Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2¢ of substrates Mattias K Andersson, Michael Thorpe and Lars Hellman Department of Cell and Molecular Biology, Uppsala University, The Biomedical Center, Sweden Keywords chymase; cleavage specificity; human chymase; mast cell; site-directed mutagenesis Correspondence L Hellman, Department of Cell and Molecular Biology, Uppsala University, The Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden Fax: +46 18 471 4382 Tel: +46 18 471 4532 E-mail: lars.hellman@icm.uu.se Website: http://www.icm.uu.se/immuno/ (Received 29 December 2009, revised March 2010, accepted March 2010) doi:10.1111/j.1742-4658.2010.07642.x Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2¢ These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively) We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2¢ of substrates In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase Analysis of the extended cleavage specificities of two single mutants (Arg143 fi Gln and Lys192 fi Met) and the combined double mutant revealed an altered specificity for P2¢ amino acids, whereas all other positions were essentially unaffected A weakened preference for acidic amino acids at position P2¢ was observed for the two single mutants, whereas the double mutant lacked this preference Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2¢ This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases Furthermore, the conservation of the preference for acidic P2¢ amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes Introduction Mast cells (MCs) are resident tissue cells that are distributed along the surfaces of the body They are frequently found in the mucosa of the airways and intestine, in connective tissue of the skin, and around blood vessels and nerves Upon activation, MCs are able to rapidly exocytose their cytoplasmic granules, resulting in the release of prestored physiologically active inflammatory mediators The majority of proteins found in these granules are serine proteases, and one subfamily of these proteases comprises the chymases Chymases cleave substrates after aromatic amino acids, and are therefore chymotrypsin-like Phylogenetic analyses of the chymases have led to the identification of two distinct subfamilies, the a-chymases and the b-chymases [1] The a-chymases are encoded by a single gene in all species investigated, except for ruminants, where two very similar a-chymase genes have been identified [2] The b-chymases Abbreviations Ang, angiotensin; DC, dog chymase; EK, enterokinase; HC, human chymase; IPTG, isopropyl thio-b-D-galactoside; MC, mast cell; mMCP, mouse mast cell protease; OC, opossum chymase; rMCP, rat mast cell protease FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ê 2010 FEBS 2255 P2Â specicity of the human mast cell chymase M K Andersson et al have only been identified in rodents Interestingly, the rodent a-chymases mouse MC protease (mMCP)-5 and rat MC protease (rMCP)-5 have changed their primary cleavage specificity from aromatic amino acids (chymotrysin-like) to aliphatic amino acids (elastaselike) A large number of in vitro substrates have been identified for the chymases However, the absolute majority of these have never been shown to also be substrates in vivo [3] Therefore, the true functions of the chymases most likely remain to be identified In order to increase our understanding of these enzymes, a necessary step is to determine the most important feature of an enzyme, the specificity-determining interactions with its substrates In a previous study, we determined the cleavage specificity in seven positions from positions P4 to P3¢ for human chymase (HC) [4] The cleavage of the peptide bond occurs between positions P1 and P1¢, where the amino acids N-terminal of this bond are designated P1, P2, P3, P4 Pn and those C-terminal P1¢, P2¢, P3¢ Pn¢ [5] The strongest preference observed, besides the primary specificity for P1 Phe or Tyr, was the preference for negatively charged (acidic) amino acids at position P2¢ An evaluation of natural substrates for HC showed that many of these also have acidic amino acids at position P2¢ [4,6] These observations suggest an important role for negatively charged amino acids at position P2¢ during substrate discrimination by HC The structure of HC has been extensively investigated, and also compared with those of MC chymases in other species These studies have provided insights into important enzyme–substrate interactions For example, molecular modeling of HC interacting with angiotensin (Ang) I has led to conclusions regarding the S2¢ binding site of HC [7–9] These studies have shown that Lys40, Arg143 and Lys192 are located close to the S2¢ binding site, which may favor negatively charged P2¢ side chains of substrates However, these data are based on structural studies of HC in complex with inhibitors, where the interaction of an acidic amino acid with the S2¢ subsite cannot be determined In addition, Ang I that was modeled into the active cleft of HC did not bring an acidic amino acid to this position Therefore, it is still uncertain which of the side chains of Lys40, Arg143 or Lys192 are able to contact the acidic side chain of a negatively charged amino acid in position P2¢ of a substrate The extended cleavage specificities of several related MC chymases have recently been determined The a-chymases opossum chymase (OC) guinea pig chymase and rMCP-5 and the b-chymase mMCP-4 have also been found to prefer acidic P2¢ amino acids (Table 1) [6,10,11,17] In contrast, the dog a-chymase and the b-chymases mMCP-1, rMCP-1 and rMCP-4 were found to prefer other amino acids than Asp or Glu in this position [6,12,13] (submitted manuscript Gallwitz et al., 2009) When the amino acids in positions 40, 143 and 192 of the above chymases are compared, the five chymases with a specificity for acidic P2¢ amino acids all have Arg143 and Lys192 However, they differ at position 40 (Table 1) Furthermore, none of the four chymases that lack the acidic P2¢ specificity has an Arg at position 143 However, three of them have Lys at position 192 On the basis of these observations, we hypothesized that Arg143 alone or in cooperation with Lys192 mediates the preference for acidic amino acids at position P2¢ In the present study, we tested the roles of Arg143 and Lys192 as P2¢ specificity-determining residues By in vitro mutagenesis, the HC coding region was modified so that Arg143 was replaced by Gln and Lys192 was replaced by Met, which are amino acids