (kcat) were as noted above. The specificity constants (kcat/KM) were calculated from kcat and KM values. Measurements were performed with an enzyme concentration of 2.8 109 M. Three to five measurements were carried out for each compound (systematic error expressed as a standard deviation never exceeded 20%). The calculated initial hydrolysis rates were used as a measure of substrate activity of the investigated peptides. All details of kinetic studies and the method of calculating kinetic parameters have been described elsewhere [22]. 2.6. Determination of the proteolytic cleavage pattern
2.2. Preparation of the peptide libraries The peptide libraries were synthesized by the portioningmixing method [18,19]. Initially, 17.1 g of the solid support (TentaGel S RAM) was used for the first library, i.e. X4-X3-X2-X1-ACC-NH2. A three-fold molar excess of the amino acid was used for the coupling. The second library, i.e. ABZ-Ile-Arg-Ala-Arg-X10 -X20 -X30 Tyr(NO2)-NH2, was synthesized on 15.2 g of the 2-chloro-chlorotrityl resin. The procedure for peptide chain elongation and the other synthetic methods employed were as described above. 2.3. Initial screening of the libraries For the enzymatic studies, MT-2 from the conditioned medium of transfected HEK cells was employed as described [7,11,20]. Briefly, the whole MT-2 construct was cloned and expressed in HEK cells with c-Myc and His6 tags at the C-terminal end of the protein. For comparison, conditioned medium of HEK cells expressing the empty vector (HEK-mock) was also used. For certain experiments, MT-2 was purified from the concentrated conditioned medium of transfected HEK cells by immunoaffinity chromatography as described [11]. MT-2-containing 10 ml-aliquots of a protein concentration of 0.7 mg/ml were applied. The enzyme concentration was determined as described below. The concentration of MT-2 used for the deconvolution of the ACC-based library ranged from 1 109 to 3.4 1010 M. For the internally quenched library, the enzyme concentration was lower, between 1.2 1010 and 5.2 1011 M. Deconvolution of the sublibraries was performed by applying the
Selected substrates were mixed with a two-fold molar excess of the enzyme in a buffer used for kinetic studies. HPLC analysis of this mixture was performed after the following incubation times: 0, 15 min and 24 h. The eluting fractions of the corresponding peaks were collected and analyzed by MALDI-TOF MS. 2.7. Molecular docking Because the X-ray structure of matriptase-2 is not available, a previously described comparative model of matriptase-2 [11] was used as template for flexible ligand docking with AutoDock 4.2 [23] to explore possible substrate binding modes. The structure of I was reduced to the optimized peptide substrate sequence H-Ile-Arg-Ala-Arg-Ser-Ala-Gly-OH to limit the number of flexible torsion angles (degrees of freedom) for flexible ligand docking. Hydrogen atoms were added to the model and the peptide substrate with the Molecular Operating Environment (MOE 2012.10) [24] and atomic partial charges were calculated using AutoDock Tools [23]. AutoDock standard docking parameter settings [23] were applied, except that the total number of genetic algorithm iterations for conformational sampling was increased, given the inherent flexibility of the peptide substrate. Plausible binding poses were selected with the aid of visual inspection taking prior knowledge of substrate and inhibitor binding characteristics into account [25]. A preferred docking pose was further adjusted by interactive model building and energy minimizations with MOE 2012.10.
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3. Results To systematically investigate the substrate specificity of MT-2 we decided to utilized iterative deconvolution of peptide libraries. As MT-2 undergoes shedding of the cell surface of transfected human embryonic kidney (HEK) cells and the activated form accumulates in the conditioned medium, the medium can be used as a source for MT-2 activity [11,20]. This was accordingly done in the course of the present study. The first part of our approach included the elucidation of the substrate specificity of MT-2 for the first four amino acids in the non-primed positions, i.e. in direction from the scissile bond to the N-terminus of the substrate, based on the Schechter & Berger notation [26]. By using a mix and split method, we synthesized a library of tetrapeptides of the general formula X4X3-X2-X1-ACC-NH2 where ACC is the C-terminal 7-aminocoumarin4-acetic acid moiety that acts as fluorophore and X1 is Lys or Arg. The latter two amino acids were placed in the P1 position by considering the known primary substrate specificity of MT-2 to cleave after basic amino acids. The tetrapeptide with the highest substrate specificity, Ile-ArgAla-Arg, was then incorporated in the internally quenched library of the general formula ABZ-Ile-Arg-Ala-Arg-X10 -X20 -X30 -Tyr(3-NO2) where 2-aminobenzoic acid (ABZ) and 3-nitrotyrosine (Tyr(3-NO2)) act as donor and acceptor of fluorescence, respectively. Finally, we report on the selection and characterization of an optimized tetrapeptide substrate, which is able to efficiently interact with the enzyme’s S4eS40 binding sites. For the synthesis of substrate candidates for matriptase-2 (MT2), the mix and split method and Fmoc-based chemistry was applied. After completing the synthesis, the 19 sublibraries were cleaved off the resin and lyophilized. Each of peptide mixture was subjected to enzymatic studies using recombinant human MT-2 from the conditioned medium of transfected HEK cells. The assay was performed in 96-well format. Extinction wavelength was 320 nm and emission 450 nm in order to detect the timedependent ACC-NH2 release.
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Deconvolution of the synthesized library with respect to position X4 reveal that MT-2 accepted most of the amino acid residues (Fig. 1A). Strong fluorescence increase was observed for sets of residues with an aliphatic side chain; however, aromatic ones were accepted as well. The lowest readout was observed for charged amino acids, in particular Glu and Lys. As the highest fluorescence was recorded for Ile, this amino acid was maintained at its position in the following experiments. In the next step, the position X3 of the libraries was inspected with fixed Ile in X4. Contrary to position X4, X3 revealed very narrow specificity since only one sublibrary with the Arg residue produced a high fluorescence increase. The remaining sublibraries displayed at least four times lower fluorescence (Fig. 1B). Then, the library with a sequence Ile-Arg-X2-X1ACC-NH2 was incubated with MT-2. In position P2 the highest proteolysis rate was observed for Ala followed by Met; all other residues were accepted with lower affinity (Fig. 1C). With respect to the known primary substrate specificity of MT-2 [8], only the basic amino acids Lys and Arg were placed in position X1. It turned out that Arg was the best accepted P1 amino acid since it produced two-fold higher fluorescence than Lys (Fig. 1D). This resulting peptide with the sequence Ile-Arg-Ala-Arg-ACC-NH2 was found to comprise the sequence best accepted by MT-2. In the next step of our study, the aforementioned peptide Ile-Arg-Ala-Arg was incorporated into the internally quenched library of the general formula ABZ-Ile-Arg-Ala-Arg-X10 -X20 -X30 Tyr(3-NO2)-NH2 where in position X10, X20 and X30 sets of the proteinogenic amino acid, except Cys, were present. The deconvolution of such a library was performed to gain insights into the substrate specificity of MT-2 with respect to the primed positions. An N-terminal 2-aminobenzoic acid moiety was introduced to act as donor of fluorescence which excited at 320 nm and the C-terminal Tyr(3-NO2) as efficient acceptor of the ABZ fluorescence [27]. The enzyme-catalyzed cleavage of any peptide bond between the ABZ and Tyr(3-NO2) results in a boost of fluorescence (monitored at 450 nm) due to reduction of quenching efficiency. The results of deconvolution show strong preference of MT-2 for the aliphatic
Fig. 1. Result of the deconvolution of the ACC-based library against matriptase-2. The fluorescence emission was monitored at 450 nm.
