Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibit
Trang 1Antimicrobial activity of histones from hemocytes of the Pacific
white shrimp
Se´verine A Patat1, Ryan B Carnegie1,*, Celia Kingsbury1,, Paul S Gross2,3, Robert Chapman3,4
and Kevin L Schey1,3
1
Department of Cell and Molecular Pharmacology,2Department of Biochemistry and3Marine Biomedicine & Environmental Sciences Center, Medical University of South Carolina, Charleston, SC, USA;4Hollings Marine Laboratory, Marine Resources Institute, SCDNR, Charleston, SC, USA
The role of vertebrate histone proteins or histone derived
peptides as innate immune effectors has only recently been
appreciated In this study, high levels of core histone proteins
H2A, H2B, H3 and H4 were found in hemocytes from the
Pacific white shrimp, Litopenaeus vannamei The proteins
were identified by in-gel digestion, mass spectrometry
ana-lysis, and homology searching The L vannamei histone
proteins were found to be highly homologous to histones of
other species Based on this homology, histone H2A was
cloned and its N-terminus was found to resemble the known
antimicrobial histone peptides buforin I, parasin, and
hip-posin Consequently, a 38 amino acid synthetic peptide
identical to the N-terminus of shrimp H2A was synthesized
and assayed, along with endogenous histones H2A, H2B,
and H4, for growth inhibition against Micrococcus luteus
Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 lMwhile a mixture of histones H2B and H4 was active at 3 lM In addition, a fraction containing a fragment of histone H1 was also found to be active The synthetic peptide similar to buforin was active at submicromolar concentrations These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a def-ense mechanism previously unreported in an invertebrate Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought
Keywords: antimicrobial peptide; histone; invertebrate; mass spectrometry; shrimp
Marine organisms, such as the shrimp Litopenaeus
vanna-mei, have developed efficient methods to survive and
prosper in a microbe-rich oceanic environment Hemocytes,
the principal effector cells in shrimp defenses, are involved in
nonself recognition, phagocytosis, melanization,
cytotoxic-ity, and cell–cell communication [1] There are three classes
of hemocytes: the hyaline, semigranular, and granular cells
The hyaline cells are associated with coagulation and
phagocytosis while the semigranular and granular cells are involved in phagocytosis, release of proteins in the prophe-noloxidase cascade, and release of antimicrobial peptides The first stage of the immune response is recognition
of nonself Microbial cell wall components [e.g lipopolysaccharides (LPS), b-1,3-glucans, or peptidogly-cans] are recognized by specific proteins in the hemolymph, namely LPS-binding proteins, b-glucan binding proteins [2],
or lectins [3] b-1,3-Glucan binding proteins have been identified in Penaeus californiensis and in L vannamei hemolymph [4,5] These molecules probably interact with the hemocytes to initiate defense responses The prophe-noloxidase cascade leading to melanization has been one of the most studied immune reactions in crustaceans [6] Melanin and its intermediates have been shown to be toxic
to microbes [7]
Antimicrobial peptides represent an essential alternative first line of defense These molecules, universally distributed
in metazoans, are usually amphipathic, carry a net positive charge, and can form a-helical or b-sheet structures in membrane-like environments [8] Antimicrobial peptides can be constitutively or inducibly expressed and have been found in different cell types including epithelial cells and phagocytes Some antimicrobial peptides are also derived from larger proteins by proteolysis For example, bovine lactoferricin is derived from lactoferrin, an iron binding protein, by pepsin cleavage of its N-terminus [9] In crustaceans, antimicrobial peptides have been described in the crab Carcinus maenas [10], in the crayfish Pacifastacus
Correspondence to K L Schey, Medical University of South Carolina,
Department of Cell and Molecular Pharmacology, 173 Ashley
Avenue, PO Box 250505, Charleston, SC 29425, USA.
Fax: +1 843 792 2475, Tel.: +1 843 792 2471,
E-mail: scheykl@musc.edu
Abbreviations: LPS, lipopolysaccharide; NET, neutrophil extracellular
trap; MAS, modified alsever solution; TFA, trifluoroacetic acid;
MIC, minimum inhibitory concentration.
*Present address: Virginia Institute of Marine Science, PO Box 1346,
Gloucester Pt., VA 23062, USA.
Present address: Pharmacopeia, Inc., PO Box 5350, Princeton, NJ
08543-5350, USA.
Note: The protein sequence data reported in this paper will appear in
the SwissProt and TrEMBL knowledgebase under the accession
numbers P83841, P83863, P83864 and P83865, and the nucleotide
sequence data has been submitted to GenBank under the accession
numbers AY576482 and AY576483.
