Báo cáo khoa học: Antimicrobial activity of histones from hemocytes of the Pacific white shrimp ppt

Antimicrobial activity of histones from hemocytes of the Pacific
white shrimp
Se
´
verine A. Patat
1
, Ryan B. Carnegie
1,
*, Celia Kingsbury
1,
, Paul S. Gross
2,3
, Robert Chapman
3,4
and Kevin L. Schey
1,3
1
Department of Cell and Molecular Pharmacology,
2
Department of Biochemistry and
3
Marine Biomedicine & Environmental Sciences
Center, Medical University of South Carolina, Charleston, SC, USA;
4
Hollings Marine Laboratory, Marine Resources Institute,
SCDNR, Charleston, SC, USA
The role of vertebrate histone proteins or histone derived
peptides as innate immune effectors h as 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 h omologous to histones o f
other species. Based on this homology, histone H2A was
cloned a nd its N-terminus was found to resemble the known
antimicrobial histon e p eptides 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, p urified to homogeneity, completely inhibited
growth of the Gram-positive bacterium at 4.5 l
M
while a
mixture of histones H2B and H4 was active at 3 l
M
.In
addition, a f raction containing a f ragment of h istone H1 was
also found to be active. The synthetic peptide similar to
buforin was active at s ubmicromolar concentrations. These
data indicate, for the first time, t hat shrimp hemocyte histone
proteins possess antimicrobial activity and represent a d ef-
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: a ntimicrobial peptide; histone; invertebrate; mass
spectrome try; 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 e ffector cells in shrimp defenses, a re 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 a nd
phagocytosis while the semigranular and granular cells are
involved in phagocytosis, release of p roteins in the prophe-
noloxidase cascade, and release o f 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 b inding 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, a nd 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 bin ding
protein, by pepsin cleavage of its N-terminus [9]. In
crustaceans, antimicrobial peptides have been described in
the c rab Carcinus maenas [10], in the crayfish Pacifastacus
Correspondence to K. L. Schey, Medical University of South Carolina,
Department of Cell and Molecular Ph armacology, 173 Ashley
Avenue, PO Box 250505, Charleston, SC 29425, USA .
Fax: +1 843 792 2475, Tel.: +1 843 792 2471,
E-mail:
Abbreviations: LPS, lipopolysaccharide; NET, neutrophil extracellular
trap; MAS, modified alsever s olution; TFA, tr ifluoroacetic acid;
MIC, minimum inhibitory concentration.
*Present add ress: Virginia Institute of Marine Science, PO Box 1346,
Gloucester Pt., VA 23062, USA.
Present add ress: Pharmacopeia, Inc., PO Box 5350, Princeton, NJ
08543-5350, USA.
Note: The protein sequence data reported in t his paper will appear in
the SwissProt and TrEMBL knowledgebase und er the accessio n
numbers P83841, P83863, P83864 and P83865, and the nucleotide
sequence data has been submitted to GenBank unde r the accession
numbers AY576482 and AY576483.
(Received 20 August 2004, r evised 29 September 2004,
accepted 21 October 2004)
Eur. J. Biochem. 271, 4825–4833 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04448.x
leniusculus [11], and in several penaeid shrimp. I n 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 a lso 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 r anging 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 (n eutrophil extracellular t raps) 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 rainb ow trout, On corh ynchus my kiss [29], a s
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 P acific white
shrimp, L. vannamei, and the r ole 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 ven tral sinus of the a nimal
under a n equal volume of modified alsever solution (MAS:
27 m
M
sodium citrate, 336 m
M
sodium chloride, 115 m
M
glucose, 9 m
M
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 (4 °C) to separate the hemocytes from the plasma.
After removal of the plasma from the cell p ellets, 600 lLof
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 (4 °C) and t he buffer was removed. The
washing a nd centrifugation steps w ere r epeated with 200 lL
of MAS. After r emoval of the buffer, 100 lLofwaterwere
added t o t he 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, H ercules, 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 lgofhemocyte
lysate proteins were mixed with r educing 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 m
M
Tris base, 192 m
M
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 m
M
Tris, pH 8.8, 0 .1% ( w/v) SDS] and staine d with 0.3
M
copper chloride for 20 min. The gel was stored in water
prior to image analysis and in-gel d igestion.
