Structural evidence of a-aminoacylated lipoproteins of Staphylococcus aureus Miwako Asanuma 1, *, Kenji Kurokawa 2 , Rie Ichikawa 1 , Kyoung-Hwa Ryu 2 , Jun-Ho Chae 2 , Naoshi Dohmae 1 , Bok Luel Lee 2 and Hiroshi Nakayama 1 1 Biomolecular Characterization Team, RIKEN Advanced Science Institute, Saitama, Japan 2 National Research Laboratory of Defense Proteins, Pusan National University, Busan, Korea Introduction Bacterial lipoproteins are lipidated proteins anchored on the outside leaflet of bacterial cell membranes and outer envelopes, and have diverse functions such as nutrient uptake, cell-wall metabolism, adhesion and transmembrane signaling. The biosynthesis pathway of bacterial lipoproteins has been established in Escheri- chia coli and consists of three sequential enzymatic reactions [1]. Following apolipoprotein translocation by Sec machinery, the first enzyme diacylglyceryl trans- ferase (Lgt) transfers a diacylglyceryl moiety from a membrane phospholipid to a sulfhydryl group of the +1 cysteine of a conserved ‘lipobox’ motif in the N-terminal signal peptide, making a thioether linkage. The lipoprotein signal peptidase (Lsp) then cleaves the signal peptide at the N-terminus of the +1 S-diacylgly- ceryl cysteine. Finally, the third enzyme apolipoprotein Keywords bacterial lipoprotein; Gram-positive bacteria; mass spectrometry; N-acyltransferase; SitC Correspondence B. L. Lee, National Research Laboratory of Defense Proteins, College of Pharmacy, Pusan National University, Jangjeon Dong, Geumjeong Gu, Busan 609-735, Korea Fax: +82 51 513 2801 Tel: +82 51 510 2809 E-mail: brlee@pusan.ac.kr H. Nakayama, Biomolecular Characterization Team, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Fax: +81 48 462 4704 Tel: +81 48 462 1419 E-mail: knife@riken.jp *Present address ERATO, Japan Science and Technology Agency (JST), Tokyo, Japan (Received 12 November 2010, revised 3 December 2010, accepted 9 December 2010) doi:10.1111/j.1742-4658.2010.07990.x Bacterial lipoproteins are known to be diacylated or triacylated and acti- vate mammalian immune cells via Toll-like receptor 2 ⁄ 6or2⁄ 1 heterodi- mer. Because the genomes of low G+C content Gram-positive bacteria, such as Staphylococcus aureus, do not contain Escherichia coli-type apoli- poprotein N-acyltransferase, an enzyme converting diacylated lipoproteins into triacylated forms, it has been widely believed that native lipoproteins of S. aureus are diacylated. However, we recently demonstrated that one lipoprotein SitC purified from S. aureus RN4220 strain was triacylated. Almost simultaneously, another group reported that another lipoprotein SA2202 purified from S. aureus SA113 strain was diacylated. The determi- nation of exact lipidated structures of S. aureus lipoproteins is thus crucial for elucidating the molecular basis of host–microorganism interactions. Toward this purpose, we intensively used MS-based analyses. Here, we demonstrate that SitC lipoprotein of S. aureus RN4220 strain has two lipo- protein lipase-labile O-esterified fatty acids and one lipoprotein lipase-resis- tant fatty acid. Further MS ⁄ MS analysis of the lipoprotein lipase digest revealed that the lipoprotein lipase-resistant fatty acid was acylated to a-amino group of the N-terminal cysteine residue of SitC. Triacylated forms of SitC with various length fatty acids were also confirmed in cell lysate of the RN4220 and Triton X-114 phase in three other S. aureus strains, including SA113 strain and one Staphylococcus epidermidis strain. Moreover, four other major lipoproteins including SA2202 in S. aureus strains were identified as N-acylated. These results strongly suggest that lipoproteins of S. aureus are mainly in the N-acylated triacyl form. Abbreviations BHI, Brain Heart Infusion; LB, Luria–Bertani; Lgt, diacylglyceryl transferase; Lnt, apolipoprotein N-acyltransferase; LPL, lipoprotein lipase; Pam3, N-palmitoyl-S- dipalmitoylglyceryl; TLR, Toll-like receptor; TX114, Triton X-114. 