(Received 20 August 2004, revised 29 September 2004,
accepted 21 October 2004)
Trang 2leniusculus[11], and in several penaeid shrimp In penaeid
shrimp, the most studied family of antimicrobial peptides is
the penaeidins These peptides range from 5.5 to 6.6 kDa
and are mostly active against Gram-positive bacteria
[12,13] They are stored in hemocyte granules and released
at the site of infection by lysis of the cells [14,15] Two other
antimicrobial peptides have been described in the shrimp:
crustin and hemocyanin-derived peptides Crustin, an
11.5 kDa antimicrobial peptide also found in the shore
crab Carcinus maenas [16,17], has not been fully
character-ized Finally, C-terminal hemocyanin fragments are active
against fungi but the mechanism by which hemocyanin is
cleaved and activated is still unclear [18]
Histone proteins or derived fragments have antimicrobial
activity in vertebrates ranging from fish to humans Histone
antimicrobial activity was first demonstrated in 1958 for
histones A and B purified from calf thymus, which exhibited
antibacterial activity against various Gram-positive and
Gram-negative bacteria [19] It was not until the 1990s that
other groups described such activity In several fish,
antimicrobial histone proteins have been detected in skin
mucus or liver tissue: H2B-like proteins in catfish skin [20],
H2A in trout skin [21], and H1 in the atlantic salmon liver
[22] In humans, histone H1 and its fragments derived from
epithelial cells in the gastrointestinal tract were active
against Salmonella typhimurium [23], and histones H2A and
H2B expressed on the surface of and secreted from amnion
epithelial cells (placenta) contributed to the antimicrobial
activity of amniotic fluid [24] A recent report identified
histone containing NETs (neutrophil extracellular traps) in
human neutrophils as a novel antimicrobial mechanism [25]
Three peptides derived from the N-terminus domain of
histone H2A: buforin I (39 amino acids), parasin I (21
amino acids), and hipposin (51 amino acids) from the Asian
toad stomach and catfish and halibut skin mucus,
respect-ively, have been described as active against bacteria [26–28]
Active antimicrobial fragments of histone H1 have been
observed in the rainbow trout, Oncorhynchus mykiss [29], as
well as in stimulated human granulocytes [30] and in the
serum and mucus of LPS-challenged Coho salmon,
Onco-rhyncus kisutch[31]
Here we present the results of a proteomic investigation
of hemocyte antimicrobial peptides of the Pacific white
shrimp, L vannamei, and the role of histones as potentially
important components of their immune system
Materials and methods
Animal handling
L vannamei individuals were obtained from Waddell
Mariculture Center, Bluffton, SC, USA They were
trans-ported to the laboratory in oxygen-saturated water and
bled within 6 h of collection
Sample collection and preparation of cells
Hemolymph was taken from the ventral sinus of the animal
under an equal volume of modified alsever solution (MAS:
27 mM sodium citrate, 336 mM sodium chloride, 115 mM
glucose, 9 mM EDTA, pH 7; according to Rodriguez
et al [32]) with 3 mL syringes and 25 gauge needles
Hemolymph from 3 to 4 animals was pooled in 1.5 mL Eppendorf tubes and immediately centrifuged at 800 g for
15 min (4C) to separate the hemocytes from the plasma After removal of the plasma from the cell pellets, 600 lL of MAS was added to the eppendorf tubes, which were then vortexed gently to wash the cells Cells were centrifuged at
800 g for 15 min (4C) and the buffer was removed The washing and centrifugation steps were repeated with 200 lL
of MAS After removal of the buffer, 100 lL of water were added to the cell pellets, followed by ultrasonication for 30 s (Branson SONIFIER 450, power 2, duty cycle 20; Branson, Danbury, CT, USA) to lyse the cells After lysis, the lysates were stored at)20 C until needed
The protein concentrations of the cells were estimated by the Bradford method (Bio-Rad, Hercules, CA, USA) with commercial BSA standards (2 mgÆmL)1, Pierce, Rockford,
IL, USA)
1D gel electrophoresis and in-gel digestion Precast 10–20% linear gradient Tris/HCl Criterion gels (Bio-Rad) were used Approximately 10 lg of hemocyte lysate proteins were mixed with reducing sample buffer [2· sample buffer (Invitrogen, Carlsbad, CA, USA)/water/ 2-mercaptoethanol; 50 : 50 : 2.5; v/v/v], boiled in a water bath for 10 min, and run on the gel with running buffer [24 mM Tris base, 192 mM glycine, 0.1% (w/v) SDS] for
