The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis ofits derivative Ac-SQLRANISH-pNA that lacks residues at P2¢–P7¢ positions,
Trang 1Processing, stability, and kinetic parameters of C5a peptidase
Elizabeth T Anderson1, Michael G Wetherell1, Laurie A Winter1, Stephen B Olmsted1,
Patrick P Cleary2and Yury V Matsuka1
1
Wyeth Research, West Henrietta, NY, USA;2Microbiology Department, University of Minnesota, Minneapolis, MN, USA
A recombinant streptococcal C5a peptidase was expressed in
Escherichia coliand its catalytic properties and thermal
sta-bility were subjected to examination It was shown that the
NH2-terminal region ofC5a peptidase (Asn32–Asp79/
Lys90) forms the pro-sequence segment Upon maturation
the propeptide is hydrolyzed either via an autocatalytic
intramolecular cleavage or by exogenous protease
strepto-pain At pH 7.4 the enzyme exhibited maximum activity in
the narrow range oftemperatures between 40 and 43C
The process ofheat denaturation ofC5a peptidase
investi-gated by fluorescence and circular dichroism spectroscopy
revealed that the protein undergoes biphasic unfolding
transition with Tmof50 and 70C suggesting melting of
different parts ofthe molecule with different stability
Unfolding of the less stable structures was accompanied by the loss ofproteolytic activity Using synthetic peptides corresponding to the COOH-terminus ofhuman comple-ment C5a we demonstrated that in vitro peptidase catalyzes hydrolysis oftwo His67-Lys68 and Ala58-Ser59 peptide bonds The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis ofits derivative Ac-SQLRANISH-pNA that lacks residues at P2¢–P7¢ positions, suggest the import-ance ofC5a peptidase interactions with the P¢ side ofthe substrate
Keywords: maturation; propeptide; streptopain; denatura-tion; substrate binding
Group A streptococcus (Streptococcus pyogenes) is a
common human pathogen causing a wide variety of
diseases These include relatively mild pathological
condi-tions such as pharyngitis and impetigo, more serious
nonsuppurative sequelae, acute rheumatic fever,
glomerulo-nephritis, deadly toxic shock syndrome and necrotizing
fasciitis S pyogenes has developed complex and
sophisti-cated molecular mechanisms that allow it to avoid human
defenses One of the important virulence factors of
strep-tococci involved in such activity is an extracellular C5a
peptidase [2] Streptococcal C5a peptidase is a
surface-associated subtilisin-like serine protease with an unusually
restricted substrate specificity The only known protein
substrate hydrolyzed by C5a peptidase is human
comple-ment fragcomple-ment C5a [3,4] C5a peptidase-generated cleavage
within the COOH-terminal region ofhuman C5a drastically
reduces the ability ofthis anaphylatoxin to bind receptors
on the surface of polymorphonuclear neutrophil leukocytes (PMNLs) and therefore abolishes its chemotactic activity [3] It is believed that C5a peptidase plays an important role
in bacterial colonization ofthe host by inhibiting the influx ofPMNLs and impeding initial clearance ofthe strepto-cocci
C5a peptidase from S pyogenes is encoded by the chromosomal scpA gene and consists of1167 amino residues (Fig 1A) C5a peptidase is first produced as a precursor, with an NH2-terminal 31 amino acid residue signal peptide responsible for exporting the protein across the membrane [2] The length ofC5a peptidase pro-sequence segment and the mechanism ofits cleavage remain unknown Homology modeling has shown that the catalytic domain ofC5a peptidase contains the structurally con-served core typical ofsubtilases, but in addition contains a number ofextra segments corresponding to various size inserts located in external loops All inserts found in the C5a peptidase catalytic domain form a total of 216 additional amino acid residues relative to subtilisin BPN¢ [5] The active site ofC5a peptidase is located within the NH2-terminal half ofits polypeptide chain and formed by catalytic residues Asp130, His193 and Ser512 (corresponding to Asp32, His64, and Ser221 in subtilisin BPN¢) Asn294 (Asn155 in subtilisin BPN¢) is involved in the formation of an oxyanion-hole and is critical for the catalytic activity of C5a peptidase [2,5] The function of the COOH-terminal region ofC5a peptidase, starting at residue 583 and representing halfofthe total polypeptide, is not known The COOH-terminal extension ofC5a peptidase is involved
in association with the surface of S pyogenes This segment
is comprised offour R1–R4 hydrophilic 17 amino acid
Correspondence to Y V Matsuka, Department ofProtein Chemistry,
Wyeth Research, WV, 211 Bailey Road, West Henrietta,
NY 14586-9728, USA.
Fax: + 1 585 273 7515, Tel.: + 1 585 273 7565,
E-mail: matsukay@wyeth.com
Abbreviations: PMNL, polymorphonuclear neutrophil leukocytes;
pNA, p-nitroanilide; T m , transition midpoint ofdenaturation; Pn, P2,
P1, P1¢, P2¢, Pn¢, protease substrate residues accommodated by
cor-responding Sn, S2, S1, S1¢, S2¢, Sn¢ subsites ofthe enzyme The scissile
peptide bond is located between the P1 and P1¢ Protease substrate
residues and subsites ofthe enzyme substrate-binding site are
desig-nated using the nomenclature ofSchechter and Berger [1].