716 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS N-acyltransferase (Lnt) transfers an acyl group from phospholipid to the newly generated a-amino group of the S-diacylglyceryl cysteine (N-acylation reaction), resulting in the generation of triacylated protein [1,2]. The N-acylation of lipoproteins is essential in Gram- negative bacteria to transport lipoproteins from the inner membrane to the outer membrane via the lipopro- tein localization pathway [3,4]. Although Lgt and LSP are widely conserved in eubacteria, Lnt has not been found in low G+C content Gram-positive bacteria (Firmicutes) [5–8]. Recently, Tschumi et al. identified an E. coli Lnt homolog in the high G+C content Gram-positive bac- terium Mycobacterium smegmatis and demonstrated its Lnt activity [9], but its homolog in Firmicutes was not identified. Several reports have presented structural and ⁄ or indirect evidence of diacylated lipoproteins, for example, dipalmitoyl macrophage-activating lipo- peptide-2 kDa (MALP-2) from Mycoplasma fermen- tans [10], SA2202 (SAOUHSC_02699) protein from S. aureus SA113 strain [11] and F 0 F 1 -type ATPase subunit b from Mycobacterium pneumoniae [12]. There- fore, Firmicutes are widely regarded as having only diacylated lipoproteins. Despite the lack of an E. coli Lnt homolog in Firmicutes, however, chemical analy- ses of lipoproteins in Bacillus subtilis and in S. aureus suggested N-acylated lipoproteins in these organisms [13,14]. We also recently used MS-based analysis to demonstrate that the SitC lipoprotein from S. aureus is triacylated [15]; however, we could not show structural evidence of N-acylation of the lipoprotein. In addition, some triacylated lipoproteins of Mollicutes, which are closely related to Firmicutes, have been reported based upon indirect evidence of nuclear factor-jB activation through Toll-like receptor (TLR)1 and TLR2 [16,17]. Therefore, evidence of the N-acylation of lipoproteins leading to triacylated forms in Firmicutes is ambiguous and controversial. Microorganism invasion activates the innate immune response in mammals. Bacterial lipoproteins as a path- ogen-associated molecular pattern [18] are sensed by the hosts through TLR2 heterodimerized with TLR1 or TLR6: this signal induces the activation of innate immunity and is necessary to control adaptive immu- nity [19]. In addition, TLR2 stimulation drives the dif- ferentiation of hematopoietic progenitor cells [20]. Although TLR2 has been considered as a receptor for various structurally unrelated pathogen-associated molecular patterns such as lipoproteins, lipoteichoic acid and peptidoglycan [18], recent studies suggest that bacterial lipoproteins function as the major, if not sole, ligand molecules for TLR2-activation [5,11,15,21,22]. To date, synthetic lipoprotein analogs, such as N-pal- mitoyl-S-dipalmitoylglyceryl (Pam3)–Cys, Pam3CSK 4 lipopeptide and MALP-2 [10], have been used to mimic the proinflammatory properties of bacterial lipoproteins, and have led to a model in which tria- cylated lipopeptides signal through TLR2 ⁄ TLR1 heterodimer, whereas diacylated lipopeptides signal through TLR2 ⁄ TLR6 heterodimer. However, recent studies have demonstrated that some synthetic lipopep- tides [23–25] and at least one native lipoprotein [15] are inconsistent with this model. Therefore, real struc- tural characterization of native lipoproteins from Gram-positive bacteria is crucial to elucidate the molecular basis of host–microorganisms interaction. Here, we carefully analyzed the structure of the lipo- peptide moiety of S. aureus lipoproteins. Analyses using lipoprotein lipase (LPL) and MS ⁄ MS revealed that S. aureus SitC was N-acylated with various length fatty acids and thus was triacylated. In addition, SitC in three other strains of S. aureus and one strain of S. epidermidis, and four other lipoproteins in S. aureus were shown to be N-acylated. These results strongly suggest that lipoproteins in S. aureus are mainly in the N-acylated triacyl form. Results N-Terminal structure of SitC from S. aureus RN4220 strain Staphylococcus aureus SitC is annotated as the sub- strate-binding component of the ATP-binding cassette transporter for iron [26], and is one of the predomi- nant lipoproteins functioning as a ligand of TLR2 [27]. Our previous report provided clear MALDI-TOF MS data representing that the N-terminal lipopeptide of SitC is triacylated [15]. Although the result is not enough to support the