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Fig. 3. Comparison of proteolysis rate for substrates: ABZeIleeArgeAlaeArgeSere AlaeGly/Ala/SereTyr(NO2)eNH2 in presence of purified MT-2 and medium from HEK-mock cells. The fluorescence increase was monitored at 450 nm.
Fig. 2. Result of the deconvolution of the library ABZ-Ile-Arg-Ala-Arg-X10 -X20 -X30 Tyr(3-NO2)-NH2 against matriptase-2. The fluorescence emission was monitored at 450 nm.
residues Ala, Met, and Leu. However, the highest fluorescent was observed in case of the sublibrary with fixed Ser in X10 position (Fig. 2A). No cleavage was observed after Glu, Asp, and Pro. Repulsive interactions of the acidic residues of Glu and Asp are assumed to account for this effect, which was also confirmed by mass spectrometry analysis (data not shown). In position X20 (Fig. 2B) of the library ABZ-Ile-Arg-Ala-ArgeSer-X20 -X30 -Tyr(3NO2)-NH2, most of the amino acid residues were accepted. Ala, Met, and Ile, in this order, arouse the highest proteolysis rate. Again, no fluorescence increase was observed for Glu, Asp and Pro. For position X30 (Fig. 2C), three peptides with Ala, Gly and Ser were identified to cause the strongest proteolytic processing, and those peptides were subjected for further studies. In order to determine the concentration of active form of enzyme, a direct, active-site titration using a ‘burst’ substrate was
performed as a prerequisite for the determination of kinetic parameters. The concentration of MT-2 in the conditioned medium of transfected HEK cells was found to be about 2.27 106 M. Next, for the peptides with Ala, Gly and Ser in P30 and the optimized amino acids in the other positions, the kinetic parameters were determined (Table 1). The highest specificity constant was observed for ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 (I; kcat/KM ¼ 4.5 105 M1 s1), followed by ABZ-Ile-Arg-Ala-ArgSer-Ala-Ser-Tyr(3-NO2)-NH2 (III; (kcat/KM ¼ 1.1 105 M1 s1) and ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Ala-Tyr(3-NO2)-NH2 (II; (kcat/ KM ¼ 6.32 104 M1 s1). We found the HPLC cleavage pattern to be identical for all three substrates studied (see Fig. 3). MS data shown that all peptides were proteolytically cleaved into the N-terminal tetrapeptide (ABZIle-Arg-Ala-Arg-) and the C-terminal one (Ser-Ala-Gly/Ala/SerTyr(3-NO2)-NH2) indicating that the Arg-Ser bond was cleaved, as it was expected. The whole deconvolution process was performed with MT-2 which accumulates in the medium of transfected HEK cells. Therefore, we checked whether the optimized substrates IeIII follow the same cleavage pattern in presence of purified protease. Purification of the c-Myc-tagged enzyme was done by immunoaffinity chromatography. Indeed, we obtained the same picture in case of fluorescence increase (Fig. 4) and proteolytic fragmentation (see Fig. 3). The peptide bond of intact peptide was cleaved and two peptide fragments appeared, corresponding to C- and N-terminal part of the analyzed compound. As a control, the peptide I was incubated with the conditioned medium of HEK-mock cells and remained stable under such conditions for 1 h (HPLC data not shown). The hypothetical binding mode of the peptide sequence of I was predicted by a docking approach (Fig. 5). It is very likely that the basic side chain of the P1 Arg forms a salt bridge interaction with the residue Asp756 of MT-2 at the bottom of the S1 pocket, which
Table 1 Kinetic parameters of selected substrates for MT-2. The enzyme concentration (3.54 1010 M) was kept constant. Sequence
KM [M] 106
kcat [s1]
kcat/KM [s1 M1] 104
ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 (I) ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Ala-Tyr(3-NO2)-NH2 (II) ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Ser-Tyr(3-NO2)-NH2 (III)
8.35 1.21 35.5 8.81 24.5 3.92
3.79 0.38 2.24 0.28 2.66 0.17
45.42 1.82 6.32 0.78 10.98 1.05
M. Wysocka et al. / Biochimie 97 (2014) 121e127 Fig. 4. Proteolytic cleavage pattern of ABZeIleeArgeAlaeArgeSereAlaeX3’ eTyr(3-NO2)eNH2 (I: X3’ ¼ Ala, II: X3’ ¼ Gly, III: X3’ ¼ Ser) in the presence of (A) MT-2 from medium of transfected HEK cells, (B) purified MT2 and (C) MT-2 from medium of HEK mock cells. The incubation mixture was analyzed using the following time points: (A) 0, 30 min, 1.5 and 2.5 h; (B) 15, 30 min, 1.5 and 2.5 h; (C) 30 min. MALDI-TOF analysis indicates that peak 1 corresponds to intact substrate, peak 2 to ABZeIleeArgeAlaeArg, and peak 3 to SereAlaeAlaeTyr(3-NO2)eNH2 (I), SereAlaeGlyeTyr(3-NO2)-NH2 (II) and SereAlaeSereTyr(3-NO2)eNH2 (III), respectively.
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Fig. 5. Putative substrate binding mode. Shown is a model of the H-Ile-Arg-Ala-ArgSer-Ala-Gly-OH substrate/MT-2 complex. The substrate is depicted in stick representation and colored in cyan. Enzyme residues forming the active site are colored in magenta. The active site is rendered as transparent surface (top) as well as a stick representation (bottom).
represents a canonical substrate/inhibitor binding characteristic for trypsin-related serine proteases. Furthermore, in the mode, the P2 Ala side chain occupies the S2 pocket and the Arg moiety at the P3 position is directed towards the S3/S4 pocket where multiple hydrogen bonding interactions are possible. Ser at the P10 position of the substrate is favorably positioned for the formation of hydrogen bonds to residues Gly760 and Ile601. In addition, the carboxy terminal P30 Gly is in interaction distance to Arg599. The side chain of the catalytic Ser762 is orientated towards the scissile bond of the substrate. This plausible binding mode is consistent with matriptase-1 and trypsin ligand binding characteristics and was largely obtained on the basis of flexible docking. Further interactive optimization of the putative binding mode was essentially limited to improving possible interactions of the P3 Arg within the S3/S4 pocket. 4. Discussion By means of molecular modeling techniques, the model of the active site of MT-2 was recently generated on the basis of the structure of matriptase, the closest homologs proteinase [11]. This model was used in a comparative mutational study to identify amino acids in the S4eS1 binding sites of MT-2 that are critical for enzymeesubstrate interactions. For example, an exchange of His665 located at the border of the S2 site and the S3/S4 pocket of MT-2 to Phe, the equivalent residue in matriptase, resulted in higher hydrophobicity and enhanced the affinity of Boc-Gln-AlaArg-para-nitroanilide; KM values of 210 mM for wild-type MT2 and 24 mM for the mutated enzyme were reported [25]. The MT-2 model was also used in this study to predict the putative binding mode of the peptide sequence of I (Fig. 5).
