Angleton, Jin Lu and Sheng-Bin Peng Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, IN, USA We have previously demonstrated that Streptococcus pneumoniae signal
Trang 1In vitro and in vivo self-cleavage of Streptococcus pneumoniae
signal peptidase I
Feng Zheng, Eddie L Angleton, Jin Lu and Sheng-Bin Peng
Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, IN, USA
We have previously demonstrated that Streptococcus
pneumoniae signal peptidase (SPase) I catalyzes a
self-cleavage to result in a truncated product, SPase37–204
[Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun,
P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L
& Wang, Q.M (2001) J Bacteriol 183, 621–627] In this
study, we investigated the effect of phospholipid on in vitro
self-cleavage of S pneumoniae SPase I In the presence of
phospholipid, the self-cleavage predominantly occurred at
one cleavage site between Gly36–His37, whereas the
self-cleavage occurred at multiple sites in the absence of
phospholipid, and two additional self-cleavage sites,
Ala65–His66 and Ala143–Phe144, were identified All
three self-cleavage sites strongly resemble the signal
pep-tide cleavage site and follow the ()1, )3) rule for SPase I
recognition Kinetic analysis demonstrated that
self-cleav-age is a concentration dependent and intermolecular event, and the activity in the presence of phospholipid is 25-fold higher than that in the absence of phospholipid Biochemical analysis demonstrated that SPase37–204, the major product of the self-cleavage totally lost activity to cleave its substrates, indicating that the self-cleavage resulted in the inactivation of the enzyme More impor-tantly, the self-cleavage was demonstrated to be happening
in vivoin all the growth phases of S pneumoniae cells The bacterial cells keep the active SPase I at the highest level
in exponential growth phase, suggesting that the self-cleavage may play an important role in regulating the activity of the enzyme under different conditions Keywords: Streptococcus pneumoniae; signal peptidase I; self-cleavage; inactivation; regulation
Many secreted and membrane proteins of both prokaryotic
and eukaryotic cells are initially synthesized as a precursor
(or preprotein) with an N-terminal extension known as a
signal (or leader) peptide This signal sequence is involved in
guiding the protein into the targeting and translocating
pathway by interacting with the membrane and other
components of the cellular secretory machinery [1] The
signal peptides of secreted proteins are normally removed by
signal peptidase (SPase) that spans in the cytoplasmic
membrane in bacteria after the proteins have been
translo-cated across the membrane Two major bacterial SPases,
SPase I and SPase II, with different cleavage specificity,
have been identified SPase I is responsible for processing
majority of the secreted proteins [2,3], whereas SPase II
exclusively processes glyceride-modified lipoproteins [4]
There is no sequence similarity and substrate overlap
between these two types of SPases
In bacteria, the majority of protein translocation
occurs post-translationally via the Sec system [5,6] The
Sec system is composed of multiple proteins SecA, SecB,
SecD, SecE, SecF, SecG and SecY In Escherichia coli,
the homotetramer SecB, a chaperone protein, interacts
with the newly synthesized precursor and targets the protein to the SecAYEG translocase at the cytoplasmic membrane surface The secretory precursor then interacts with the membrane-associated homodimer SecA, which contains an ATP binding domain and utilizes the energy from ATP hydrolysis to translocate the precursor through the membrane protein channel thought to be formed from components SecYEG SPase I is a membrane-bound endopeptidase that presumably is localized in close proximity to Sec YEG it is typically anchored to the cytoplasmic membrane by one transmembrane segment in most of the gram-positive enzymes, or two transmem-brane segments in most of the gram-negative enzymes in the N-terminus Topological analysis demonstrated that the C-terminal catalytic domain of E coli SPase I resides
on the outer surface of the cytoplasmic membrane, and is thus localized in the periplasm of the cells [7–9] SPase I functions to cleave away the signal peptides from the translocated precursors, thereby releasing the mature proteins from the membrane and allowing them to their final destinations in the periplasm, outer membrane, or extracellular milieu Inhibition of SPase I leads to the accumulation of secretory precursors in the cell mem-brane and eventual cell death [10–13] Therefore, SPase I
is an essential component for bacterial growth, and a potential target for development of novel antibacterial agents
Proteases in general are divided into four classes accord-ing to their mechanism of action, they are serine, cysteine, metallo- and aspartyl proteases However, recent investiga-tions have unambiguously demonstrated that SPase I is not
a member of any of these four traditional classes, it is not
Correspondence to S B Peng, Lilly Research Laboratories, Lilly
Corporate Center, Indianapolis, IN 46285, USA.
Fax: + 1317 2769086, Tel.: + 1317 4334549,
E-mail: Peng_Sheng-Bin@lilly.com
Abbreviations: SPase, signal peptidase; IPTG,
isopropyl-b-thiogalactopyranoside; BHI, brain heart infusion.
(Received 8 April 2002, revised 20 June 2002, accepted 28 June 2002)
Trang 2sensitive to any of the standard protease inhibitors [2,14].