(Received 28 May 2002, revised 8 August 2002,
accepted 15 August 2002)
Trang 2residues cell wall repeats, followed by the LPTTN motif,
hydrophobic membrane-spanning region and a cytoplasmic
tail [2] The presence ofthe LPTTN motifthat precedes the
hydrophobic membrane-spanning region and cytoplasmic
charged tail suggests covalent linkage ofC5a peptidase to
the peptidoglycan [6,7]
Despite the recognized role ofC5a peptidase as an
important streptococcal virulence factor [8,9], there is a lack
ofdata on its biochemical and catalytic properties Studies
with synthetic peptides corresponding to the
COOH-terminus ofhuman C5a suggested that C5a peptidase
generates a single cleavage site between histidine 67 and
lysine 68 residues, resulting in the release ofthe
KDMQLGR heptapeptide from the C5a fragment [4]
The identified His67-Lys68 peptide bond within human C5a
represented the only known cleavage site for streptococcal
C5a peptidase Several proteins including human
comple-ment C5, C3, human serum albumin, myosin, ovalbumin,
and cytochrome c were tested as substrates for the C5a
peptidase, but none ofthem underwent hydrolysis [3,4]
Such a highly restricted substrate specificity ofC5a
pepti-dase is in striking contrast to the broad specificity
ofwell-studied bacterial serine proteases ofthe subtilisin family
Thus, it is ofgreat interest to investigate the biochemical and
catalytic properties ofC5a peptidase In the present study,
we have focused on the mode of C5a peptidase maturation
and determination ofthe exact borders ofits pro-sequence
region We have also evaluated the thermal stability and
kinetic parameters ofC5a peptidase and the results are
discussed with regard to the structural organization and biological activity
E X P E R I M E N T A L P R O C E D U R E S
Construction of expression vectors Wild-type C5a peptidase The region ofthe scpA gene (bases 94–3112) encoding C5a peptidase amino acid residues 32–1038 (Fig 1A) was produced by PCR ampli-fication using chromosomal DNA from S pyogenes M1 strain 90–226 as a template To clone the scpA gene, we designed the following forward 5¢-CCC GAA TTC AAT ACT GTG ACA GAA GAC ACT CCT GC-3¢ and reverse 5¢-CCC GGA TCC TTA TTG TTC TGG TTT ATT AGA GTG GCC-3¢ PCR primers The forward primer incorpor-ated the EcoRI restriction site, while the reverse primer included a BamHI site (underlined) The reverse primer also incorporated a TAA stop codon immediately after the coding segment The amplified PCR product was first ligated into TA cloning vector pCR2.1 (Invitrogen Corp.) and then subcloned into pTrc99a expression vector (Amer-sham Pharmacia Biotech) using the EcoRI and BamHI restriction sites Incorporation ofthe EcoRI restriction site within the forward primer for subsequent ligation of the amplified scpA gene into pTrc99a expression vector yielded three NH2-terminal extra residues MEF that are not part of the natural protein The resulting plasmid pTrc99a (wild-type C5a peptidase) was transformed into E coli DH5a host cells for protein expression
S512A C5a peptidase mutant Site-directed mutagenesis was performed using inverse PCR amplification [10] using expression plasmid pTrc99a (wild-type C5a peptidase) as a template For this purpose we designed two PCR primers,
so that they would abut each other in opposite orientations: 5¢-ACT GCT ATG TCT GCG CCA TTA G-3¢ (forward) and 5¢-TCC AGA AAG TTT GGC ATA CTT GTT GTT AGC C-3¢ (reverse) The forward primer contains GCT codon that replaced AGT to produce the desired SerfiAla mutation at position 512 The GCTfiAGT mutation also resulted in elimination ofa SpeI restriction site The inverse PCR was performed using ExpandTMLong Template PCR System (Boehringer Mannheim Corp.) The resulting blunt ended PCR product was self-ligated and transformed into TOP10F¢ E coli cells (Invitrogen Corp.) Clones were screened and selected for the presence of the desired mutation by loss ofa SpeI restriction site The presence of SerfiAla mutation at position 512 was also confirmed by sequencing the scpA gene The resultant plasmid was digested with NcoI and BamHI restriction enzymes and isolated scpA gene containing GCTfiAGT mutation was then subcloned into a pTrc99a expression vector where a kanamycin resistance cassette had been inserted into the ampicillin gene The plasmid pTrc99a (S512A C5a pepti-dase) was transformed into E coli DH5a host cells for protein expression
Expression and purification of recombinant C5a peptidase proteins
DH5a cells were grown overnight at 37C in HSY medium (10 mM potassium phosphate, pH 7.2, 20 gÆL)1 HySoy,
Fig 1 Schematic representation of C5a peptidase (panel A) and SDS/
PAGE analysis of purified recombinant C5a peptidase species (panel B).
Panel A depicts location ofthe major regions ofC5a peptidase The
signal sequence (presequence), catalytic triad residues, cell wall,
membrane, and cytoplasmic segments are indicated The recombinant
C5a peptidase (residues Asn32 through Gln1038) expressed in E coli is
boxed Panel B shows the relative mobility ofisolated recombinant
S512A mutant (lane 2) and the wild-type (lane 3) C5a peptidase species
on 10–20% gradient gel The outer lanes 1 and 4 in the gel contain
molecular mass standards as indicated.
Trang 35 gÆL)1yeast extract, 10 mMNaCl) For expression ofthe
wild-type C5a peptidase or S512A mutant, 100 lgÆmL)1
ampicillin or 50 lgÆmL)1 kanamycin, respectively, was
incorporated into HSY medium Overnight cultures were
diluted 1 : 100 with fresh HSY medium, grown to
D600¼ 1.5, and induced with 3 mM isopropyl
thio-b-D-galactoside After 3 h of induction, DH5a cells were
harvested by centrifugation and lysed by the freeze-thaw