Our results are in good agreement with the MT-2 model and also with respect to the crystal structure of the catalytic domain of matriptase [28]. MT-2 reveals strong preference towards substrates with a basic amino acid either in position P4 or P3 due to the presence of a large hydrophobic pocket negatively charged at the top formed by Glu662, Asp663 and Ser664 residues. A corresponding interaction is expected for Arg in P3 position of peptides IeIII. In position P2, MT-2 accepts small aliphatic residues, Ala in case of the identified peptides IeIII, which are accommodated in the small hydrophobic S2 pocket. This finding correlates with the situation found in the complex between one of the strongest trypsin inhibitors isolated from sunflower seed (SFTI-1) and matriptase [29]. The primary specificity of MT-2, and of the trypsinlike proteases in general, to cleave after basic amino acids is caused by the conserved residue Asp756 at the bottom of the S1 pocket. In direct proximity, Ala757 in MT-2 takes the place of a Ser residue in matriptase. The occurrence of Ala757 in MT-2 is thought to account for a preferred binding of Arg, rather than Lys in P1 position [2] as it was also found in our study. MT-2 prefers aliphatic residues in position P10 and Ser seems to be best accepted. Interestingly, several serine proteases prefer Ser in position P10, e.g. trypsin, cathepsin G, neutrophil elastase [30]. When comparing the sequences of substrates IeIII with the potential site of the MT-2-catalyzed cleavage of hemojuvelin, IleIle-Ile-Arg-Gln-Thr-Ala-Gly, [6] I and III also possess Ile and Arg in P4 and P1, and II shares three amino acids (bold). The sequence of the activation site which is cleaved in the course of an autoprocessing of MT-2, i.e. Pro-Ser-Ser-Arg-Ile-Val-Gly-Gly, as well as the two autoshedding sites, i.e. Pro-Gly-Val-Arg-Val-His-Tyr-Gly and Cys-Gly-Lys-Arg-Ile-Leu-Gln-Pro [7], showed a lower similarity with IeIII. With the exception of Gly in P30 of I, the three peptides only have the P1 amino acid Arg in common with these autoprocessing sites of MT-2. These findings reflect the promiscuity of MT-2 with respect to the cleavage of peptidic substrates as it was already noticed by others [12]. To study the substrate specificity of TTSPs, Béliveau et al. designed internally quenched fluorescent substrates whose sequences were based in the P4eP40 autoactivation sequence of matriptase, Arg-Gln-Ala-Arg-Val-Val-Gly-Gly. Positions P4, P3, P2 and P10 were replaced by representative residues with different physicochemical properties. The resulting octapeptides were flanked by the ABZ/Tyr(3-NO2) pair. Some of the resulting 18 individual peptides showed substrate inhibition of MT-2. For three peptides, the specificity constants for the MT-2-catalyzed cleavage were obtained. Noteworthy, the best substrate, Arg-Arg-Ala-ArgVal-Val-Gly-Gly, exhibiting a kcat/KM value of 9.6 104 M1 s1 shares four amino acids (bold) with peptide I. Further six P4eP40 peptides were derived from known cleavage sequences of natural matriptase substrates. When inspecting the corresponding internally quenched fluorescent peptides with respect to the MT-2catalyzed cleavage, the sequence from the potential matriptase cleavage motif in filaggrin, Arg-Lys-Arg-Arg-Gly-Ser-Arg-Gly, resulted in the highest kcat/KM value of 2.3 105 M1 s1 [12]. The reactive-center loop of antithrombin III (serpin C1) contains the P4 to P40 tetrapeptide Ile-Arg-Gly-Arg-Ser-Leu-Asn-Pro [12]. This serpin was a more efficient inhibitor of MT-2 than other investigated serpins and, again, its sequence shares four amino acids (bold) with peptide I. So far, for assaying the proteolytic activity of matriptase-2, the chromogenic or fluorogenic tripeptide Gln-Ala-Arg was used as a substrate, containing the P3 to P1 amino acids. Following enzymecatalyzed hydrolysis of the amide bond, para-nitroaniline or 7-amino-4-methylcoumarin is released [2,7,11,12,25,31e33]. These tripeptide-based substrates are kinetically poor and not specific for matriptase-2. In this study, we introduce the internally quenched
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substrate I which was obtained by the combinatorial approach of iterative deconvolution. To the best of our knowledge, I has the highest specificity constant reported so far for MT-2 substrates. The identification of preferred P4 to P30 sequences might provide further insights into the physiological function of MT-2. For the closely related enzyme matriptase, such an approach was carried out. Based on the sequence of adequate peptide ligands, a scan for natural substrates in a protein database was performed and aEb7 integrin was suggested as a new putative substrate of matriptase [12]. Inhibition of MT-2 is considered as a new strategy for the therapeutic treatment of b-thalassemia or hereditary hemochromatosis. The results of our study are expected to prove beneficial for the design of improved MT-2 inhibitors and also for the development of activity-based probes. Such probes would be useful as tools to further characterize the role of matriptase-2 in iron homeostasis. Acknowledgments This work was supported by grant of Ministry of Science and Higher Education 530-8290-D373-13. N.F. is supported by a fellowship from the Jürgen Manchot Foundation, Düsseldorf, Germany. References [1] R. Szabo, T.H. Bugge, Type II transmembrane serine proteases in development and disease, Int. J. Biochem. Cell. Biol. 40 (2008) 1297e1316. [2] G. Velasco, S. Cal, V. Quesada, L.M. Sánchez, C. López-Otín, Matriptase-2, a membrane bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins, J. Biol. Chem. 277 (2002) 37637e37646. [3] K.E. Finberg, M.M. Heeney, D.R. Campagna, Y. Aydinok, H.A. Pearson, K.R. Hartman, M.M. Mayo, S.M. Samuel, J.J. Strouse, K. Markianos, N.C. Andrews, M.D. Fleming, Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA), Nat. Genet. 40 (2008) 569e571. [4] X. Du, E. She, T. Gelbart, J. Truksa, P. Lee, Y. Xia, K. Khovananth, S. Mudd, N. Mann, E.M. Moresco, E. Beutler, B. Beutler, The serine protease TMPRSS6 is required to sense iron deficiency, Science 320 (2008) 1088e1092. [5] L. Silvestri, A. Pagani, A. Nai, I. De Domenico, J. Kaplan, C. Camaschella, The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin, Cell Metab. 8 (2008) 502e511. [6] J.E. Maxson, J. Chen, C.A. Enns, A.S. Zhang, Matriptase-2- and proprotein convertase-cleaved forms of hemojuvelin have different roles in the downregulation of hepcidin expression, J. Biol. Chem. 285 (2010) 39021e39028. [7] M. Stirnberg, E. Maurer, A. Horstmeyer, S. Kolp, S. Frank, T. Bald, K. Arenz, A. Janzer, K. Prager, P. Wunderlich, J. Walter, M. Gütschow, Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface, Biochem. J. 430 (2010) 87e95. [8] M. Stirnberg, M. Gütschow, Matriptase-2, a regulatory protease of iron homeostasis: possible substrates, cleavage sites and inhibitors, Curr. Pharm. Des. 19 (2013) 1052e1061. [9] E. Maurer, M. Gütschow, M. Stirnberg, Hepatocyte growth factor activator inhibitor type 2 (HAI-2) modulates hepcidin expression by inhibiting the cell surface protease matriptase-2, Biochem. J. 450 (2013) 583e593. [10] C.J. Farady, C.S. Craik, Mechanisms of macromolecular protease inhibitors, ChemBioChem 11 (2010) 2341e2346. [11] M.T. Sisay, T. Steinmetzer, M. Stirnberg, E. Maurer, M. Hammami, J. Bajorath, M. Gütschow, Identification of the first low-molecular-weight inhibitors of matriptase-2, J. Med. Chem. 53 (2010) 5523e5535.