The catalytic mechanism of the bacterial SPase I has been
studied by site-directed mutagenesis using E coli enzyme
[15,16], Bacillus subtilis SipS [17] and S pneumoniae enzyme
[18] In these cases, a conserved serine and a conserved lysine
were identified to be critical for enzymatic activity These
results suggest that these enzymes belong to a novel class of
serine proteases that utilize a serine and a lysine to form a
catalytic dyad Therefore, this class of protease is a unique
serine protease that does not utilize a histidine as a catalytic
base, but may instead employ a lysine side chain to fulfill
this role [15,16,19] This serine–lysine catalytic dyad
struc-ture has been recently confirmed by structural analysis in
E coliSPase I [20]
A precedent for a mechanism involving a serine–lysine
dyad for a peptidase has been previously reported [21–23]
The LexA protein, which is involved in the SOS response in
E coli, undergoes a specific self-cleavage reaction that
inactivates the protein The self-cleavage of LexA protein is
important in the SOS response in bacteria [21–23] In vitro
self-cleavage was also observed in all investigated bacterial
SPase I including enzymes from E coli [24], B subtilis [25]
and S pneumoniae [18] In E coli, the self-cleavage of
SPase I occurred in a hydrophilic domain connecting the
two transmembrane segments at the N-terminus [24] In
B subtilis, the self-cleavage of SPase I (SipS) occurred
immediately after the active residue serine [25] In S
pneu-moniae, the self-cleavage site was identified between Gly36
and His37, one residue away from the active residue Ser38
[18] Although in vitro self-cleavage is common in all
investigated bacterial SPase I, the further studies on this
basic biochemical property of the enzyme are very limited so
far
We have previously demonstrated that S pneumoniae
SPase I catalyzes a self-cleavage reaction in the presence
of phospholipid After self-cleavage, the N-terminal 36
residues are removed from the protein, the major
product (SPase37–204) consisting of amino acids
37–204, contains the two critical residues, Ser38 and
Lys76, required for the formation of the catalytic dyad
However, we do not know if the self-cleavage is
activating or inactivating the enzyme, and if the
self-cleavage is happening in vivo within the bacterial cells
In the current study, we have shown that phospholipid
enhances the activity of self-cleavage, and self-cleavage
results in the inactivation of the enzyme More
impor-tantly, we have demonstrated for the first time that
the self-cleavage of SPase I is happening within the
S pneumoniae cells in all the growth phases, and the
cells maintain the active SPase I at the highest level in
the exponential growth phase
M A T E R I A L S A N D M E T H O D S
Materials and bacterial strains
Restriction enzymes, T4 DNA ligase and Taq DNA
polymerase were purchased from Life Technologies, BRL
Inc The peptide substrate (KLTFGTVKPVQAIA
GYEWL) was developed and synthesized based upon the
signal peptide of prestreptokinase of S pyogenes, as
described previously [26,27] CM- and DEAE-Sepharose
and an ECL kit for Western blot analysis were obtained
from Amershan-Pharmacia Ni-nitrilotriacetic acid agarose was from Qiagen E coli lipid extract and pure phospho-lipids were purchased from Avanti Polar Lipid, Inc Trifluoroacetic acid, EDTA, and acetonitrile were pur-chased from Fisher Scientific Chemicals for SDS/PAGE were from Nova All HPLC measurements were performed
on a HP 1100 system using C18 reversed-phase column
E coli strain Bl21(DE3)pLysS was from Novagen, and
S pneumoniaeR6 strain was from American Type Culture Collection
Purification ofS pneumoniae SPase I The expression vector, pET16b-spi, that directs the synthesis of the full length S pneumoniae SPase I, was constructed and transformed into E coli strain BL21(DE3) pLysS The transformed E coli cells were grown and induced with IPTG for protein expression at
30C The overexpressed protein was purified as described previously [18] Typically, 1 L of IPTG-induced
E coli cells were lysed by sonication in 20 mL of lysis buffer containing 300 mM NaCl and 50 mM Na2HPO4 (pH 8.0) The lysate was then centrifuged at 50 000 g for
1 h at 4C The resultant supernatant was discarded, and the pellet was resuspended and sonicated in 20 mL of lysis buffer with 1% Triton X-100 After centrifugation at
50 000 g for 1 h, the supernatant was diluted with 80 mL
of lysis buffer, and loaded to a 2mL of Ni-nitrilotriacetic acid column, that was then washed with 50 mL of lysis buffer with 0.1% Triton X-100 and 15 mM imidazole Finally, the protein was eluted with 10 mL of elution buffer containing 20 mMTris/HCl (pH 8.0), 20% glycerol, 0.1% Triton X-100 and 100 mM imidazole The purity of the protein was analyzed by SDS/PAGE, and selected fractions were utilized for enzyme assays