60 min at 200 V The gel was stained with copper Briefly, the gel was washed for 5 min with washing buffer [200 mM Tris, pH 8.8, 0.1% (w/v) SDS] and stained with 0.3M copper chloride for 20 min The gel was stored in water prior to image analysis and in-gel digestion
Bands of interest were cut out of the gel and sliced into
1 mm pieces The copper stained pieces were destained with 500 lL of copper destain solution for 20 min After destaining, the gel pieces were washed three times for
20 min with 100 mM ammonium bicarbonate, once for
15 min with acetonitrile/100 mM ammonium bicarbonate (50 : 50, v/v), and once for 15 min with acetonitrile, before being dried in a speed vac (5 min) Dried gel pieces were rehydrated with 10 lL of trypsin or chymotrypsin (100 ng in 100 mM ammonium bicarbonate, pH 7.8) and then covered with ammonium bicarbonate (100 mM,
30 lL) Digestion was carried out overnight at 37C Supernatants were removed and the same volume of acetonitrile/water/formic acid (50 : 45 : 5, v/v/v) was added to the gel pieces followed by sonication for
20 min Supernatants were added to the previous super-natants These steps were repeated with acetonitrile/water/ formic acid (85 : 10 : 5, v/v/v) Supernatants were then dried in a speed vac and resuspended in acetonitrile/water (10 : 90, v/v) Zip tips (C18 Millipore, Billerica, MA, USA) were used to desalt the samples before analysis Elution from the Zip tips was accomplished with 3.5 lL
of acetonitrile/water/acetic acid (49 : 49 : 2, v/v/v) Protein purification and quantification
After thawing, 300 lL of hemocyte lysate supernatant was subjected to HPLC by injection onto a C18 column (Alltech, C18 Prosphere, 4.6· 250 mm, 300 A˚, 5 lm; Alltech, Deerfield, IL, USA) The solvent system included
Trang 30.1% trifluoroacetic acid (TFA) in water (solvent A) and
0.08% TFA in acetonitrile (solvent B) Proteins were eluted
from the column with a gradient of 5–30% solvent B over
13 min followed by 30–60% solvent B over 67 min at a flow
rate of 0.7 mLÆmin)1 Protein elution was monitored by UV
absorbance at 225 nm and peak fractions were collected
Fractions were subsequently lyophilized, reconstituted in
100 lL water, and analyzed by MALDI MS Histones H4
and H2B coeluted and were not further separated prior to
protein assay and antimicrobial assay Fractions containing
only histone H2A were pooled Fractions containing impure
histone H2A were repurified over the same C18 column and
solvent system but with a shallower gradient (40–60%
solvent B over 60 min) Pure histone H2A fractions from
multiple runs were pooled as well as H2B/H4 fractions and
the resulting samples were lyophilized, and reconstituted in
100 lL of deionized water for protein assay and liquid
growth inhibition assay (see below)
The concentrations of the endogenous histone protein
fractions were estimated by the Bradford method (Bio-Rad)
with commercial BSA standards (2 mgÆmL)1, Pierce)
Proteolytic treatments
After HPLC fractionation, histone proteins were digested
by trypsin (Promega, Madison, WI, USA), chymotrypsin
(Roche Applied Science, Indianapolis, IN, USA), or
endoproteinase Glu-C (Roche Applied Science) Tryptic
or chymotryptic digestions were carried out overnight at
37C in 25 mMammonium bicarbonate/10% (v/v)
aceto-nitrile, pH 7.8 with 200 ng of trypsin or 1 lg of
chymo-trypsin Glu-C digests were carried out overnight at room
temperature in 25 mMammonium bicarbonate/10% (v/v)
acetonitrile, pH 7.8 with 150 ng of enzyme
Mass spectrometry analysis and peptide sequencing
Matrix assisted laser desorption ionization mass
spectro-metry (MALDI MS) was carried out on an Applied
Biosystems Voyager-DE STR (Applied Biosystems, Foster
City, CA, USA) The matrix a-cyano-4-hydroxycinnamic
acid (10 mgÆmL)1) in 70% (v/v) acetonitrile/0.1% (v/v)
TFA was used Peptides and matrix solutions were mixed
(1 : 3 lL) and 0.5 lL of the mixture was placed on top of
0.5 lL of dried matrix on the sample plate and allowed to
dry Typically, 250 laser shots were averaged to produce a
mass spectrum
Nanospray tandem mass spectrometry was carried out on
a quadrupole/time of flight instrument (QSTAR, Applied
Biosystems) or on an ion trap intrument (LCQ Classic,
Finnigan West Palm Beach, FL, USA) using a custom built
nanospray source (LCQ) or a Protana source (QSTAR)
