Prediction of Extracellular Proteases of the Human Pathogen Helicobacter pylori Reveals Proteolytic Activity of the Hp1018/19 Protein HtrA
et al. (2008) Prediction of Extracellular Proteases of the Human Pathogen Helicobacter
pylori Reveals Proteolytic Activity of the Hp1018/19 Protein HtrA. PLoS ONE 3(10): e3510. doi:10.1371/journal.pone.0003510
Prediction of Extracellular Proteases of the Human Pathogen Helicobacter pylori Reveals Proteolytic Activity of the Hp1018/19 Protein HtrA
Martin Lo wer 0
Christiane Weydig 0
Dirk Metzler 0
Andreas Reuter 0
Anna Starzinski-Powitz 0
Gisbert Schneider 0
Raphael H. Valdivia, Duke University Medical Center, United States of America
0 1 Goethe-University, Institute of Cell Biology and Neuroscience / CMP , Frankfurt am Main, Germany , 2 Junior Research Group, Paul-Ehrlich Institute , Langen, Germany , 3 Goethe-University, Institute of Computer Science , Frankfurt am Main, Germany , 4 Paul-Ehrlich Institute, Department of Allergology , Langen , Germany
Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.
Funding: This research was supported by the Beilstein-Institut zur F orderung der Chemischen Wissenschaften Frankfurt am Main (Germany), the Centre for
Membrane Proteomics (CMP) Frankfurt am Main (Germany), the J urgen-Manchot Stiftung, the Paul-Ehrlich Institut Langen (Germany), and the Deutsche
Forschungsgemeinschaft (SFB-579, project A11).
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The mucosal epithelium in the human stomach forms the first
barrier that prevents infiltration of pathogens into the host
organism. The human pathogen H. pylori developed efficient
strategies to colonize the gastric epithelium as a unique niche,
where it induces the disruption of the epithelial layer contributing
to inflammatory diseases (e.g. chronic gastritis, ulceration),
mucosaassociated lymphoid tissue (MALT) lymphoma and gastric cancer
in humans [1,2]. More virulent H. pylori strains express a
combination of key disease-associated virulence factors allowing
successful colonization in the stomach . Among those, H. pylori
harbors cag pathogenicity island (cagPAI), which encodes a type IV
secretion system (T4SS) to inject the bacterial CagA
(cytotoxinassociated gene A) oncoprotein into host cells . In vitro,
translocated CagA can strongly enhance the disruption of
intercellular adhesions [4,5]. This process is believed to contribute
to inflammation, carcinogenesis and invasive growth. Although the
cellular aspects of CagA have been investigated intensively, the
complex mechanisms of the actual interaction of H. pylori and the
human epithelium are not fully understood yet.
Many pathogens developed elegant mechanisms for tissue
destruction by secreting proteins with proteolytic activity.
Exported bacterial enzymes can directly activate host
pro-matrixmetalloproteinases (pro-MMPs) representing a biochemical
efficient way for matrix degradation. An example is set by the wide
range of proteases of the thermolysin family secreted by
Pseudomonas aeruginosa and Vibrio cholera that activate pro-MMP-1,
8, and -9 . It has been further observed that serine proteases
associated with lipopolysaccharides can induce MMP-9 activity in
macrophages . MMP-9 cleavage was also detected by a secreted
zinc metalloproteinase (ZmpC) from Streptococcus pneumoniae, which
indicates that ZmpC may play a role in pneumococcal virulence
and pathogenicity in the lung .
Proteases might also play a role in H. pylori pathogenesis, and
protease secretion has already been described for this organism
. H. pylori sheds an unknown protease that efficiently degrades
PDGF (platelet derived growth factor) and TGF-b (transforming
growth factor beta), which can be inhibited with sulglycotide .
Some features present in the primary sequence of H. pylori
virulence factor vacuolating cytotoxin A (VacA) are reminiscent of
serine proteases , although the predicted proteolytic activity of
VacA has not been detected yet. In 1997, a H. pylori
metalloproteinase with a native molecular size of approximately
200 kDa was discovered, which was secreted when H. pylori was
grown in liquid culture . The authors hypothesized that
surface expression of this metalloprotease activity may be involved
in proteolysis of a variety of host proteins in vivo and thereby
contribute to gastric pathology . Importantly, H. pylori secretes
a collagenase, encoded by hp0169, which might represent an
essential virulence factor for H. pylori stomach colonization .