Purification of truncated SPase37–204 from self-cleaved SPase I
Purified SPase I (5 mg) was incubated for 2h at 37C in
5 mL of reaction buffer containing 20 mM Tris/HCl (pH 8.0), 100 mM imidazole, 20% glycerol, 0.1% Triton X-100 and 1 lgÆlL)1of E coli lipid extract The reaction mixture was diluted to 50 mL with 20 mM Tris/HCl (pH 8.0), and loaded into a 2mL of Ni-nitrilotriacetic acid agarose column The pass flow from Ni-nitrilotriace-tic acid agarose column was loaded to a 1 mL of CM-Sepharose column, and subsequently to a 1 mL of DEAE-Sepharose column that was preequilibrated with buffer A consisting of 20 mM Tris/HCl (pH 8.0), 20% glycerol After washing the DEAE-Sepharose column with
5 mL of buffer A, the protein was eluted with 10 mL of 0–400 mM NaCl gradient prepared in buffer A, 1 mL of fractions were collected, and selected fractions were utilized for functional assay
Overexpression and purification of truncated
S pneumoniae SPase37–204
To develop a simpler method to purify S pneumoniae SPase37–204, we constructed an expression vector, pET23b-spiD1-36, that directs the expression of SPase I lacking the N-terminal 36 amino acids with a C-terminal
Trang 3histidine tag Briefly, the encoding region for residues
37–204 of S pneumoniae SPase I was amplified by PCR
using genomic DNA as the template and two
oligonucleo-tides as primers (5¢-GCAATGTTCGCGTACATATGCA
TTCCATGGATCCGACC-3¢ and 5¢-TGGTGGTGCTC
GAGAAATGTTCCGATACGGGTGATTGGCCAGA
AGCG-3¢), which were designed to contain NdeI and XhoI
restriction sites at the 5¢ ends, respectively The primers were
synthesized in accordance with the published sequence of
S pneumoniae SPase I [28] The PCR product was
puri-fied and cloned into the NdeI and XhoI sites of vector
pET23b, resulting in pET23b-spiD1-36 The identity of the
cloned gene was confirmed by DNA sequencing For
expression of SPase37–204, E coli strain
BL21(DE3)-pLysS was transformed with pET23b-spiD1-36, grown
and induced with 0.4 mM IPTG at 30C, as described
previously [29] For purification, 1 L of the IPTG-induced
E colicells were harvested by centrifugation, resuspended
in 40 mL of lysis buffer, and sonicated for 5 min on ice
The lysate was then centrifuged at 50 000 g for 1 h The
resultant supernatant was loaded onto a 2mL
preequil-ibrated Ni-nitrilotriacetic acid column, which was then
washed with 50 mL of lysis buffer with 15 mMimidazole
The protein was finally eluted out with 10 mL of elution
buffer, and 1 mL of fractions were collected and analyzed
by SDS/PAGE
In vitro self-cleavage of S pneumoniae SPase I
For in vitro self-cleavage of S pneumoniae SPase I in the
presence of phospholipid, 20 lL of reaction containing
5 lg of purified SPase I was incubated in 37C for 2 h in
2 0 mM Tris/HCl (pH 8.0), 0.05% Triton X-100, 10%
glycerol, and 50 lg of E coli lipid extract For
self-cleavage in the absence of phospholipid, 20 lL of reaction
containing 5 lg of purified SPase I was incubated at
37C in the same buffer without phospholipid Typically,
the reactions were terminated by the addition of SDS
sample buffer, and separated on a 4–20%
SDS/poly-acrylamide gel; the gel was then stained with Coomassie
Brilliant Blue For kinetic analysis of self-cleavage,
reac-tions (20 lL) containing different concentrareac-tions of
SPase I were incubated at 37C for 30 min in the
presence of 50 lg phospholipid or 4 h in the absence of
phospholipid Densitometer analysis was performed using
a Personal Densitometer SI and IMAGE QUANT5.0
soft-ware from Molecular Dynamics Specific activities of
self-cleavage in the presence or absence of phospholipid were
calculated according to the cleavage of SPase I at
concentration of 1 mgÆmL)1
N-Terminal peptide sequencing
To determine the self-cleavage sites of S pneumoniae
SPase I, the proteolytic products of the self-cleavage were
fractionated on a 4–20% SDS/polyacrylamide gel, and
transferred to a poly(vinylidene difluoride) (PVDF)
mem-brane by electroblotting The memmem-brane was then briefly
stained by Commassie bright blue and destained by 50%
methanol The visualized protein bands on the membrane
were excised and the N-terminal amino acid sequence
of each protein was determined by automated Edman
degradation
Cleavage of prestreptokinase byS pneumoniae SPase I and SPase37–204
We have previously demonstrated that prestreptokinase is a native substrate of S pneumoniae SPase I The gene encoding S pyogenes prestreptokinase was amplified by PCR based upon the published sequence [26], and cloned into the expression vector pET23b to result in pET23b-ska For the expression of the prestreptokinase, E coli strain BL21(DE3)pLysS was transformed with pET23b-ska, grown and induced by IPTG The overexpressed prestrep-tokinase was then solubilized with 1% Zwittergent 3–16, and purified with Ni-nitrilotriacetic acid column, as described previously [18] For the cleavage of prestrepto-kinase, typically, reactions (20 lL) containing 0.1 lg SPase I