Bands of interest were cut out of the g el and sliced into
1 m m p ieces. The copper stained pieces were d estained
with 500 lL of copper destain solution for 20 min. After
destaining, the gel pieces were washed three times for
20 min w ith 100 m
M
ammonium bicarbonate, once f or
15 min with acetonitrile/100 m
M
ammonium bicarbonate
(50 : 50, v/v), and once for 15 min with acetonitrile,
before being dried in a spee d vac ( 5 min). Dried gel pieces
were rehydrated with 10 lL of trypsin or chymotrypsin
(100 ng in 100 m
M
ammonium bicarbonate, pH 7.8) and
then covered with ammonium bicarbonate (100 m
M
,
30 lL). Digestion was carried out overnight at 37 °C.
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 r esuspended 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/wat er/acetic acid (49 : 49 : 2, v/v/v).
Protein purification and quantification
After thawing, 300 lL of h emocyte lysate supernatant was
subjected to HPLC by injection onto a C18 column
(Alltech, C18 Prosphere, 4.6 · 250 mm, 300 A
˚
,5lm;
Alltech, Deerfield, I L, USA). T he solvent s ystem included
4826 S. A. Patat et al.(Eur. J. Biochem. 271) Ó FEBS 2004
0.1% trifluoroacetic acid (TFA) in water (solvent A) and
0.08% TFA in ace tonitrile (solvent B). Proteins were eluted
from the column with a gradient of 5–30% solvent B o ver
13 min followed by 30–60% solvent B over 67 min at a flow
rate of 0.7 mLÆmin
)1
. Protein elution was monito red b y U V
absorbance at 225 nm and peak fractions were collected.
Fractions were subsequently lyophilized, reconstituted in
100 lL water, and analyzed by MALDI MS. Histones H 4
and H2B co eluted and were not further separated prior to
protein assay and antimicrobial assay. Fractions containing
only histone H2A were pooled. F ractions containing impure
histone H2A were repurified over the same C 18 colum n 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 estima ted b y t he 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 A pplied Science). T ryptic
or chymotryptic digestions were carried out overnight at
37 °Cin25m
M
ammonium bicarbonate/10% (v/v) aceto-
nitrile, pH 7.8 with 200 ng of trypsin or 1 lgofchymo-
trypsin. Glu-C digests were carried out overnight at room
temperature in 25 m
M
ammonium 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 M S) 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 7 0% (v/v ) acetonit rile/0.1% ( v/v)
TFA was used. Peptides and matrix solutions were mixed
(1 : 3 lL) and 0.5 lL of t he 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 t o produce a
mass spectrum.
Nanospray tandem mass spectrometry w as 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 f or 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 a nd, 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 t o m atrix. Sequences from tandem m ass
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 ( 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
CRA-3¢;R3:5¢-CTTKGGDAGRACTGC-3¢)were
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), a nd Asellus aqu aticus
(AJ238321). Several combinations of forward and reverse
primers were used in a ttempts to amplify a 3¢ histone H2A
gene fragment f rom L. vannamei gill cDNA library. Reac-
tion volumes of 20 lL i ncluded PCR buffer, nucleotides at
1.25 m
M
, primers at 100 ngÆlL
)1
, Taq DNA polymerase
(Advantage kit, Clontech, Palo Alto, CA, USA), and
0.4 lL o f g ill 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 94 °Cfor
1min,48°C for 30 s and 72 °C for 30 s, and ended with a
5 m in final extension at 72 °C. DNA products were
separated on a 1.2% agarose g el and stained with ethidium
bromide. Bands of interest were excised and extracted u sing
a Nucleospin k it (Clontech), and then cloned into Escheri-
chia coli XL1 blue w ith ampicillin. Plate colonies were
picked randomly and grown in 2 mL of Luria broth with
200 lg of ampicillin overnight at 37 °C. Plasmid DNA was
extracted using NucleoSpin Plus Minipreps (Clontech), and
inserts were sequenced at the MUSC DNA Sequencing
Facility (Medical U niversity of South Carolina, Charleston,
SC, USA). Based on the initial sequence, a new reverse
primer (5¢-GGATGGCCAGCTGCAAGTGACGG-3¢)
was designed. To determine the 5¢ histone H2 A gene
sequence, the new reverse primer, a vector-specific fo rward
primer, an d 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 3 0 cycle s o f
94 °Cfor1min,68°C for 30 s, and 72 °Cfor30s,and
ended w ith a 5 min final exten sion at 72 °C. Products were
cloned and sequenced as above. DNA sequences were
translated for comparison with described histone sequences
using the ExPASy translate tool ( />Peptide synthesis
A peptide similar in length to buforin [26] was s ynthesized
at the MUSC P eptide Synthesis Facility. The sequence was
based o n the N-terminus of the shrimp H 2A DNA sequence
(SGRGKGGKVKGKSKSRSSRAGLQFPVGRIHRLL
RKGNY). After synthesis, the peptide, named H2A 2–39,

was purifiedby HPLC.Approximately 0 .2 mg ofpeptide was
Ó FEBS 2004 Antimicrobial activity of shrimp histone proteins (Eur. J. Biochem. 271) 4827
loaded onto a C18 column ( Alltech, C18 Prosphere, 4.6 ·
25 mm, 300 A
˚
,5lm). 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 o ff the c olumn
with a gradient of 5–75% B in 150 min at a flow rate
of 0.7 mLÆmin
)1
. T he purified synthetic peptide’s concentra-
tion was measured by a mino acid analysis by C. Schwabe,
MUSC Biochemistry Department, Charleston, SC, U SA.
