Báo cáo khoa học: Antibacterial peptides in stimulated human granulocytes Characterization of ubiquitinated histone H1A doc

Antibacterial peptides in stimulated human granulocytes Characterization of ubiquitinated histone H1A Yuqin Wang, William J.. Keywords: antibacterial peptides; histone; post-translation

Antibacterial peptides in stimulated human granulocytes

Characterization of ubiquitinated histone H1A

Yuqin Wang, William J Griffiths, Hans Jérnvall, Birgitta Agerberth and Jan Johansson

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Antibacterial peptides were isolated from human peripheral

granulocytes of a healthy donor who had been treated with

granulocyte-colony stimulating factor (G-CSF) and cortisol

Peptides were solubilized in acidified chloroform/methanol,

and partitioned in chloroform/methanol/water Water-

soluble polypeptides were separated by cation-exchange and

reversed-phase chromatography Several previously char-

acterized antibacterial polypeptides were identified; defen-

sins 1-3, defensin 4, lysozyme, eosinophil cationic protein,

and calgranulin A In addition, several histone fragments

were isolated and exhibited activity against the Gram-

positive bactertum Bacillus megaterium strain Bm11 These

fragments included two C-terminal fragments of histone

HIA, three C-terminal fragments of histone HID, one

fragment of histone H1B, and two fragments of histone H4

The molecular masses of both histone H1A fragments, as

determined by electrospray (ES) MS, were 270 Da higher

than those calculated from their amino acid sequences The two histone HIA fragments corresponded to Lys152- Lys222 (7527 + 1 Da)and Lysl67—Lys222 (6023 + 1 Da) Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa frag- ments indicated that the post-translational modification is

on Lys222, the s-amino group of which was conjugated with the a-carboxyl group of the tripeptide Arg-Gly-Gly This finding was substantiated by digestion of the 7.5-kDa poly- peptide with trypsin and analysis of the resulting peptides by

ES MS and MS/MS The tripeptide Arg-Gly-Gly corre- sponded uniquely to the three C-terminal residues of ubiquitin, demonstrating the presence of ubiquitinated histone H1A

Keywords: antibacterial peptides; histone; post-translational modification; ubiquitination; tandem mass spectrometry

In innate immunity, antibacterial peptides are important

effector molecules, and exhibit broad-spectrum antimicro-

bial activities [1] Over the last 20 years, more than 500

peptides with bactericidal properties have been isolated

from plants, insects and mammals, as well as from bacteria

[2] Despite apparent diversity in structure, most of these

peptides are amphipathic and cationic, and can therefore

interact with phospholipid membranes, in particular with

membranes rich in anionic phospholipids Differing from

classical antibiotics, these peptide antibiotics are gene-

encoded, and the mature active peptides are derived from

inactive precursors by proteolysis Post-translational mod-

ifications can occur during processing, and include

N-terminal formation of pyroglutamate or C-terminal

amidation [3,4]

Many mammalian antibacterial peptides, like a-defensins

and cathelicidins [5], were initially found in granulocytes; of

these the o-defensins are the most studied The œ-defensins

are 29-35 residues long, have three disulfide bridges, and

Correspondence to Y Wang, Department of Medical, Biochemistry

and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Fax: + 46 8337462, Tel.: + 46 87287681,

E-mail: yuqin.wang@mbb.ki.se

Abbreviations: G-CSF, granulocyte-colony stimulating factor; ES,

electrospray; TFA, trifluoroacetic acid; CID, collision induced disso-

ciation; MS/MS, tandem mass spectrometry; uH1A, ubiquitinated

histone H1A; D2S, dextrin-2-sulfate

(Received 21 May 2001, revised 14 November 2001, accepted 15

November 2001)

constitute > 5% of the total cellular protein in human neutrophil granulocytes [6]