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[12] F. Béliveau, A. Désilets, R. Leduc, Probing the substrate specificities of matriptase, matriptase-2, hepsin and DESC1 with internally quenched fluorescent peptides, FEBS J. 276 (2009) 2213e2226. [13] K.E. Finberg, Striking the target in iron overload disorders, J. Clin. Invest. 123 (2013) 1424e1427. [14] L. De Falco, M. Sanchez, L. Silvestri, C. Kannengiesser, M.U. Muckenthaler, A. Iolascon, L. Gouya, C. Camaschella, C. Beaumont, Iron refractory iron deficiency anemia, Haematologica 98 (2013) 845e853. [15] M. Wysocka, B. Spichalska, A. Lesner, M. Jaros, K. Brzozowski, A. qegowska, K. Rolka, Substrate specificity and inhibitory study of human airway trypsinlike protease, Bioorg. Med. Chem. 18 (2010) 5504e5509. [16] N.A. Sole, G. Barany, Optimization of solid-phase synthesis of [Ala8]dynorphin A, J. Org. Chem. 57 (1992) 5399e5403. [17] J.L. Harris, B.J. Backes, F. Leonetti, S. Mahrus, J.A. Ellman, C.S. Craik, Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7754e7759. [18] A. Furka, F. Sebestyn, M. Asgedom, G. Dibó, General method for rapid synthesis of multicomponent peptide mixtures, Int. J. Pept. Protein Res. 37 (1991) 487e493. [19] R.A. Houghten, C. Pinilla, S.E. Blondelle, J.R. Appel, C.T. Dooley, J.H. Cuervo, Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery, Nature 354 (1991) 84e86. [20] S. Dosa, M. Stirnberg, V. Lülsdorff, D. Häußler, E. Maurer, M. Gütschow, Active site mapping of trypsin, thrombin and matriptase-2 by sulfamoyl benzamidines, Bioorg. Med. Chem. 20 (2012) 6489e6505. [21] T. Chase Jr., E. Shaw, p-Nitrophenyl-p’-guanidinobenzoate HCl: a new active site titrant for trypsin, Biochem. Biophys. Res. Commun. 29 (1967) 508e514. [22] M. Wysocka, A. Lesner, N. Gruba, B. Korkmaz, F. Gauthier, M. Kitamatsu, A. qe˛ gowska, K. Rolka, Three wavelength substrate system of neutrophil serine proteinases, Anal. Chem. 84 (2012) 7241e7248. [23] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.S. Olson, AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility, J. Comput. Chem. 16 (2009) 2785e2791. [24] Molecular Operating Environment (MOE), 2012.10, Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada H3A 2R7, 2012. [25] E. Maurer, M.T. Sisay, M. Stirnberg, T. Steinmetzer, J. Bajorath, M. Gütschow, Insights into matriptase-2 substrate binding and inhibition mechanisms by analyzing active-site-mutated variants, ChemMedChem 7 (2012) 68e72. [26] I. Schechter, A. Berger, On the size of the active site in proteases I. Papain, Biochem. Biophys. Res. Commun. 27 (1967) 157e162. [27] M. Meldal, K. Breddam, Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin, Anal. Biochem. 195 (1991) 141e147. [28] R. Friedrich, P. Fuentes-Prior, E. Ong, G. Coombs, M. Hunter, R. Oehler, D. Pierson, R. Gonzalez, R. Huber, W. Bode, E.L. Madison, Catalytic domain structures of MT-SP1/matriptase, a matrix-degrading transmembrane serine proteinase, J. Biol. Chem. 277 (2002) 2160e2168. [29] C. Yuan, L. Chen, E.J. Meehan, N. Daly, D.J. Craik, M. Huang, J.C. Ngo, Structure of catalytic domain of matriptase in complex with sunflower trypsin inhibitor-1, BMC Struct. Biol. 11 (2011) 30e42. [30] N.D. Rawlings, A.J. Barrett, A. Bateman, MEROPS: the database of proteolytic enzymes, their substrates and inhibitors, Nucl. Acids Res. 40 (2012) D343e D350. [31] F. Guillem, S. Lawson, C. Kannengiesser, M. Westerman, C. Beaumont, B. Grandchamp, Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency, Blood 112 (2008) 2089e2091. [32] F. Béliveau, C. Brulé, A. Désilets, B. Zimmerman, S.A. Laporte, C.L. Lavoie, R. Leduc, Essential role of endocytosis of the type II transmembrane serine protease TMPRSS6 in regulating its functionality, J. Biol. Chem. 286 (2011) 29035e29043. [33] D. Meynard, V. Vaja, C.C. Sun, E. Corradini, S. Chen, C. López-Otín, L. Grgurevic, C.C. Hong, M. Stirnberg, M. Gütschow, S. Vukicevic, J.L. Babitt, H.Y. Lin, Regulation of TMPRSS6 by BMP6 and iron in human cells and mice, Blood 118 (2011) 747e756.