Liquid growth inhibition assays
Antibacterial activity was tested against the Gram-positive
bacterium Micrococcus l uteus. Stocks of bacteria were kept
at )80 °C in 50% (v/v) glycerol ( 200 lL). One stock of
bacteria was a dded to 5 mL of Luria–Bertani (LB) media
andgrownovernightat37 °C with s haking, and was used to
spread a LB agar p late. A fter overnight i ncubation at 37 °C,
theplatewaskeptat4°C and served as a stock for the
experiments. One colony was picked and incubated in 5 mL
of LB media overnight at 37 °C 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 (D ¼ 0.01) were added to
each we ll of a 96 well plate with 10 lL of the appropriate
control or sample. Negative controls were water or a
synthetic peptide (MIP peptide: CVTGEPVELD TQAL,
10 l
M
) chosen from a pool of synthetic peptides used for
vision research and without known antimicrobial activity,
and t he positive c ontrol was Ala-magainin (Sigma, S t Louis,
MO, USA, 5 l
M
). Samples were fractionated histone
proteins or the N-terminal H 2A synthetic peptide (H2A 2–
39) at various concentrations (less than 10 l
M
) diluted in
water. The plate was then i ncubated at 37 °Covernightwith
shaking and the D at 595 nm or 570 nm was read in a plate
reader (M olecular Devices).
Results
Identification of histone proteins in the hemocytes
In the course of an initial proteomic inve stigation 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 c hymotrypsin, and the resulting p eptides were
sequenced by nanospray tandem mass spectrometry. The
peptides were identified using a

BLAST
homology search of
the obtained sequences. The lower band yielded peptides
similar to histone H4, the upper b ands 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 H PLC 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 h igh number o f 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 m asses. Also, t he 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 a cids, 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 p eptides.
Purification of histone proteins for liquid growth
inhibition assays
The hemocyte lysate supernatants wer e s eparated b y r everse
phase HPLC on a C18 column. Histone proteins eluted
between 40 and 5 5% 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 solu ble prote ins. Copp er stained
1D SDS 10–20% Tris/HCl gel of h emocyte l ysate p roteins. Molec ular
markers are indicated on the left in kDa. Histone proteins and
hemocyanin protein bands indicated on the right.
4828 S. A. Patat et al.(Eur. J. Biochem. 271) Ó FEBS 2004
with hemocyanin, the abundant oxygen carrier p rotein, at
56% solvent B (not shown). Approximately 61 lgof
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 a nd H4 are the two major components in t heir 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 s hown to be histone H2A by n anospray mass
spectrometry sequencing a fter tryptic d igestion.
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 c ompletely inhibited growth was 4.5 l
M
and the lowest concentration of H2B/H4 fraction that
completely inhibited growth was 3 l
M
(Fig. 6). Partial
inhibition of growth was observed w ith 2 l
M
of the H2B/H4
fraction and no inhibition was observed with 0.5 l
M
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 sequenc e 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, QSTAR
a
QLAIRNDEELNKLLSGVTIAQGGVLPNIQAVL C H2A 84–115

4099.91 (1
+
) 4100.87 (1
+
) MALDI-STR,
0.96, QSTAR
a
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.
b
Mox 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.