Two microliters of sample were loaded into the nanospray
tip for analysis Precursor ions were selected from the MS
survey scan About 20 mass spectra were averaged to
increase the signal to noise ratio on the LCQ On the
QSTAR, tandem mass spectra were acquired and averaged
for 3 min and, when necessary, the enhancer mode was used
to increase intensities of specific regions of the spectrum
MS/MS was also acquired on a MALDI-TOF-TOF
instrument (4700 Proteomics Analyzer, Applied
Biosys-tems) with matrix a-cyano-4-hydroxycinnamic acid
(10 mgÆmL)1) in 70% (v/v) acetonitrile/0.1% (v/v) TFA in
a 1 : 1 ratio sample to matrix Sequences from tandem mass spectrometry were determined manually and homologies to known proteins were searched using a BLAST search for short, nearly exact matches available online from the NCBI server (http://www.ncbi.nlm.nih.gov/blast) with the non-redundant database
Histone H2A N-terminal cDNA cloning Two degenerate forward primers (F1: 5¢-AACMGKGCM GGACTCCAG-3¢; F2: 5¢-TMCGYAARGGMAACTA TG-3¢) and three degenerate reverse primers (R1: 5¢-TTCG TCRTTMCKGATGGC-3; R2: 5¢-CACCTCCTTGRG
designed based on a CLUSTALW alignment (http://www ebi.ac.uk/clustalw/) of histone H2A DNA sequences from the crustaceans Tigriopus californicus (GenBank acc no S49144), Artemia (X14815), and Asellus aquaticus (AJ238321) Several combinations of forward and reverse primers were used in attempts to amplify a 3¢ histone H2A gene fragment from L vannamei gill cDNA library Reac-tion volumes of 20 lL included PCR buffer, nucleotides at 1.25 mM, primers at 100 ngÆlL)1, Taq DNA polymerase (Advantage kit, Clontech, Palo Alto, CA, USA), and 0.4 lL of gill library cDNA Reactions were carried out in a RoboCycler Gradient 96 thermocycler (Stratagene, La Jolla, CA, USA) The reaction profile began with an initial denaturation for 3 min, followed by 30 cycles of 94C for
1 min, 48C for 30 s and 72 C for 30 s, and ended with a
5 min final extension at 72C DNA products were separated on a 1.2% agarose gel and stained with ethidium bromide Bands of interest were excised and extracted using
a Nucleospin kit (Clontech), and then cloned into Escheri-chia coli XL1 blue with ampicillin Plate colonies were picked randomly and grown in 2 mL of Luria broth with
200 lg of ampicillin overnight at 37C Plasmid DNA was extracted using NucleoSpin Plus Minipreps (Clontech), and inserts were sequenced at the MUSC DNA Sequencing Facility (Medical University of South Carolina, Charleston,
SC, USA) Based on the initial sequence, a new reverse
was designed To determine the 5¢ histone H2A gene sequence, the new reverse primer, a vector-specific forward primer, and hemocyte cDNA library were used in a second PCR Briefly, 50 ng plasmid DNA was used in a 20 lL reaction volume as above The reaction profile began with
an initial denaturation for 3 min, followed by 30 cycles of
94C for 1 min, 68 C for 30 s, and 72 C for 30 s, and ended with a 5 min final extension at 72C Products were cloned and sequenced as above DNA sequences were translated for comparison with described histone sequences using the ExPASy translate tool (http://www.expasy.org/) Peptide synthesis
A peptide similar in length to buforin [26] was synthesized
at the MUSC Peptide Synthesis Facility The sequence was based on the N-terminus of the shrimp H2A DNA sequence (SGRGKGGKVKGKSKSRSSRAGLQFPVGRIHRLL RKGNY) After synthesis, the peptide, named H2A 2–39, was purified by HPLC Approximately 0.2 mg of peptide was
Trang 4loaded onto a C18 column (Alltech, C18 Prosphere, 4.6·
25 mm, 300 A˚, 5 lm) The solvent system included 0.1%
(v/v) TFA in water (Solvent A) and 0.08% (v/v) TFA in
acetonitrile (Solvent B) Peptide was eluted off the column
with a gradient of 5–75% B in 150 min at a flow rate
of 0.7 mLÆmin)1 The purified synthetic peptide’s
concentra-tion was measured by amino acid analysis by C Schwabe,
MUSC Biochemistry Department, Charleston, SC, USA
Liquid growth inhibition assays
Antibacterial activity was tested against the Gram-positive
bacterium Micrococcus luteus Stocks of bacteria were kept
at)80 C in 50% (v/v) glycerol ( 200 lL) One stock of
bacteria was added to 5 mL of Luria–Bertani (LB) media
and grown overnight at 37C with shaking, and was used to
spread a LB agar plate After overnight incubation at 37C,
the plate was kept at 4C and served as a stock for the
experiments One colony was picked and incubated in 5 mL