The predicted serine protease and chaperone HtrA (Hp1019) was
previously identified as an extracellular protein of H. pylori ,
but its proteolytic role and substrates are still unknown.
As 658 of the 1,576 identified genes of the H. pylori genome 
are annotated as hypothetical or as bearing a hypothetical
function , we aimed at the identification of H. pylori genes
possibly coding for secreted proteases by combining genomic data
analysis with zymography. Indeed, we found that H. pylori secretes
unknown proteins exhibiting caseinolytic activity. By calculating
similarities to known proteases and using localization prediction
methods, we inferred function and localization of these
hypothetical H. pylori proteins. We also identified a sequencing error in the
hp1018 gene, which after correction encodes for a signal peptide
for the putative serine protease HtrA (Hp1019). Eventually, we
verified proteolytic activity of HtrA in biochemical approaches.
The present study demonstrates the usefulness of sequence-based
genome mining for potential drug targets representing one possible
route for the prevention of matrix degradation of the mucosal
epithelium by H. pylori and other pathogens.
Results and Discussion
H. pylori secretes caseinolytic proteases
Data are accumulating that bacteria secrete proteases with
functional roles in microbial pathogenesis, but knowledge of H.
pylori-secreted proteases and their functions is still limited. To
analyze whether H. pylori actually secretes proteases, we performed
casein zymography to monitor proteolytic activity in the
supernatants of H. pylori lysates (Figure 1A, lane1) and H. pylori
culture medium (Figure 1A, lane 2). At least three casein-cleaving
proteases were exported by H. pylori exhibiting apparent molecular
weights of approximately 170 kDa, 140 kDa, and 50 kDa
(Figure 1A, lane 2). Interestingly, the protein band pattern present
in the supernatant of the H. pylori medium obviously differs from
the equivalent H. pylori lysate (Figure 1A lanes 1). The detected
170 kDa protease present in the supernatant of H. pylori (BHI Hp)
consistently migrated slightly faster than in the H. pylori lysate,
while the 140 kDa protein was only present in the supernatant, but
absent in the lysate of H. pylori (Hp son). In contrast to the double
band detected in the lysates, we observed only a single proteolytic
activity in the supernatant (Figure 1A, lanes 12). These data
indicate that the export of the proteases might occur via active
signal peptide-dependent translocation, rather than being an
artifact of bacterial autolysis in the H. pylori liquid culture.
Since H. pylori encodes a well-described T4SS and the
T4SSindependently secreted pathogenic factor VacA with a
hypothesized protease function , we also included supernatants of
isogenic H. pylori mutants which are deficient of T4SS and CagA
(DPAI, Figure 1B, lane 3), or VacA (DVacA, Figure 1B, lane 4),
and compared them with the H. pylori wildtype strain (wt,
Figure 1B, lane 2) and H. pylori-free culture medium (-,
Figure 1B, lane 1). Compared to the wildtype strain, the DPAI
mutant showed the same secretion pattern of proteins with
caseinolytic activity in the extracellular space suggesting that their
secretion might occur independently from the T4SS (Figure 1B,
lane 3). Although initial publications indicated a predicted serine
protease activity of the pathogenic factor VacA , we can
exclude a caseinolytic effect of VacA since the isogenic
vacAdeficient H. pylori mutant showed a similar pattern of proteases
(Figure 1B, lane 4). Gelatin zymographies were also performed by
us and clearly demonstrated the lack of gelatinolytical H. pylori
proteases (not shown). A positive result here would have
demonstrated a closer link to matrix degaradation, as gelatin is a
product of collagen, a major extacellular matrix protein.
So far, the identity of the detected H. pylori proteases was
unknown. A previously described multi-metalloprotease-like
complex secreted by H. pylori with a molecular weight of about
200 kDa  might be an explanation for the largest protein seen
in the zymogram, since its size is four to six times greater than
comparable proteases of other Gram-negative bacteria . Also,
protease DegP of Escherichia coli, which is a homolog of Hp1019
from H. pylori, was shown to form hexamers when crystallized .
Therefore, as zymography was performed under non-reducing
conditions, the upper band(s) might result from smaller proteins
forming a macromolecular complex.
In silico genome screening for candidates of H. pylori
secreted hypothetical proteases
Based on the finding that H. pylori actively secretes proteases, we
then aimed to identify suitable candidates by in silico analysis.