found in the same positions of chymases that lack acidic P2¢ specificity Our results clearly show that positions 143 and 192 have an effect in mediating the acidic P2¢ specificity Arg143 and Lys192 are essential in conferring a strong preference for acidic amino acids at position P2¢ of the substrates Table P2¢ specificity and amino acids found in positions 40, 143 and 192 of nine different chymases Chymase P2¢ specificity Residue 40 Residue 143 Residue 192 Reference Human a-chymase Opossum a-chymase rMCP-5 (a-chymase) mMCP-4 (b-chymase) Guinea pig chymase Dog a-chymase Asp ⁄ Glu Asp Glu > Leu Glu ⁄ Asp Glu ⁄ Asp > Gln > Ala Ser ⁄ Leu > Glu ⁄ Asp Lys His Ser Ala Lys Ala Arg Arg Arg Arg Arg Lys Lys Lys Lys Lys Lys Lys rMCP-1 (b-chymase) rMCP-4 (b-chymase) mMCP-1 (b-chymase) Ser Gly > Ser ⁄ Leu Leu > Val > Ala Ala Thr Asp Gln Gln Lys Lys Lys Met 10 11 17 (Gallwitz et al 2009, unpublished results) 13 12 2256 FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS M K Andersson et al P2¢ specificity of the human mast cell chymase Results Production and purification of recombinant HC mutants Two single mutants of HC, Arg143 fi Gln and Lys192 fi Met, and a double mutant, Arg143 fi Gln + Lys192 fi Met, were produced by in vitro mutagenesis Following control sequencing of the full coding regions, the three different pCEP-Pu2 vector constructs were transfected into HEK 293 EBNA cells for protein production Recombinant protein was purified from conditioned media on Ni2+–nitrilotriacetic acid agarose, by binding through the N-terminal His6-tag The protein yield was 100–150 lg recombinant protein from L of medium for all three mutants Activation and further purification of the recombinant HC mutants Following the initial Ni2+–nitrilotriacetic acid agarose purification, the three different recombinant HC mutants were activated by removal of the His6-tag by proteolytic cleavage with enterokinase (EK) Approximately 30 lg of each mutant was treated with EK for h at 37 °C Samples of inactive and activated proteases were separated on SDS ⁄ PAGE gels, in order to ensure successful removal of the His6-tag and the EK-susceptible cleavage site (Fig 1) Like the wildtype enzyme, the mutated inactive proteases migrated R143Q K192M R143Q + K192M kDa –EK +EK Hep –EK +EK Hep –EK +EK Hep 97 66 45 30 20 14 Fig Purification and activation of recombinant HC mutants Three different recombinant HC mutants were expressed with an N-terminal His6-tag followed by an EK-susceptible sequence replacing the signal peptide These proenzymes were first purified on Ni2+–nitrilotriacetic acid beads ()EK), and then activated by removal of the His6-tag by EK digestion (+EK) Following activation, the enzymes were further purified on heparin–Sepharose columns (Hep) Proenzymes and activated enzymes before and after heparin–Sepharose purification were analyzed by separation on SDS ⁄ PAGE gels and visualized with Coomassie Brilliant Blue staining as 35 kDa bands, and the EK-digested enzymes migrated as 33 kDa bands (Fig 1) This is somewhat over the theoretical value of 25 kDa for wild-type HC, which indicates glycosylation at two sites of these proteases To purify the activated proteases from contaminating serum and cellular proteins, imidazole, and EK, they were purified over a heparin–Sepharose column The heparin–Sepharose-purified fractions were separated on SDS ⁄ PAGE gels, and no contaminating bands could be detected (Fig 1) The proteolytic activities of the eluted fractions of the three mutated HCs were analyzed by cleavage of the chymotrypsin-sensitive chromogenic substrate S-2586 (MeO-Suc-ArgAla-Tyr-pNA, Chromogenix, Molndal, Sweden, data ă not shown) Determination of the extended cleavage specificity of the three HC mutants by phage display technology The phage library used to determine the extended cleavage specificity of the HC mutants contains  · 107 phage clones Each phage clone expresses a unique sequence of nine random amino acids, followed by a His6-tag in the C-terminus of capsid protein 10 Thereby, the phages display a random nonamer on their surface, and by interactions of the His6-tag the phages can be immobilized on Ni2+–nitrilotriacetic acid agarose beads The three HC mutants were used to screen the phage library for peptides susceptible to cleavage After the first selection step (biopanning), the phages, released by digestion of nonapeptides, were amplified in Escherichia coli and subjected to additional biopannings Selection of nonamers susceptible to cleavage by the Lys192 fi Met and Arg143 fi Gln + Lys192 fi Met HC mutants was performed over five biopannings, after which they induced the release of 47 and 46 times more phages, respectively, than an NaCl ⁄ Pi control (Fig 2) Peptides sensitive to cleavage by the Arg143 fi Gln mutant were selected over six biopannings, which resulted in an 81-fold greater release of phages as compared with an NaCl ⁄ Pi control (Fig 2) After the last biopanning, 44 individual phage clones were isolated for each of the three HC mutants, and the sequences encoding the randomly synthesized nonapeptides were determined The nucleotide sequences were then translated into nonapeptides, which were aligned on the basis of similarities to the cleavage specificity of wild-type HC [4] For the Arg143 fi Gln mutant, 41 sequences were determined in total, two of which were obvious background sequences and therefore not included in the FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2257 P2¢ specificity of the human mast cell chymase Cleaved/control ratio 100 M K Andersson et al R143Q K192M R143Q + K192M 80 60 40 20 0 Biopannings Fig Amount of released T7 phages after digestion with HC mutants, as compared with an NaCl ⁄ Pi control A library of randomly synthesized nonamers expressed at the C-terminus of T7 phages were subjected to cleavage by HC mutants Selection for nonamers susceptible to cleavage by the HC mutants was performed in five or six rounds of selection (biopannings) After each biopanning, the amount of released phages was determined and compared to an NaCl ⁄ Pi control The ratio of phages released by enzyme digestion over the NaCl ⁄ Pi control for each biopanning is shown The Arg143 fi Gln mutant is indicated by a dashed line, the Lys192 fi Met mutant by a dotted line, and the Arg143 fi Gln + Lys192 fi Met double mutant by an unbroken line alignment The 39 remaining sequences were aligned (Fig 3A) Twelve of these sequences were derived from the same phage clone (Trp-Trp-Ala-Ile-Glu-Met-PheAsp-Met), four sequences from