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Research paper
Substrate specificity of human matriptase-2 M. Wysocka a, N. Gruba a, A. Miecznikowska a, J. Popow-Stellmaszyk a, M. Gütschow b, M. Stirnberg b, N. Furtmann b, c, J. Bajorath c, A. Lesner a, *, K. Rolka a a
Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk, Poland Pharmaceutical Institute, Rheinische Friedrich-Wilhelms-Universität, An der Immenburg 4, 53121 Bonn, Germany c Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität, Dahlmannstr. 2, 53113 Bonn, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2013 Accepted 1 October 2013 Available online 22 October 2013
Human matriptase-2 is an enzyme that belongs to the family of type II transmembrane serine proteases. So far there is a limited knowledge regarding its specificity and protein substrate(s). One of the identified natural substrates is hemojuvelin, a protein involved in the control of iron homeostasis. In this work, we describe the synthesis and evaluation of internal quenched substrates using a combinatorial approach. The iterative deconvolution of two libraries to define the specificity of matriptase-2 yielded to the identification of the substrate ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 with a kcat/Km value of 4.5 105 M1 s1, i.e. the highest specificity constant reported so far for matriptase-2. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Matriptase-2 Combinatorial chemistry Fluorogenic substrates Internally quenched substrates
1. Introduction Matriptase-2 (MT-2, also referred to as TMPRSS6) belongs to the class of type II transmembrane serine proteases (TTSPs) [1] located at the plasma membrane with a C-terminal protease domain directed to the extracellular space and a stem region with domains of different types, between the transmembrane domain near the N-terminus and the protease domain [2]. Since mutations in MT-2 have been linked to iron-refractory iron deficiency anemia (IRIDA), MT-2 was identified as a regulatory protease controlling iron homeostasis [3,4]. MT-2 was shown to cleave the BMP (bone morphogenetic protein) co-receptor hemojuvelin [5] and thereby to downregulate the expression of hepcidin, the systemic iron regulatory peptide hormone. The cleavage site within hemojuvelin targeted by MT-2 was determined [6]. So far, hemojuvelin represents the only known putative substrate of MT-2 beside the protease itself. In a transfected cell system, MT-2 is cleaved at two shedding sites within the stem region to yield a released form and at the activation site at the junction of the protease domain [7]. From these four reported processing sites an attempt was made to draw a conclusion about the substrate specificity of MT-2 [8]. Recently, the Kunitz-type serine protease inhibitor HAI-2 (hepatocyte growth factor activator inhibitor type 2) has been shown to inhibit the proteolytic activity of MT-2 [9]. Since Kunitz-type
* Corresponding author. Tel.: þ48 585235095; fax: þ48 585235472. E-mail addresses: [email protected], [email protected] (A. Lesner). 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.10.001
inhibitors bind to their target proteases in a substrate-like manner [10], HAI-2 can also be considered to bear a processing site attacked by MT-2. For instance, Kunitz domain I exhibited two critical Arg residues which might be recognized by MT-2. As a typical trypsin-like serine protease, MT-2 bears a conserved Asp located at the bottom of the S1 pocket of the active site determining the primary substrate specificity to cleave behind positively charged residues like Arg and Lys [11]. Recently, a set of FRET peptides was used to define the substrate specificity of matriptase, a TTSP closely related to MT-2. Other members of transmembrane serine proteases, such as MT-2, hepsin, and DESC1, a human airway trypsin like serine protease were also investigated in this study [12]. There is, however, a strong need to elucidate the substrate specificity of MT-2 more in detail, in particular with respect to the identification of further endogenous substrates and to the structural optimization of peptide-derived inhibitors for MT-2. Such compounds are expected to be of great value as pharmacological tools and, even more, as potential therapeutic agents for the treatment of iron overload diseases and b-thalassemia [8,13,14]. 2. Materials and methods 2.1. Peptide synthesis The peptide libraries and individual peptides were synthesized manually by the solid-phase method applying Fmoc chemistry,
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as described previously [15]. TentaGel S RAM (substitution 0.25 meq/g) (RAPP Polymere, Germany) was used as a solid support. The a-amino groups of the amino acids were Fmoc-protected and these amino acids were attached to the resin using N,N’-diisopropylcarbodiimide (DIPCI) and N-hydroxybenzotriazole (HOBt). The synthesis of the ACC-based library was initiated by deprotection of the amino groups of the resin with 20% piperidine in the mixture of DMF/N-methyl-2-pyrrolidone (NMP) (1:1, v/v) and coupling of Fmoc-7-aminocoumarin-4-acetic acid (Fmoc-ACC) using the N,N,N0 ,N0 -tetramethyl-O-(benzotriazol-1-yl)uranium tetrafluoroborate (TBTU) and HOBt [16]. Briefly, two equiv of Fmoc-ACC and two equiv of TBTU/HOBt were dissolved in 5 ml DMF and the solution was added to the resin. After 30 s, four equiv of N,N-diisopropylethylamine (DIPEA) were added and the mixture was stirred for 5 h. The solution was filtered off and the resin was washed with DMF. The procedure was repeated three times. Next, the whole resin was incubated with five-fold molar excess of N-acetylimidazole in DMF to cap the remaining amino groups. After deprotection with 20% piperidine in a mixture of DMF/NMP (1:1, v/v), the peptide chain was elongated as follows. A mixture of the N-protected amino acid derivative, DIPCI and HOBt (molar ratio, 1:1:1) was dissolved in DMF:NMP solution (1:1, v/v) and added to the resin. A three-fold excess over the resin active sites was used. The library of the internally quenched ABZ/Tyr(3-NO2) peptides was similarly synthesized as described [15], also on TentaGel S RAM resin, starting with of Fmoc-protected 3-nitrotyrosine. After completing the synthesis, the peptides were cleaved from the resin using a TFA/phenol/triisopropylsilane/H2O mixture (88:5:2:5, v/v) [17]. Purity of the peptides was checked on RP-HPLC Pro Star system (Varian, Australia) equipped with a Kromasil 100 C8 column (8 250 mm) (Knauer, Germany) and a UVeVIS detector. A linear gradient from 20 to 80% B within 40 min was applied (A: 0.1% TFA; B: 80% acetonitrile in A). The peptides were monitored at 226 nm. Mass spectra of the synthesized peptides were recorded using a Biflex III MALDI TOF mass spectrometer (Bruker, Germany) and a-cyano-4-hydroxycinnamic acid as a matrix.