Thus, we compared the H. pylori proteome to a set of known
proteases from various organisms using sequence alignment
techniques. A reference set of known proteases containing 3,566
amino acid sequences was compiled from the UniProtKB/
SwissProt database (version 6.7) , which served as queries
for exhaustive pairwise alignment to genomic and protein
sequence data of H. pylori strain 26695 with 1,576 annotated
genes from the NCBI RefSeq database (accession number
NC_000915) . For the 1,576 putative H. pylori proteins,
75,524 local alignments were returned by the BLAST algorithm
. Alignments yielding an E-value#0.5 were selected and
divided into four classes:
The latter (class D) were not further examined. Information
about the localization of the active sites was retrieved from the
feature tables of the respective SwissProt entries .
Then, we predicted protein localization using prediction
systems, which are publicly available on the World Wide Web:
SignalP , SecretomeP , Phobius , CELLO ,
PASUB , and PSORTb . All systems are capable or explicitly
designed to analyze amino acid sequences from Gram-negative
bacteria. Alignments were selected for further examination when
the corresponding predictions for a protein sequence matched one
or more of the following criteria:
i) predicted extracellular localization (CELLO, PSORTb,
ii) predicted signal peptide (SignalP),
iii) predicted signal peptide, but no transmembrane helices
(Phobius), and a SecretomeP score$0.5.
By filtering the alignments with respect to the active residues
marked in the sequence of reference protease and the localization
prediction, we obtained 47 class A, 39 class B and 32 class C
proteins (vide supra) and their corresponding genes. The
bestscoring alignments of those proteins to proteases of the reference
set were manually inspected. Among those, nine genes have not
been described to code for H. pylori proteases yet, but can be
aligned with a statistically significant score to proteases of the
reference protease sequences (Table 1). Interestingly, the putative
translation products of genes hp0289, hp0609, and hp0922 form a
group of paralogs to VacA cytotoxin , which can be seen in a
multiple sequence alignment (not shown). Structural similarities of
VacA to extracellular IgA proteases of Haemophilus influenzae have
been described previously . Pairwise sequence identity of these
VacA paralogs to VacA ranges between 25% and 30% which also
include the C-terminal autotransporter sequence . As this
sequence is sufficient to transclocate the N-terminal part of the
protein across the outer membrane 2 which is often followed by
an autoproteolytic event to release the translocated part into the
extracellular space  2 it seems likely that some of these
proteins possess a proteolytical function.
Finally, although we could not detect caseinolytic activity of
VacA in our casein zymography study, we cannot exclude an
effect of VacA and its paralogs on other substrates per se. However,
the alignments do not reveal conserved active site residues in VacA
paralogs. Still they might represent autoproteolytic autotransporter
proteins without common protease motifs which have been
reported already . Notably, their precursor proteins have a
molecular weight of 136 to 311 kDa (according to SwissProt
entries O25063, O25330 and O25579) which is in accordance
with the molecular weights we observed in the zymography after a
possible cleavage of the N-terminal signal peptide and the
H. pylori harbors five genes that are described in the literature
and/or database annotations to code for potential extracellular
proteases (Table 1). Processing protease YmxG (Hp0657) and
protease pqqE (Hp1012) are predicted to possess a signal peptide
(Table 1) and to be extracellular or outer membrane-bound. The
protease coded by the gene hp1350 could be extracellular, as
SecretomeP and PA-Sub vote for this localization and the
existence of a signal peptide is also predicted (Table 1). The
product of hp1019, which is annotated as a serine protease in the
respective GenBank file, seems to be a homologue to heat shock
protein HtrA from Escherichia coli. Its active site is fully conserved,
and the extracellular localization has been determined previously
. The gene product of hp1584 is annotated as a
sialoglycoprotease (gcp). Its amino acid sequence does not contain known
export motifs, and the amino acid composition is predicted to be
cytoplasmic. However, the PA-SUB and PSORTb predictors
categorized the protein as extracellular (Table 1) based on the
extracellular localization of the homologous o-sialoglycoprotein
endopeptidase of Mannheimia haemolytica (SwissProt identifier
GCP_PASHA), which also lacks an N-terminal targeting signal
. In fact, very recently Hp0657, Hp1012, Hp1019, and
Hp1350 have been identified in the extracellular H. pylori
proteome  indicating the high specificity of our bioinformatical
prediction of hypothetical extracellular H. pylori proteases (Table 1).