the clone Trp-Phe-ValThr-Phe-Tyr-Asp-Ser-Leu, and two from the clone Val-Val-Ser-Tyr-Gly-Gly-Val-Leu-Glu From the 44 phage clones isolated for the Lys192 fi Met mutant, 40 sequences were determined, of which three were background phages The remaining 37 sequences were aligned Among these sequences, one of the phage clones (Pro-Met-Leu-Tyr-Ser-Leu-Asn-Asp-Ser) was found twice (Fig 3B) Of the phage clones from the double mutant, 36 clones were analyzed Three of these were background phages and the remaining 33 were aligned (Fig 3C) Two of the aligned sequences were derived from the same clone (Thr-Leu-Phe-TyrTrp-Gly-Ala-Thr-Gly) On the basis of the alignments, the distribution of amino acids in positions P4–P3¢ were calculated for each HC mutant (Fig 4) In order to normalize for the uneven occurrence of individual phage clones in the alignment, all clones that were found more than once were calculated as one As expected, the three HC mutants showed very similar preferences for amino acids flanking the cleaved peptide bond In the positions N-terminal of the cleaved bond, positions P2–P4, a clear overrepresentation of aliphatic amino acids, particularly Val and Leu, was observed In position P1, Phe and Tyr were more frequently seen 2258 than Trp The three mutants also shared preferences in the positions on the C-terminal side of the scissile bond The aliphatic Gly and Ala, and the hydrophilic Ser, dominate in position P1¢ Similar preferences were identified in position P3¢, where aliphatic amino acids were generally preferred However, in this position, the hydrophilic amino acids Ser and Thr were also frequently found All of the positions mentioned above fit very well with the cleavage specificity of wild-type HC, as previously determined by our group [4] The only position where we detected an altered specificity between the wild-type HC and the HC mutants was in position P2¢ The strong preference for acidic amino acids found in wild-type HC had disappeared in the Arg143 fi Gln and Lys192 fi Met mutants and the double mutant, and instead we observed a preference for aliphatic amino acids A comparison between the wild-type HC and the HC mutants regarding the specificity for acidic amino acids at position P2¢ is shown in Fig In a previous study, where the same phage-displayed nonapeptide library was used to determine the cleavage specificity of wild-type HC, a negatively charged amino acid at position P2¢ was seen in 58% of the sequences For the Arg143 fi Gln mutant, this figure was reduced to 25%, and for the Lys192 fi Met mutant, 19% of the sequences were aligned with an acidic amino acid at position P2¢ When these mutations were combined in the double mutant, we could only observe acidic amino acids at position P2¢ in 6% of the sequences Thus, Arg143 and Lys192 contribute almost equally to the P2¢ specificity of HC Mutating either of the two basic amino acids in these positions partially disrupts the acidic P2¢ preference, and mutating both of them totally removes this preference The negatively charged amino acids Asp and Glu are roughly equally distributed in this position in the wildtype HC and the three HC mutants Therefore, Arg143 and Lys192 not distinguish between Asp or Glu, but attract these residues equally well, probably solely on the basis on the negative charge of their side chains Verifying the consensus sequence by the use of a new type of recombinant protein substrate In order to verify the results from the phage display analysis, a new type of recombinant substrate was developed The consensus sequence obtained from the phage display analysis was inserted in the linker region between two E coli thioredoxin molecules by ligating a double-stranded oligonucleotide encoding the actual sequence into a BamHI and a SalI site of the vector construct (Fig 6A) For purification purposes, a His6-tag FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS M K Andersson et al P2¢ specificity of the human mast cell chymase R143Q A K192M B T M W W W W W 12 W W W G W T V V L T G R A V M M T T V L P M E V T E S V A F S L L V G R L S P V T V A Y F Y L V D M F T Y F Y F F Y W F Y F Y Y F F Y F F F F F F F W F F F G S S S S P F T S G L R G S A L A G S D A Y L G G T S E R V V G L S A S G A D V L W T M D D D E V G G H M S T T T Y T L E D V G A L N H V S F H L E S E R E D L V L R P V G G M G F I G V V Y P I I V W R W T A W V E G W E Q I P Y W G L L Aromatic Negatively charged R L R G W W A W G Y M W A A I V W V Positively charged Large aliphatic Small aliphatic P4 P3 P2 P1 P1′P2′ P3′ P4 P3 P2 P1 P1′P2′ P3′ P4 P3 P2 P1 P1′P2′ P3′ V V G E I P R Q V A V G V E V E W G I S A T M S P W E I W R A I V V W F W A V D R143Q + K192M C A W Y W F E V A A P P L V I A V T V W K R K R V V L V L P W V L L V W A P V F P P R T V V V M M V L V V L S V F A I I W T S R W G Q V F Y F R W L T I V W W F A G M L M A P F D F F V V L P S L L V K R G S S A S T F L V F S S L S Y Y Y Y Y Y Y Y F Y F F Y Y F W F F F F F F Y F F F F Y F Y F Y F F F Y F A A E S A G A L S S S S S F G A A W S T L I L V A A G A E S S G F A S A A A M L L L R E M L L A L G S G G G H D L G H G L E D E A G L F S L D E G T V N E D S L L L A S E Q S E S T H M C R H H P R L L F F F T W H E W R V L D E C D M S A V M A V S R K E G S G S L T L L W V F I L P A C L W R P L L A A L L A V W M M M A M V L A V V L S S R G A V E V T V V R S P T G G G R V P V A V G W V T W G E V G M M V V V V A L I L A L L L T A A L L V V V S F L V V T L R R I L A A G L S M S S L G F T F L F F L L A V S V L F P A A F D L L Y F F Y Y F Y Y Y W W F F F F F F Y F Y F F F F W F F F Y W Y F S S S S A G G G V H T G A M W G S A F W F F W A A G F W W T S Y A S S A A G G G G H H D A G G G G F C M A G A S E F M G G R W A A L V R L R V S I H H V I S T L E V C C R L V A T V Y V A G G G L L P R R L L L K E G L L Q V G G L P R T R G V L T I G W W G G T W R A R A A L R R R H P E T T G Y P F Fig Phage-displayed nonamers susceptible to cleavage by HC mutants after five or six biopannings After the last selection step, phages released by proteolytic cleavage of the HC mutants were isolated, and the sequences encoding the nonamers were determined The general sequence of the T7 phage capsid proteins is PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers The protein sequences were aligned into a P4–P3¢ consensus, where cleavage occurs between positions P1 and P1¢ If the sequence was found more than once, this is indicated by the corresponding number to the left of the sequence The amino acids are color coded according to the side chain