of LB media overnight at 37C with shaking After
incubation, bacteria were diluted 1 : 100 or 1 : 50 and
incubated until the attenuance at 595 nm was about 0.1 The
dilution medium was either LB broth or 1% (w/v)
bacto-tryptone/1% (w/v) sodium chloride at pH 7.2 When an
attenuance (D) of 0.1 was reached, bacteria were further
diluted with poor broth [1% (w/v) bactotryptone/0.5%
(w/v) sodium chloride, pH 7.2] to a D of 0.01 at 595 nm
Ninety microliters of bacteria (D¼ 0.01) were added to
each well of a 96 well plate with 10 lL of the appropriate
control or sample Negative controls were water or a
synthetic peptide (MIP peptide: CVTGEPVELDTQAL,
10 lM) chosen from a pool of synthetic peptides used for
vision research and without known antimicrobial activity,
and the positive control was Ala-magainin (Sigma, St Louis,
MO, USA, 5 lM) Samples were fractionated histone
proteins or the N-terminal H2A synthetic peptide (H2A 2–
39) at various concentrations (less than 10 lM) diluted in
water The plate was then incubated at 37C overnight with
shaking and the D at 595 nm or 570 nm was read in a plate
reader (Molecular Devices)
Results
Identification of histone proteins in the hemocytes
In the course of an initial proteomic investigation of shrimp
immune function, simple one-dimensional SDS/PAGE
analysis of the hemocytes soluble proteins was carried out
The analysis (Fig 1) revealed four intense bands in the
region of 17 kDa They were cut out of the gel and digested
by trypsin or chymotrypsin, and the resulting peptides were
sequenced by nanospray tandem mass spectrometry The
peptides were identified using aBLASThomology search of
the obtained sequences The lower band yielded peptides
similar to histone H4, the upper bands contained peptides
from histones H2A and H2B, and the highest molecular
mass band produced peptides homologous to histone H3
(Table 1) After fractionation of the hemocyte soluble
proteins by C18 reverse phase HPLC and enzyme digestion,
additional peptides were sequenced from the histone
proteins (Table 1) Furthermore, intact protein molecular
masses were determined by MALDI MS of the histone
containing fractions Direct MALDI MS analysis of the unfractionated hemocyte supernatant shows the molecular masses of the histone proteins: histone H4 is 11.3 kDa, H2A 13.2 kDa, H2B 13.5 kDa, and histone H3 is 15.3 kDa (Fig 2) Histone proteins have a high number of basic residues, which make them run more slowly on a SDS/ PAGE gel thus explaining the molecular mass differences observed between the 1D gel and the MALDI MS data Because histone sequences are highly conserved between species, the identification of these proteins was straightfor-ward, based on primary amino acid sequences and the measured molecular masses Also, the H2A N-terminal cDNA cloning (see below) and an expressed sequence tag from L vannamei similar to histone H4 (http://www marinegenomics.org; EST #7381) confirmed our assign-ments to histone proteins Both the 1D gel and the direct MALDI MS spectrum show very strong signals for the hemocyte core histone proteins, suggesting a high abun-dance of histones in these cells
Histone H2A N-terminal cDNA cloning and C-terminal mass spectrometry determination
The forward primer F2 and the reverse primers R1 and R2 successfully amplified a 161 base DNA sequence (F2 and R1) corresponding to 53 amino acids, and a 207 base sequence (F2 and R2) corresponding to 69 amino acids, of histone H2A From the nucleic acid sequence one new reverse primer was generated to obtain the 5¢ end of the cDNA sequence Figure 3A shows the translated amino acid sequence using the translating tool of the ExPASy website The C-terminal sequence was determined by tandem mass spectrometry of chymotryptic peptides
Purification of histone proteins for liquid growth inhibition assays
The hemocyte lysate supernatants were separated by reverse phase HPLC on a C18 column Histone proteins eluted between 40 and 55% solvent B More specifically, a histone H1 fragment eluted at 42.5% solvent B, histones H2B and H4 coeluted at 51% solvent B, and histone H2A eluted at 52% solvent B as depicted in Fig 4 Histone H3 coeluted
Fig 1 1D SDS/PAGE of hemocyte soluble proteins Copper stained 1D SDS 10–20% Tris/HCl gel of hemocyte lysate proteins Molecular markers are indicated on the left in kDa Histone proteins and hemocyanin protein bands indicated on the right.
