Báo cáo khoa học: Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2¢ of substrates pdf

We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2¢ of substrates.. Analysis of the extended cleavage speciﬁci

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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

Introduction

Mast cells (MCs) are resident tissue cells that are

dis-tributed along the surfaces of the body They are

fre-quently 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

pro-teins 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-chy-mases and the b-chya-chy-mases [1] The a-chya-chy-mases are encoded by a single gene in all species investigated, except for ruminants, where two very similar a-chym-ase genes have been identiﬁed [2] The b-chyma-chym-ases

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 0 18 471 4382

Tel: +46 0 18 471 4532

E-mail: lars.hellman@icm.uu.se

Website: http://www.icm.uu.se/immuno/

(Received 29 December 2009, revised 2

March 2010, accepted 4 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 posi-tion P1, among the chymases Furthermore, the conservaposi-tion of the prefer-ence 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

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.

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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

pri-mary cleavage speciﬁcity from aromatic amino acids

(chymotrysin-like) to aliphatic amino acids

(elastase-like)

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

inter-actions 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

pep-tide 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

sub-strates for HC showed that many of these also have

acidic amino acids at position P2¢ [4,6] These

observa-tions suggest an important role for negatively charged

amino acids at position P2¢ during substrate

discrimi-nation by HC The structure of HC has been

exten-sively investigated, and also compared with those of

MC chymases in other species These studies have

pro-vided insights into important enzyme–substrate

interac-tions For example, molecular modeling of HC

interacting with angiotensin (Ang) I has led to

conclu-sions 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 posi-tion [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 chy-mases with a specificity for acidic P2¢ amino acids all have Arg143 and Lys192 However, they differ at posi-tion 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-determin-ing residues By in vitro mutagenesis, the HC codspecificity-determin-ing 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 essen-tial in conferring a strong preference for acidic amino acids at position P2¢ of the substrates

Table 1 P2¢ specificity and amino acids found in positions 40, 143 and 192 of nine different chymases.

unpublished results)

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Production and purification of recombinant HC

mutants

Two single mutants of HC, Arg143ﬁ Gln and Lys192

ﬁ Met, and a double mutant, Arg143 ﬁ Gln +

Lys192ﬁ 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

aga-rose, by binding through the N-terminal His6-tag The

protein yield was 100–150 lg recombinant protein

from 1 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)

Approxi-mately 30 lg of each mutant was treated with EK for

5 h at 37C Samples of inactive and activated

prote-ases 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

wild-type enzyme, the mutated inactive proteases migrated

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 activ-ities of the eluted fractions of the three mutated HCs were analyzed by cleavage of the chymotrypsin-sensi-tive chromogenic substrate S-2586 (MeO-Suc-Arg-Ala-Tyr-pNA, Chromogenix, Mo¨lndal, Sweden, data 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

5 · 107phage 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 addi-tional biopannings Selection of nonamers susceptible

to cleavage by the Lys192 ﬁ Met and Arg143

fi Gln + Lys192 fi Met HC mutants was per-formed over five biopannings, after which they induced the release of 47 and 46 times more phages, respec-tively, than an NaCl⁄ Picontrol (Fig 2) Peptides sensi-tive 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⁄ Picontrol (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 nucle-otide sequences were then translated into nonapep-tides, 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 deter-mined in total, two of which were obvious back-ground sequences and therefore not included in the

–EK +EK Hep –EK +EK Hep –EK +EK Hep

R143Q +

97

66

45

30

20

14

kDa

Fig 1 Purification and activation of recombinant HC mutants Three

different recombinant HC mutants were expressed with an

N-termi-nal His6-tag followed by an EK-susceptible sequence replacing the

signal peptide These proenzymes were first purified on Ni 2+

–nitrilo-triacetic 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)

Proen-zymes and activated enProen-zymes before and after heparin–Sepharose

purification were analyzed by separation on SDS ⁄ PAGE gels and

visualized with Coomassie Brilliant Blue staining.