fluorescent method with peptides solution at concentration: of 1 mg/ml (1.6 107 M) (ACC-based library) and 3 mg/ml (2.76 107 M) for the internally quenched library, respectively. Stock solutions of the peptides were prepared in DMSO. All measurements were performed using the Omega plate reader (BMG, Germany) with an excitation wavelength of 360 nm and emission wavelength 450 nm for ACC-based library, while for the internally quenched library wavelengths of 320 nm and 450 nm were used. Enzymatic hydrolysis of the peptide was performed in 50 mM Trise HCl (pH 8.0) buffer with 150 mM NaCl at 37 C and followed over 30 min. 2.4. Determination of the matriptase-2 concentration using a ‘burst’ substrate The concentration of MT-2 was determined by spectrophotometric titration with 4-nitrophenyl-40 -guanidinobenzoate (NPGB) [21]. Briefly, 10 ml of an NPGB solution (3.36 mg/ml) was added into 1500 ml of veronal buffer (0.1 M with 20 mM CaCl2, pH 8.3) followed by adding 20 ml of the conditioned medium of transfected HEK cells, or purified enzyme solution, respectively. 2.5. Determination of KM and kcat Assay conditions for the determination of the Michaelis constants (KM) and catalytic constants (kcat) were as noted above. The specificity constants (kcat/KM) were calculated from kcat and KM values. Measurements were performed with an enzyme concentration of 2.8 109 M. Three to five measurements were carried out for each compound (systematic error expressed as a standard deviation never exceeded 20%). The calculated initial hydrolysis rates were used as a measure of substrate activity of the investigated peptides. All details of kinetic studies and the method of calculating kinetic parameters have been described elsewhere [22]. 2.6. Determination of the proteolytic cleavage pattern
2.2. Preparation of the peptide libraries The peptide libraries were synthesized by the portioningmixing method [18,19]. Initially, 17.1 g of the solid support (TentaGel S RAM) was used for the first library, i.e. X4-X3-X2-X1-ACC-NH2. A three-fold molar excess of the amino acid was used for the coupling. The second library, i.e. ABZ-Ile-Arg-Ala-Arg-X10 -X20 -X30 Tyr(NO2)-NH2, was synthesized on 15.2 g of the 2-chloro-chlorotrityl resin. The procedure for peptide chain elongation and the other synthetic methods employed were as described above. 2.3. Initial screening of the libraries For the enzymatic studies, MT-2 from the conditioned medium of transfected HEK cells was employed as described [7,11,20]. Briefly, the whole MT-2 construct was cloned and expressed in HEK cells with c-Myc and His6 tags at the C-terminal end of the protein. For comparison, conditioned medium of HEK cells expressing the empty vector (HEK-mock) was also used. For certain experiments, MT-2 was purified from the concentrated conditioned medium of transfected HEK cells by immunoaffinity chromatography as described [11]. MT-2-containing 10 ml-aliquots of a protein concentration of 0.7 mg/ml were applied. The enzyme concentration was determined as described below. The concentration of MT-2 used for the deconvolution of the ACC-based library ranged from 1 109 to 3.4 1010 M. For the internally quenched library, the enzyme concentration was lower, between 1.2 1010 and 5.2 1011 M. Deconvolution of the sublibraries was performed by applying the
Selected substrates were mixed with a two-fold molar excess of the enzyme in a buffer used for kinetic studies. HPLC analysis of this mixture was performed after the following incubation times: 0, 15 min and 24 h. The eluting fractions of the corresponding peaks were collected and analyzed by MALDI-TOF MS. 2.7. Molecular docking Because the X-ray structure of matriptase-2 is not available, a previously described comparative model of matriptase-2 [11] was used as template for flexible ligand docking with AutoDock 4.2 [23] to explore possible substrate binding modes. The structure of I was reduced to the optimized peptide substrate sequence H-Ile-Arg-Ala-Arg-Ser-Ala-Gly-OH to limit the number of flexible torsion angles (degrees of freedom) for flexible ligand docking. Hydrogen atoms were added to the model and the peptide substrate with the Molecular Operating Environment (MOE 2012.10) [24] and atomic partial charges were calculated using AutoDock Tools [23]. AutoDock standard docking parameter settings [23] were applied, except that the total number of genetic algorithm iterations for conformational sampling was increased, given the inherent flexibility of the peptide substrate. Plausible binding poses were selected with the aid of visual inspection taking prior knowledge of substrate and inhibitor binding characteristics into account [25]. A preferred docking pose was further adjusted by interactive model building and energy minimizations with MOE 2012.10.
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3. Results To systematically investigate the substrate specificity of MT-2 we decided to utilized iterative deconvolution of peptide libraries. As MT-2 undergoes shedding of the cell surface of transfected human embryonic kidney (HEK) cells and the activated form accumulates in the conditioned medium, the medium can be used as a source for MT-2 activity [11,20]. This was accordingly done in the course of the present study. The first part of our approach included the elucidation of the substrate specificity of MT-2 for the first four amino acids in the non-primed positions, i.e. in direction from the scissile bond to the N-terminus of the substrate, based on the Schechter & Berger notation [26]. By using a mix and split method, we synthesized a library of tetrapeptides of the general formula X4X3-X2-X1-ACC-NH2 where ACC is the C-terminal 7-aminocoumarin4-acetic acid moiety that acts as fluorophore and X1 is Lys or Arg. The latter two amino acids were placed in the P1 position by considering the known primary substrate specificity of MT-2 to cleave after basic amino acids. The tetrapeptide with the highest substrate specificity, Ile-ArgAla-Arg, was then incorporated in the internally quenched library of the general formula ABZ-Ile-Arg-Ala-Arg-X10 -X20 -X30 -Tyr(3-NO2) where 2-aminobenzoic acid (ABZ) and 3-nitrotyrosine (Tyr(3-NO2)) act as donor and acceptor of fluorescence, respectively. Finally, we report on the selection and characterization of an optimized tetrapeptide substrate, which is able to efficiently interact with the enzyme’s S4eS40 binding sites. For the synthesis of substrate candidates for matriptase-2 (MT2), the mix and split method and Fmoc-based chemistry was applied. After completing the synthesis, the 19 sublibraries were cleaved off the resin and lyophilized. Each of peptide mixture was subjected to enzymatic studies using recombinant human MT-2 from the conditioned medium of transfected HEK cells. The assay was performed in 96-well format. Extinction wavelength was 320 nm and emission 450 nm in order to detect the timedependent ACC-NH2 release.
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Deconvolution of the synthesized library with respect to position X4 reveal that MT-2 accepted most of the amino acid residues (Fig. 1A). Strong fluorescence increase was observed for sets of residues with an aliphatic side chain; however, aromatic ones were accepted as well. The lowest readout was observed for charged amino acids, in particular Glu and Lys. As the highest fluorescence was recorded for Ile, this amino acid was maintained at its position in the following experiments. In the next step, the position X3 of the libraries was inspected with fixed Ile in X4. Contrary to position X4, X3 revealed very narrow specificity since only one sublibrary with the Arg residue produced a high fluorescence increase. The remaining sublibraries displayed at least four times lower fluorescence (Fig. 1B). Then, the library with a sequence Ile-Arg-X2-X1ACC-NH2 was incubated with MT-2. In position P2 the highest proteolysis rate was observed for Ala followed by Met; all other residues were accepted with lower affinity (Fig. 1C). With respect to the known primary substrate specificity of MT-2 [8], only the basic amino acids Lys and Arg were placed in position X1. It turned out that Arg was the best accepted P1 amino acid since it produced two-fold higher fluorescence than Lys (Fig. 1D). This resulting peptide with the sequence Ile-Arg-Ala-Arg-ACC-NH2 was found to comprise the sequence best accepted by MT-2. In the next step of our study, the aforementioned peptide Ile-Arg-Ala-Arg was incorporated into the internally quenched library of the general formula ABZ-Ile-Arg-Ala-Arg-X10 -X20 -X30 Tyr(3-NO2)-NH2 where in position X10, X20 and X30 sets of the proteinogenic amino acid, except Cys, were present. The deconvolution of such a library was performed to gain insights into the substrate specificity of MT-2 with respect to the primed positions. An N-terminal 2-aminobenzoic acid moiety was introduced to act as donor of fluorescence which excited at 320 nm and the C-terminal Tyr(3-NO2) as efficient acceptor of the ABZ fluorescence [27]. The enzyme-catalyzed cleavage of any peptide bond between the ABZ and Tyr(3-NO2) results in a boost of fluorescence (monitored at 450 nm) due to reduction of quenching efficiency. The results of deconvolution show strong preference of MT-2 for the aliphatic
Fig. 1. Result of the deconvolution of the ACC-based library against matriptase-2. The fluorescence emission was monitored at 450 nm.
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Fig. 3. Comparison of proteolysis rate for substrates: ABZeIleeArgeAlaeArgeSere AlaeGly/Ala/SereTyr(NO2)eNH2 in presence of purified MT-2 and medium from HEK-mock cells. The fluorescence increase was monitored at 450 nm.