N-acylation of SitC protein, it is still surprising because of the presumed absence of E. coli Lnt homologs in the S. aureus genomes [28]. Contrary to our findings, Tawaratsumida et al. reported that another lipoprotein SA2202 of S. aureus SA113 had a diacylated (dipalmitoylated) N-terminus, based on MS ⁄ MS data [11]. To clarify this discrep- ancy, we decided to determine the bona fide structure of lipoproteins in S. aureus. Also, determination of the exact structure of the Gram-positive bacterial native lipoproteins is essential for the elucidation of the molecular mechanism of host–microorganism interac- tions. To characterize the acylated structure of S. aureus lipoproteins, we used commercially available LPL which is known to degrade bacterial lipoprotein and reduce the TLR2-stimulating activity of lipoproteins M. Asanuma et al. Triacylated lipoproteins in S. aureus FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 717 [21]. At first, we characterized the specificity of the enzyme using synthetic Pam3CSK 4 triacyl lipopeptide as a substrate, and found that the enzyme hydrolyzes O-esterified fatty acids of the (di)acylglyceride moiety, but not N-acylated fatty acids. To examine the cleav- age patterns of native SitC lipoproteins by LPL, SitC protein prepared by Triton X-114 (TX114) phase partitioning was separated by SDS ⁄ PAGE and then subjected to in-gel digestion with trypsin to make the N-terminal lipopeptide of SitC in the presence of n-decyl-b-D-glucopyranoside, followed by chloro- form ⁄ methanol extraction. Figure 1A shows MALDI- TOF MS of the chloroform ⁄ methanol organic phase representing a series of 14-Da interval peaks, explained by an increasing number of methylene (CH 2 ) groups in their fatty acids between m ⁄ z 1297 and 1409, which corresponds with triacylated N-terminal lipopeptides of SitC (Table 1). The result is consistent with the MS data from our previous study [15], and suggests that the N-terminal lipopeptides of SitC were highly puri- fied, because other peptides generated by the tryptic digest were rarely detected in the organic phase. As a positive control, we confirmed that S-dipalmitoylglyce- ryl–CSK 4 and Pam3CSK 4 lipopeptides were also recovered in the organic phase using this extraction method and represented specific signals on MALDI- TOF MS (data not shown). Recovery of TLR2-stimu- lating activity in the organic phase was also confirmed using TLR2-expressing Chinese hamster ovary cells (Fig. S1), suggesting that the N-terminal lipopeptides of SitC are responsible for the TLR2 stimulation. After 5 h incubation with LPL and lipopeptides, a new series of 14-Da interval peaks was detected between m ⁄ z 1044 and 1115 (Fig. 1B). The mass values of these ions corresponds to those of the diacyl-glyceryl CGTGGK (Table 1) generated by the release of one O-esterified fatty acid from the original triacylated lipopeptide. After 17 h incubation, another series of peaks between m ⁄ z 835 and 891 was detected (Fig. 1C), corresponding to monoacyl-glyceryl CGTGGK generated by releasing of two O-esterified fatty acids from the triacylated lipopeptide (Table 1). On further incubation, no additional series of peaks was detected. Thus, the triacylated SitC lipoprotein 1086.75 2 1114.78 1381.02 1 1352.99 1409.05 A B 1338.98 1072.73 b1 y5 y4 y3 [MH-thioglycerol] + [M+H] + [MH-2H 2 O] + [MH-H 2 O] + b°5 b°4 b5 y°5 b°1 0 50 100 Relative intensity D [MH-C 2 H 6 O 2 ] + 862.5 754.4 m/z 800 900 100011001200 1300 14001500 862.60 3 890.63 C 300 400 500 600 700 800 900 m/z NCH O C (C 18 H 35 O) S CHHO CH 2 CH 2 CH 2 HO H G T G G K 716.3 698.3 444.5 426.5 362.2 419.3 401.3 b b° 261.1 y y° E 754.4 844.3 826.4 800.5 641.2 Fig. 1. Lipoprotein lipase analysis determines triacylated structures of SitC lipoprotein in Staphylococcus aureus RN4220 cells. SitC lipopro- tein in the TX114 fraction prepared from exponential-growth phase RN4220 cells was separated by SDS ⁄ PAGE and digested in-gel with tryp- sin. The resulting N-terminal lipopeptides of SitC were extracted to the organic phase, dried and resolved in water (A) or further incubated with LPL for 5 h (B) or 17 h (C). MALDI-TOF MS of each fraction is shown. Group 1, 2 or 3 in the figures is consistent