Histones are the major proteins in chromatin, where their function is to assistin DNA folding and packaging However, the presence of histones on the plasma membrane of activated human peripheral blood lymphocytes suggests that histones are not confined to the nucleus [7] Similarly, histones H2A and H2B have been found extracellularly in preparations of homeostatic thymus hormone [8] Furthermore, the histone- derived antibacterial peptides parasin and buforin have been isolated from the epithelial layer of the catfish [9], and the stomach tissue of the Asian toad [10], respectively

All histones are basic proteins, containing a relatively large amount of lysine and/or arginine Several post-translational modifications of histones have been found, including acetyl- ation, methylation, phosphorylation, ADP-ribosylation, glycosylation and ubiquitination [11] Histone H2A was the first histone found to be modified by covalent linkage to ubiquitin; the carboxyl end of ubiquitin is linked to the s-amino group of Lys119 in H2A [12] Subsequently, histone H2B was found to be modified in the same manner on Lys120, but to a lesser extent (I-2%) than in histone H2A (10%) [13,14] Recently, ubiquitination of histone H3 was reported [15] The covalent linkage of ubiquitin targets most proteins for proteasome degradation, whereas the ubiquiti- nation of histones has been suggested to be important for chromatin remodelling during transcription [11] In this study, we isolated antibacterial peptides from human gran- ulocytes obtained from a donor after treatment with granulocyte-colony stimulating factor (G-CSF) and cortisol,

a treatment which gives a maximum number of peripheral

blood granulocytes In addition to several known antibac-

terial peptides, histone fragments were isolated and showed

activity against the Gram-positive bacterrum, Bacillus mega-

terium Bm11 Here we present evidence for ubiquitination of

histone H1A using electrospray (ES) MS The tripeptide Arg-

Gly-Gly, corresponding to the three C-terminal residues of

ubiquitin, was found to be linked to Lys222 of histone H1A

MATERIALS AND METHODS

Extraction and purification of proteins from stimulated

human granulocytes

Granulocytes from a healthy donor treated with G-CSF

(Neupogen, 5 pgkg' body weight, 1 day before harvest)

and cortisol (100 mg Solucortef, 15 min before start of

harvest) were obtained from the Department of Transfusion

Medicine at the Karolinska Hospital

Harvested human _ peripheral blood = granulocytes

(4.5 x 10'° cells, 82% granulocytes) were centrifuged at

3000 g for 30 min and the pellet (69 g) was homogenized 1n

1330 mL chloroform/methanol (2:1, v/v), containing

0.1% (v/v) trifluoroacetic acid Next 332 mL 0.1 m NaCl

was added, yielding a two-phase system of chloroform/

methanol/0.1 m NaCl, 8: 4: 3 (v/v) After phase separa-

tion, the aqueous phase was lyophilized, redissolved in 30%

(v/v) acetic acid, and desalted by RP chromatography using

a Sep-Pak Cig cartridge (Waters), which had been equili-

brated in 0.1% (v/v) aqueous trifluoroacetic acid Salts were

removed by washing with 10% (v/v) acetonitrile containing

0.1% (v/v) trifluoroacetic acid, after which proteins were

eluted with 80% (v/v) acetonitrile containing 0.1% (v/v)