properties as indicated in the key For the Arg143 fi Gln mutant (A), 24 unique sequences were aligned; for the Lys192 fi Met mutant (B), 36 unique sequences were aligned; and for the Arg143 fi Gln + Lys192 fi Met double mutant (C), 32 unique sequences were aligned The sequences with one aromatic amino acid (potential cleavage site) are placed on the top, followed by sequences containing two, three or four aromatic amino acids was added to the C-terminus of this protein (Fig 6A) A number of related and unrelated substrate sequences were also produced with this system, by ligating the corresponding oligonuclotides into the BamHI ⁄ SalI sites of the vector All of these substrates were expressed as soluble proteins in a bacterial host, E coli, and purified on immobilized metal affinity chromatography columns to obtain a protein with a purity of 90–95% These recombinant proteins were then used to study the preference of HC and the double mutant for these different sequences (Fig 6B–E) The results showed that HC very efficiently cleaved the HC consensus sequence (VVLFSEVL) [4] When the Glu in position P2¢ of the HC consensus sequence was replaced by a Gly (VVLFSGVL), the efficiency of cleavage by HC dropped by a factor of 4–5 (Fig 6B) In contrast, the HC double mutant preferred this substrate over the HC consensus site by a factor of 2–5 (Fig 6C and data not shown) This latter experiment shows that HC has a marked preference for negatively charged amino acids in position P2¢ and that the double mutant has lost this preference, instead preferring aliphatic amino acids in this position The consensus site for dog chymase (DC) has recently been determined (submitted manuscript Gallwitz et al., 2009) This site (VVRFLSLL) shows similarities with the preferred site for the HC double mutant, and neither sequence contains an acidic P2¢ residue Consequently, HC was found to cleave the DC consensus sequence five-fold to seven-fold less FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2259 Occurrence (%) P2¢ specificity of the human mast cell chymase 40 P4 M K Andersson et al P3 40 WT 30 30 10 10 Occurrence (%) 10 Occurrence (%) 20 40 Occurrence (%) 20 40 40 F YWG A V L I P S T CMNQ H K R D E 40 R143Q 30 F YWG A V L I P S T CMNQ H K R D E 40 R143Q 30 F YWG A V L I P S T CMNQ H K R D E R143Q 30 20 20 20 10 10 10 0 F YWG A V L I P S T CMNQ H K R D E 40 40 K192M 30 F YWG A V L I P S T CMNQ H K R D E F YWG A V L I P S T CMNQ H K R D E K192M 30 K192M 30 20 20 20 10 10 10 0 20 F YWG A V L I P S T CMNQ H K R D E 40 40 R143Q+ K192M 30 F YWG A V L I P S T CMNQ H K R D E F YWG A V L I P S T CMNQ H K R D E R143Q+ K192M 30 20 10 20 10 Occurrence (%) F YWG A V L I P S T CMNQ H K R D E 70 70 WT 50 40 40 30 30 20 20 10 Occurrence (%) K192M 50 10 F YWG A V L I P S T CMNQ H K R D E 70 P1 60 F YWG A V L I P S T CMNQ H K R D E 70 R143Q P1 60 50 40 40 30 30 20 20 10 10 F Y WG A V L I P S T C M N Q H K R D E P1´ 40 WT 30 P2´ 40 WT 10 0 Occurrence (%) 10 F YWG A V L I P S T CMNQ H K R D E 20 Occurrence (%) 20 40 F YWG A V L I P S T CMNQ H K R D E 40 30 R143Q+ K192M 50 30 P1 60 40 F YWG A V L I P S T CMNQ H K R D E F YWG A V L I P S T CMNQ H K R D E P1 60 R143Q+ K192M 30 10 Occurrence (%) WT 30 20 R143Q 40 30 30 P3´ WT 20 10 F YWG A V L I P S T CMNQ H K R D E R143Q 40 30 20 20 10 10 R143Q 10 F YWG A V L I P S T CMNQ H K R D E 20 30 F YWG A V L I P S T CMNQ H K R D E F YWG A V L I P S T CMNQ H K R D E Occurrence (%) P2 40 WT 40 K192M 30 20 10 K192M 20 10 F YWG A V L I P S T CMNQ H K R D E 40 30 20 R143Q+ K192M 10 R143Q+ K192M F YWG A V L I P S T CMNQ H K R D E 40 10 K192M 10 40 20 30 20 F YWG A V L I P S T CMNQ H K R D E 30 F YWG A V L I P S T CMNQ H K R D E 40 30 20 F YWG A V L I P S T C MN Q H K R D E R143Q+ K192M 10 F YWG A V L I P S T CMNQ H K R D E F YWG A V L I P S T CMNQ H K R D E Fig Distribution of amino acids at positions P4–P3¢ in phage-displayed nonamers cleaved by wild-type (WT) HC or HC mutants after five or six biopannings On the basis of the alignment in Fig and previously published data on wild-type HC, the percentage of each amino acid present in each position, P4 to P3¢, was calculated The amino acids are ordered from left to right: aromatic, aliphatic, hydrophilic, basic (positively charged), and acidic (negatively charged) 2260 FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS M K Andersson et al P2¢ specificity of the human mast cell chymase Discussion Acidic amino acids in P2′ (%) 70 Glu Asp 60 50 40 30 20 10 HC wt R143Q K192M R143Q + K192M Fig Distribution of acidic amino acids in position P2¢ of wild-type (wt) HC and HC mutants The occurrence of acidic amino acids aligned in position P2¢ was compared between wild-type HC and the Arg143 fi Gln mutant, the Lys192 fi Met mutant, and the Arg143 fi Gln + Lys192 fi Met double mutant Glu residues in this position are depicted as open bars, and Asp residues as filled bars The occurrence of acidic amino acids in position P2¢ of wildtype HC was determined in a previous study [4] efficiently than the HC consensus sequence, whereas the HC double mutant cleaved this substrate almost as efficiently as its own consensus site (Fig 6B,C) A few additional substrates were also included in this study The optimal sequence for cleavage by OC has recently been determined [10] As compared with HC, this enzyme was found to have a preference for Trp over Phe and Tyr at position P1 When we analyzed the cleavage of this sequence (VGLWLDRV), we observed that HC cleaved this sequence  50-fold less efficiently than the HC consensus sequence (Fig 6B and data not shown) Similarly, the HC double mutant cleaved this sequence with a very low cleavage rate (Fig 6C) We also tested three additional sequences, the human thrombin consensus (LTPRGVRL), which we recently determined by phage display analysis, and the rat granzyme B (LIETDSGL) [14] and EK consensus sequences (LDDDDKGL) Neither the granzyme B, the EK nor the thrombin substrate was cleaved at all by HC and the HC double mutant, even after 150 at room temperature (Fig 6D,E) OC was included here as reference and to compare its preferences for the different sequences used to study HC and the HC double mutant (Fig 6F) OC was found to cleave the OC consensus sequence fivefold to eight-fold more efficiently than the HC or the DC consensus sites This verifies its preference for Trp over Phe and Tyr at position P1 and the accuracy of the information obtained by the phage display analysis Site-directed mutagenesis has previously been used to study the effect on cleavage