Trang 5with hemocyanin, the abundant oxygen carrier protein, at
56% solvent B (not shown) Approximately 61 lg of
histone H2A proteins and 152 lg of mixed histones H2B/
H4 proteins were purified from 300 shrimp Histone H2A protein was purified close to homogeneity, and histones H2B and H4 are the two major components in their fraction
as evidenced by MALDI MS (Fig 5A,B) and gel electro-phoresis (Fig 5D) The fraction containing histone H2A shows two peaks by MALDI MS analysis (Fig 5B) with a
78 Da difference The two forms were separated by HPLC and both were shown to be histone H2A by nanospray mass spectrometry sequencing after tryptic digestion
Liquid growth inhibition assays with endogenous histone proteins purified from the hemocytes
When tested for antimicrobial activity against the Gram-positive bacterium M luteus, the lowest concentration of histone H2A that completely inhibited growth was 4.5 lM and the lowest concentration of H2B/H4 fraction that completely inhibited growth was 3 lM (Fig 6) Partial inhibition of growth was observed with 2 lMof the H2B/H4 fraction and no inhibition was observed with 0.5 l of
Table 1 Litopenaeus vannamei histone peptides sequenced by mass spectrometry Predicted m/z is monoisotopic except for peptides from MS on MALDI-STR (average) Underlined sequence indicates sequence determined from MS/MS data Enzyme indicates digestion with trypsin (T), chymotrypsin (C) or Glu-C (G) Amino acid positions of histones H2A and H4 are reported based on the L vannamei cDNA sequences Observed m/z
(charge state)
Predicted m/z
(charge state)
Instrument, Dm/z Sequence Enzyme Identification 425.73 (2+) 425.77 (2+) QSTAR, 0.04 HLQLAIR T H2A 82–88 454.21 (2+) 454.24 (2+) QSTAR, 0.03 YLAAEVLE G H2A 57–64 472.73 (2 + ) 472.77 (2 + ) QSTAR, 0.04 AGLQFPVGR T H2A 21–29 545.30 (2 + ) 545.29 (2 + ) LCQ, 0.01 AERVGAGAPVY C H2A 40–50 1092.58 (1+) 1092.68 (1+) LCQ, 0.1 PNIQAVLLPK T H2A 109–118 476.90 (3 + ) 476.60 (3 + ) LCQ, 0.3 AIRNDEELNKLL C H2A 86–97 760.34 (2 + ) 760.40 (2 + ) QSTAR, 0.06 RVGAGAPVYLAAVMoxE b G H2A 42–56 767.74 (3+) 767.80 (3+) QSTAR, 0.06 LLSGVTIAQGGVLPNIQAVLLPK T H2A 96–118 3146.99 (1+) 3147.67 (1+) MALDI-STR,
0.68, QSTAR a
AIRNDEELNKLLSGVTIAQGGVLPNIQAVL C H2A 86–115 3388.22 (1+) 3388.96 (1+) MALDI-STR,
0.74, QSTARa
QLAIRNDEELNKLLSGVTIAQGGVLPNIQAVL C H2A 84–115 4099.91 (1 + ) 4100.87 (1 + ) MALDI-STR,
0.96, QSTARa
AIRNDEELNKLLSGVTIAQGGVLPNIQAVLLPKKTEKK C H2A 86–123 953.58 (1+) 953.60 (1+) LCQ, 0.02 LLLPGELAK T H2B 795.61 (2 + ) 795.44 (2 + ) LCQ, 0.17 AKHAVSEGTKAVTKY C H2B 895.68 (2 + ) 895.42 (2 + ) LCQ, 0.26 AMoxSIMNSFVNDIFER b T H2B 993.70 (2+) 993.54 (2+) LCQ, 0.16 PGELAKHAVSEGTKAVTKY C H2B 1106.91 (2 + ) 1106.62 (2 + ) LCQ, 0.29 LLPGELAKHAVSEGTKAVTKY C H2B 537.24 (1 + ) 537.33 (1 + ) QSTAR, 0.09 KPHR T H3
330.67 (2+) 330.70 (2+) QSTAR, 0.03 LPFQR T H3
358.18 (2+) 358.21 (2+) QSTAR, 0.03 DIQLAR T H3
394.71 (2 + ) 394.74 (2 + ) QSTAR, 0.03 KLPFQR T H3
416.21 (2+) 416.25 (2+) QSTAR, 0.04 STELLIR T H3
425.68 (2+) 425.72 (2+) QSTAR, 0.04 EIAQDFK T H3
516.76 (2 + ) 516.80 (2 + ) QSTAR, 0.04 YRPGTVALR T H3
1335.65 (1 + ) 1335.69 (1 + ) 4700, 0.04 EIAQDFKTDLR T H3
989.54 (1+) 989.58 (1+) 4700, 0.04 VFLENVIR T H4 61–68 1180.58 (1 + ) 1180.62 (1 + ) 4700, 0.04 ISGLIYEETR T H4 47–56 647.14 (2 + ) 646.85 (2 + ) LCQ, 0.29 LENVIRDAVTY C H4 63–73 1325.71 (1+) 1325.75 (1+) 4700, 0.04 DNIQGITKPAIR T H4 25–36 734.22 (2 + ) 733.91 (2 + ) LCQ, 0.31 TVTAMDVVYALKR T H4 81–93 584.65 (3 + ) 584.35 (3 + ) LCQ, 0.3 RDNIQGITKPAIRRL C H4 24–38
a
MS determined on MALDI-STR, MS/MS acquired on QSTAR.bMox indicates an oxidized methionine.
Fig 2 MALDI spectrum of hemocyte soluble proteins Histone
pro-teins are indicated by their abbreviations: H2A, H2B, H3 and H4.