Fig. 2. Result of the deconvolution of the library ABZ-Ile-Arg-Ala-Arg-X10 -X20 -X30 Tyr(3-NO2)-NH2 against matriptase-2. The fluorescence emission was monitored at 450 nm.
residues Ala, Met, and Leu. However, the highest fluorescent was observed in case of the sublibrary with fixed Ser in X10 position (Fig. 2A). No cleavage was observed after Glu, Asp, and Pro. Repulsive interactions of the acidic residues of Glu and Asp are assumed to account for this effect, which was also confirmed by mass spectrometry analysis (data not shown). In position X20 (Fig. 2B) of the library ABZ-Ile-Arg-Ala-ArgeSer-X20 -X30 -Tyr(3NO2)-NH2, most of the amino acid residues were accepted. Ala, Met, and Ile, in this order, arouse the highest proteolysis rate. Again, no fluorescence increase was observed for Glu, Asp and Pro. For position X30 (Fig. 2C), three peptides with Ala, Gly and Ser were identified to cause the strongest proteolytic processing, and those peptides were subjected for further studies. In order to determine the concentration of active form of enzyme, a direct, active-site titration using a ‘burst’ substrate was
performed as a prerequisite for the determination of kinetic parameters. The concentration of MT-2 in the conditioned medium of transfected HEK cells was found to be about 2.27 106 M. Next, for the peptides with Ala, Gly and Ser in P30 and the optimized amino acids in the other positions, the kinetic parameters were determined (Table 1). The highest specificity constant was observed for ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 (I; kcat/KM ¼ 4.5 105 M1 s1), followed by ABZ-Ile-Arg-Ala-ArgSer-Ala-Ser-Tyr(3-NO2)-NH2 (III; (kcat/KM ¼ 1.1 105 M1 s1) and ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Ala-Tyr(3-NO2)-NH2 (II; (kcat/ KM ¼ 6.32 104 M1 s1). We found the HPLC cleavage pattern to be identical for all three substrates studied (see Fig. 3). MS data shown that all peptides were proteolytically cleaved into the N-terminal tetrapeptide (ABZIle-Arg-Ala-Arg-) and the C-terminal one (Ser-Ala-Gly/Ala/SerTyr(3-NO2)-NH2) indicating that the Arg-Ser bond was cleaved, as it was expected. The whole deconvolution process was performed with MT-2 which accumulates in the medium of transfected HEK cells. Therefore, we checked whether the optimized substrates IeIII follow the same cleavage pattern in presence of purified protease. Purification of the c-Myc-tagged enzyme was done by immunoaffinity chromatography. Indeed, we obtained the same picture in case of fluorescence increase (Fig. 4) and proteolytic fragmentation (see Fig. 3). The peptide bond of intact peptide was cleaved and two peptide fragments appeared, corresponding to C- and N-terminal part of the analyzed compound. As a control, the peptide I was incubated with the conditioned medium of HEK-mock cells and remained stable under such conditions for 1 h (HPLC data not shown). The hypothetical binding mode of the peptide sequence of I was predicted by a docking approach (Fig. 5). It is very likely that the basic side chain of the P1 Arg forms a salt bridge interaction with the residue Asp756 of MT-2 at the bottom of the S1 pocket, which
Table 1 Kinetic parameters of selected substrates for MT-2. The enzyme concentration (3.54 1010 M) was kept constant. Sequence
KM [M] 106
kcat [s1]
kcat/KM [s1 M1] 104
ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 (I) ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Ala-Tyr(3-NO2)-NH2 (II) ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Ser-Tyr(3-NO2)-NH2 (III)
8.35 1.21 35.5 8.81 24.5 3.92
3.79 0.38 2.24 0.28 2.66 0.17
45.42 1.82 6.32 0.78 10.98 1.05
M. Wysocka et al. / Biochimie 97 (2014) 121e127 Fig. 4. Proteolytic cleavage pattern of ABZeIleeArgeAlaeArgeSereAlaeX3’ eTyr(3-NO2)eNH2 (I: X3’ ¼ Ala, II: X3’ ¼ Gly, III: X3’ ¼ Ser) in the presence of (A) MT-2 from medium of transfected HEK cells, (B) purified MT2 and (C) MT-2 from medium of HEK mock cells. The incubation mixture was analyzed using the following time points: (A) 0, 30 min, 1.5 and 2.5 h; (B) 15, 30 min, 1.5 and 2.5 h; (C) 30 min. MALDI-TOF analysis indicates that peak 1 corresponds to intact substrate, peak 2 to ABZeIleeArgeAlaeArg, and peak 3 to SereAlaeAlaeTyr(3-NO2)eNH2 (I), SereAlaeGlyeTyr(3-NO2)-NH2 (II) and SereAlaeSereTyr(3-NO2)eNH2 (III), respectively.
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Fig. 5. Putative substrate binding mode. Shown is a model of the H-Ile-Arg-Ala-ArgSer-Ala-Gly-OH substrate/MT-2 complex. The substrate is depicted in stick representation and colored in cyan. Enzyme residues forming the active site are colored in magenta. The active site is rendered as transparent surface (top) as well as a stick representation (bottom).
represents a canonical substrate/inhibitor binding characteristic for trypsin-related serine proteases. Furthermore, in the mode, the P2 Ala side chain occupies the S2 pocket and the Arg moiety at the P3 position is directed towards the S3/S4 pocket where multiple hydrogen bonding interactions are possible. Ser at the P10 position of the substrate is favorably positioned for the formation of hydrogen bonds to residues Gly760 and Ile601. In addition, the carboxy terminal P30 Gly is in interaction distance to Arg599. The side chain of the catalytic Ser762 is orientated towards the scissile bond of the substrate. This plausible binding mode is consistent with matriptase-1 and trypsin ligand binding characteristics and was largely obtained on the basis of flexible docking. Further interactive optimization of the putative binding mode was essentially limited to improving possible interactions of the P3 Arg within the S3/S4 pocket. 4. Discussion By means of molecular modeling techniques, the model of the active site of MT-2 was recently generated on the basis of the structure of matriptase, the closest homologs proteinase [11]. This model was used in a comparative mutational study to identify amino acids in the S4eS1 binding sites of MT-2 that are critical for enzymeesubstrate interactions. For example, an exchange of His665 located at the border of the S2 site and the S3/S4 pocket of MT-2 to Phe, the equivalent residue in matriptase, resulted in higher hydrophobicity and enhanced the affinity of Boc-Gln-AlaArg-para-nitroanilide; KM values of 210 mM for wild-type MT2 and 24 mM for the mutated enzyme were reported [25]. The MT-2 model was also used in this study to predict the putative binding mode of the peptide sequence of I (Fig. 5).