Since we demonstrated that several caseinolytic proteases are
secreted by H. pylori independently of functional T4SS, it is likely
that other secretion systems exist. This is underlined by our
observation that nine out of 14 genes either contain a signal
peptide, which only explains a transportation to the periplasm, or
receive a high SecP prediction score (Table 1). We stress that these
predicted features are common for extracellular proteins but do
not explain a possible transport pathway. Thus one can speculate
that a secretory machinery not yet attributed to H. pylori, or
entirely novel ones, might be involved which require export signals
of an unknown nature. For example, H. pylori might involve a
specific type I (ABC) or a type III transportation system.
H. pylori HtrA is an active protease
We were then interested in answering the question whether one
of the predicted H. pylori proteases accounts for the observed
proteolytic activity. In a first step, concentrated H. pylori lysates
were separated by zymography under non-reducing conditions
followed by protein eluation of proteins from the negatively
stained protein bands I and II (Figure 2A). Then, eluated proteins
were concentrated and separated by a denaturating SDS PAGE
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(Figure 2B). We detected four different proteins in the
Coomassiestained SDS PAGE, which were isolated from protein band I in
the zymogram (compare Figure 2A, band I and Figure 2B, lane I).
Electrophoretic separation of proteins from protein band II
(Figure 2A) resulted in two different proteins (Figure 2B, lane II).
The identity of these proteins was determined by
MALDI-TOFMS. The accession number, denomination and a summary of the
MS data are presented in Table 2. The results of the MS analyses
are shown for a single database entry for each band. However, due
to the high degree of sequence identity between proteins isolated
from different H. pylori strains significant hits were obtained also
for other urease and serine proteases, e.g. serine protease from H.
pylori strain J99 or Ure B from database entry gi/51989332.
Hp1018 encodes a signal peptide for an active Hp1019
Hp1019 has been previously predicted as a secreted H. pylori
protease with unknown function [14,29]. However, its proteolytic
activity had not been demonstrated. Considering the protein
sequence of H. pylori HtrA, it lacks an annotated N-terminal signal
peptide, in contrast to HtrA of E. coli. The gene hp1019 has an
Nterminal overlap with the adjoining gene hp1018, which is 147
bases long and in a different reading frame. It has been suggested
before that those genes might belong together . Thus, we
resequenced the gene hp1018 and aligned it to the published
genomic data of H. pylori Hp26695 (Figure 3). Here, we
demonstrate that hp1018 reveals a wrongly sequenced guanidine
at position 1081558 of the published genome of H. pylori strain
26695. We conclude from our data that the translation of Hp1018
actually contains a signal peptide-like sequence (SignalP
score.0.99) at its N-terminus, and it is most likely that Hp1018
represents the N-terminal part of Hp1019 resulting in a new
sequence with 475 amino acids.
To prove proteolytic activity of Hp1018/19 for the first time, we
fused the hp1018/19 gene lacking the putative signal peptide to the
glutathione-S-transferase (gst) gene and transformed the construct
into E. coli BL21 to express the recombinant protein (Figure 4A).
Both, induction and enrichment of GST-Hp1018/19Dsp protein
were analyzed by Coomassie-stained SDS PAGEs (Figure 4B).
During GST-Hp1018/19Dsp preparation, contaminating proteins
were co-purified, which were identified by MALDI-TOF-MS as
glutathione-S-transferase and degradation products of HtrA.
Accordingly, it had been demonstrated that E. coli encoded HtrA
is an endopeptidase . To remove the GST tag from the fusion
protein, GST-Hp1018/19Dsp coupled to GST sepharose was
incubated with PreScission protease resulting in the release of
Hp1018/19Dsp protein (Figure 4B, lane 6).
Purified proteins were then probed for proteolytic activity
(Figure 4C). The GST-Hp1018/19Dsp proteins were bound to
GST sepharose, washed and eluated using reduced glutathione. As
a control, we cloned and purified the Hp1018/19DspS205A protein
in which serine-205 was mutated to alanine in the presumable
active center of HtrA. As expected, we observed casein
degradation by GST-Hp1018/19Dsp protein (Figure 4C, lane
3), but not by the GST-Hp1018/19DspS205A (Figure 4C, lanes 1
2). This finding demonstrates that H. pylori HtrA actually is an
active protease, which can be inactivated by mutation of
serine205. In parallel, we cloned and purified Hp0506, Hp0657,
Hp1012, Hp1037, Hp1543, and Hp0169, which previously had
been described as a collagenase . With the exception of
Hp1019, we did not detect any proteolytical activities using casein
as a substrate in zymography studies (data not shown). Therefore,
we conclude that the observed caseinolytic activities were actually
mediated by Hp1018/19.
urease B subunit [H.pylori]
Serine protease (htra) [H.pylori 26696]
Serine protease (htra) [H.pylori 26696]
aresults of two independently processed samples; bprotein score was below the level that indicates a p-value of ,0,05, cnot determined.