Trang 6histone H2A (data not shown) Also, a fraction containing a
histone H1 fragment inhibited growth of M luteus (data
not shown), but it is not known if the H1 fragment is the
active antimicrobial component in the fraction Note that
the liquid growth inhibition assay does not distinguish
between bacteriocidal or bacteriostatic mechanisms
Generation of the synthetic peptide H2A 2–39
and liquid growth inhibition assays
Based on the N-terminal sequence of H2A from
L vannamei, a synthetic peptide (H2A 2–39) similar in
length to buforin I was synthesized (Fig 3B) and purified by
HPLC on a C18 column for antimicrobial assays The
MALDI MS of the tested fraction is depicted in Fig 5C
The synthetic peptide H2A 2–39 was tested for anti-microbial activity against the Gram-positive bacterium
M luteus and has a minimum inhibitory concentration (MIC) value (inhibition of 50% growth compared to control) in the range of 0.5–1.0 lM (Fig 7) Peptide antimicrobial activity was also observed against the Gram-positive bacteria Bacillus subtilis and Bacillus mega-teriumat concentrations between 1.5 and 5 lM(data not shown)
Discussion
Histone proteins are primarily involved in DNA pack-aging and regulation of DNA replication and transcrip-tion These proteins form the basic building blocks of chromatin structure when the four core histone proteins, H2A, H2B, H3 and H4, come together as heterodimers
to constitute the nucleosome The core histone proteins are highly conserved between species Histone H1 is the linker that condenses the nucleosomes and exhibits greater sequence variability Many reports have shown that histone proteins or histone-derived peptides from various vertebrates possess antimicrobial activity [19– 21,24,26,28] Our results indicate that histones in L van-namei have antimicrobial activity in vitro Their high abundance in hemocytes suggests that the shrimp may be using histones in antimicrobial defense To the best of our knowledge, this would be unprecedented in an invertebrate
Shrimp histone H2A protein completely inhibited growth
of the test bacterium M luteus at a concentration of 4.5 lM Histone H2A from a variety of vertebrate species has been shown to possess antimicrobial activity, but its activity appears to be variable Fernandes et al [21] showed that the trout histone H2A protein was active against several Gram-positive bacteria (including M luteus) with MIC values between 0.08 and 1.2 lM, but activity against Gram-negative bacteria was not observed at those concentrations However, Kim et al [24] tested the human histone H2A protein against the Gram-negative E coli bacteria and
Fig 3 Histone H2A alignments and synthetic peptide sequence (A) Litopenaeus vannamei histone H2A alignment with histone H2A from other species The shrimp histone H2A sequence was determined by cloning of the cDNA (bold sequence) and by mass spectro-metry sequencing (underlined sequence) (B) Synthetic peptide H2A 2–39, based on the length of buforin I and the shrimp N-terminus sequence.
Fig 4 Chromatogram at 225 nm of hemocyte soluble proteins on a C18
column The acetonitrile concentration gradient is indicated by the
dashed line The peaks containing the histone proteins are indicated
with the arrows.
Trang 7observed a complete inhibition of growth at 10 lgÆmL)1 ( 1.2 lM)
Peptides derived from the N-terminus of histone H2A of different organisms have activity against Gram-positive, Gram-negative bacteria and fungi at concentrations ranging from 0.5 to 5.0 lM[26–28] In the toad, the intact protein is secreted into the stomach [33] and is then cleaved by pepsin
A similar mechanism was demonstrated in the catfish where the protein was secreted in the skin mucus and then cleaved
by cathepsin D [34] to produce the active peptides These data suggest the possibility that the N-terminus of shrimp histone H2A could be an active antimicrobial peptide At this point in time however, there is no evidence that the shrimp H2A fragment 2–39 exists in vivo; but, a synthetic peptide identical to the first 38 amino acids of the N-terminus of H2A is active at submicromolar range against M luteus and at concentrations lower than 5 lM against B subtilis and B megaterium Examination of shrimp hemolymph for histone peptides is an obvious extension of this work
The shrimp histone H2B/H4 fraction completely inhibited growth of the test bacterium M luteus at 3 lM Clearly the shrimp fraction H2B/H4 needs to be further fractionated to determine if the active component is H2B, H4, or both Histone H2B protein has been reported as having antimicrobial activity in the channel catfish skin [20], in human placenta [24], and in murine macrophages [35] Also, an active fragment of histone H2B is expressed
in T cells and natural killer cells [36] Histone H4-derived fragments were reported to be present in an active fraction against B megaterium, however, the peptides were not purified to homogeneity (histone H1 fragments coeluted),
so it could not be concluded that they were the active components [30]
The L vannamei H1 fragment found in an active fraction
is smaller than 11 kDa and contains peptides similar to the central domain of the Drosophila histone H1 protein It is necessary to purify this peptide to homogeneity to confirm its activity A histone H1 protein fragment from rainbow trout skin secretions [37] has recently been described to inhibit growth of various Gram-positive and Gram-negative bacteria including M luteus This fragment is 69 amino acids long and is derived from the C-terminus of histone H1 Histone proteins undergo several post-translational mod-ifications (acetylation at several lysines, at their N-terminus
or methylation) during regulation of DNA transcription and packaging Kim et al demonstrated by
immunocyto-Fig 6 Liquid growth inhibition assay of endogenous histones H2A and
H2B/H4 against M luteus *Significant difference (P < 0.001) in
growth compared to the water control growth using a Student’s t-test
assuming equal variances n ¼ 4 for each treatment except for H2A
(n ¼ 2) MIP, CVTGEPVELDTQAL.