trifluoroacetic acid The eluate was lyophilized and redis-

solved in 0.02 mM sodium phosphate buffer pH 6.4, and

applied to a cation-exchange column (Express-ion exchang-

er C, Whatman) that had been equilibrated in the same

buffer Proteins were eluted with: (a) 0.02 m phosphate

buffer pH 6.4; (b) 0.2 m NaCl mm 0.02 m phosphate buffer

pH 6.4; and (c) 0.2 mM HCL The contents of the three eluates

from the cation-exchange column were further separated by

RP-HPLC on a Resource column (10 x 2.0 cm, Pharma-

cia), with a water/acetonitrile gradient (see Fig 1A) Each

fraction was lyophilized and redissolved in 100 uL 0.1%

(v/v) aqueous trifluoroacetic acid, and screened for antibacte-

rial activity Fractions containing activity were purified by

RP-HPLC on a Cj, column (Vydac, 250 x 4.6 mm) with a

water/acetonitrile gradient and an inhibition zone assay

Components showing activity were characterized

Antibacterial assay

Aninhibition zone assay [16] with B megaterium strain Bm 11

or Escherichia coli strain D21 was used to detect antibacterial

activity Lyophilized samples were redissolved in 100 th

0.1% (v/v) aqueous trifluoroacetic acid, and 3-uL aliquots

were loaded into wells (3 mm) punched into agarose plates

seeded with the test bacteria After incubation at 30 °C

overnight, the diameters of the inhibition zones were recorded

Protein analysis

The HPLC-purified polypeptides demonstrating activity

were characterized by Edman degradation on an Applied

A

5 ¥ Eosinophil cationic protein ⁄ a | : oan a | & "8

g Histones (+37 W\ Lysozyme {40 §

0

Time (min)

B

0.5

480

—

b-

°

$

=

=

Time (min) Fig 1 Chromatography data (A) RP chromatography (Resource column 10 x 2.0 cm) of the 0.2 m HCl eluate from cation-exchange chromatography The flow rate was 6 mL-min! and fractions of 6 mL were collected The dashed line indicates the gradient of acetonitrile containing 0.1% (v/v) trifluoroacetic acid The line with asterisks indicates the activity (inhibition zone diameter) against B megaterium

Bm 11 and the line with circles indicates the activity against FE coli D21 (B) Analytical RP-HPLC (Vydac Cj) of the fraction containing histone fragments from Resource column (indicated in A) The flow rate was | mL-min™' The two uH1A fragments (Lys152—Lys222 and Lysl67-Lys222) were eluted in peaks 2 and 5, respectively Three histone HID fragments (A179-—K212, K175-K212 and T166—-K212) were found in peaks 1, 3 and 4, respectively

Biosystems 494 Prosice sequencer and MALDI-TOF MS Some peptides were further analysed by ES MS and MS/

MS Amino acid analysis was performed on a 4151 Alpha Plus amino acid analyser (LKB Biochrom) using the ninhydrin method Tryptic digestion was performed as described previously [4]

Mass spectrometry For MALDI-TOF MS, aliquots of the samples were mixed with 0.5 wL matrix (saturated o-cyano-4-hydroxy-cinnamic- acid in 70% acetonitrile containing 0.1% trifluoroacetic acid) on the target plate and left to dry Mass spectra were

