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. Grif®ths, Hans JoÈ rnvall, 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 d onor who had been treated with granulocyte-colony stimulating factor (G-CSF) and cortisol. Peptides were solubilized in acidi®ed 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 identi®ed; defen- sins 1±3, defensin 4, lysozyme, eosinophil cationic protein, and calgranulin A. In addition, several histone fragm ents were isolated and exhibited activity against the Gram- positive bacterium Bacillus megaterium strain Bm11. These fragments included two C-terminal fragments of histone H1A, three C-terminal fragments of histone H1D, 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 H1A fragments corresponded to Lys152± Lys222 (75 27  1 Da) and Lys167±Lys222 (6023  1 Da). Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa frag- ments indicated that the post-translational modi®cation is on Lys222, the e-amino group of which was conjugated with the a-carboxyl group of the tripeptide Arg-Gly-Gly. This ®nding 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. T he tripeptide Arg-Gly-Gly c orre- sponded uniquely to the t hree C-terminal residues of ubiquitin, demonstrating the presence of ubiquitinated histone H1A. Keywords: antibacterial peptides; histone; post-translational modi®cation; ubiquitination; tandem mass spectrometry. In innate immunity, antibacterial peptides are important effector molecules, and exhibit broad-spectrum antimicro- bial activities [1 ]. Over the last 2 0 y ears, more t han 500 peptides with bactericidal properties have been isolated from pla nts, insects and mammals, as well as from bacteria [2]. Despite apparent diversity in structure, most of these peptides are a mphipathic and c ationic, and can therefore interact with p hosp holipid membranes, in particular with membranes rich i n 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 m od- i®cations can occur during processing, and include N-terminal formation o f p yroglutamate or C-terminal amidation [3,4]. Many mammalian antibacterial peptides, like a-defensins and cathelicidins [5], were initially found in granulocytes; of these the a-defensins are the most studied. The a-defensins are 29±35 residues long, have th ree disul®de bridges, and constitute > 5% of t he total cellular protein in human neutrophil granulocytes [6]. Histones are the major proteins in chromatin, where their function is toassist in DNA folding a nd packaging. However, the presence o f histones on t he plasma membrane of activated human peripheral blood lymphocytes suggests t hat histones are not con®ned 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 t he cat®sh [9], and the stomach tissue of the Asian toad [10], respectively. All h istones are basic proteins, containing a relatively large amount of lysine and/or arginine. Several post-translational modi®cations of histones have been found, including acetyl- ation, methylation, phosphorylation, ADP-ribosylation, glycosylation and ubiquitination [11]. Histone H2A was the ®rst histone found to be modi®ed by covalent linkage to ubiquitin; the carboxyl end of ubiquitin is linked to the e-amino group of Lys119 in H2A [12]. Subsequently, histone H2B was found to be mo di®ed in the same manner o n Lys120, but to a l esser extent (1±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 i mportant for chromatin r emodelling during transcription [11]. In this study, we isolated antibacterial peptides from human gran- ulocytes obtained from a donor after t reatment with granulocyte-colony stimulating factor (G-CSF) and cortisol, a treatment which gives a m aximum number of peripheral 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, tri¯uoroacetic 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) Eur. J. Biochem. 269, 512±518 (2002) Ó FEBS 2002 blood granulocytes. In addition to several known antibac- terial peptides, histone fragments were isolated and showed activity against the Gram-positive bacterium, Bacillus mega- terium Bm11. Here we present evidence for ubiquitination of histone H1Ausing electrospray ( ES) MS. The tripeptide A rg- 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 puri®cation of proteins from stimulated human granulocytes Granulocytes from a healthy donor treated w ith G -CSF (Neupogen, 5 lgákg )1 body weight, 1 day before harvest) and c ortisol (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 ´ 10 10 cells, 8 2% granulocytes) were centrifuged at 3000 g for 30 min and the pellet (69 g) was homogenized in 1330 mL chloroform/methanol (2 : 1 , v/v), containing 0.1% (v/v) tri¯uoroacetic 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 