method Isolated soluble fractions of bacterial lysate were
sequentially fractionated with 50% and then 70% of
ammonium sulfate Material precipitated with 70%
ammo-nium sulfate was collected by centrifugation, dissolved in
20 mMTris, pH 8.5, 25 mMNaCl and dialyzed overnight at
4C against the same buffer Dialyzed samples were diluted
1 : 1 (v/v) with 20 mMTris, pH 8.5, 2Murea, and applied
to a Q-Sepharose ion exchange column (Amersham
Phar-macia Biotech), equilibrated with 20 mMTris, pH 8.5, 1M
urea Material bound to the anion exchange resin was eluted
with a linear gradient ofNaCl and pH using 20 mMTris,
pH 7.0, 1M Urea, 1MNaCl buffer Fractions containing
C5a peptidase species were collected, pooled, and dialyzed
against NaCl/Tris, pH 7.4 Purified recombinant C5a
peptidase samples (wild-type enzyme and S512A mutant)
were aliquoted and stored frozen at)20 C
Protein concentration determination
Protein concentrations were determined
spectrophotomet-rically, using extinction coefficients (E280, 0.1%) calculated
from the amino acid composition The extinction coefficients
were estimated using the equation: E280, 0.1%¼ (5690 W +
1280Y + 120S-S)/M, where W, Y, and S-S represent the
number ofTrp and Tyr residues and disulfide bonds,
respectively, and M represents the molecular mass [11,12]
Molecular masses ofthe proteins were calculated on the
basis oftheir amino acid composition The following
molecular masses and E280, 0.1% values were obtained:
wild-type C5a peptidase, 104.2 kDa and 0.92; S512A C5a
peptidase, 109.9 kDa and 0.87 Alternatively, protein
con-centrations were estimated using bicinchoninic acid assay
[13] according to BCA protein assay kit instructions (Pierce
Chemical Company) Concentration ofstreptococcal
cys-teine protease was also determined using active-site titration
with E64 (Roche Molecular Biochemicals) [14] Titration of
the cysteine protease active-sites was performed in NaCl/
Tris, pH 7.4, 10 mMdithiothreitol, using resorufin-labeled
casein (Roche Molecular Biochemicals) as a substrate
SDS/PAGE
SDS/PAGE was performed with the Bio-Rad
electrophor-esis system (Bio-Rad Laboratories) using precast 10–20%
gradient gels All SDS-polyacrylamide gels in this study
were stained with Coomassie Brilliant Blue R (Bio-Rad
Laboratories) or Coomassie R-350 (Amersham Pharmacia
Biotech) solutions
Amino terminal sequence analysis
NH2-terminal sequence analysis was performed with an
Applied Biosystems model 490 sequenator The NH2
-termini ofthe proteins and peptides were determined by
direct sequencing for 10 or more cycles
Synthetic peptides Peptides corresponding to the COOH-terminal region ofthe human C5a fragment (VVASQLRANISHKDMQLGR, SQLRANISHKDMQLGR, and VVASQLRANISH) and
NH2-terminal segment ofthe C5a peptidase (QTPDEAAE ETI and AEETIADDANDL) were synthesized as C-ter-minal amides on a Gilson AMS422 Multiple Peptide Synthesizer using Fmoc chemistry with pentrafluorophenyl amino acid active esters and a polyethylene glycol polysty-rene support with a 5-(4¢-Fmoc-aminomethyl-3¢-5¢-dimeth-oxyphenoxy)valeric acid linker After synthesis, peptides were purified by reverse-phase HPLC and lyophilized Homogeneity ofsynthesized peptides was assessed by NH2 -terminal sequence and mass spectral analysis Peptide solutions ofknown concentration were prepared by weigh-ing and dissolvweigh-ing purified lyophilized peptide in a known volume ofdistilled water to give concentrated stock solutions Two chromogenic p-nitroanilide (pNA) peptide derivatives were used in this study: the Ac-SQLRANISH-pNA was custom synthesized by New England Peptide, Inc and the Suc-AAPF-pNA was obtained from Sigma To prepare concentrated stock solutions, the Ac-SQLRAN ISH-pNA was dissolved in distilled water and Suc-AAPF-pNA was dissolved in dimethylsulfoxide
Mass spectral analysis Determination ofthe molecular masses ofproteins and peptides was performed using MALDI-TOF mass spectro-meter Voyager DE-STR (Perseptive Biosystems) Ions formed by laser desorption at 337 nm (N2 laser) were recorded at an acceleration voltage of20 kV in the linear mode for proteins and 25 kV in the reflector mode for peptides In general, 200 single spectra were accumulated for improving the signal/noise ratio and analyzed by use ofthe
DATA EXPLORER software supplied with the spectrometer Sinapinic acid and a-cyano-4-hydroxycinnamic acid were used as ultraviolet-absorbing matrices for proteins and peptides, respectively 1 lL of a 10-mgÆmL)1solution ofthe matrix compounds in 70% acetonitrile/0.1% trifluoroacetic acid was mixed with 1 lL analyte solution (5–10 pmolÆ
lL)1) For MALDI-TOF MS, 1 lL ofthis mixture was spotted on a stainless steel sample target and dried at room temperature The mass spectra were calibrated using external standards: serum albumin (bovine), Glu1-fibrino-peptide B (human), angiotensin I (human), and des-Arg1-bradykinin (synthetic) The mass accuracy was in the range of0.1%
Hydrolysis of protein and peptide substrates Treatment ofthe S512A C5a peptidase precursor with C5a peptidase was performed at 25C in NaCl/Tris, pH 7.4,
5 mM CaCl2 For this purpose 33 lM ofthe S512A C5a peptidase precursor was incubated with 3.3 lMofwild-type C5a peptidase resulting in an enzyme/substrate ratio of
1 : 10 (M/M) Proteolysis ofS512A C5a peptidase precursor (50 lM) with streptococcal cysteine protease (1.8 lM) was performed at 25C in NaCl/Tris, pH 7.4, 10 mM dithio-threitol at enzyme/substrate ratio of1 : 25 (M/M) Samples from each reaction mixture were removed at 15, 30, 45, 60,
120, 240, 360, and 1320 min, mixed with SDS, heated and
Trang 4later analyzed by SDS/PAGE using 10–20% gradient gel.
