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Supplementary Methods
Homology modeling of the structures of CrHMC and Hb subunits
The known 3D structure of hemocyanin (HMC) subunit 2 from Limulus polyphemus,
1nolA (Hazes et al, 1993), was used for modeling the structure of CrHMC2, 3a and 3b
subunits. The hemoglobin (Hb), 1bzlC (Hui et al, 1999) and 1dxtD (Kavanaugh et al,
1992), were used for modeling the structure of Hb α1 and Hb β, respectively. The protein
structures
were
modeled
by
using
SWISS-MODEL
server
(available
at
http://swissmodel.expasy.org/).
MALDI-TOF-TOF identification and protein N-terminal sequencing
The protein bands of interest separated on the PAGE gel were excised and in-gel digested
with trypsin (Promega) according to the protocols of Shevchenko et al (Shevchenko et al,
1997). The trypsinized peptide samples were analysed by matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) with Voyager-DE STR Biospectrometry
Workstation (Applied Systems) in the Proteins and Proteomics Centre, National
University of Singapore. The peptide mass and sequences were analysed using Matrix
Sciences Mascot search at http://www.matrixscience.com/search_form_select.html. For
N-terminal sequencing, the protein was trans-blotted onto a PVDF membrane (Bio-rad)
followed by Coomassie blue R250 staining. The recovered protein band was subjected to
Edman degradation and analysed by ABI Procise 494 Protein Sequencer. At least 5
amino acid residues were collected for each protein sample.
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Optimization of partial proteolysis of HMC and metHb
Subtilisin A from Bacillus licheniformis, proteinase K from Tritirachium album, trypsin
from bovine pancreas were obtained from Sigma-Aldrich. The Pseudomonas aeruginosa
elastase was from Elastin Product Company (USA). The respiratory proteins: microbial
proteases was in a pathophysiological range of µg:ng, and the duration of the reaction for
partial proteolysis were primarily determined by the cleavage of metHb with subtilisin A
(Supplementary Figure S9A & B). Partial proteolysis of HMC and metHb by different
proteases were performed at room temperature for 1 h at varying ratio of 100: 1 for
subtilisin A and trypsin, 200:1 for elastase and 300:1 for proteinase K. These different
ratios used was to achieve controlled limited proteolysis of the Hb/HMC (not to allow
complete proteolysis) in order to observe progressive cleavage products of the proteins on
the SDS-PAGE. The reaction mixtures were in 100 mM Tris-HCl (pH 7.0), 5 mM CaCl2
and 5 mM MgCl2 for HMC, and phosphate buffered saline (PBS), pH 7.3 for metHb.
Zymography of metHb-POX cycle and HMC-PO
The proteolysed metHbwithout boiling, was separated on 15% SDS-PAGE and transblotted onto PVDF membrane in the absence of methanol. The membrane was incubated
with SuperSignal West Pico Chemiluminescent substrate (Pierce) for 5 min before
exposure onto an X-ray film. This was to detect the production of superoxide
representing the activation of metHb-POX cycle. The proteolysed HMC-PO resolved on
12% native PAGE was zymographically stained with phenol substrate containing 10 mM
4-hydroxyanisole, 10 mM 3,4–dihydroxyphenylpropionic acid and 10 mM 3-methyl-2-
2
benzothiazolinone hydrazone in 50 mM phosphate buffer saline (pH 6.0) according to
Dicko et al (Dicko et al, 2002).
The chemiluminescence (CLA-CL) assays for Hb-POX cycle activity
The Cypridina luciferin analog, a specific substrate for the superoxide (Kawano et al,
2002; Nakano, 1990) was used in the chemiluminiscence assay (CLA-CL) to determine
the production of superoxide, which represents the POX cycle activity (Jiang et al, 2007).
A reaction mixture of 100 µl was assembled with stipulated amounts of metHb or
proteolyzed metHb, 10 µM CLA and 5 mM H2O2 in PBS, pH 7.3. Immediately after
assembling the reaction mixtures, the chemiluminescence was continuously monitored at
1 s intervals for 1 min, by using GloMaxTM 20/20 luminometer (Promega). The POX
cycle activity was designated as the relative luminescence units per second (RLU.S-1).
Quantification of HMC-PO activity
The PO enzyme activity was determined according to Jiang et al. (Jiang et al, 2007).
Briefly, the HMC or proteolyzed HMC in 50 mM Tris-HCl, pH 7.0 containing 0.05 M
NaCl was incubated at room temperature for 10 min followed by the addition of 1 mM 4methylcatechol in 0.1 M potassium phosphate, pH 6.0 as substrate. The absorbance at
405 nm was then monitored continuously by using a microplate reader (Molecular
Devices, USA). The PO activity is presented as A405 at 10 min after the addition of
substrate.