or SPase37–204 were incubated with 5 lg of purified prestreptokinase at 37C for 1 h in the buffer containing
2 0 mM Tris/HCl (pH 8.0), 0.02% Triton X-100, 5% glycerol and 50 lg of E coli total lipid extract The reactions were then terminated by the addition of SDS sample buffer, and the proteins were separated on a 4–20% SDS/poly-acrylamide gel, and stained by Coomassie Brilliant Blue
Cleavage of a peptide substrate byS pneumoniae SPase I and SPase37–204
A peptide substrate, KLTFGTVKPVQAIAGYEWL was developed and synthesized based upon the signal peptide
of the prestreptokinase of S pyogene [26,27] Typically, cleavage reactions were performed in 50 lL reaction mixtures containing 20 mM Tris/HCl (pH 8.0), 50 lg of E.coli lipid extract, 0.1 lM SPase I and 100 lM of the peptide substrates Reactions were incubated at 37C for 2h and terminated by the addition of an equal volume of
8M urea Cleavage of the peptide substrate was deter-mined by HPLC using a Hewlett Packard Series 1100 system equipped with an autosampler The reaction mixtures were injected into a reversed-phase column (Vydac C18) and the fragments were separated using a 0–67% linear gradient of buffer B in buffer A (buffer
A¼ 0.1% trifluoroacetic acid in water, buffer B ¼ 90% acetonitrile and 0.1% trifluoroacetic acid) with a flow rate
of 1 mLÆmin)1 Peak detection was accomplished by monitoring the absorbance at 214 nm
Preparation of a polyclonal antibody against
S pneumoniae SPase I Two S pneumoniae SPase I-specific peptides, SP-Ab1 (CHEEDGNKDIVKRVIG) and SP-Ab2(CLADYIK RFKDDKLQS), were synthesized based upon the deduced amino acid sequence [28] A cysteine was artificially added
to the N-terminus of each peptide to increase the coupling efficiency After purification, the synthetic peptides were coupled to keyhole limpet hemocyanin, and utilized for immunization of New Zealand white rabbits to generate polyclonal antibodies, as described previously [30]
Detection ofin vivo self-cleavage of S pneumoniae SPase I by Western blot analysis
Fresh S pneumoniae cells were prepared by growing the cells in 5 mL of brain–heart infusion (BHI) broth in a
Trang 4series of dilution at 37C with 5% CO2 overnight The
culture with D620< 0.3 was carefully centrifuged, washed
once with fresh BHI broth The cells were then
resuspended in fresh BHI broth with an initial
D620¼ 0.075, and grown at 37 C and 5% CO2 for a
period up to 5 h 2mL of culture was harvested at
different time points by centrifugation and immediately
lysed with a solution containing 1· SDS sample buffer,
B-PER II bacterial extraction reagent from Pierce and a
protease inhibitor cocktail from Roche to protect proteins
from nonspecific proteolysis After boiling for 10 min, the
samples (20 lg of total protein each) were separated on a
4–20% SDS/polyacrylamide gel and transferred
electroph-oretically to a PVDF membrane Immunodetection was
performed using immune serum against S pneumoniae
SPase I at 1 : 2000 dilution or a polyclonal antibody
against S pneumoniae Era at 1 : 1000 dilution
Densitom-eter analysis was performed using a Personal DensitomDensitom-eter
S1 and IMAGE QUANT5.0 software The ratio of the
full length SPase/cleaved SPase was calculated based upon
the relative intensity of the two protein bands from
Western blot analysis
R E S U L T S
In vitro self-cleavage of S pneumoniae SPase I
We have previously reported that S pneumoniae SPase I
catalyzes a specific self-cleavage reaction in vitro A
similar self-cleavage reaction was also observed in
purified E coli SPase I [24] The self-cleavage of E coli
SPase I was initially speculated to be protected within the
bacterial cells by the interaction between the enzyme and
the cytoplasmic membrane In this study, we were
interested in investigating the effect of phospholipid on
the self-cleavage of S pneumoniae SPase I In the
pres-ence of phospholipid, the self-cleavage occurred
predom-inantly at one cleavage site to produce two protein
bands, b1 and b4, as demonstrated in Fig 1A; no other
cleavage site was observed In the absence of
phosphol-ipid, the highly purified S pneumoniae SPase I, when
incubated at 37C, also resulted in the self-cleavage of the enzyme Interestingly, the self-cleavage was somewhat different, it occurred at multiple sites and resulted in at least five identifiable protein bands, b1, b2, b3, b4, and b5 with molecular masses ranging from 8 to 19 kDa, as shown in Fig 1B To confirm the specificity of the self-cleavage, we also tested the self-cleavage of two SPase I mutants, S38A and K76A that lost their activity to catalyze substrate cleavage as described previously [18] Results demonstrated that the purified S38A and K76A were unable to catalyze self-cleavage in the presence or absence of phospholipid, confirming that the self-cleavage