with a series of tria- cylated, diacylated or monoacylated S-glyceryl CGTGGK peptides of N-terminal SitC, respectively, as described in Table 1. The series of mass signals harboring 14-Da mass differences [an increasing number of methylene (CH 2 ) groups] is due to various length saturated fatty acids. (D) MALDI-IT MS ⁄ MS using 2,5-dihydroxybenzoic acid as a matrix was carried out for an LPL-resistant lipopeptide of m ⁄ z 862.5, as described in (C). Isolation width was ± 2 Da. (E) The elucidated structure of N-octadecanoly-S-glycerylcysteinyl GTGGK from the observed fragment ions in (D). Peaks designated y° or b° correspond to y-type or b-type ions that have lost an H 2 O moiety, respectively. Triacylated lipoproteins in S. aureus M. Asanuma et al. 718 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS purified from RN4220 cells is modified by two O-ester- ified fatty acids and one LPL-resistant fatty acid. To determine the exact modification site of the LPL-resistant fatty acid, one of the peaks after LPL digestion corresponding to the octadecanoyl-glyceryl CGTGGK with m ⁄ z 862.60 shown in Fig. 1C was further analyzed by MALDI-ion trap (IT) MS ⁄ MS. Figure 1D,E show the MS ⁄ MS spectrum and the elucidated structure of the lipopeptide, respectively. The C-terminus-containing y-series ions at m ⁄ z 261.1 (y 3 ), 362.2 (y 4 ), 401.3 (y° 5 ;y 5 -H 2 O) and 419.3 (y 5 )in the spectrum confirmed the amino acid sequence of GTGGK, which is complemented by ions at m ⁄ z 444.5, 641.2 and 698.3, which are assigned as N- terminus-containing b-series ions (b 1 ,b° 4 and b° 5 , respectively). This result strongly suggests that the fatty acid modification site is at the N-terminal cyste- ine residue. Importantly, a characteristic fragment ion for N-acyl-dehydroalanyl peptide generated by neutral loss of thioglycerol was observed at m ⁄ z 754.4, indi- cating that the octadecanoyl group is linked at the a-amino group of cysteine via an amide bond. These results are consistent with our previous observation that the N-terminus of SitC was blocked when Edman degradation sequencing was performed [15]. Therefore, the results of both LPL digestion and MS ⁄ MS of the N-terminal peptides demonstrated that the N-terminal cysteine of SitC from S. aureus RN4220 cells is N-acylated with a saturated C16 to C20 fatty acid in addition to the expected S-diacylgly- cerylation with two saturated fatty acids. Triacylated SitC is the major form in the cell lysate of S. aureus RN4220 strain We next examined whether the triacylated forms are the major molecular species of SitC in S. aureus cells. To evaluate the overall molecular species of SitC and prevent the loss of specific molecular species during isolation of SitC through the TX114 phase-partitioning method, the cell lysate of S. aureus RN4220 strain was directly subjected to SDS ⁄ PAGE (Fig. 2A). When an approximately 33-kDa band was in-gel digested and the resulting peptides were analyzed by LC-MS ⁄ MS, the 33-kDa band was identified as SitC (data not shown). The digest was then extracted with chloro- form ⁄ methanol and analyzed by MALDI-TOF MS. As shown in Fig. 2B, the triacylated N-terminal lipopeptides of SitC were detected. The arrows indicate peaks corresponding to triacylated lipopeptides with the sum of the carbon number for three fatty acids of 47–55 in Table 1. By contrast, any significant peaks with mass values corresponding to the diacylated N-terminal lipopeptides of SitC referred to in Table 1 were not detected in the MALDI mass spectrum (Fig. 2C). These results indicate that the triacylated forms are the major forms of SitC in S. aureus RN4220 strain. Table 1. Calculated and observed masses of the lipid-modified N-terminal peptides of SitC and those generated by lipoprotein lipase diges- tion shown in Fig. 1. Modified peptide Calculated [M + H] + Observed m ⁄ z D (ppm) Triacyl(C47) + CGTGGK 1296.94 1296.95 6.4 Triacyl(C48) 1310.96 1310.94 )19.4 Triacyl(C49) 1324.98 1324.95 )21.9 Triacyl(C50) 1338.99 1338.98 )6.5 Triacyl(C51) 1353.01 1352.99 )10.6 Triacyl(C52) 1367.02 1367.01 )12.4 Triacyl(C53) 1381.04 1381.02 )10.6 Triacyl(C54) 1395.05 1395.05 )4.5 Triacyl(C55) 1409.07 1409.05 )12 Diacyl(C30) + CGTGGK 1044.70 