aSep-PakC 18 cartridge (Waters), which had been equili- brated in 0.1% (v/v) aqueous tri¯uoroacetic acid. Salts were removed by washing with 10% (v/v) acetonitrile containing 0.1% (v/v) tri¯uoroacetic acid, after which proteins were eluted with 80% (v/v) a cetonitrile containing 0.1% (v/v) tri¯uoroacetic acid. The e luate was lyophilized and r edis- solved in 0.02 M sodium phosphate buffer pH 6.4, and applied to a cation-exchange column (Express-ion exchang- er C, Whatman) that had b een equilibrated in the same buffer. Proteins were eluted with: (a) 0.02 M phosphate buffer pH 6.4; (b) 0.2 M NaCl in 0.02 M phosphate buffer pH 6.4; and (c) 0.2 M HCl. The contents of the three eluates from the cation-exchange column were further separated by RP-HPLC on a Resource column (10 ´ 2.0 cm, Pharma- cia), with a water/acetonitrile gradient (see Fig. 1A). Each fraction was lyophilized and redissolved in 100 lL0.1% (v/v) a queous tri¯uoroacetic a cid, and screened for a ntibacte- rial activity. F ractions containing activity were puri®ed by RP-HPLC on a C 18 column (Vydac, 250 ´ 4.6 mm) with a water/acetonitrile gradient and an inhibition zone assay. Components showing activity were characterized. Antibacterial assay An inhibition zoneassay [16] with B. megaterium strain Bm11 or Escherichia coli strain D21 was used to detect antibacterial activity. Lyophilized samples were redissolved in 100 lL 0.1% (v/v) aqueous tri¯uoroacetic acid, and 3-lL a liquots were loaded into wells (3 mm) punched into agarose plates seeded with the test bacteria. After incubation at 30 °C overnight,thed iametersofth ei nhibitionzo neswererec orded. Protein analysis The HPLC-puri®ed polypeptides demonstrating activity were characterized by Edman degradation on an Applied Biosystems 494 Prosice sequencer and M ALDI-TOF MS. Some peptides were further analysed by ES MS and MS/ MS. Amino acid analysis was performed on a 4151 Alpha Plus amino a cid analyser (LKB Biochrom) using the ninhydrin method. Tryptic digestion was performed as described previously [4]. Mass spectrometry ForMALDI-TOFMS,aliquotsofthesamplesweremixed with 0.5 lL matrix (saturated a-cyano-4-hydroxy-cinnamic- acid in 70% acetonitrile containing 0.1% tri¯uoroacetic acid) on the target plate and left to dry. Mass spectra were Fig. 1. Chromatography data. (A) RP chromatography (Resource column 10 ´ 2.0 cm) of the 0.2 M HCl e luate from cation-exchange chromatography. The ¯ow rate w as 6 mLámin )1 and fractions of 6 mL were collected. The dashed line indicates the gradient of acetonitrile containing 0.1% (v/v) tri¯uoroacetic a cid. The line with asterisks indicates the activity (inhibition z one diameter) against B. megaterium Bm11 and the line with circles indicates the activity a gainst E. coli D21. (B) Analytical RP-HPLC (Vydac C 18 ) of the fraction containing histone fragments from Resource column (indicated in A). The ¯ ow rate was 1 mLámin )1 . The two uH1A fragments (Lys152±Lys222 and Lys167±Lys222) we re eluted in peaks 2 a nd 5, respectively. Thr ee histone H1D fragments (A179±K212, K175±K212 and T166±K212) were found in peaks 1, 3 and 4, respectively. Ó FEBS 2002 Ubiquitination of histone H1A (Eur. J. Biochem. 269) 513 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 re solution double focusing magnetic sector-orthogonal acceleration TOF tan- dem mass spectrometer [17]. Mass spectra were recorded at both low (3500 resolution, 10% valley de®nition) and high resolution (12500 resolution, 10% valley de®nition) 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 r ecording o f m ass spectra, the quadrupole w as 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 ®lter and used to select the d esired 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 Identi®cation of antibacterial polypeptides in stimulated granulocytes Water-soluble polypeptides were obtained from G-CSF- and cortisol-treated human granulocytes, by homogeniza- tion in chloroform/methan ol 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 colu mn 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 ®nally 0.2 M HCl. The antibacterial activity of each fraction was determined by an inhibition zone assay. While all three fractions exhibited antibacterial ac tivity, most antibacterial polypeptides were found after elution with 0.2 M HCl (Table 1). Components i n the fractions were subjected to RP-HPLC on a Resource column and fractions that had antibacterial activity were puri®ed by RP-HPLC on a Vydac C18 column. Fig. 1 shows the Resource RP separa- tion of the 0.2 M HCl eluate and the C18 RP separation of fractions eluting with  20% acetonitrile during Resource chromatography and showing