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Reconstitution of in vitro simulated infection microenvironment
To confirm that proteolysis of Hb and HMC by microbial proteases exposes dual-action
antimicrobial centres in these respiratory proteins, the isolated Hb/HMC or their
respective endogenous counterparts contained in red blood cells (Hb-from-RBC), in
blood (Hb-in-blood) or in the hemolymph (HMC-in-hemolymph), were examined in a
reconstituted in vitro “infection-microenvironment” mimicking the in vivo bacterial
infection, where the S. aureus V8 (+)/(-) or P. aeruginosa elastase (+)/(-) bacteria were
introduced. For the HMC, a 150 μl reaction mixture containing P. aeruginosa elastase
(+/-) or S. aureus V8 (+/ -), 1 mM 4-methylcatechol (4-ME), 60 μg of isolated HMC or
10% (v/v) cell-free hemolymph in 100 mM Tris-HCl pH 7.0, 5 mM CaCl2 and 5 mM
MgCl2 was assembled to test the antimicrobial consequence of the proteolyticallyactivated HMC. Bacterial culture alone or bacterial culture incubated with 4-ME alone
were used as two negative controls. For the isolated metHb or endogenous Hb-fromRBC, or Hb-in-blood, 100 μl of reaction mixtures were reconstituted with hemolytic S.
aureus V8 (+/-), 0.5µmole H2O2, 100 μg metHb or 0.5% (v/v) rabbit RBC or 1% (v/v)
rabbit blood in PBS, pH 7.3. The 0.5µmole H2O2 was only added after 20 min of the
incubation when partial proteolysis of RBC or blood was achieved. The incubation of
bacterial culture alone and bacterial culture with 0.5µmole H2O2 were used as negative
controls. In examining the inhibitory effect of catalase (CAT), superoxide dismutase
(SOD), reduced glutathione (GSH), CAT inhibitor: 3-amino-1,2,4-triazole (3AT) and
SOD inhibitor: diethyldithiocarbamate (DDC), the incubation of bacterial culture alone,
bacterial culture with 0.5µmole H2O2 and bacterial culture with respective antioxidants
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or antioxidant inhibitors served as negative controls. The mixtures were incubated at
room temperature for 1 h.
In preparation for the above experiments, the bacteria were incubated overnight at
37°C according to Drapeau et al (Drapeau et al, 1972) to obtain the respective protease
(+/-) cultures. The protease activity was determined at 37oC by using 1% azocasein assay
(Lee et al, 2005). One Unit (U) of protease activity was defined as an absorbance of 0.1 at
OD366.
Determination of catalase and superoxide dismutase activities and GSH level
To determine the level of antioxidants present in the in vitro simulated infection reactions
on the activated Hb-POX mediated free radical formation, the level of catalase (CAT),
superoxide dismutase (SOD) and reduced glutathione (GSH) & oxidized glutathione
disulfide (GSSG), contained in RBC and/or bacterial cultures, were determined according
to the procedures described by Aebi (Aebi, 1984), Flohe & Otting (Flohe & Otting, 1984)
and Baker et al. (Baker et al, 1990), respectively. The known concentrations of CAT,
SOD and GSSG were used to generate the standard curves for calculation.
The cloning, expression and purification of recombinant Hbα p12 and Hbβ p10
The E. coli expression vector pET22b (Novagen, ampicillinR) was used for constructing
the recombinant clones. The human hemoglobin cDNA clones (Accession Numbers:
BC101846 and BC007075, encoding Hb α1 and Hb β, respectively) purchased from
Open Biosystems (USA) were used as templates for PCR amplification. The forward
primer: 5’ CGGGATCCG (BamHI)-CTGGAGAGGATGTTCCTGT 3’ and reverse
primer: 5’ CCGCTCGAG (XhoI)-ACGGTATTTGGAGGTCAGC 3’ were used to
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amplify the 342 bp Hbα p12; while the forward primer: 5’ ACGCGTCGAC (SalI)TCCACCCCTGATGCTGTTA 3’ and reverse primer: 5’ CCGCTCGAG (XhoI)GTGATACTTGTGGGCCAGG 3’ were used to amplify the 297 bp Hbβ p10. The
cloning map is shown in Supplementary Figure S9C. The recombinant expression
constructs were verified by DNA sequencing, and transformed into E. coli BL-21 (DE3)
for the protein expression. The overnight cultures of the respective clones were separately
resuspended in M9Y broth (Uechi et al, 2005) supplemented with 100 μg/ml ampillicin
and 0.3 mM heme precursor, δ-aminolevulinate (Sigma). The inoculated broth culture
was shaken at 30°C until the OD600 reached 0.5 – 0.6. The cultures were then induced
overnight with 0.2 mM IPTG. The harvested cells were used for purification and
assessment of the recombinant proteins. It was found that both recombinant proteins were
mainly present in the inclusion bodies (Supplementary Figures S9D & E), hence, the
purification was achieved under denaturing conditions of 6 M urea followed by TALON®
polyhistidine-tagged affinity chromatography (Clontech) according to the manufacturer’s
instructions. The purified denatured recombinant proteins were refolded and bufferexchanged by using the Amicon Ultra centrifugal filter device (Millipore). The refolded
proteins were then electrophoretically resolved on 10% Tris-Tricine SDS-PAGE, and
verified by immuno-blotting followed by CL-CLA assay to determine the POX enzyme
activity. The purified recombinant proteins are henceforth referred to as rHbα p12 and
rHbβ p10.
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