in both conditions was specific and not due to the possibly contaminating proteases (data not shown) Additionally, all the major protein bands from the self-cleavage of SPase I were separated, excised and subjected for N-terminal peptide sequencing The sequences obtained from five cleaved protein bands were summa-rized in Table 1 In the absence of phospholipid, three self-cleavage sites, Gly36–His37, Ala65–His66, and Ala143–Phe144 were identified as indicated in Fig 1C
In the presence of phospholipid, only one self-cleavage site (Gly36–His37) was identified The peptide sequence GHHHHHHHHHHSSG from products b2and b4 was the histidine tag fused to the N-terminus of the enzyme
Kinetics of SPase I self-cleavage Kinetic analysis demonstrated that self-cleavage was a protein concentration dependent event Titration experi-ments revealed that the specific activities of self-cleavage in the presence of phospholipid were increasing when SPase I concentrations were increased (Fig 2A) A similar protein concentration-dependent self-cleavage of SPase I was also observed in the absence of phospholipid (Fig 2B) It suggests that self-cleavage of SPase I is catalyzed through
an intermolecular mechanism The activities of self-cleav-age in the presence or absence of phospholipid were calculated to be 0.025 or 0.001 min)1, respectively, at SPase I concentration of 1 mgÆmL)1, and a 2 5-fold
Fig 1 Self-cleavage of S pneumoniae SPase I Reactions (20 lL) containing 5 lg of wild type SPase I were incubated at 37 C in the presence (A) or absence (B) of phospholi-pid The samples were separated on 4–20% SDS/polyacrylamide gels, and stained with Coomassie Brilliant Blue Lane 1, purified full length SPase I before incubation; lane 2, full length SPase I after incubation Protein bands corresponding to degradation products of
S pneumoniae SPase I were indicated as b1, b2, b3, b4 and b5 (C) Amino acid sequence of
S pneumoniae SPase I The self-cleavage sites, identified by automated Edman degradation, are marked with arrows The peptides utilized for antibody preparation are underlined.
Trang 5stimulation by phospholipid was observed Therefore,
phospholipid greatly stimulates the self-cleavage of
SPase I
Self-cleavage sites ofS pneumoniae SPase I resemble
signal peptide cleavage sites
Although signal peptides of secreted proteins do not
show a great deal of sequence identity, they do share
some common structural properties Statistical analysis of
the amino acid sequences surrounding signal peptide
cleavage sites has led to the so called ()1, )3) rule that
states that the residues at the)1 and )3 positions relative
to the SPase I cleavage site must be small and neutral
residues [31–33] The residues at )1 position are usually
Ala, Gly, and Ser, and at )3 position are usually Ala,
Val, Gly, Ser, and Thr Sequence analysis around the
three self-cleavage sites identified from S pneumoniae
SPase I revealed that all these self-cleavage sites strongly
resemble the signal peptide cleavage sites, and follow the
()1, )3) rule well with the most common alanine or
glycine in the)1 position, and an alanine or a valine at
)3 position as demonstrated in Fig 3 This result further
supports that the self-cleavage of SPase I with or without
phospholipid is specific and not caused by possibly
contaminating proteases from the purification or by
careless handling of the protein Similarly, the
self-cleavage sites identified from E coli and B subtilis SPases
also follow the ()1, )3) rule for signal peptidase
recognition as aligned in Fig 3
Purification of truncatedS pneumoniae SPase37–204 from self-cleaved products and overexpressedE coli cells
We have demonstrated that S pneumoniae SPase I pre-dominantly catalyzes self-cleavage at one cleavage site between Gly36 and His37 in the presence of phospholipid The major product of this cleavage, SPase37–204 still contains residues Ser38 and Lys76, which are two active residues to form a catalytic dyad [18] Therefore, we are interested in comparing the enzymatic activity of the full length enzyme with this truncated product For this purpose, we developed a procedure to purify SPase37–204 from the reaction mixture of the self-cleavage When
Fig 2 Kinetic analysis of Self-cleavage of S pneumoniae SPase I Reactions (20 lL) containing different concentrations of SPase I as indicated were incubated at 37 C for 30 min or 4 h in the presence (A) or absence (B) of phospholipid The samples were separated on 4–20% SDS/ polyacrylamide gels and stained with Coomassie Brilliant Blue Densitometer analysis was performed with a Personal Densitometer SI and IMAGE QUANT 5.0 software from Molecular Dynamics Percentage of the self-cleavage was calculated according to the decrease of the full length SPase I in each reaction.