a ntibacterial activity. Each fraction was tested by an antibacterial assay, and active peptides were identi®ed by amino acid sequence analysis. Several known antibacterial polypeptides w ere identi®ed (Table 1). Most of the a-defensins (1±3) were eluted from the cation-exchange column in 0.2 M NaCl in 0.02 M phosphate buffer pH 6.4, while a-de fensin 4, l ysozyme, 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. Puri®cation and identi®cation of ubiquitinated histone H1A (uH1A) fragments Two h istone H1A f ragments and t hree histone H1D fragments were puri®ed by C18 R P-HPLC (Fig. 1B) and identi®ed by Edman degradation for 20 cycles. As indicated in Fig. 1B, peak 2, eluting at 16% acetonitrile, contained a polypeptide corresponding to Lys167±Lys222 of histone H1A, and peak 5, eluting at 18% acetonitrile, contained a longer f ragment o f h istone H1A (Lys152±Lys222). How- ever, the masses o f 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 H1A. Three C-terminal histone H1D fragments were contained i n p eaks 1, 3 and 4 (Fig. 1B), respectively, corresponding to Ala179±Lys212, Lys175± Lys212 and Thr166±Lys212. ES mass spectra con®rmed these assignments and showed no covalent modi®cations. Tandem m ass spectra were recorded on a Q-TOF instrument of the [M + 10H] 10+ ±[M+12H] 12+ ions from the mod i®ed histone H1A fragments. The resultant CIDspectrumofthe[M+10H] 10+ ion from the shorter histone H1A(Lys167±Lys222) polypeptide is shown i n 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 i n this CID spectrum. Series of b-type and y-type ions were observed (Scheme 1 ), which, combined, cover the complete sequence of t he polypeptide. T his means that it was possible to localize the modi®cation to the C-terminal lysine residue. The interpretation of the CID spectrum presented in Fig. 3 was con®rmed by comparison w ith spectra of the [M + 11H] 11+ and [M + 12H] 12+ ions, and also with the CID spectra of the longer histone H1A polypeptide (data not shown). T he two histone H1A polypeptides gave similar y-type ion series, however, the b-type ion series was displaced b y 15 residues in t he 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 modi®cation was at the C-terminal lysine residue. Histones H2A, H2B, H3 are known to be reversibly ubiquitinated by c ovalent a ddition o f ubiquitin, where the carboxyl end of ubiquitin is linked to t he e-amino group of a lysine residue. A 270-Da modi®cation could correspond to the conjugation of the tripeptide Arg-Gly-Gly to the Lys222 side chain e-amino group. Signi®cantly, a database Table 1. Identi®ed polypeptides with antibacterial activity contained in the three cation-exchange column fractions. Eluate a, 0.02 M phosphate buer pH 6.4; e luate b, 0.2 M NaCl in 0.02 M phosphate b uer p H 6.4; eluate c, 0.2 M HCl. Cation-exchange column eluates Polypeptides Elution from RP-HPLC (% acetonitrile) Eluate a a-Defensins 1±3 33% Eluate b a-Defensins 1±3 33% Eluate c Histone H1A, H1D, H4 20% fragments Eluate c a-Defensin 4 40% Eosinophil cationic protein Histone H1B fragment Eluate c Lysozyme 48% Calgranulin A Eluate c Histone H4 fragment 50% 514 Y. Wang et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (Swissprot v ersion 39) search revealed that i n humans, a C-terminal tripeptide segment Arg-Gly-Gly corresponds uniquely to the three C-terminal residues of ubiquitin. Thus, it would seem probable that histone H1A is ubiquitinated, and that the 270 Da modi®cation corre- sponds to the addition of A rg-Gly-Gly to the Lys222 side chain amino group (Scheme 2). To con®rm this hypo- thesis, a modi®ed histone fragment was digested with trypsin and the resultant tryptic peptides were characterized by ES MS. 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 s pectra on a true mass scale. Fig. 3. MS/MS spectra. MS/MS spectrum of the [M + 10H] 10+ ion at m/z 603 s hown in Fig. 2A. The inset shows the y 1 ion corre- sponding to the Arg-Gly-Gly-eLys fragment. See Table 2 for description of all fragment ions identi®ed. Ó FEBS 2002 Ubiquitination of histone H1A (Eur. J. Biochem. 