specificity of changing a single amino acid in an enzyme As an example, the primary specificity of the serine protease mouse granzyme B was altered so that it cleaved after an aromatic amino acid instead of Asp by a single Arg226 fi Gly mutation [15] A preference for basic amino acids was seen when Arg226 was replaced by Glu in the same enzyme [16] In the present study, we used the same strategy to investigate the effect of two amino acids on the extended substrate interactions of HC We showed that we could change the cleavage specificity of HC for position P2¢ of substrates by mutating positions 143 and 192 in the enzyme By replacing Arg143 by Gln or Lys192 by Met, which are amino acids commonly found in these positions in related rodent chymases, the preference for acidic amino acids at position P2¢ was markedly reduced, whereas the cleavage specificity for all other positions was essentially unaffected The basic amino acids at positions 143 and 192 attract and stabilize the interaction of acidic amino acid side chains at position P2¢ When either of these positively charged residues was replaced by an amino acid with an uncharged side chain, a preference for mainly aliphatic amino acids was observed However, a weak preference for acidic P2¢ amino acids still remained for the two single mutants In the double mutant, the preference for negatively charged P2¢ amino acids was lost completely In order to put our hypothesis to a stringent test, we aligned the enzyme-selected peptides with acidic P2¢ amino acids, when an aromatic amino acid could be aligned at position P1 We then aligned the sequences according to the cleavage specificity of the wild-type enzyme, considering the remaining positions We could thus be certain that we were not overestimating the effect of the mutations However, a large fraction of the acidic amino acids aligned at position P2¢ belong to sequences with three or four possible aromatic P1 amino acids In these cases, it is difficult to know where the cleavage actually occurred or if they were selected owing to multiple cleavage sites Therefore, the effects of the two single mutants may be slightly underestimated On the other hand, when the enzymeselected sequences are examined overall, fewer negatively charged amino acids are found among the sequences cleaved by the HC double mutant than among those cleaved by the single mutants (nine, 21, and 21, respectively) This is an indication that the single mutants are more tolerant of acidic amino acids in cleavable substrates More importantly, we can FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2261 P2¢ specificity of the human mast cell chymase M K Andersson et al A B C D E F 2262 FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS M K Andersson et al conclude from our data that the preference of HC for acidic amino acids at position P2¢ is mediated by the combined effects of both Arg143 and Lys192 A similar combined effect has, to our knowledge, never been identified for a serine protease By the use of a new type of recombinant substrate, we were also able to verify this marked change in preference for a negatively charged amino acid at position P2¢ by mutating Arg143 and Lys192 Wildtype HC was found to cleave the consensus substrate four-fold to five-fold more efficiently than the substrate in which the amino acid at position P2¢ had been exchanged for a Gly In contrast, the HC double mutant showed a two-fold to five-fold greater preference for the substrate in which position P2¢ was not negatively charged These results clearly show that the double mutant had lost its preference for negatively charged amino acids, and instead preferred aliphatic or noncharged amino acids at this position The identification of Arg143 and Lys192 as P2¢ preference-determining amino acids facilitates predictions about other related chymases As stated earlier, we have identified three other chymases with the HC-like preference for acidic amino acids at position P2¢, namely the opossum a-chymase, rMCP-5, and mMCP-4 [6,10,11] All of these chymases have Arg143 and Lys192 Other chymases that have Arg143 and Lys192 are the a-chymases from the macaque and the baboon, the sheep MC protease, mMCP-5, and hamster chymase-2 Furthermore, the b-chymases rMCP-3, hamster chymase-1 and gerbil chymase-1 also have Arg143 and Lys192 The guinea pig a-chymase was recently cloned and shown to have Arg143 and Lys192 [17] In agreement with our prediction, screening of a combinatorial library indicated a preference for acidic P2¢ amino acids [17] The preference for acidic P2¢ amino acids seems to be highly preserved among the a-chymases However, there may be minor exceptions The dog a-chymase has Lys143 and Lys192, and according to our analysis this chymase has only a weak preference for acidic amino acids at position P2¢ (submitted P2¢ specificity of the human mast cell chymase manuscript Gallwitz et al., 2009) Apparently, a minor change from the positively charged Arg to a slightly smaller but still positively charged side chain of a Lys at position 143 is enough to partially affect the preference for acidic P2¢ amino acids The gerbil chymase-2 also has Lys143 and Lys192, and may therefore also have a lower preference for acidic amino acids at position P2¢ HC efficiently converts Ang I to Ang II (cleavage of the Phe8-His9 bond of Ang I), and the structural requirements for this specificity have been addressed in several studies Synergistic interactions of positions P4–P1, together with the dipeptidyl leaving group of Ang I, were found to be important for efficient conversion by HC [18,19] However, the side chains on the leaving group of Ang I not seem to be important for the selectivity of HC in converting Ang I [19] Instead, the negatively charged C-terminal carboxyl group of Ang I probably interacts with the Lys40 side chain of HC to stabilize the substrate [8] Lys40 and Arg143 of HC have previously been analyzed for their role in the selective Ang I conversion by HC Analysis of Lys40 fi Ala and Arg143 fi Gln mutants of HC showed that Lys40 but not Arg143 contributed to the high specificity of HC in converting Ang I to Ang II [20] The Arg143 fi Gln mutant was actually shown to be more active than the wild type in converting Ang I, indicating a minor role or no role at all of Arg143 in Ang I conversion The lack of function for Arg143 and Lys192 in Ang I conversion is also substantiated by the fact that mMCP-1, which has Lys143 and Met192, and has a P2¢ specificity for Leu, is a good Ang I