Table 1 N-terminal sequences of self-cleaved products of S pneumoniae SPase I N-terminal sequences of two cleaved products, b1 and b4 in the presence of phospholipid, and five cleaved products, b1, b2, b3, b4 and b5 in the absence of phospholipid were determined by automated Edman degradation.
Peptide sequences
b1
b2
19 18
GHHHHHHHHHHSS
Fig 3 Self-cleavage sites of S pneumoniae SPase I resemble signal peptide cleavage sites The three self-cleavage sites of S pneumoniae SPase I identified by automated Edman degradation were aligned along with the self-cleavage sites identified from E coli and B subtilis enzymes Self-cleavage sites are marked with arrow The )1 and )3 positions relative to the cleavage sites are highlighted.
Trang 6reaction mixture was passed through a Ni-nitrilotriacetic
acid agarose column, the uncleaved SPase I and the
N-terminal product containing a histidine tag bound to
the Ni-nitrilotriacetic acid column The C-terminal product,
SPase37–204, passed through the Ni-nitrilotriacetic acid
column, was further purified by chromatography utilizing a
CM- and a DEAE-Sepharose column as described under
the Materials and methods The purified SPase37–204, we
called it the self-cleavage-generated SPase37–204, was
utilized for activity analysis As the self-cleavage-generated
SPase37–204 may have lost its activity due to a relatively
complicated and lengthy purification protocol, we also
constructed an expression vector, pET23b-spiD1-36 to
direct the overexpression of SPase37–204 with a C-terminal
His tag The overexpressed SPase37–204 was easily
solubi-lized by a simple salt extraction and purified to near
homogeneity by one step Ni-nitrilotriacetic acid agarose
chromatography as described under the Materials and
methods Approximately 5 mg of purified protein was
obtained from 1 L of IPTG-induced E coli cells
SPase37–204 loses its ability to cleave its native
substrate, prestreptokinase
We have previously identified prestreptokinase, an
extra-cellular protein in pathogenic streptococci to be cleaved
between Ala26 and Ile27 by S pneumoniae SPase I [18] To
evaluate the activity of SPaseD37–204, we incubated the
substrate with the self-cleavage-generated SPase37–204 in
the presence of phospholipid As demonstrated in Fig 4,
lane 2, the purified SPase37–204 was unable to cleave
prestreptokinase It indicated that the major product of the
SPase I from self-cleavage was not active Similarly, the
overexpressed SPase37–204, that was purified simply by one
step Ni column was unable to cleave prestreptokinase either
as shown in Fig 4, lane 3, whereas the full length SPase I
cleaved the substrate effectively (Fig 4, lane 4) These
results indicate that SPase I lacking the N-terminal 36
amino acids has lost its activity to cleave its native substrate, prestreptokinase
SPase37–204 loses its ability to cleave a peptide substrate
Based upon the signal peptide sequence of prestreptokinase,
we developed a peptide substrate, KLTFGTVKPVQAIA GYEWL that was effectively and specifically cleaved between Ala and Ile by the full length S pneumoniae SPase I [27] As demonstrated by HPLC analysis in Fig 5, this 19 amino acid peptide substrate had a retention time of 4.25 min When it was incubated with the full length SPase I in the presence of phospholipid, two products were generated with retention times of 3.32and 3.88 min, respectively (Fig 5A) Mass spectrum analysis of the two products confirmed that the cleavage specifically occurred between residues Ala–Ile as expected (data not shown) However, when the peptide substrate was incubated with
Fig 4 SDS/PAGE analysis of purified prestreptokinase and its
cleav-age by full length SPase I and SPase37–204 Reactions containing
0.1 lg SPase I or SPase37–204 were incubated with 5 lg of purified
prestreptokinase at 37 C for 1 h in the buffer containing 20 m M Tris/
HCl (pH 8.0), 0.02% Triton X-100, 5% glycerol and 50 lg
phos-pholipid The reactions were terminated by the addition of SDS sample
buffer, and the proteins were separated on a 4–20%
SDS-poly-acrylamide gel, and stained by Coomassie Brilliant Blue Lane 1,
prestreptokinase (pre-Ska); lane 2, prestreptokinase plus
self-cleavage-generated SPase37–204; lane 3, prestreptokinase plus overexpressed
SPase37–204; and lane 4, prestreptokinase plus full length SPase I.
Prestreptokinase was processed to mature streptokinase (mSka) upon
incubation with full length SPase I, as demonstrated in lane 4.
Fig 5 HPLC analysis of the peptide substrate cleavage by full length SPase I and SPase37–204 The peptide substrate, KLTFGTVK PVQAIAGYEWL was incubated at 37 C for 2 h with full length SPase I (A), self-cleavage-generated SPase37–204 (B), or overex-pressed SPase37–204 (C) The cleavage of the peptide substrate was determined by HPLC using a Hewlett Packard Series 1100 system with
a reversed-phase column (Vydac C18) as described under the Materials and methods The peaks labeled 1, 2and 3 correspond to the substrate, the C-terminal cleavage product and the N-terminal cleavage product, respectively.