Ó FEBS 2004 Antimicrobial activity of shrimp histone proteins (Eur. J. Biochem. 271) 4829
histone H2A (data no t 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 o n t he N-ter minal sequence of H 2A from
L. vannamei, a synthetic peptide (H2 A 2–39) s imilar i n
length to buforin I w as synthesized (Fig. 3B) and pu rified 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 inhib itory concentration
(MIC) value (inhibition of 50% growth compared to
control) in the range of 0.5–1.0 l
M
(Fig. 7). Peptide
antimicrobial activity was also observed against the
Gram-positive bacteria Bacillus subtilis and Bacillus mega-
terium at concentrations between 1.5 and 5 l
M
(data not
shown).
Discussion
Histone protein s 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 c ore histone proteins
are highly conserved between species. Histone H1 is the
linker t hat 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 i n antimicrobial defense. To the best o f
our knowledge, this would be unprecedented in an
invertebrate.
Shrimp histone H2A protein completely inhibited growth
of the test bacterium M. luteus at a c oncentration of 4.5 l
M
.
Histone H2A from a variety of v ertebrate species has been
shown to poss ess an timicrobial activity, but its activity
appears to be variable. Fernandes et al.[21]showedthatthe
trout histone H2A protein was active against several G ram-
positive bacteria (including M. luteus) w ith M IC value s
between 0.08 and 1.2 l
M
, 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 h istone 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 22 5 nm of hemocyte soluble proteins on a C18
column. The acetonitrile concentration gradient is indicated by the
dashed line. The peaks containing t he histone prote ins are indicated
with the arrows.
4830 S. A. Patat et al.(Eur. J. Biochem. 271) Ó FEBS 2004
observed a complete inhibition of growth at 10 lgÆmL
)1
( 1.2 l
M
).
Peptides derived from the N-terminus of histone H2A of
different organisms have activity against Gram-positive,
Gram-negative b acteria a nd fungi at concentrations ranging
from 0.5 to 5.0 l
M
[26–28]. In the toad, t he intact protein is
secreted into the s tomach [33] and is then cleaved by p epsin.
A similar m echanism w as demo nstrated 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 p ossibility 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 l
M
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 l
M
.
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 c hannel catfish skin
[20], in human placenta [24], and in murine macrophages
[35]. Also, an active fragment of histone H2B is e xpressed
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 o f t he 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 g rowth of v arious Gram-positive and Gram-negative
bacteria including M. luteus.Thisfragmentis69amino
acids l ong and i s derived from the C-terminus of histone H1.
Histone proteins undergo s everal post-translational mod-
ifications (acetylation at s everal 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). M IP, CVTGEPVELD TQAL.
Fig. 7. Growth inhibitio n assa y of M. luteus after 25 h incubation with
the synthetic peptide H2A 2–39 at 37 °C, n ¼ 4, D 59 5 nm.
A
B
C
D
Fig. 5. MALDI spectra (A–C) a n d 1D SDS/PAGE gel (D) of fraction s
tested for inhibition of g rowth of M. luteus. (A) Histones H 2B 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).
Ó FEBS 2004 Antimicrobial activity of shrimp histone proteins (Eur. J. Biochem. 271) 4831
chemistry t hat buforin I is unacetylated and that it is derived
from a cytoplasmic unacetylated histone H2A p rotein [33].
However, in the rainbow trout, the histon e H2A active
protein is a cetylated at its N-terminus [21]. The N-terminal
20 amino acids of shrimp his tone 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 m ass spectrometry sequencing o f 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 t hat
the protein with a molecular m ass higher b y 7 8 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 natu ral killer cells when stimulated with
interleukin-2 [36]. I n 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]. T heir 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 H 4, identified by m ass
spectrometry sequencing, are abundant proteins in
L. vannamei hemocytes. Histone H2A as well as a mixture
of histones H2B and H4 p revented growth of the t est
bacterium M. luteus at concentrat ions 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 l
M
. 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. lute us. Further work is necessary to deter mine 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 t hank 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 en couraging discussi ons; the MUSC Mass Spectrometry
facility f or the use of their instruments; D r Christian Schwabe from t he
MUSC Biochemistry and Molecular Biology Department, and the
MUSC Biotechnology f acility f or D NA sequencing a nd 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 t he authors
and do not necessarily reflect the views of the National Scienc e
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 Div ision of the
South Carolina Department o f Natural Resources and num ber 9 to the
MUSC Marine Biomedicine & Environmental Sciences Center.
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