converter [21] Furthermore, the rat vascular chymase, which has Arg143 and Thr192 and thus does not have a predicted P2¢ specificity for acidic amino acids, also has a very good Ang I conversion capability [22] However, our results clearly show that Arg143 and Lys192 of HC are of major importance in mediating the specificity for acidic P2¢ side chains of substrates, but not when the negative charge is situated at a C-terminal carboxyl group of a P2¢ amino acid of Fig Analysis of cleavage specificity by the use of new types of recombinant protein substrate (A) The overall structure of the recombinant protein substrates used for analysis of the efficiency of cleavage by HC and the HC Arg143 fi Gln + Lys192 fi Met double mutant In these substrates, two thioredoxin molecules are positioned in tandem, and the proteins have His6-tags positioned in their C-termini The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one BamHI site and one SalI site, which are indicated at the bottom of (A) (B, D) The cleavage of a number of substrates by HC The names and sequences of the different substrates are indicated above the pictures of the gels The times of cleavage in minutes are also indicated above the corresponding lanes of the different gels The uncleaved substrates have a molecular mass of  25 kDa, and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa (C, E) The cleavage of the same substrates as for HC in (B) and (D), but now after cleavage with the HC double mutant (F) The cleavage of a number of the above selected substrates with OC On the right side of the figure, the scanned and quantified protein bands are summarized in individual diagrams The quantification was performed with IMAGEJ FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2263 P2¢ specificity of the human mast cell chymase M K Andersson et al the substrate The marked difference in the importance of Lys40, Arg143 and Lys192 in determining substrate specificity between peptides and long substrates is striking This clearly shows the importance of analyzing a broad range of different substrates, with different biochemical characteristics, when looking for the natural in vivo substrates The high degree of conservation of the preference for negatively charged amino acids at position P2¢ is also a strong indicator that substrates other than Ang I are evolutionarily conserved targets for the MC a-chymases or their rodent counterpart, the b-chymase mMCP-4 The search for potential in vivo substrates is being performed using bioinformatic screening However, the identification of these substrates may be challenging, mainly because of the ability of HC to interact with different amino acid side chains in each subsite of the enzyme, which leads to a very large number of potential substrates during the screening of the full human proteome This highlights the importance of factors other than the extended cleavage specificity in determining whether a protein will be a biologically significant substrate for HC For example, the local concentration of the protease and availability of the potential substrate in the immediate environment are significant factors The extended cleavage specificity predicts those sequences (and hence substrates) that would be preferentially cleaved within a shorter time frame These substrates can theoretically be cleaved before the protease encounters a protease inhibitor The influence of the cleavage sequence position in the protein is also important It is likely that surfaceexposed, flexible regions would be cleaved efficiently Conversely, nonexposed, more rigid regions would remain uncleaved, regardless of whether the preferred sequence for the protease was present or not In the event of substrate cleavage, whether this leads to a biological effect also needs to be determined With knowledge of the extended cleavage specificity and other parameters in hand, the search for biologically significant substrates for HC is more conceivable Experimental procedures In vitro mutagenesis of HC The HC wild-type sequence has previously been cloned and inserted into the pCEP-Pu2 vector for expression in mammalian cells [4,23,24] This vector construct was used for in vitro mutagenesis of HC In this construct, the 5¢-end of the coding region contains a signal sequence followed by a sequence encoding a His6-tag and an EK-susceptible site (Asp-Asp-Asp-Asp-Lys) The N-terminal His6-tag and EK 2264 site in the translated protein facilitated purification and activation of the enzyme Mutagenesis of HC was performed using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) The Arg143 fi Gln mutant was produced by cycling the wild-type sequence in pCEP-Pu2, according to the manufacturer’s recommendations, using the following primers: sense primer, 5¢-CGGGTGGCTGG CTGGGGACAGACAGGTGTGTTGAAGC-3¢; and antisense primer, 5¢-GCTTCAACACACCTGTCTGTCCCCA GCCAGCCACCCG-3¢ These primers had a melting temperature of 79.5 °C, and the mismatches resulting in replacement of the Arg codon with a Gln codon are underlined To produce the Lys192 fi Met mutant, the following primers were used: sense primer, 5¢-CAGGAA GACAAAATCTGCATTTATGGGAGACTCTGG-3¢; and antisense primer, 5¢-CCAGAGTCTCCCATAAATGCAGA TTTTGTCTTCCTG-3¢ These primers had a melting temperature of 78 °C, and the mismatches resulting in replacement of the Lys codon with a Met codon are underlined The Arg143 fi Gln + Lys192 fi Met double mutant was produced by introducing the Lys192 fi Met mutation into the Arg143 fi Gln mutant All primers were purchased from Sigma-Aldrich (Steinheim an Albuch, Germany) in a PAGE-purified form Thermal cycling was performed with PfuUltra high-fidelity DNA polymerase (provided by the manufacturer) After this PCR step, nonmutated parental DNA constructs were digested with Dpn1 endonuclease for h at 37 °C The remaining nondigested and mutated DNA vector constructs were ethanol precipitated The salt and ethanol concentration during precipitation was 75 mm NaAc (pH 5.2) in 75% ethanol After precipitation, the DNA was resuspended in 15 lL of double-distilled H2O This DNA was then used to transform XL10-Gold ultracompetent E coli cells (provided by the manufacturer) All mutants were sequenced to confirm the inserted mutations and the absence of unintended additional mutations, by using an ABI PRISM 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) and vector-specific primers Production and purification of recombinant HC mutants The vector constructs encoding the HC mutants were transfected into a human