Streptococcal cysteine protease or streptopain (EC
3.4.422.10) was prepared as described elsewhere [15]
Operational molarity ofcysteine protease preparations used
in this study corresponded to 98% ofthat expected on a
protein concentration basis The specificity ofstreptopain
catalyzed cleavage was confirmed using the specific cysteine
protease inhibitor E64 (Roche Molecular Biochemicals)
The NH2-terminal truncation ofS512A C5a peptidase
precursor by streptococcal cysteine protease was completely
blocked in the presence of20 lME64
Caseinolytic activity ofC5a peptidase was evaluated
using resorufin-labeled casein (Roche Molecular
Biochemi-cals) Briefly, increasing amounts ofwild-type C5a
pepti-dase, S512A C5a peptidase mutant, and subtilisin from
Bacillus subtilis(EC 3.4.21.14) (Fluka) ranging from 0 to
10 lg were incubated for 60 min at 37C in the presence of
0.4% resorufin-labeled casein in NaCl/Tris, pH 7.8, 5 mM
CaCl2 Undigested substrate was removed by 5%
trichlo-roacetic acid precipitation, and followed by centrifugation
the absorbance ofreleased resorufin-labeled peptides in the
supernatant fractions was measured spectrophotometrically
at 574 nm
The C5a peptidase-catalyzed hydrolysis ofthe 19-mer
synthetic peptide VVASQLRANISHKDMQLGR was
performed at 25C in NaCl/Tris, pH 7.4, 5 mM CaCl2
Incubation of345 lM VVASQLRANISHKDMQLGR
peptide with 0.28 lMofC5a peptidase was carried out for
5, 10, 15, 20, 30, 40, 60, 80, 100, 120, and 140 min and
reactions were terminated by the addition oftrifluoroacetic
acid to 0.05% At each time point, the presence ofspecific
peptide in reaction mixture was monitored at 210 nm by
reverse-phase HPLC (Hewlett Packard model 1090 Liquid
Chromatograph) using C4 reverse-phase column (Vydac)
Buffer A was 0.1% trifluoroacetic acid in distilled water,
and buffer B was 0.1% trifluoroacetic acid in 100%
acetonitrile Peptides were eluted with 0–40% linear
gradi-ent ofBuffer B during a 20-min interval The relative
amount ofeach peptide in the reaction mixture was
determined using the area beneath the peak corresponding
to this peptide and then plotted as a function of time The
identity ofpeptides was determined by NH2-terminal
sequence- and mass spectral analysis
Treatment ofthe QTPDEAAEETI and AEETIADD
ANDL synthetic peptides with C5a peptidase was
per-formed either at 25 or 37C in NaCl/Tris, pH 7.4, 5 mM
CaCl2and in 100 mMTris, pH 8.6, 5 mMCaCl2 Each of
these peptides (100 or 200 lM) was incubated with 0.1 or
1 lM ofC5a peptidase for 1, 18, and 71 h Reaction
mixtures were analyzed using reverse-phase HPLC as
described above At tested conditions, cleavage ofthe
QTPDEAAEETI and AEETIADDANDL peptides was
not detected
Effect oftemperature on the proteolytic activity ofC5a
peptidase was evaluated using 19-mer synthetic peptide
VVASQLRANISHKDMQLGR At each tested
tempera-ture, 0.1 lMofthe C5a peptidase in NaCl/Tris, pH 7.4 was
preincubated for 3 min The reactions were started by
addition of200 lM ofthe 19-mer peptide to the
preincu-bated solution ofC5a peptidase followed by another 10-min
incubation at the same temperature After termination of
hydrolysis with 0.05% trifluoroacetic acid, the reaction
mixtures were analyzed using HPLC as described above
The percentage ofhydrolyzed peptide substrate (Shydr%) was determined using the equation: Shydr%¼ P/(P + S), where P represents area ofthe product peaks and S represents area ofthe uncleaved substrate peak
Assays revealing the pH dependence ofthe hydrolysis of VVASQLRANISHKDMQLGR peptide were performed
at 25C in 100 mM NaAc (pH 4.5–5.0), 100 mM Mes (pH 5.5–6.5), 100 mM Hepes (pH 7.0–8.0), 100 mM Tris (pH 7.0–9.0), 100 mMAmpso (pH 8.5–9.5), 100 mMCaps (pH 10.0–11.0) At each tested pH, 0.1 lMofC5a peptidase was incubated with 200 lMofthe 19-mer peptide for 10 min followed by HPLC analysis The percentage of hydrolyzed peptide was determined and plotted as a function of pH Hydrolysis ofthe peptide substrate in both temperature and
pH dependence experiments did not exceed 25% The effect ofpH on hydrolysis ofpNA substrate Ac-SQLRANISH-pNA was determined at 25C in 100 mMMes (pH 6.0–6.5),
100 mM Hepes (pH 7.0–8.0), 100 mM Tris (pH 7.0–9.0),
100 mM Ampso (pH 8.5–9.5), 100 mM Caps (pH 10.0– 11.0) At each tested pH, 200 lM ofthe pNA peptide substrate was incubated in quartz cell for 60 min either alone or in the presence of1 lM ofC5a peptidase while monitoring hydrolysis by measurement ofabsorbance at
405 nm using Spectronic Genesis 2 Spectrophotometer (Spectronic Instruments, Inc.)
Kinetic measurements All kinetic data were obtained by incubating various concentrations ofpeptide with a constant enzyme concen-tration to achieve between 5 and 20% cleavage ofthe substrate in each reaction The concentration ofC5a peptidase in each reaction was 0.1 lM, while peptide concentrations ranged from 50 lM to 600 lM (16-mer SQLRANISHKDMQLGR) and from 50 lMto 2000 lM
(12-mer VVASQLRANISH) Concentration ofC5a pepti-dase in each reaction was at least 500-fold lower than the lowest substrate concentration All reactions were per-formed at 25C in NaCl/Tris, pH 7.4, 5 mM CaCl2 Reactions were carried out for 5 or 100 min with 16-mer and 12-mer peptide, respectively, and stopped by the addition oftrifluoroacetic acid to 0.05% Cleavage of peptides by C5a peptidase was monitored at 210 nm by reverse-phase HPLC and percentage ofhydrolyzed peptide was determined as described above Initial velocities (V) were determined and plotted against substrate concentra-tion [S] The data were fitted to the Michaelis–Menten equation V¼ Vmax[S]/(Km+ [S]) with a nonlinear regres-sion analysis program The best fits ofthe data produced
Vmaxand Kmvalues, where Vmaxrepresents the maximum rate ofhydrolysis and Kmis the Michaelis constant The turnover number (kcat) values were calculated from Vmax/[E], where [E] represents enzyme concentration The identity of hydrolyzed peptide fragments was determined by NH2 -terminal sequence and mass spectral analysis
Kinetic studies ofC5a peptidase using chromogenic pNA substrate Ac-SQLRANISH-pNA were performed with enzyme present at concentrations between 0.01 and 0.59 lM The concentration ofAc-SQLRANISH-pNA was varied from 50 to 2000 lM Reactions were performed at
25C in 100 mMTris, pH 8.6, 5 mMCaCl2buffer Assays were carried out in 1-cm path length quartz cells and reaction rates were monitored by continuous measurement
Trang 5ofabsorbance at 405 nm for 180–900 min using a
Spec-tronic Genesys 2 Spectrophotometer (SpecSpec-tronic
Instru-ments, Inc.) The concentration ofreleased p-nitroaniline