Our results are in good agreement with the MT-2 model and also with respect to the crystal structure of the catalytic domain of matriptase [28]. MT-2 reveals strong preference towards substrates with a basic amino acid either in position P4 or P3 due to the presence of a large hydrophobic pocket negatively charged at the top formed by Glu662, Asp663 and Ser664 residues. A corresponding interaction is expected for Arg in P3 position of peptides IeIII. In position P2, MT-2 accepts small aliphatic residues, Ala in case of the identified peptides IeIII, which are accommodated in the small hydrophobic S2 pocket. This finding correlates with the situation found in the complex between one of the strongest trypsin inhibitors isolated from sunflower seed (SFTI-1) and matriptase [29]. The primary specificity of MT-2, and of the trypsinlike proteases in general, to cleave after basic amino acids is caused by the conserved residue Asp756 at the bottom of the S1 pocket. In direct proximity, Ala757 in MT-2 takes the place of a Ser residue in matriptase. The occurrence of Ala757 in MT-2 is thought to account for a preferred binding of Arg, rather than Lys in P1 position [2] as it was also found in our study. MT-2 prefers aliphatic residues in position P10 and Ser seems to be best accepted. Interestingly, several serine proteases prefer Ser in position P10, e.g. trypsin, cathepsin G, neutrophil elastase [30]. When comparing the sequences of substrates IeIII with the potential site of the MT-2-catalyzed cleavage of hemojuvelin, IleIle-Ile-Arg-Gln-Thr-Ala-Gly, [6] I and III also possess Ile and Arg in P4 and P1, and II shares three amino acids (bold). The sequence of the activation site which is cleaved in the course of an autoprocessing of MT-2, i.e. Pro-Ser-Ser-Arg-Ile-Val-Gly-Gly, as well as the two autoshedding sites, i.e. Pro-Gly-Val-Arg-Val-His-Tyr-Gly and Cys-Gly-Lys-Arg-Ile-Leu-Gln-Pro [7], showed a lower similarity with IeIII. With the exception of Gly in P30 of I, the three peptides only have the P1 amino acid Arg in common with these autoprocessing sites of MT-2. These findings reflect the promiscuity of MT-2 with respect to the cleavage of peptidic substrates as it was already noticed by others [12]. To study the substrate specificity of TTSPs, Béliveau et al. designed internally quenched fluorescent substrates whose sequences were based in the P4eP40 autoactivation sequence of matriptase, Arg-Gln-Ala-Arg-Val-Val-Gly-Gly. Positions P4, P3, P2 and P10 were replaced by representative residues with different physicochemical properties. The resulting octapeptides were flanked by the ABZ/Tyr(3-NO2) pair. Some of the resulting 18 individual peptides showed substrate inhibition of MT-2. For three peptides, the specificity constants for the MT-2-catalyzed cleavage were obtained. Noteworthy, the best substrate, Arg-Arg-Ala-ArgVal-Val-Gly-Gly, exhibiting a kcat/KM value of 9.6 104 M1 s1 shares four amino acids (bold) with peptide I. Further six P4eP40 peptides were derived from known cleavage sequences of natural matriptase substrates. When inspecting the corresponding internally quenched fluorescent peptides with respect to the MT-2catalyzed cleavage, the sequence from the potential matriptase cleavage motif in filaggrin, Arg-Lys-Arg-Arg-Gly-Ser-Arg-Gly, resulted in the highest kcat/KM value of 2.3 105 M1 s1 [12]. The reactive-center loop of antithrombin III (serpin C1) contains the P4 to P40 tetrapeptide Ile-Arg-Gly-Arg-Ser-Leu-Asn-Pro [12]. This serpin was a more efficient inhibitor of MT-2 than other investigated serpins and, again, its sequence shares four amino acids (bold) with peptide I. So far, for assaying the proteolytic activity of matriptase-2, the chromogenic or fluorogenic tripeptide Gln-Ala-Arg was used as a substrate, containing the P3 to P1 amino acids. Following enzymecatalyzed hydrolysis of the amide bond, para-nitroaniline or 7-amino-4-methylcoumarin is released [2,7,11,12,25,31e33]. These tripeptide-based substrates are kinetically poor and not specific for matriptase-2. In this study, we introduce the internally quenched
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substrate I which was obtained by the combinatorial approach of iterative deconvolution. To the best of our knowledge, I has the highest specificity constant reported so far for MT-2 substrates. The identification of preferred P4 to P30 sequences might provide further insights into the physiological function of MT-2. For the closely related enzyme matriptase, such an approach was carried out. Based on the sequence of adequate peptide ligands, a scan for natural substrates in a protein database was performed and aEb7 integrin was suggested as a new putative substrate of matriptase [12]. Inhibition of MT-2 is considered as a new strategy for the therapeutic treatment of b-thalassemia or hereditary hemochromatosis. The results of our study are expected to prove beneficial for the design of improved MT-2 inhibitors and also for the development of activity-based probes. Such probes would be useful as tools to further characterize the role of matriptase-2 in iron homeostasis. Acknowledgments This work was supported by grant of Ministry of Science and Higher Education 530-8290-D373-13. N.F. is supported by a fellowship from the Jürgen Manchot Foundation, Düsseldorf, Germany. References [1] R. Szabo, T.H. Bugge, Type II transmembrane serine proteases in development and disease, Int. J. Biochem. Cell. Biol. 40 (2008) 1297e1316. [2] G. Velasco, S. Cal, V. Quesada, L.M. Sánchez, C. López-Otín, Matriptase-2, a membrane bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins, J. Biol. Chem. 277 (2002) 37637e37646. [3] K.E. Finberg, M.M. Heeney, D.R. Campagna, Y. Aydinok, H.A. Pearson, K.R. Hartman, M.M. Mayo, S.M. Samuel, J.J. Strouse, K. Markianos, N.C. Andrews, M.D. Fleming, Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA), Nat. Genet. 40 (2008) 569e571. [4] X. Du, E. She, T. Gelbart, J. Truksa, P. Lee, Y. Xia, K. Khovananth, S. Mudd, N. Mann, E.M. Moresco, E. Beutler, B. Beutler, The serine protease TMPRSS6 is required to sense iron deficiency, Science 320 (2008) 1088e1092. [5] L. Silvestri, A. Pagani, A. Nai, I. De Domenico, J. Kaplan, C. Camaschella, The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin, Cell Metab. 8 (2008) 502e511. [6] J.E. Maxson, J. Chen, C.A. Enns, A.S. Zhang, Matriptase-2- and proprotein convertase-cleaved forms of hemojuvelin have different roles in the downregulation of hepcidin expression, J. Biol. Chem. 285 (2010) 39021e39028. [7] M. Stirnberg, E. Maurer, A. Horstmeyer, S. Kolp, S. Frank, T. Bald, K. Arenz, A. Janzer, K. Prager, P. Wunderlich, J. Walter, M. Gütschow, Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface, Biochem. J. 430 (2010) 87e95. [8] M. Stirnberg, M. Gütschow, Matriptase-2, a regulatory protease of iron homeostasis: possible substrates, cleavage sites and inhibitors, Curr. Pharm. Des. 19 (2013) 1052e1061. [9] E. Maurer, M. Gütschow, M. Stirnberg, Hepatocyte growth factor activator inhibitor type 2 (HAI-2) modulates hepcidin expression by inhibiting the cell surface protease matriptase-2, Biochem. J. 450 (2013) 583e593. [10] C.J. Farady, C.S. Craik, Mechanisms of macromolecular protease inhibitors, ChemBioChem 11 (2010) 2341e2346. [11] M.T. Sisay, T. Steinmetzer, M. Stirnberg, E. Maurer, M. Hammami, J. Bajorath, M. Gütschow, Identification of the first low-molecular-weight inhibitors of matriptase-2, J. Med. Chem. 53 (2010) 5523e5535.
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