Figure 3. Blastn alignment of the re-sequenced nucleotide sequence (query) with the original genomic sequence (subject) of
hp1019. The annotated gene hp1018 is marked in grey. The letter r represents (a OR g), while the letter k represents (t OR g). The inserted
guanidine is printed white on black. Numbers give residue positions. The amino acid translation is given in single letter code for Hp1018, starting at
position 1081440, and for Hp1019, starting at position 1081537. The predicted most likely signal peptidase cleavage site between the amino acids
LNA and GNI is marked with an asterisk. The underlined part of the amino acid sequences will not be part of the translation if the marked guanidine is
As shown my mass-spectrometry, we also co-purified processed
HtrA variants with GST-Hp1018/19Dsp (Figure 4B). We detected
proteolytic activity of these proteins in casein zymography
(Figure 4C). We therefore assume that processed variants of HtrA
formed multimers with GST-Hp1018/19Dsp during the
purification steps. This suggestion is supported by the finding that removal
of the GST tag from GST-Hp1018/19Dsp protein led to the
formation of the 170 kDa protease (Figure 4C, lane 4), which was
not detected after purification of Hp1018/19DspS205A (Figure 4C,
lane 2). Together with our analysis showing that HtrA was present
in the upper and lower protein bands (Figure 2), we conclude from
our data that HtrA might also be active as a multimer.
The complex mechanisms how H. pylori strongly induce
inflammatory responses and invasive growth leading to the
disruption of the human epithelium are still unclear. Although
exported proteases of pathogens represent extensively studied
virulence factors, not much is known about their involvement in
H. pylori-associated pathogenesis. This comprehensive analysis of the
H. pylori strain 26695 genome by sequence analysis and activity
prediction methods revealed several genes coding for putative
proteases. Among those, we identified the HtrA from H. pylori as a
secreted enzyme exhibiting proteolytic activity. We also found that
HtrA forms proteolytically active multimers, which is consistent
with an earlier report of Windle and colleagues who demonstrated
that H. pylori secretes a metalloprotease with a native molecular size
of approximately 200 kDa and speculated whether this
metalloprotease activity may be involved in proteolysis of a variety of host
proteins in vivo and thereby contributes to gastric pathology .
The E. coli homologue HtrA functions as a heat shock protein,
although it cannot be excluded that Hp1019 represents a so-called
moonlighting protein , serving a function both in the
periplasm in heat shock degradation and the extracellular matrix
as a virulence factor. In fact, a secreted collagenase Hp0169 was
identified as an important virulence factor for H. pylori colonization
. Although the biological function of Hp0169 and the recently
detected extracellular proteases Hp0657, Hp1012, and Hp1350
 are unknown, it underscores the potential importance of
secreted bacterial proteases in H. pylori mediated pathogenesis,
which represent attractive vaccine and drug target candidates.
Materials and Methods
Proteases were compiled for the reference data set by selecting all
entries from the UniProtKB/SwissProt database (version 6.7) 
containing the keyword protease, but lacking the phrases
inhibitor*, probable*, fragment*, hypothetical*,
putative*, possible* or predicted* in the keyword and description
fields, where the asterisk is a wildcard for any arbitrary suffix. The
NCBI BLAST package was employed for pairwise sequence
alignment . The Blastp program was used for protein-protein
comparison. Tblastn was used to compare the whole DNA sequence
of H. pylori strain 26695 with the protease sequence set. The
substitution matrices PAM30, PAM70, PAM250, BLOSUM45,
BLOSUM62 and BLOSUM80 were used with default parameter
settings (e.g. scoring penalties, window size).
H. pylori wildtype strains 26695 and P12, its isogenic mutant
strains DVacA, and DPAI had been described before [11,33].
Bacteria were grown in protein-free liquid brain heart infusion
(BHI) medium (Merck, Darmstadt, Germany) supplemented with
b-cyclodextrin for 48 hours, which has been previously optimized
for minimal autolysis of H. pylori cells . Lysates of H. pylori were
obtained by sonification in PBS containing 0.1% Triton X-100.
Supernatants of H. pylori BHI cultures were sterilized by filtration
(pore size 0.22 mm).