embryonic kidney cell line (HEK 293 EBNA) at  80% confluence, using Lipofectamine (Invitrogen, Carlsbad, CA, USA) as previously described [23,24] Selection of transfected cells was initiated by the addition of 1.5 lgỈmL)1 puromycin to the cell culture medium (DMEM supplemented with 5% fetal bovine serum, 50 lgỈmL)1 gentamicin, and lgỈmL)1 heparin) The level of puromycin was decreased to 0.5 lgỈmL)1 after  days of selection Conditioned medium was collected and centrifuged at 1600 g to remove cell debris, and this was followed by the FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS M K Andersson et al addition of 300 lL of Ni2+–nitrilotriacetic acid agarose beads (Qiagen, GmbH, Hilden, Germany) per liter of conditioned medium After h of incubation with gentle agitation at °C, the beads were pelleted by centrifugation at 135 g and transferred to 1.5 mL reaction tubes (Trefflab, Degersheim, Switzerland) The collected Ni2+–nitrilotriacetic acid beads were washed five times with washing buffer (1 m NaCl, 0.2% Tween in NaCl ⁄ Pi) Bound protein was then eluted with elution buffer (100 mm imidazole, 0.2% Triton X-100 in NaCl ⁄ Pi) Protein purity and concentration was estimated by separation on 12.5% SDS ⁄ PAGE gels Protein samples were mixed with sample buffer, and b-mercaptoethanol was added to a final concentration of 5% To visualize the protein bands, the gel was stained with Coomassie Brilliant Blue Activation and further purification of recombinant HC variants Approximately 30 lg of each HC mutant was diluted : in double-distilled H2O and digested for h at 37 °C with EKMax EK (Invitrogen), using one unit per 10 lg of recombinant protease In order to remove EK and other impurities, the EK-digested HC mutants were purified by affinity chromatography on heparin–Sepharose columns as described previously [13] PolyPrep Chromatography columns containing 0.2 mL of heparin–Sepharose beads (SigmaAldrich) were equilibrated with NaCl ⁄ Pi (pH 7.2) Each EK-cleaved HC mutant was applied to a column, and this was followed by washing with 0.3 m NaCl in NaCl ⁄ Pi and subsequent elution with m NaCl in NaCl ⁄ Pi Enzymatic activity towards the chromogenic substrate S-2586 (MeOSuc-Arg-Ala-Tyr-pNA) (Chromogenix) was measured Measurements were performed in 96-well microtiter plates with a substrate concentration of 0.18 mm in 200 lL of NaCl ⁄ Pi S-2586 hydrolysis was monitored spectrophotometrically at 405 nm in a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA) The protein content of flow through, wash and eluted fractions was analyzed on SDS ⁄ PAGE gels Determination of cleavage specificity with a phage-displayed nonapeptide library A library of · 107 unique phage-displayed nonameric peptides was used to determine the cleavage specificity of the HC mutants, as previously described [6,11,13] In these T7 phages, the C-terminus of capsid protein 10 was modified to contain a nine amino acid random peptide followed by a His6-tag [13] An aliquot of the amplified phages ( 109 plaque-forming units) was bound to 100 lL of Ni2+–nitrilotriacetic acid beads by their His6-tags for h at °C under gentle agitation Unbound phages were removed by washing 10 times in 1.5 mL of m NaCl and P2¢ specificity of the human mast cell chymase 0.1% Tween-20 in NaCl ⁄ Pi (pH 7.2), and two subsequent washes with 1.5 mL of NaCl ⁄ Pi The beads were finally resuspended in mL of NaCl ⁄ Pi Activated and heparin– Sepharose-purified HC mutant ( 0.1 lg) was added to the resuspended beads and left to digest susceptible phage nonapeptides under gentle agitation at room temperature overnight NaCl ⁄ Pi without protease was used as control Phages with a random peptide that was susceptible to protease cleavage were released from the Ni2+–nitrilotriacetic acid matrix, and the supernatant containing these phages was recovered To ensure that all of the released phages were recovered, the beads were resuspended in 100 lL of NaCl ⁄ Pi (pH 7.2) and the supernatant, after mixing and centrifugation at 10 000 g, was added to the first supernatant To ensure that the His6-tags had been hydrolyzed on all phages recovered after protease digestion, 15 lL of fresh Ni2+–nitrilotriacetic acid agarose beads was added to the combined phage supernatant, and the mixture was agitated for 15 and then centrifuged at 135 g A control elution of the phages still bound to the beads, using 100 lL of 100 mm imidazole, showed that at least · 108 phages were attached to the matrix during each selection Ten microliters of the supernatant containing the released phages was used to determine the amount of phage detached in each round of selection Dilutions of the supernatant were plated in 2.5 mL of 0.6% top agarose containing 300 lL of E coli (BLT5615), 100 lL of diluted supernatant, and 100 lL of 100 mm isopropyl thio-b-d-galactoside (IPTG) The remaining volume of the supernatant was added to a 10 mL culture of BLT5615 (D  0.6) Thirty minutes prior to phage addition IPTG was added to the bacterial culture to a final concentration of 1mm to induce production of the native T7 phage capsid protein The bacteria lysed  75 after phage addition The lysate was centrifuged at 10 000 g to remove cell debris, and 500 lL of the phage sublibrary was added to 100 lL of fresh Ni2+–nitrilotriacetic acid beads to start the next round of selection After binding of the sublibrary for h at °C under gentle agitation, the Ni2+– nitrilotriacetic acid beads were washed 15 times in 1.5 mL of m NaCl and 0.1% Tween-20 in NaCl ⁄ Pi (pH 7.2), and then twice in 1.5 mL of NaCl ⁄ Pi Following five or six rounds of selection, 44 plaques for each HC mutant were isolated from LB plates after plating in top agarose Each phage plaque, corresponding to a phage clone, was dissolved in phage extraction buffer (100 mm NaCl and mm MgSO4 in 20 mm Tris ⁄ HCl, pH 8.0) and vigorously shaken for 30 in order to extract the phages from the agarose The phage DNA was then amplified by PCR, using primers flanking the variable region of the gene encoding the modified T7 phage capsid protein After amplification, PCR fragments were purified using the E.Z.N.A Micro Elute Cycle-Pure kit (Omega biotek, Doraville, GA, USA) Purified PCR fragments were then sequenced (Macrogen Inc., Seoul, Korea) using an ABI PRISM 3730 DNA Analyzer (Applied Biosystems) FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2265 P2¢ specificity of the human mast cell chymase M K Andersson et al Generation of a consensus sequence from sequenced phage inserts Phage insert sequences were aligned