recorded on a Finnigan MAT Lasermat 2000 operated in

the positive ion mode

Nano-ES mass spectra were recorded on an AutoSpec-

OATOFFPD (Micromass) hybrid high resolution double

focusing magnetic sector-orthogonal acceleration TOF tan-

dem mass spectrometer [17] Mass spectra were recorded at

both low (3500 resolution, 10% valley definition) and high

resolution (12500 resolution, 10% valley definition) as

magnet and voltage scans, respectively Accurate mass

measurements were made as voltage scans at high resolution

Nano-ES mass and MS/MS spectra were also recorded

on a Q-TOF (Micromass), quadrupole (Q)-TOF instru-

ment [18] Both mass and collision induced dissociation

(CID) spectra were recorded For the recording of mass

spectra, the quadrupole was operated in the radio

frequency only mode and all transmitted ions accelerated

orthogonally into the TOF For the recording of MS/MS

spectra the quadrupole was operated as a mass filter and

used to select the desired precursor ion isotope cluster

Precursor ions were directed into a hexapole collision cell

preceding the orthogonal acceleration region The collision

gas was argon and the collision voltage was in the range

10-40 V

RESULTS

Identification of antibacterial polypeptides in stimulated

granulocytes

Water-soluble polypeptides were obtained from G-CSF-

and cortisol-treated human granulocytes, by homogeniza-

tion in chloroform/methanol followed by phase separation

in chloroform/methanol/0.1 m NaCl Polypeptides in the

water phase were separated by cation-exchange chroma-

tography Three fractions were obtained from the cation-

exchange column by elution with 0.02 m phosphate buffer

pH 6.4; 0.2 M NaCl in 0.02 m phosphate buffer pH 6.4; and

finally 0.2 m HCI The antibacterial activity of each fraction

was determined by an inhibition zone assay While all three

fractions exhibited antibacterial activity, most antibacterial

polypeptides were found after elution with 0.2m HCl

(Table 1) Components in the fractions were subjected to

RP-HPLC on a Resource column and fractions that had

antibacterial activity were purified by RP-HPLC on a

Vydac C18 column Fig 1 shows the Resource RP separa-

tion of the 0.2 mM HCl eluate and the C18 RP separation of

fractions eluting with ~ 20% acetonitrile during Resource

chromatography and showing antibacterial activity Each

fraction was tested by an antibacterial assay, and active

peptides were identified by amino acid sequence analysis

Several known antibacterial polypeptides were identified

(Table 1) Most of the o-defensins (1-3) were eluted from

the cation-exchange column in 0.2m NaCl in 0.02 mM

phosphate buffer pH 6.4, while o-defensin 4, lysozyme,

eosinophil cationic protein and calgranulin A and several

fragments of histones H1 and H4 were found in the fraction

eluted with 0.2 m HCL

Purification and identification of ubiquitinated histone

H1A (uH1A) fragments

Two histone H1IA fragments and three histone HID

fragments were purified by C18 RP-HPLC (Fig 1B) and

Table 1 Identified polypeptides with antibacterial activity contained in the three cation-exchange column fractions Eluate a, 0.02 m phosphate buffer pH 6.4; eluate b, 0.2 m NaCl in 0.02 m phosphate buffer pH 6.4; eluate c, 0.2 m HCI

Elution from

fragments

Eosinophil cationic protein Histone H1B fragment

Calgranulin A

identified by Edman degradation for 20 cycles As indicated

in Fig 1B, peak 2, eluting at 16% acetonitrile, contained a polypeptide corresponding to Lysl67—Lys222 of histone HIA, and peak 5, eluting at 18% acetonitrile, contained a longer fragment of histone HIA (Lys152—Lys222) How- ever, the masses of both polypeptides (6023 + 1 Da and

7527 + 1 Da, respectively) determined by ES MS (Fig 2), were 270 Da higher than predicted from the primary structure of histone HIA Three C-terminal histone HID fragments were contained in peaks 1, 3 and 4 (Fig 1B), respectively, corresponding to Alal79-Lys212, Lys175— Lys212 and Thrl66—Lys212 ES mass spectra confirmed these assignments and showed no covalent modifications Tandem mass spectra were recorded on a Q-TOF

instrument of the [M + 10H]!°* —[M + 12H]'?~ ions

from the modified histone H1A fragments The resultant CID spectrum of the [M + 10H]'°~ ion from the shorter histone H1A(Lys167—Lys222) polypeptide is shown in Fig 3 Prominent fragment ion peaks were observed as a result of peptide bond cleavage N-terminal to a proline residue Table 2 describes the fragment ions observed in this CID spectrum

Series of b-type and y-type ions were observed (Scheme 1), which, combined, cover the complete sequence of the polypeptide This means that it was possible to localize the modification to the C-terminal lysine residue The interpretation of the CID spectrum presented in Fig 3 was confirmed by comparison with

spectra of the [M + 11H]''* and [M + 12H]'?~ ions,

and also with the CID spectra of the longer histone HIA polypeptide (data not shown) The two histone HIA polypeptides gave similar y-type ion series, however, the b-type ion series was displaced by 15 residues in the larger polypeptide, as expected from its N-terminal elongation

by 15 residues

Thus MS/MS spectra of the H1A polypeptides indicated that the 270 Da modification was at the C-terminal lysine residue Histones H2A, H2B, H3 are known to be reversibly ubiquitinated by covalent addition of ubiquitin, where the carboxyl end of ubiquitin is linked to the s-amino group

of a lysine residue A 270-Da modification could correspond

to the conjugation of the tripeptide Arg-Gly-Gly to the Lys222 side chain s-amino group Significantly, a database