product was estimated based on the molar absorption
coefficient e405¼ 10500M )1Æcm)1 The Km value ofC5a
peptidase hydrolysis ofAc-SQLRANISH-pNA was too
high (Km [S]) for accurate measurement Therefore, the
kcatand Kmconstants were not individually determined The
specificity constant kcat/Kmfor hydrolysis of Ac-SQLRAN
ISH-pNA was determined using equation: kcat/Km¼
V/([E]Æ[S]) During extended kinetic runs, no detectable loss
ofcatalytic activity ofthe C5a peptidase was observed The
background (nonenzymatic) hydrolysis ofAc-SQLRAN
ISH-pNA was evaluated by incubating blank substrate
solutions At tested conditions, nonenzymatic hydrolysis
was not detectable
Fluorescence measurements
Thermal unfolding was monitored by observing the change
in ratio ofthe intrinsic fluorescence intensity at 350 nm to
that at 320 nm with excitation at 280 nm [16] in an SLM
Aminco-Bowman Series 2 spectrofluorometer Temperature
was controlled with circulating water bath programmed to
raise the temperature at 1CÆmin)1 and monitored with
Omega DP81 thermocouple probe inserted into a dummy
cuvette All fluorescence measurements were performed at
protein concentration ranging from 0.04 to 0.05 mgÆmL)1
Circular dichroism measurements
CD spectra were recorded on a Jasco J-810
spectropola-rimeter equipped with a Peltier PTC-423S/L unit for
temperature control CD measurements were performed in
20 mM NaCl/Pi, pH 7.4 using protein concentration of
0.2 mgÆmL)1 in 0.1 cm path length cells Spectra were
recorded at 25 and 98C Four scans were accumulated per
each spectrum Spectra were averaged and expressed as
mean residue ellipticity [Q], in units ofdegreesÆcm2Ædmol)1
Thermal denaturation was monitored by changes in
ellip-ticity at 205 nm while heating cell at 1CÆmin)1
R E S U L T S
Preparation of recombinant C5a peptidase and analysis
of its NH2-terminal truncation
The recombinant wild-type C5a peptidase comprising
residues Asn32 through Gln1038 (Fig 1A) was produced
in DH5a E coli cells using pTrc99A expression vector as
described in Experimental procedures During isolation of
C5a peptidase from E coli lysate, its mobility on SDS/
PAGE was slightly but progressively increasing, indicating
possible proteolytic degradation Purified recombinant C5a
peptidase exhibited a single band on SDS/PAGE with a
relative mobility close to its expected molecular mass
(Fig 1B, lane 3) However, all preparations offreshly
isolated wild-type C5a peptidase consistently displayed
truncated NH2-terminus starting at Ala72 and suggesting
the loss of43 amino acid residues Subsequent analysis of
the same protein samples stored either at 4C or samples
that underwent freeze-thaw cycle(s) revealed NH2-terminal
sequence starting at Asp79, indicating the loss ofa total of
50 amino acid residues This observation was reproducible, suggesting that the Asp79 residue represented a final point ofprogressive NH2-terminal truncation ofthe wild-type C5a peptidase The NH2-terminal cleavage ofthe wild-type C5a peptidase may be caused by E coli proteases, or alternatively might be a result ofautocatalytic cleavage (maturation) reaction In order to investigate the nature of the C5a peptidase truncation, we expressed in E coli a mutated and enzymatically inactive form of C5a peptidase Based on homology analysis ofsubtilisin family ofserine proteases, it was reported earlier that Ser residue at position
512 is involved in the formation ofcatalytic site ofC5a peptidase [2,5] When S512A C5a peptidase mutant was expressed in DH5a E coli cells using pTrc99A vector and isolated using the same procedure used for purification of the wild-type enzyme, sequence analysis revealed the presence ofan intact NH2-terminus starting at MEFNTV TEDT The three NH2-terminal extra residues, MEF, are not part ofthe natural protein and originated from the cloning strategy as described in Experimental procedures Electrophoretic mobility ofS512A C5a peptidase mutant was slightly decreased compared to that ofthe wild-type enzyme (Fig 1B, lane 2) consistent with the presence ofan extra 50 amino acid residues Since both proteins were produced using the same expression vectors, host cells, and isolated using the same purification procedure, it is highly unlikely that the NH2-terminus ofthe wild-type form but not that ofthe S512A mutant was cleaved by E coli proteases upon protein expression and subsequent isolation Given the absence ofNH2-terminal truncation in the S512A mutant, these results indicate that C5a peptidase undergoes autocatalytic processing resulting in cleavage ofits 50 amino acid residue propeptide segment To further investigate the mechanism ofNH2-terminal autocatalytic processing, we incubated S512A C5a peptidase mutant in the presence of the wild-type enzyme Upon treatment ofthe S512A C5a peptidase precursor with the wild-type C5a peptidase, there was no evidence for NH2-terminal truncation (Fig 2A) Similarly, no cleavage was detected upon incubation ofthe wild-type C5a peptidase with synthetic peptides corres-ponding to its propeptide region These experiments were performed with QTPDEAAEETI (Gln67–Ile76) and AEETIADDANDL (Ala72–Leu83) overlapping peptides containing Glu71–Ala72 and Asp78–Asp79 autocatalytic cleavage sites, respectively Peptides were incubated with, or without C5a peptidase and followed by HPLC monitoring using a C4 reverse phase column (not shown) Failure ofthe C5a peptidase to cleave the propeptide segment ofS512A C5a peptidase precursor or synthetic peptides correspond-ing to its propeptide region and containcorrespond-ing autocatalytic cleavage sites indicates that autoprocessing proceeds via an intramolecular route
In this study we also investigated the role ofsecreted streptococcal cysteine protease in the maturation ofC5a peptidase precursor Streptococcal cysteine protease, or streptopain, is an extracellular thiol endopeptidase pro-duced by Streptococcus pyogenes [17,18] Cysteine protease has been shown to release biologically active fragments from the bacterial surface such as M protein, protein H and C5a peptidase [19,20] Released by streptopain, the 116 kDa fragment of C5a peptidase inhibited granulocyte migration into the infectious site, and therefore exhibited characteristic peptidase activity [20] The ability ofstreptopain to release
Trang 6an active C5a peptidase fragment from the surface of
streptococci makes this secreted protease an interesting
candidate for evaluation of its role in processing of C5a
peptidase precursor To test this hypothesis, the S512A C5a
peptidase precursor was incubated in the presence of
streptococcal cysteine protease and analyzed by SDS/