by hand, assuming a preference for aromatic amino acids at position P1 Sequences with only one aromatic amino acid were aligned first, and sequences with more than one possible cleavage site were then aligned to fit this pattern Amino acids with similar characteristics were grouped together as follows: aromatic amino acids (Phe, Tyr, and Trp); negatively charged amino acids (Asp and Glu); positively charged amino acids (Lys and Arg); small aliphatic amino acids (Gly and Ala); larger aliphatic amino acids (Val, Leu, Ile, and Pro); and hydrophilic amino acids (Ser, Thr, His, Asn, Gln, Cys, and Met) The nomenclature of Schechter and Berger [5] was adopted to designate the amino acids in the substrate cleavage region, where P1–P1¢ corresponds to the scissile bond Generation of recombinant substrates for analysis of cleavage specificity A new type of substrate was developed to verify the results obtained from the phage display analysis Two copies of the E coli thioredoxin gene were inserted in tandem into the pET21 vector for bacterial expression (Fig 6A) In the C-terminal end, a His6-tag was inserted for purification on Ni2+ immobilized metal affinity chromatography columns In the linker region, between the two thioredoxin molecules, the different substrate sequences were inserted by ligating double-stranded oligonucleotides into two unique restriction sites, one BamHI site and one SalI site (Fig 6A) The sequences of the individual clones were verified after cloning by sequencing of both DNA strains The plasmids were then transformed into the E coli Rosetta gami strain for protein expression (Novagen; Merck, Darmstadt, Germany) A 10 mL overnight culture of the bacteria harboring the plasmid was diluted 10 times in LB + ampicillin, and grown at 37 °C for 1–2 h until D600 nm reached 0.5 IPTG was then added to a final concentration of mm The culture was then grown at 37 °C for an additional h with vigorous shaking, after which the bacteria were pelleted by centrifugation at 1600 g for 12 The pellet was washed once with 25 mL of NaCl ⁄ Pi + 0.05% Tween-20 The pellet was then dissolved in mL of NaCl ⁄ Pi and sonicated six times for 30 s each to open the cells The lysate was centrifuged at 10 000 g for 10 min, and the supernatant was transferred to a new tube Five hundred microliters of Ni2+–nitrilotriacetic acid slurry (50 : 50) (Qiagen, Hilden, Germany) was added, and the sample was slowly rotated for 45 at room temperature The sample was then transferred to a mL column, and the supernatant was allowed to slowly pass through the filter, leaving the Ni2+– nitrilotriacetic acid beads with the bound protein in the column The column was then washed four times with mL 2266 of washing buffer (NaCl ⁄ Pi, 0.05% Tween-20, 10 mm imidazole, and m NaCl) Elution of the protein was performed by adding 150 lL of elution buffer followed by five 300 lL fractions of elution buffer (NaCl ⁄ Pi, 0.05% Tween-20, and 100 mm imidazole) Each fraction was collected individually Ten microliters from each of the eluted fractions was then mixed with one volume of · sample buffer and lL of b-mercaptoethanol, and heated for at 80 °C The samples were analyzed on an SDS Bis ⁄ Tris 4–12% PAGE gel, and the second and third fractions, which contained the most protein, were pooled The protein concentration of the combined fractions was determined with a Bio-Rad DC Protein assay kit (Bio-Rad Laboratories Hercules, CA, USA) Approximately 60 lg of recombinant protein was added to each 120 lL of cleavage reaction (in NaCl ⁄ Pi) Twenty microliters from this tube was removed before addition of the enzyme (the time point) The active enzyme was then added ( 35 ng of HC or the HC double mutant), and the reaction was kept at room temperature for the entire experiment Twenty-microliter samples were removed at the indicated time points (15, 45, and 150 min), and prevented from reacting further by addition of one volume of · sample buffer One microliter of b-mercaptoethanol was then added to each sample, and this was followed by heating for at 80 °C Twenty microliters from each of these samples was then analyzed on 4–12% precast SDS ⁄ PAGE gels (Invitrogen) The gels were stained overnight in colloidal Coomassie staining solution, and destained for several hours according to previously described procedures [25] The intensities of the individual bands on the gel were determined from scanned high-resolution pictures by densitometric scanning of the gels and using imagej (rsb.info.nih.gov ⁄ nih-image ⁄ ) In order to obtain good estimates of the differences in activity towards different substrates, different concentrations of the enzyme were used in several individual experiments The combined results from these different gels were then used to obtain a good estimate of the differences in activity against the various substrates Acknowledgement This study was supported by grants from the Swedish National Research Council (VR-NT) References Chandrasekharan UM, Sanker S, Glynias MJ, Karnik SS & Husain A (1996) Angiotensin II-forming activity in a reconstructed ancestral chymase Science 271, 502– 505 Gallwitz M, Reimer JM & Hellman L (2006) Expansion of the mast cell chymase locus over the past 200 million years of mammalian evolution Immunogenetics 58, 655– 669 FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS M K Andersson et al Pejler G, Abrink M, Ringvall M & Wernersson S (2007) Mast cell proteases Adv Immunol 95, 167–255 Andersson MK, Enoksson M, Gallwitz M & Hellman L (2009) The extended substrate specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile Int Immunol 21, 95–104 Schechter I & Berger A (1967) On the size of the active site in proteases I Papain Biochem Biophys Res Commun 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(1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250 Electrophoresis 9, 255–262 FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2267 ... strong preference for acidic amino acids at position P2¢ of the substrates Table P2¢ specificity and amino acids found in positions 40, 143 and 192 of nine different chymases Chymase P2¢ specificity... alone or in cooperation with Lys192 mediates the preference for acidic amino acids at position P2¢ In the present study, we tested the roles of Arg143 and Lys192 as P2¢ specificity-determining residues... 6% of the sequences Thus, Arg143 and Lys192 contribute almost equally to the P2¢ specificity of HC Mutating either of the two basic amino acids in these positions partially disrupts the acidic P2¢