Amplification and sequencing of hp1018
The gene hp1018 was amplified from the genomic DNA of H.
pylori strain 26695 by standard PCR using the Pfx
DNApolymerase (Invitrogen, Karlsruhe, Germany). The following
primers were used: hp1018for: 59-GGC TAT GGA TAA GGA
TCA ACG C-39, hp1018rev: 59-CCA CCG CCT TAA TAG
AGT CCT T-39. The PCR product, having a calculated length of
333 bases, was submitted to a commercial provider (GENterprise,
Mainz, Germany) for sequencing.
Cloning, mutation and purification of HtrA
The construct Hp1018/19Dsp was amplified from genomic
DNA of H. pylori strain 26695 using the primers
59-aagaattcgacccacccctatcatttcacc39 with Pfx DNA polymerase in supplied buffer with 26 PCR
Enhancer (Invitrogen). The amplified BamH1/EcoR1 flanked
PCR product was then ligated into the pGEM-T Easy plasmid
(Promega), subcloned into the pGEX-6P-1 plasmid DNA (GE
Healthcare Life Sciences) and transformed in E. coli BL21. The
construction of the protease-inactive Hp1018/19DspS205A protein,
serine 205 was mutated to alanine using the QuikChangeH
Lightning Site-Directed Mutagenesis Kit (Stratagene) according to
the manufacturers instructions. For heterologous overexpression
and purification of GST-Hp1018/19Dsp, transformed E. coli was
grown in 500 ml TB medium to an OD550 of 0.6 and the
expression was induced by the addition of 0.1 mM
isopropylthiogalactosid (IPTG). The bacterial culture was pelleted at 40006g
for 30 minutes and lysed in 25 ml PBS by sonification. The lysate
was cleared by centrifugation and the supernatant was incubated
with glutathione sepharose (GE Healthcare Life Sciences) at 4uC
over night. The fusion protein was either eluted with 10 mM
reduced glutathione for 10 minutes at room temperature or
cleaved with 180 U Prescission Protease for 16 h at 4uC (GE
Healthcare Life Sciences). Elution and cleavage products were
analyzed by SDS PAGE and zymography.
Zymography and protein eluation
Undiluted aliquots were loaded onto 8% SDS-PAGE
containing 0.1% casein (Invitrogen, Germany) and separated by
electrophoresis. After separation, the gel was re-naturated in
2.5% Triton X-100 solution at room temperature for 60 min with
gentle agitation, equilibrated in developing buffer (50 mM
TrisHCl, pH 7.4; 200 mM NaCl, 5 mM CaCl2, 0.02% Brij35) at
room temperature for 30 min with gentle agitation, and incubated
overnight at 37uC in fresh developing buffer. Transparent bands
of caseinolytic activity were visualized by staining with 0.5%
Coomassie Blue R250. For identification of proteases present in
zymographies, negatively stained bands were excised from a
preparative casein zymogram and proteins were eluted twice for
6 hours via D-TubeTM Dialyzers Maxi MWCO 68 kDa
(Novagene). Eluated proteins were desalted and concentrated
using Vivaspin columns from Sartorius (Germany).
Eluated proteins from zymograms were separated by means of
SDS-PAGE and stained with Coomassie for subsequent MS
analysis in two independent experiments (A+B). In gel digestion
was performed as previously described with several minor
modifications . Peptide mixtures were additionally purified
and concentrated by using ZipTipC-18 tips (Millipore) according
to the manufacturers instructions. The identity of HtrA and
urease B was proven by mass spectrometry as described [35,36].
Briefly, Samples were mixed with peptide standard (peptide
standard II, Bruker) and matrix (a saturated solution of HCCA in
50% ACN+0,5%TFA) at a ratio of 1:1:2; v:v:v), and with matrix
only at a ratio of 1:1; v:v, and transferred on a ground steel target.
Mass analysis was done on a Bruker Reflex II mass spectrometer
with predefined default instrument settings. Proteins were
identified by running MASCOT (http://www.matrixscience.
com) against the entire NCBI database. Peptide tolerance was
set to 50 ppm and a maximum of one missed cleavage site was
allowed. A hit was considered as significant at a probability value
Conceived and designed the experiments: ML ASP SW GS. Performed the
experiments: ML CW AR. Analyzed the data: ML CW DM AR ASP SW
GS. Contributed reagents/materials/analysis tools: DM AR. Wrote the
paper: ML SW GS.
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