PAGE (Fig 2B) Upon incubation, the band corresponding
to C5a peptidase precursor steadily increased its mobility on
SDS/PAGE resulting in the appearance ofhigher mobility
truncated specie The truncated form of S512A C5a
peptidase was a terminal product ofproteolysis, since no
further degradation was observed even after prolonged
incubation with cysteine protease (Fig 2B) After treatment
with streptococcal cysteine protease, the S512A C5a
pep-tidase exhibited an NH2-terminal sequence starting at Lys90
(KTADTPATSK) suggesting the cleavage of61 amino
acids from its NH2-terminus The same NH2-terminal
sequence was found in C5a peptidase released from the
surface of Streptococcus pyogenes by the action ofsecreted
streptococcal cysteine protease [20] These data suggest that
in addition to COOH-terminal cleavage ofC5a peptidase,
resulting in the release ofthe anchored enzyme,
streptococ-cal cysteine protease is involved in the maturation ofC5a
peptidase precursor Thus, the NH2-terminal segment
comprising of47–58 amino acid residues forms the
pro-sequence peptide region ofC5a peptidase Cleavage ofthe
pro-sequence peptide and maturation ofC5a peptidase
precursor is realized via an intramolecular autoprocessing
mechanism Alternatively, processing ofC5a peptidase
precursor can be achieved by an exogenous protease
streptopain
Proteolytic activity and thermal stability of the C5a peptidase
The earlier reported highly restricted substrate specificity of C5a peptidase for human C5a fragment was further investigated in this study Proteolytic activity ofC5a peptidase was tested using resorufin-labeled casein In the control reaction, treatment ofresorufin-casein with increas-ing amounts ofsubtilisin was accompanied by a dose-dependant increase ofabsorbance at 574 nm indicating effective cleavage of the substrate In contrast, incubation of resorufin-labeled casein with increasing amounts ofwild-type or S512A C5a peptidase mutant resulted in absence of detectable hydrolysis (Fig 3A) Cleavage ofcasein was not detected even upon prolonged incubation with high con-centrations ofC5a peptidase These results suggest that C5a peptidase does not exhibit caseinolytic activity typical for classical subtilisins The results also indicate the absence of contaminating E coli proteases in the C5a peptidase preparations To gain insight into the mode ofC5a peptidase catalysis, we examined cleavage ofsynthetic peptide corresponding to the COOH-terminus ofthe human complement fragment C5a First, we tested the 19-mer VVASQLRANISHKDMQLGR synthetic peptide contain-ing previously described His67-Lys68 cleavage site [4] This peptide was incubated alone and in the presence ofeither wild-type C5a peptidase or S512A C5a peptidase mutant Incubation with the S512A C5a peptidase mutant did not result in detectable cleavage ofthe peptide In contrast, treatment ofthe 19-mer peptide with the wild-type C5a peptidase resulted in progressive hydrolysis ofthe substrate (Fig 3B) As seen from Fig 4A and B, incubation of the 19-mer peptide with C5a peptidase produced several smaller peptide products suggesting the presence within the tested substrate ofmore than one cleavage site The products of hydrolysis were examined by mass spectroscopy and NH2 -terminal sequence analysis, and the exact positions of cleavage sites were identified Results ofboth mass spectral and NH2-terminal sequence analysis suggested that in addition to the earlier reported cleavage site His67-Lys68 [4], C5a peptidase also hydrolyzed the peptide bond between Ala58-Ser59 Time course digestion ofthe 19-mer peptide monitored by HPLC revealed that the first cleavage occurs between His67-Lys68, resulting in the formation of VVASQLRANISH and KDMQLGR peptides Gradual depletion ofthe initial VVASQLRANISHKDMQLGR substrate and accumulation ofVVASQLRANISH product was accompanied by detection ofthe second cleavage between Ala58-Ser59, resulting in production ofSQLR ANISH and presumably VVA (Fig 4C) Upon elution from reverse-phase column; peaks corresponding to VVA peptide product were not detected, probably as a result of the precipitation ofthe highly hydrophobic VVA product followed by its release from the parent VVASQLRANISH peptide
To assess the thermal stability ofC5a peptidase, we investigated the effect of temperature on both proteolytic activity and structural integrity ofthe enzyme (Fig 5) All experiments were performed at neutral pH, where C5a peptidase exhibited maximum activity towards peptide substrate (Fig 5A, inset) As illustrated in Fig 5A, raising the temperature from 5C to 40 C resulted in a 10-f old increase ofthe relative proteolytic activity ofC5a peptidase
Fig 2 Incubation of S512A C5a peptidase precursor with wild-type C5a
peptidase (panel A) and streptococcal cysteine protease (panel B)
ana-lyzed by SDS/PAGE using 10–20% gradient gel Lanes 0 contain the
starting material Lanes 15, 30, 45, 60, 120, 240, 360, and 1320
repre-sent increasing times ofincubation The outer lanes in the gels contain
molecular mass standards as indicated Arrows show relative mobility
ofS512A C5a peptidase bands after incubation with wild-type C5a
peptidase (panel A) or streptococcal cysteine protease (panel B).
Trang 7towards the 19-mer peptide Maximum activity ofC5a
peptidase was observed in the narrow range oftemperatures
between 40 and 43C A further increase in temperature
caused a sharp decline in C5a peptidase activity and
subsequently its complete inactivation at 60C
Heat-induced unfolding of the C5a peptidase was studied
using fluorescence and circular dichroism spectroscopy
(Fig 5B,C,D) Figure 5B presents a melting curve obtained
by heating C5a peptidase while monitoring the ratio of
fluorescence intensity at 350 nm to that at 320 nm as a
measure ofthe spectral shift that accompanies unfolding At
neutral pH, in response to heating, the protein exhibited a
high magnitude sigmoidal denaturation transition with a midpoint (Tm) of 50C The midpoint ofthe less pro-nounced second transition was observed at 70C The biphasic nature ofdenaturation curve suggested that the compact structure ofC5a peptidase is formed by at least two
Fig 3 Treatment of casein-resorufin (panel A) and 19-mer synthetic
peptide VVASQLRANISHKDMQLGR (panel B) with recombinant
C5a peptidase species Casein-resorufin or 19-mer peptide substrate
were incubated in the presence ofthe wild-type C5a peptidase (empty
squares) and S512A C5a peptidase mutant (filled diamonds) Subtilisin
from B subtilis (filled squares) was used as a positive control in
caseinolytic experiments.