Trang 7the self-cleavage-generated and the overexpressed SPase37–
204, there was no product peak formed at the expected
retention time in the HPLC profiles (Fig 5B,C) The result
was in accordance with that observed with the native
substrate, prestreptokinase Taken together, these results
confirmed that S pneumoniae SPase37–204, the major
product of the self-cleavage lost its ability to cleave its
substrates Therefore, the self-cleavage of SPase I is believed
to inactivate the activity of the enzyme
In vivo self-cleavage of S pneumoniae SPase I
As the in vitro self-cleavage inactivates the protease activity
of S pneumoniae SPase I, and this self-cleavage is actually
stimulated, not protected by phospholipid, we are interested
in exploring the reality of the self-cleavage within the
bacterial cells Polyclonal antibody against S pneumoniae
SPase I was obtained after immunization of rabbits with
synthetic peptides To detect SPase I and its cleavage within
the bacterial cells, we performed a Western blot analysis on
S pneumoniaecells The whole cell lysates were prepared
from submerged cultures grown to different points
through-out their exponential and stationary growth phases as
indicated in Fig 6A, separated on a SDS/polyacrylamide
gel, and transferred to a PVDF membrane for
immunode-tection with antidodies against S pneumoniae SPase I and
Era, an essential membrane associated GTP binding protein
from S pneumoniae [34] As demonstrated in Fig 6C, the
full length S pneumoniae SPase I and its cleaved product were detected in all the growth phases This result confirmed for the first time that the self-cleavage of SPase I is indeed happening within the bacterial cells throughout all the growth phases The cleaved product reacting with the peptide antibody against SPase I has a molecular mass of
11 kDa, equivalent to the molecular mass of peptides from residues 36–143, a possible self-cleaved product based upon self-cleavage sites identified As expected, this protein band reacted specifically with both peptide antibodies against SPase I It should be noted that the cell lysates were prepared immediately after harvest by adding SDS sample buffer and protease inhibitor cocktail, and boiling for
10 min to protect proteins from nonspecific proteolysis
S pneumoniae cells maintain the full length SPase I
in the highest level in exponential growth phase
As demonstrated by Western blot analysis in Fig 6C,
S pneumoniaecells appeared to produce the overall SPase I
in the same level in all growth phases However, differences
in full length and cleaved SPase I were observed in different growth phases In lag and stationary phases, the cells showed lower levels of full length SPase I and higher levels
of cleaved product In contrast, the cells had a higher level
of full length protein and a lower level of cleaved product
in exponential growth phase In general, the bacterial cells maintained the active SPase I in the highest level in
Fig 6 Cell growth of S pneumoniae and Western blot analysis of in vivo self-cleavage of S pneumoniae SPase I (A) Growth curve of
S pneumoniae cells The cells were grown at 37 C in BHI broth, and harvested at different time points as indicated The cell growth was monitored by the measurement of absorbence at 620 nm (B) Densitometer analysis of full length SPase I and the cleaved product in different growth phases The analysis was performed based upon the results of Western blot analysis using a Personal Densitometer SI and IMAGE QUANT
5.0 software from Molecular Dynamics The ratio of full length/cleaved SPase I was calculated based upon the relative intensity of the two protein bands reacting with antibody (C) Western blot analysis of in vivo self-cleavage of SPase I 20 lg of whole cell lysate from different growth phases was separated on a 4–20% SDS/polyacrylamide gel, and transferred to a PVDF membrane Immunodetection was performed using immune serum against S pneumoniae SPase I at 1 : 2000 dilution Lanes 1–9 were the whole cell lysate prepared from S pneumoniae R6 cells grown in BHI broth for different time as indicated (D) Western blot analysis with Era antibody.