Fig 7 Growth inhibition assay of M luteus after 25 h incubation with
the synthetic peptide H2A 2–39 at 37 °C, n¼ 4, D 595 nm.
A
B
C
D
Fig 5 MALDI spectra (A–C) and 1D SDS/PAGE gel (D) of fractions
tested for inhibition of growth of M luteus (A) Histones H2B and H4.
(B) Histone H2A (C) Synthetic peptide H2A 2–39 (D) Silver stained
1D SDS 10–20% Tris/HCl gel D1, histones H2B and H4 ( 80 ng
loaded); D2, histone H2A fraction ( 60 ng loaded).
Trang 8chemistry that buforin I is unacetylated and that it is derived
from a cytoplasmic unacetylated histone H2A protein [33]
However, in the rainbow trout, the histone H2A active
protein is acetylated at its N-terminus [21] The N-terminal
20 amino acids of shrimp histone H2A have not been
observed by mass spectrometry, and therefore the status of
the N-terminus is unknown Based on the cDNA partial
sequence and the mass spectrometry sequencing of the
C-terminal, the predicted molecular mass of histone H2A is
13 266 Da If Met1 is cleaved and the protein acetylated on
the N-terminus then the predicted molecular mass is
13 177 Da Taking into account the oxidized methionine
at position 55, the predicted molecular mass becomes
13 193 Da The observed molecular mass for histone H2A
by MALDI MS is 13 189 Da, which is within the
experi-mental error of the instrument Furthermore, two forms of
histone H2A were identified in the shrimp It is possible that
the protein with a molecular mass higher by 78 Da is
phosphorylated The role of modification on the
antimicro-bial properties remains to be elucidated
Many questions remain regarding the in vivo mechanism
of antimicrobial histone action, particularly in the shrimp
Originally, histone proteins were thought to be localized in
the nucleus; however, they have been shown to occur in the
cytoplasm of various cell types [24,35] as well as, most
recently, in NETs from neutrophils [25] Histone proteins
or histone-derived peptides can also be secreted, as
demonstrated by a fragment of histone H2B found in the
secretions of natural killer cells when stimulated with
interleukin-2 [36] In both the toad and catfish, histone
H2A is secreted prior to enzymatic cleavage to active
peptides [33,34] We hypothesize that the shrimp histone
proteins from the hemocytes are localized in the cytoplasm
and more specifically in the granules along with other
antimicrobial peptides Antimicrobial action could take
place after release into the hemolymph upon infection or
inside the cell phagosome The well characterized
antimi-crobial peptides in shrimp, the penaeidins, are produced
and stored in the granular and semigranular hemocytes
[14] Their release into the hemolymph is not thought to be
through exocytosis but rather through a release of the
granular content into the cytoplasm followed by hemocyte
lysis [15]
In summary, the present study demonstrates that the core
histone proteins H2A, H2B, H3 and H4, identified by mass
spectrometry sequencing, are abundant proteins in
L vannameihemocytes Histone H2A as well as a mixture
of histones H2B and H4 prevented growth of the test
bacterium M luteus at concentrations in the same range as
measured for vertebrate histones A synthetic peptide
containing the shrimp H2A sequence homologous to the
known antimicrobial peptide buforin was antimicrobial
with a MIC of 0.5–1.0 lM Intact histone H1 protein was
not found in this study; however, an H1-derived fragment
was found in an HPLC fraction that was active against
M luteus Further work is necessary to determine the in vivo
defense mechanisms of shrimp hemocyte histone proteins
The present report suggests that multifunctional histone
proteins are a conserved feature of innate immunity,
probably not limited to vertebrates, such that all organisms
in which histones are present are potentially able to utilize
them as antimicrobial agents
Acknowledgements
The authors thank Dr Gregory Warr for helpful and insightful discussions; Dr Craig Browdy, Sarah Prior and Adrienne Metz for their help with the live shrimp; Javier Robalino and Brandon Cuthbertson for their encouraging discussions; the MUSC Mass Spectrometry facility for the use of their instruments; Dr Christian Schwabe from the MUSC Biochemistry and Molecular Biology Department, and the MUSC Biotechnology facility for DNA sequencing and peptide synthesis This study was funded by NSF grants IBN 0317303 to K.L.S and EPS 0083102 Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation This work was also supported in part by grant NA03NMF4720362 from the National Marine Fisheries Service and
is contribution number 550 to the Marine Resources Division of the South Carolina Department of Natural Resources and number 9 to the MUSC Marine Biomedicine & Environmental Sciences Center.
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