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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-Phe-Asp-Met), four sequences from the clone

Trp-Phe-Val-Thr-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-Tyr-Trp-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

posi-tions N-terminal of the cleaved bond, posiposi-tions 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

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 fre-quently 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 speci-ficity for acidic amino acids at position P2¢ is shown in Fig 5 In a previous study, where the same phage-dis-played 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 muta-tions 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 mutat-ing both of them totally removes this preference The negatively charged amino acids Asp and Glu are roughly equally distributed in this position in the wild-type HC and the three HC mutants Therefore, Arg143 and Lys192 do 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 con-struct (Fig 6A) For puriﬁcation purposes, a His6-tag

R143Q K192M R143Q + K192M

Biopannings

0

20

40

60

80

100

Fig 2 Amount of released T7 phages after digestion with HC

mutants, as compared with an NaCl ⁄ P i control A library of

ran-domly 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

per-formed in five or six rounds of selection (biopannings) After each

biopanning, the amount of released phages was determined and

compared to an NaCl ⁄ P i control The ratio of phages released by

enzyme digestion over the NaCl ⁄ P i 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.

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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

dou-ble 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 sub-strate 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 dou-ble 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

Y S A R R

V G V M

F S S V

F S S

M M A L L E

Y V G I K G S

I L

F G G S V

Y G G

V A M V L Q

Y G G L

W T H H

G

G A R S G S

Y S A A

M

M M

Y A A L R L

L V L

F F

T L M G S L R F

L

V T W G T P A

F F L V

F T L V E

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V A G G L R R F

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R A S G E T A Y

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S A F V R A

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L L

P T

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G A R L R R

F W V S

F A L

V F S A G E

F F

G V S G L T R H

W G R D T R G

Y W

W E V R L S G

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F W

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F F W V

V

F L V

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G I L A G P

2 T L F Y W G A T G

W H H H

L L

S S G E K R

P4 P3 P2 P1 P1 ′P2′ P3′

R143Q + K192M

R I S F A G E V F

Y E M V L M M

V P

R

Y A A G C A

V A

Y A L E E A M

M L

Y A A T V D G

V A

F T D M K

R V G W

P

P G G Y I G R L W R L

Y S L E L T V

F K

W E K I P W G S S

W T I L P Y A R S

F F

V S S L A V S

F

F G Q L L

R A L

F A G S I L L

V W

F

F S L

V T L F W G T P A

Y S A S

V V W G E

S F S H H

L V

P Y

Q I

F F A

A V L E L V

L L W R F L L C C

Y S M L

V V V

F L H H

S Q G R L

F A E

Y S

F A L P

F A

W G L

Y G D L F

V T A W

F A E L

R F W I A

F S G F

L V P V W

A

Y S L F A F

V T

F I S F G F T L L

V

V V S F A

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Y E A F L L

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F

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F

V T E

W R V V A Y G L D

2 P M L Y S L N D S

P4 P3 P2 P1 P1 ′P2′P3′

K192M

Negatively charged Positively charged Aromatic

Large aliphatic Small aliphatic

P G G W S R M L V L R P

V

V L F S T G S E E

M

T I T V Y S E H

R Q R R F P V S V

G L L F S S G R E D

V A L Y F V T G G

Y F

I S L S A D I

G M P Y S L T M G F

W A V V S F T G T

Y F M

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T V

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W E V T T F L A T

Y

Y G G E V P

V A

G

V F F

A T P A D I

W E

W P L F A L A

W V V S F W A D V

T

W W R D F S T N

G W A A Y F L D F

T V

W D F F FG E H

W E I V V F G W L

12 W W A I E M F D M H

W F V T F Y D S L

4

2 V V S Y G G V L E

P4 P3 P2 P1 P1 ′P2′ P3′

R143Q

Fig 3 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)9indicates 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.