Fig 2 ES mass spectra (A) ES mass spec-

trum of the polypeptide in peak 2 (see

Fig 1B) (B) ES mass spectrum of the poly-

peptide in peak 5 (see Fig 1B) The large

panels show the raw data, and the insets show

the deconvoluted spectra on a true mass scale

Fig 3 MS/MS spectra MS/MS spectrum of

the [M + 10H]!°~ ion at m/z 603 shown in

Fig 2A The inset shows the y,; 1on corre-

sponding to the Arg-Gly-Gly-eLys fragment

See Table 2 for description of all fragment

ions identified

(Swissprot version 39) search revealed that in humans, a

C-terminal tripeptide segment Arg-Gly-Gly corresponds

uniquely to the three C-terminal residues of ubiquitin

Thus, 1t would seem probable that histone HIA 1s

ubiquitinated, and that the 270 Da modification corre-

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sponds to the addition of Arg-Gly-Gly to the Lys222 side chain amino group (Scheme 2) To confirm this hypo- thesis, a modified histone fragment was digested with trypsin and the resultant tryptic peptides were characterized

by ES MS

Table

Gly-Gly

3444 bis

bịa A

bio A

bị K

bị” KI

5+6+

bạo K

bist

bog A

P 5+6-+ J3

S+9+

bs A Y5

P 2434 #11

P4I yee

0 ; 0} O ' O

HạN——CH—C——N——CH—C——N——CH—C—:-N——CH—C——COH

~< < <

Scheme 1 Peptide fragmentation nomenclature Protonated peptides fragment in low energy CID reactions predominantly at amide bonds

to give b ions with the charge residing on the N-terminus of the pep- tide, and y ions with the charge residing on the C-terminus of the peptide

Histone H1A fragments `

(CH2)3

Arg-Gly-Gly ~ e NH

Tee, ;

Scheme 2 Schematic structure of uH1A fragment The o-carboxy group of Gly is linked to the ¢-amino group of Lys222 of histone H1A

The tryptic digest of modified histone H1A(Lys152— Lys222) was analysed by ES MS on both Q-TOF and AutoSpec-OATOFFPD instruments Tryptic digestion was not complete, resulting in both the regularly expected peptides and those in which a cleavage site had been

missed When both sets of data were taken into account,

100% coverage of the polypeptide was achieved (Fig 4)

By recording accurate mass spectra at high resolution (12500, 10% valley definition) on the AutoSpec-OAT- OFFPD it was also possible to confirm the elemental composition of the Ts, peptide, which contains the Arg- Gly-Gly modification of the C-terminal lysine residue Further experiments were performed in which tryptic peptide T>4 containing the Arg-Gly-Gly modification was subjected to CID To achieve sufficient product ion sensitivity it was necessary to record these spectra on the Q-TOF instrument, which 1s not capable of high resolution precursor ion selection The unit mass resolution available was not sufficient to uniquely select the desired precursor, and thus a mixture of precursor ions was fragmented However, the immonium ions characteristic of Arg and

the accurate mass data, identified the modification as

Arg-Gly-Gly

Quantitation and antibacterial activity of uH1A fragments

The concentration of uH1A fragments measured by amino acid analysis corresponded to 2.5 pmol per 10° cells Antibacterial assays showed that the uH1A fragments were effective against B megaterium Bm11, but inactive against

E coli D21 The diameter of the inhibition zone against

B megaterium Bm11 was 8 mm when 67 um uHIA frag- ment (Lys152—Lys222) was applied The corresponding