Trang 8exponential growth phase compared to lag and stationary
growth phases Densitometer analysis demonstrated that
the ratio of the full length SPase I/the cleaved SPase I was
0.81–1.0 in exponential phase, whereas the ratio was 0.36–
0.51 in lag phase and 0.39–0.55 in stationary phase
(Fig 6B) This result is very reproducible, Fig 6C shows
one example of several experiments Clearly, a more
quantitative methodology, other than Western blot analysis,
needs to be developed to quantify the SPase I and its
cleaved products more accurately In addition, Western blot
analysis with an antibody against S pneumoniae Era
demonstrated that approximately an equal amount of
protein was loaded in each lane (Fig 6D) The Era antibody
was selected because of its equally expression in all the
growth phases of S pneumoniae
D I S C U S S I O N
A previous study demonstrated that S pneumoniae SPase I
catalyzes a self-cleavage In the presence of phospholipid,
the enzyme predominantly cleaves itself at one cleavage site
between Gly36 and His37 [18] In this study, we found that
the self-cleavage occurred at multiple sites in the absence of
phospholipid, and two additional self-cleavage sites, Ala65–
His66 and Ala143–Phe144, were identified All three
self-cleavage sites strongly resemble the signal peptide self-cleavage
site and follow the ()1, )3) rule for signal peptidase
recognition Phospholipid was demonstrated to stimulate
the self-cleavage of S pneumoniae SPase I We also
dem-onstrated that the major product of the self-cleavage,
SPase37–204, totally lost its activity to cleave a native
substrate prestreptokinase and a peptide substrate,
indicat-ing that the self-cleavage inactivates the enzyme More
importantly, we found that the self-cleavage of S
pneumo-niae SPase I is also happening in vivo in all the growth
phases, and that the bacterial cells maintain the active
SPase I at the highest level in the exponential growth phase
These results suggest that the self-cleavage of SPase I may
play an important role in regulating the activity of the
enzyme within the cells
A number of genes encoding SPase I have been cloned
and sequenced from both negative and
Gram-positive bacteria including E coli [9], Salmonella enterica
serovar Typhimurium [35], Haemophilus influenzae [36],
Staphylococcus aureus[37], Bacillus subtilis [38–40], S
pneu-moniae[18,28], and Streptomyces lividans [41] To date, the
in vitro biochemical studies on SPase I were performed
using enzymes from three species, E coli, S pneumoniae,
and Bacillus subtilis Although significant differences in
primary sequences exist, these three enzymes share a
common biochemical property, i.e in vitro self-cleavage
In E coli, the self-cleavage of SPase I occurs between the
residues Ala40 and Ala41, which are located in a
hydro-philic domain connecting the two transmembrane segments
at the N-terminus of the enzyme Although the major
product of this self-cleavage is still active, its specific activity
is 100-fold less than the native enzyme [24] In S
pneumo-niae, the purified full length SPase I catalyzes an
intermo-lecular self-cleavage The major product of this self-cleavage
totally lost its activity as demonstrated in this study In
B subtilis, the self-cleavage of SPase I (SipS) was observed
recently, the soluble form of SipS that lacks the N-terminal
membrane anchor is prone to self-cleavage, and the
self-cleavage also results in complete inactivation of the enzyme [25] Taken together, the self-cleavage was observed
in all bacterial SPases investigated so far, suggesting that it is most likely a common biochemical property shared by bacterial SPases Another common biochemical property is that the self-cleavage of the SPase I resulted in the complete lose or dramatic decrease of the enzymatic activity, implying that the self-cleavage, if occurring in vivo, may play an important role in the regulation of the enzymatic activity within the cells
Interestingly, phospholipid was demonstrated to affect self-cleavage of SPase I dramatically In the absence of phospholipid, SPase I cleaves itself at multiple sites, whereas the self-cleavage predominantly occurs at one cleavage site
in the presence of phospholipid We believe that the interaction between SPase I and phospholipid somehow changes the conformation of the enzyme, and makes SPase I preferentially cleave itself at one specific site More importantly, phospholipid was shown to stimulate the self-cleavage about 25-fold This phospholipid stimulation was also observed for substrate cleavage of the SPase I [18] Therefore, we believe that the interaction of SPase I and phospholipid may play an important role in the catalytic mechanism of the enzyme
Self-cleavage of the E coli SPase I was previously described [24] Scientists working on this enzyme specu-lated that the self-cleavage might be protected in vivo by the interaction of the enzyme with cytoplasmic membrane Therefore, the possible physiological role of self-cleavage was basically ignored However, our investigation revealed that the self-cleavage of S pneumoniae SPase I was not protected by the phospholipid mixture from E coli lipid extract In contrast, the phospholipid mixture, which composed mainly of phosphatidylethanolamine, phosphat-idylglycerol, and cardiolipin, actually stimulated the self-cleavage of the S pneumoniae SPase I These results intrigued us to investigate the cleavage of S pneumoniae SPase I in vivo As shown in Fig 6C, Western blot analysis demostrated that the self-cleavage of SPase I is indeed occuring in vivo in S pneumoniae throughout all the growth phases Although, at this moment, we can not conclusively explain why self-cleavage is happening in vivo, one speculation is that it may be involved in regulating the activity of the enzyme It is not difficult to imagine that bacteria may secrete proteins at different levels at different growth phases and various conditions, and thus may require differential SPase I activity Indeed, as we have shown in this study, S pneumoniae cells maintain the full length SPase I in the highest level in exponential phase compared to lag and stationary growth phases Bacterial cells in exponential phase secrete more proteins, therefore a higher level of SPase activity may be required for increasing the secretion capacity It appears that the self-cleavage of SPase I may play a role in bacterial cells to control the overall activity of SPase I at a certain level Further investigation to establish this hypothesis is needed
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