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0 10 20 30 40

F Y W G A V L I P S T C M N Q H K R D E

0 10 20 30 40

WT

R143Q

K192M

R143Q+

K192M

P2

0 10 20 30 40

WT

R143Q

K192M

R143Q+

K192M

P3

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R143Q

K192M

R143Q+

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P4

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WT

R143Q

K192M

R143Q+

K192M

P3´

0 10 20 30 40

WT

R143Q

K192M

R143Q+

K192M

P2´

0 10 20 30 40

0

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WT

R143Q

K192M

R143Q+

K192M

P1´

0 10 20 30 40 50 60 70

R143Q+

K192M

P1

0 10 20 30 40 50 60 70

R143Q

P1

0 10 20 30 40 50 60 70

WT

0 10 20 30 40 50 60 70

K192M

P1

Fig 4 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 3 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 (pos-itively charged), and acidic (negatively charged).

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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

ana-lyzed 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 min

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

five-fold to eight-five-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

accu-racy of the information obtained by the phage display

analysis

Discussion

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 gran-zyme 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 posi-tions 143 and 192 in the enzyme By replacing Arg143

by Gln or Lys192 by Met, which are amino acids com-monly found in these positions in related rodent chy-mases, the preference for acidic amino acids at position P2¢ was markedly reduced, whereas the cleav-age 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 enzyme-selected sequences are examined overall, fewer nega-tively 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

Glu

HC wt R143Q K192M R143Q +

K192M 0

10

20

30

40

50

60

70

Fig 5 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

wild-type HC was determined in a previous study [4].

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B

C

D

E

F

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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

simi-lar 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

Wild-type 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

prefer-ence 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¢

pref-erence-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

chym-ase-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

agree-ment 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

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 prefer-ence 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 posi-tions P4–P1, together with the dipeptidyl leaving group

of Ang I, were found to be important for efﬁcient con-version by HC [18,19] However, the side chains on the leaving group of Ang I do 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 Lys40fi 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 Arg143fi 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 mediat-ing the specificity for acidic P2¢ side chains of sub-strates, but not when the negative charge is situated at

a C-terminal carboxyl group of a P2¢ amino acid of

Fig 6 Analysis of cleavage specificity by the use of new types of recombinant protein substrate (A) The overall structure of the recombi-nant 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 per-formed with IMAGEJ

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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

analyz-ing a broad range of different substrates, with different

biochemical characteristics, when looking for the

natu-ral 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

poten-tial substrates during the screening of the full human

proteome This highlights the importance of factors

other than the extended cleavage speciﬁcity in

deter-mining whether a protein will be a biologically

signiﬁ-cant 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

surface-exposed, 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

bio-logical effect also needs to be determined With

knowl-edge of the extended cleavage speciﬁcity and other

parameters in hand, the search for biologically

signiﬁ-cant 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

mam-malian cells [4,23,24] This vector construct was used for

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

site in the translated protein facilitated puriﬁcation and activation of the enzyme

Mutagenesis of HC was performed using the Quik-Change II XL Site-Directed Mutagenesis Kit (Stratagene,

pro-duced 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 anti-sense primer, 5¢-GCTTCAACACACCTGTCTGTCCCCA GCCAGCCACCCG-3¢ These primers had a melting

replacement of the Arg codon with a Gln codon are

following primers were used: sense primer, 5¢-CAGGAA GACAAAATCTGCATTTATGGGAGACTCTGG-3¢; and antisense primer, 5¢-CCAGAGTCTCCCATAAATGCAGA TTTTGTCTTCCTG-3¢ These primers had a melting

replace-ment of the Lys codon with a Met codon are underlined

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

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

This DNA was then used to transform XL10-Gold ultra-competent 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 Biosys-tems, Foster City, CA, USA) and vector-speciﬁc primers

Production and purification of recombinant HC mutants

The vector constructs encoding the HC mutants were trans-fected into a human embryonic kidney cell line (HEK 293

(Invitro-gen, Carlsbad, CA, USA) as previously described [23,24] Selection of transfected cells was initiated by the addition

(DMEM supplemented with 5% fetal bovine serum,

of selection

Conditioned medium was collected and centrifuged at

1600 g to remove cell debris, and this was followed by the

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