288.3

100-

Fig 4 ES mass spectrum of the tryptic

digestion of modified histone H1A fragment

(Lys152-Lys222) The inset shows the tryptic

peptide T>4 corresponding to Arg-Gly-Gly-

data for 10-fold concentrated (670 um) human antibacterial

peptide LL-37 was 10 mm

DISCUSSION

Several histone fragments were purified from human

granulocytes from a donor stimulated with G-CSF and

cortisol and found to exhibit strong antibacterial activity

against the Gram-positive bactertum B megaterium Bm11

The mass spectrometric results showed that the two

C-terminal histone HIA fragments Lysl52-Lys222 and

Lys167—Lys222 were modified by Arg-Gly-Gly at Lys222

via an isopeptide bond between that s-amino group and the

a-carboxyl group of the terminal Gly of Arg-Gly-Gly This

conclusion was arrived at upon consideration of the MS/

MS spectra of the 6.2- and 7.5-kDa histone HIA polypep-

tides, and of the accurate mass and MS/MS spectra of the

tryptic digest of the 7.5-kDa polypeptide The confirmation

of the primary structures of large polypeptides by MS/MS 1s

comparatively rare [19] In the present study the interpre-

tation of the CID spectra was greatly facilitated by the

tendency of the protein to fragment at amide bonds

N-terminal to proline, giving intense y-type ions and

complimentary b-type ions This made it possible to locate

the modification of Lys222 of histone HIA This so called

‘proline effect’ has been noted previously [20,21] and 1s an

important factor in the interpretation of CID spectra of

polypeptides

Ubiquitin contains 76 residues and adopts a stable

globular conformation with the evolutionarily conserved

three C-terminal residues, Arg-Gly-Gly, flexibly disordered

[22] Interestingly, both of the two histone H1A fragments

now found were derived by cleavage of a Val—Lys peptide

bond A similar site between an aliphatic residue and a basic

residue (i.e the peptide segment Leu—Arg) must be cleaved

in ubiquitin to release the C-terminal tripeptide Arg-Gly-

Gly This suggests that the modified histone fragments were

derived from ubiquitinated histone H1A by proteolysis

taking place between one hydrophobic and one basic

residue Ubiquitination has been found in histones H2A,

H2B and H3, and the amount of ubiquitinated histone was

found to vary during the cell cycle [23] At present, it 1s not

known whether the presence of uHIA 1n human granulo-

346.3

417.3

371.2 ° o 4183

445.3

cytes after treatment with G-CSF and cortisol 1s an effect of the stimulation, or if histone HIA 1s also ubiquitinated in nonstimulated cells It 1s also not known whether histone HIA 1s ubiquitinated so as to target it to a degradation pathway or for other reasons

Several recent studies have indicated extra-nuclear loca- tions of histones, with some studies suggesting immunolog- ical roles of histones, in addition to the classical function of histones 1n chromatin conformation In phytothaemagglu- tinin-activated human peripheral lymphocytes, histone H2B was found to move from the nucleus to the plasma membrane and interact with dextrin-2-sulfate (D2S) [7] D25 1s a sulfated polysaccharide that binds to histone H2B

on the cell surface and there it can inhibit human immunodeficiency virus type 1 infection [7] Moreover, nonacetylated histone H2A was found in the cytoplasm of the gastric gland cells of Asian toads, from which the antibacterial peptide buforin I 1s derived by pepsin-mediated cleavage between Tyr39 and Ala40 [10] Whether the uH1A fragments described in this paper are derived from the nucleus, or from another cellular compartment remains to

be established

ACKNOWLEDGEMENTS

We thank E Cederlund and C Palmberg for technical assistance and

O Wrange and J Lundahl for constructive comments on this manuscript This study was supported by the Swedish Medical Research Council, Hagbergs stiftelse Aners stiftelse, and Magn Bergvalls stiftelse, and by the Research funds of the Karolinska Institutet

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