269) 515 The tryptic digest of modi®ed 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% cov erage of the polypeptide was a chieved ( Fig. 4). By recording accurate mass spectra at high resolution (12500, 10% valley de®nition) on the AutoSpec-OAT- OFFPD it was also possible to con®rm the elemental composition o f the T 24 peptide, which contains the Arg- Gly-Gly modi®cation of the C-terminal lysine residue. Further e xperiments were performed i n w hich tryptic peptide T 24 containing the Arg-Gly-Gly modi®cation was subjected to CID. To achieve suf®cient product ion sensitivity it was necessary to record these spectra on the Q-TOF instrument, which is not capable of high resolution precursor ion selection. The unit mass resolution available was not suf®cient to uniquely select the desired precursor, and thus a m ixture of precursor i ons was fragmented. However, the immonium ions characteristic of Arg and Lys were o bse rved, and t his, in combination with the accurate mass data, identi®ed the modi®cation 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 8 cells. Antibacterial assays showed that the uH1A fragments were effective a gainst B. megaterium Bm11, but inactive against E. coli D21. The diameter o f t he inhibition zone against B. me gaterium Bm11 was 8 mm when 67 l M uH1A frag- ment (Lys152±Lys222) was applied. The corresponding Table 2. Fr agment ions observed in the CID spectrum of the [M + 10H] 10+ ion at m/z 603 of modi®ed histone H1A fragment Lys167±Lys222. K* is modi®ed by the addition of 270 Da corresponding to Arg- Gly-Gly. b 1 + b 2 + b 3 + b 4 + b 5 + b 6 + b 7 + b  2  8 b 9 2+ b 10 2+ b 11 2+ b 12 2+ b 14 2+ b 15 2+ b 17 3+ b 3  4 18 K1 KVAKSPKKAKAAAKPKKAT20 y 55 9+ y 50 8+ y 41 7+ b 3  4 21 b 22 4+ b 24 4+ b 4  5 25 b 27 5+ b 30 5+ b 32 5+ b 5  6 35 K21 S PAKPKAVKPKAAKPKAAK40 y 34 6+ b 45 7+ y 5  6  31 y 26 5+ b 8  9  52 y 21 3+ y 19 3+ P41 KAAKPKAKKAAAKKK* y 2  3  4  16 y 14 3+ y 2  3  11 y 10 2+ y 9 2+ y 5 + y 4 + y 2 + y 1 + Scheme 1. Pe ptide fragmentation nomenclature. Protonated peptides fragment in low energy CID reactions predominantly at amide bonds to give b ions with the charge resid ing on the N-terminus of the pep- tide, and y ions with the c harge residing on t he C-termin us of the peptide. Scheme 2. Sc hematic structure of uH1A fragment. The a-carboxy group of Gly is linked to the e-amino group of Lys222 of hi stone H1A. 516 Y. Wang et al. (Eur. J. Biochem. 269) Ó FEBS 2002 data for 10-fold concentrated (670 l M ) human antibacterial peptide LL-37 was 10 mm. DISCUSSION Several histone fragments were puri®ed from human granulocytes from a donor stimulated with G-CSF and cortisol and found to exhibit strong antibacterial activity against the Gram-positive bacterium B. me gate rium Bm11. The mass spectrometric results showed that the t wo C-terminal histone H1A fragments Lys152±Lys222 and Lys167±Lys222 were modi®ed by Arg-Gly-Gly at Lys222 via an isopeptide bond between that e-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 H1A polypep- tides, and o f the accurate mass and MS/MS spectra of the tryptic digest of the 7.5-kDa polypeptide. The con®rmation of the primary structures of large polyp eptides by MS/MS is comparatively rare [19]. In the present study the interpre- tation of the CID spectra was greatly facilitated by the tendency of the protein to f ragment at amide bonds N-terminal to proline, giving intense y-type ions and complimentary b-type ions. T his made it possible t o locate the m odi®cation of Lys222 of h istone H1A. This so called Ôproline effectÕ has been noted previously [20,21] and is a n important factor in the interpretation of C ID spectra of polypeptides. Ubiquitin contains 76 residues and adopts a stable globular conformation with the evolutionarily conserved three C-terminal residues, Arg-Gly-Gly, ¯exibly disordered [22]. I nterestingly, both of the two histone H1A fragments now found were derived by c leavage 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 modi®ed 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 is not known whether the presence of uH1A in human granulo- cytes after treatment with G-CSF and cortisol is an effect of the stimulation, or if h istone H1A is also ubiquitinated i n nonstimulated cells. It is also not known whether histone H1A i s ubiquitinated so as t o target it t o 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 in 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 d extrin-2-sulfate ( D2S) [7]. D2S is a sulfated polysaccharide that binds to histone H2B on the cell surface and there it can inhibit human immunode®ciency virus type 1 infection [7]. Moreover, nonacetylated histone H2A was found in the cytoplasm of the gastric gland c ells of Asian toads, from which the antibacterial peptide buforin I is derived by pepsin-mediated cleavage between Tyr39 and Ala40 [10]. Whether the uH1A fragments d escribed 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 a nd O È . Wrange and J. Lundahl for constructive comments on this manuscript. This study was supported by the Swedish Medical Research Council, Hagberg s stiftelse Ane  rs stiftelse, and Magn. 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Keywords: antibacterial peptides; histone; post-translational modi®cation;