Fig 4 HPLC analysis of C5a peptidase-catalyzed cleavage of 19-mer synthetic peptide VVASQLRANISHKDMQLGR The19-mer peptide (345 l M ) was incubated in the absence (panel A) or presence (panel B) ofC5a peptidase (0.28 l M ) in NaCl/Tris, pH 7.4, 5 m M CaCl 2 for
120 min at 25 C The products ofenzymatic hydrolysis were identified
by NH 2 -terminal sequence and mass spectral analysis Peaks corres-ponding to each peptide are labeled as peak 1 – (filled diamonds) VVASQLRANISHKDMQLGR, peak 2 – (empty triangles) VVASQLRANISH, peak 3 – (empty diamonds) SQLRANISH, and peak 4 – (empty squares) KDMQLGR The VVA product ofhydro-lysis was not recovered from the reaction mixture Accumulation of peptide products in reaction mixture was monitored and plotted as a function of incubation time (panel C).
Trang 8domains with different stability Melting of C5a peptidase in
the presence of5 mM CaCl2 did not affect denaturation
profile and transition midpoints (Fig 5C, curve a) The
addition of2 mM EDTA and heating under these
condi-tions again produced a biphasic denaturation curve with a
high amplitude first transition and low amplitude second
transition The Tmofthe major transition, however, was
shifted about 6C to lower temperature (Fig 5C, curve b),
suggesting that the compact structure ofC5a peptidase in
the presence ofEDTA was destabilized Heat induced
denaturation data obtained in the presence or absence of
EDTA demonstrated that C5a peptidase contains high
affinity metal-binding site(s) that is (are) presumably
occupied Circular dichroism spectroscopy measurements
revealed that C5a peptidase has a spectrum in the far UV region that exhibits characteristic positive band at 194 nm and negative bands at 210 nm and 215 nm Heating ofC5a peptidase up to 98C abolished these features (Fig 5D inset) Monitoring ofthe ellipticity at 205 nm during heating produced a sigmoidal biphasic transition curve indicative of cooperative unfolding for a multidomain protein (Fig 5D) Again, the midpoints for low and high temperature transitions were observed near 50 and 70C, respectively Results ofdenaturation experiments are consistent with peptidase activity data and suggest that the decrease of activity ofC5a peptidase at temperatures above 43C is associated with the beginning ofthermal unfolding
Kinetic parameters of C5a peptidase: hydrolysis
of peptide andpNA substrates
In order to investigate the kinetic parameters for peptide cleavage by C5a peptidase, we synthesized two derivatives of the 19-mer parent peptide These include the 16-mer SQLRANISHKDMQLGR and the 12-mer VVASQLR ANISH peptides Based on the data obtained with the 19-mer peptide, each ofthese peptides contains a single potential cleavage site, and therefore can be easily utilized for kinetic studies This was confirmed by HPLC assay with subse-quent mass-spectroscopic evaluation ofgenerated products (Figs 6A,B and 7A,B) To determine kinetic constants for the hydrolysis ofpeptides by C5a peptidase, time course experiments using different substrate concentrations were performed in NaCl/Tris, pH 7.4, 5 mMCaCl2at 25C In each case, the initial velocity ofhydrolysis ofthe peptide bond was obtained Using nonlinear regression analysis, these data were fitted to the Michaelis–Menten equation to yield Vmaxand apparent Kmvalues (Figs 6C and 7C) The kinetic parameters for C5a peptidase-catalyzed cleavage of 16- and 12-mer synthetic peptides corresponding to the COOH-terminus ofhuman complement fragment C5a are summarized in Table 1 As can be seen from Table 1, C5a peptidase hydrolyzes the 16-mer peptide with catalytic efficiency (k /K ) about 14-fold higher than the 12-mer
Fig 5 Effect of temperature on proteolytic activity of C5a peptidase (panel A) and heat-induced unfolding of C5a peptidase detected by fluorescence (panels B, C) and circular dichroism (panel D) spectroscopy Panel A shows relative proteolytic activity ofC5a peptidase at various temperatures in NaCl/Tris, pH 7.4 Reactions were performed using 19-mer synthetic peptide VVASQLRANISHKDMQLGR as a sub-strate The pH dependence ofC5a peptidase relative activity is shown
in the inset Reactions were performed at 25 C in 100 m M NaAc (pH 4.5–5.0), 100 m M Mes (pH 5.5–6.5), 100 m M Hepes (pH 7.0–8.0),
100 m M Ampso (pH 8.5–9.5), 100 m M Caps (pH 10.0–11.0) (filled squares) and 100 m M Tris (pH 7.0–9.0) (empty squares) Panel B illustrates fluorescence-detected thermal denaturation ofC5a peptidase
in NaCl/Tris, pH 7.4 Fluorescence-detected melting curves ofC5a peptidase in NaCl/Tris, pH 7.4, 5 m M CaCl 2 (a) and NaCl/Tris,
pH 7.4, 2 m M EDTA (b) are presented in panel C Protein solutions were heated while monitoring the ratio offluorescence at 350 nm to that at 320 nm with excitation at 280 nm Panel D shows changes in ellipticity at 205 nm upon heating ofC5a peptidase sample; the CD spectra ofC5a peptidase at 25 and 98 C are presented in the inset All
CD measurements were performed in NaCl/P i , pH 7.4.
Trang 9This is consistent with our data generated using 19-mer
parent peptide that contains both cleavage sites (Fig 4C)
The low efficiency ofhydrolysis ofthe 12-mer peptide
compared to its 16-mer counterpart was a result ofa sixfold
reduction in kcatvalue and threefold increase in Kmvalue
When 5 m EDTA was included into reaction buffer,
hydrolysis ofboth 16-mer and 12-mer peptides was significantly inhibited (Figs 6C and 7C), again suggesting existence ofmetal-binding site(s) within C5a peptidase Based on the sequence of16-mer SQLRANISHKDMQ LGR peptide, we designed a water-soluble chromogenic pNA substrate, Ac-SQLRANISH-pNA Incubation of
Fig 6 HPLC analysis of C5a peptidase-catalyzed cleavage of 16-mer
synthetic peptide SQLRANISHKDMQLGR The 16-mer peptide
(410 l M ) was incubated in the absence (panel A) or presence (panel B)
ofC5a peptidase (0.28 l M ) for 120 min The products of enzymatic
hydrolysis were identified by NH 2 -terminal sequence and mass spectral
analysis Peaks corresponding to each peptide are labeled as peak
1 – SQLRANISHKDMQLGR, peak 2 – SQLRANISH, and peak 3 –
KDMQLGR Initial rate ofhydrolysis V was plotted vs the
concen-tration ofthe substrate SQLRANISHKDMQLGR [S] (panel C).
Experiments were performed in NaCl/Tris, pH 7.4, containing either
5 m M CaCl 2 (filled squares) or 5 m M EDTA (empty squares) at 25 C.
Fig 7 HPLC analysis of C5a peptidase-catalyzed cleavage of 12-mer synthetic peptide VVASQLRANISH The12-mer peptide (550 l M ) was incubated in the absence (panel A) or presence (panel B) ofC5a peptidase (0.28 l M ) for 360 min The products of enzymatic hydrolysis were identified by NH 2 -terminal sequence and mass spectral analysis Peaks corresponding to each peptide are labeled as peak 1 – VVASQLRANISH, and peak 2 – SQLRANISH The VVA product ofhydrolysis was not recovered from the reaction mixture Initial rate ofhydrolysis V was plotted vs the concentration ofthe substrate VVASQLRANISH [S] (panel C) Experiments were performed in NaCl/Tris, pH 7.4, containing either 5 m M CaCl 2 (filled squares) or
5 m M EDTA (empty squares) at 25 C.
Trang 10Ac-SQLRANISH-pNA in the presence ofC5a peptidase
was accompanied by increase ofabsorbance at 405 nm,
suggesting enzymatic release of p-nitroaniline The linear
dependence ofAc-SQLRANISH-pNA cleavage with
enzyme concentration is demonstrated in Fig 8 In contrast,
upon incubation ofC5a peptidase with Suc-AAPF-pNA, a
substrate commonly used for kinetic analysis of subtilisins
[21,22], hydrolysis was not detected This observation is
consistent with limited substrate specificity ofC5a peptidase
and further illustrates significant differences in the
organ-ization ofits substrate-binding site compared to that ofthe
classical subtilisins The pH-dependence ofthe hydrolysis of
Ac-SQLRANISH-pNA reveals the optimum activity of
C5a peptidase in the alkaline region (pH 8.5–9.5) (Fig 8,
inset) At pH 8.6 activity ofC5a peptidase towards
Ac-SQLRANISH-pNA was about 60% higher than that
observed at pH 7.4 Analysis ofAc-SQLRANISH-pNA cleavage by C5a peptidase revealed that estimated Michaelis constant value is too high and exceeds maximum substrate concentration (2 mM) used in the experiments and therefore prevents accurate determination ofindividual kinetic parameters Instead, the specificity constant kcat/Km was determined directly Specificity ofC5a peptidase towards Ac-SQLRANISH-pNA was only 13M )1Æs)1(Table 1) This value is about 230-fold lower than that obtained for the parent SQLRANISHKDMQLGR extended peptide sub-strate Thus, substitution ofthe lysine residue at P1¢ position
to pNA moiety and the lack ofresidues at the P2¢ through P7¢ positions, resulting in a drastic reduction ofcatalytic efficiency, indicate the importance of C5a peptidase inter-actions with the P¢ side ofthe substrate
D I S C U S S I O N
In Gram-positive bacteria, extracellular proteases are syn-thesized initially as inactive precursors containing an amino-terminal extension that is composed ofthe signal peptide and propeptide The signal sequence, or prepeptide, is involved in translocation ofprecursor through the cyto-plasmic membrane One ofthe major functions ofthe pro-sequence region is to prevent unwanted protein degradation and to enable spatial and temporal regulation ofproteolytic activity The pro-sequence region associates to the protease module, thus preventing access ofsubstrate(s) to the active site [23] Zymogen conversion to the active enzyme occurs
by limited proteolysis ofthe inhibitory pro-sequence segment and may be either autocatalytic or involve acces-sory molecules The length ofpropeptide may vary and range from short polypeptide segments to independently folded domains comprising more than 100 residues [24,25] Often the precise length of the mature, active enzyme is not known due to the fact that the NH2-terminal processing site(s) has not been mapped Such information was not available for C5a peptidase from pathogenic Streptococcus pyogenes One ofthe aims ofthis study was to map the pro-sequence region ofC5a peptidase and to investigate the mechanism(s) ofits maturation Recombinant wild-type C5a peptidase and its S512A mutant, both lacking NH2 -terminal signal sequence and COOH terminal membrane anchor sequence (Asn32 – Gln1038), were overexpressed in
E coliand isolated from the soluble fraction of cell lysate Mobility ofpurified S512A C5a peptidase mutant was slightly decreased on SDS/PAGE compared to that ofthe wild-type enzyme, suggesting partial proteolytic degrada-tion ofthe latter Sequence analysis ofwild-type C5a peptidase confirmed the loss ofits NH2-terminal 50 amino residues, while the enzymatically inactive S512A mutant
Table 1 Kinetic parameters for the hydrolysis of peptide and pNA substrates by C5a peptidase The arrow (fl) represents location ofscissile bond.
ND, not determined The standard errors ofthe given k cat and K m values did not exceed 20% Kinetic constants for hydrolysis of peptide substrates were obtained in NaCl/Tris, pH 7.4, 5 m M CaCl 2 Specificity constant for hydrolysis of pNA substrate was obtained in 100 m M Tris/HCl, pH 8.6,
5 m M CaCl 2
Substrate
(Pn…, P2, P1 fl P1¢, P2¢,… Pn¢)
k cat
(s)1)
K m
(l M )
k cat /K m
( M )1 Æs)1)
Fig 8 Incubation of Ac-SQLRANISH-pNA and Suc-AAPF-pNA with
different amounts of C5a peptidase Reactions were carried out at 25 C
for 180 min in 100 m M Tris, pH 8.6, 5 m M CaCl 2 in the presence of
220 l M Ac-SQLRANISH-pNA and in 100 m M Tris, pH 8.6, 5 m M
CaCl 2 (containing 2% dimethylsulfoxide) in the presence of 400 l M
Suc-AAPF-pNA The pH-activity profile ofC5a peptidase f or
Ac-SQLRANISH-pNA is shown in the inset Reactions were
per-f ormed in 100 m M Mes (pH 6.0–6.5), 100 m M Hepes (pH 7.0–8.0),
100 m M Ampso (pH 8.5–9.5), 100 m M Caps (pH 10.0–11.0) in the
presence (filled squares) or absence (circles) ofthe enzyme Reactions
were also carried out in the presence (empty squares) or absence
(tri-angles) ofC5a peptidase in 100 m Tris (pH 7.0–9.0).