1044.71 a 6.3 Diacyl(C31) 1058.72 1058.70 a )15.2 Diacyl(C32) 1072.73 1072.74 a 12.4 Diacyl(C33) 1086.75 1086.75 a )0.3 Diacyl(C34) 1100.76 1100.78 a 11.8 Diacyl(C35) 1114.78 1114.78 a )2.4 Monoacyl(C16) + CGTGGK 834.50 834.57 b 87.4 Monoacyl(C17) 848.52 848.57 b 67.5 Monoacyl(C18) 862.53 862.60 b 78.4 Monoacyl(C19) 876.55 876.61 b 73.0 Monoacyl(C20) 890.56 890.63 b 76.7 Lipoprotein lipase digestion for a 5 h and b 17 h. M. Asanuma et al. Triacylated lipoproteins in S. aureus FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 719 Characterization of the N-terminal structure of SitC in other strains of S. aureus and S. epidermidis Because one lipoprotein in S. aureus SA113 strain was reported to be diacylated [11], we then asked whether the SitC lipoproteins of three other strains of S. aur- eus, including SA113 strain, and of S. epidermidis ATCC12228 strain are diacylated or triacylated. The organic phase of the in-gel-digested SitC isolated from the TX114 fraction of exponential-growth phase S. aureus SA113 cells grown in Luria–Bertani (LB) medium was analyzed by MALDI-TOF MS. As shown in Fig. 3A and Table 2, a series of 14-Da interval peaks between m ⁄ z 1283 and 1381, corresponding to the triacylated N-terminal lipopeptides of SitC modi- fied with saturated fatty acids (the sum of the carbon AB C Fig. 2. SitC prepared from a crude cell lysate of S. aureus RN4220 cell is also triacylated. (A) SDS ⁄ PAGE profile visualized with Coomassie Brilliant Blue of a crude cell lysate or its TX114 fraction of S. aureus RN4220 cells is shown. The arrowhead indicates the migration position of SitC that was identified at 33 kDa in either the cell lysate or TX114 phase. (B,C) MALDI-TOF mass spectrum of the in-gel tryptic digests of a 33-kDa region of the cell lysate. Arrows indicate the calculated mass positions of the triacylated (B) or diacylated (C) N-terminal lipopeptides of SitC. Calculated mass values for the lipopeptides are shown in Table 1. B C A 1367.05 1381.11 1353.05 1339.03 1325.02 1310.99 1296.98 1282.91 * * * * * * C46 C47 C48 C50 C49 C51 C52 C53 1352.99 1381.02 1367.00 1338.98 1324.96 1395.04 1310.95 1409.04 C49 C51 C52 C53 C48 C50 C54 C55 m/z 1367.02 1339.01 1353.01 1324.96 1381.02 1310.97 1280 1300 1320 1340 1360 1380 1400 1420 1409.04 C49 C51 C52 C53 C48 C50 C55 Fig. 3. Triacylated structures of N-terminal peptides of SitC of other three S. aureus strains. MALDI-TOF mass spectra of the organic phase of in-gel tryptic digests of SitC protein, which was isolated through TX114 phase extraction from exponentially growing S. aureus cells in LB medium of laboratory strain SA113 (A), clinically isolated methicillin-resistant strain MW2 (B) or clinically isolated methicillin- sensitive strain MSSA476 (C). Asterisks in (A) are signals 2-Da smaller than the triacylated peptides filled with saturated fatty acids. Triacylated lipoproteins in S. aureus M. Asanuma et al. 720 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS number for three fatty acids is 46–53), was detected. In the case of clinically isolated S. aureus strains MW2 and MSSA476, a series of 14-Da interval peaks between m ⁄ z 1311 and 1409, corresponding to the tria- cyl lipopeptides of SitC with the sum of the carbon number of the fatty acids equal to 48–55 (see Table 1), was also detected (Fig. 3B,C). Because Tawaratsumida et al. used Brain Heart Infusion (BHI) medium when they determined the N-terminal lipopeptide structure of SA2202 protein of SA113 strain [11], we used this medium for the SA113 strain. As in case of LB med- ium, SitC lipopeptides isolated from SA113 cells grown in BHI medium had a series of peaks corresponding to the triacylated forms (data not shown). The total carbon number of the modified fatty acids in the most abundant peak of the SitC lipopeptides derived from the SA113 strain was smaller than that from RN4220, MW2 or MSSA476 strain, indicating that shorter fatty acids were mainly attached to the tria- cylated lipopeptides of SitC of the SA113 strain (Figs 1A and 3). The usage of shorter fatty acids was also detected in the spectrum of the SitC lipopeptides isolated from SA113 cells grown in BHI medium (data not shown). Moreover, triacyl peptides 2 Da smaller than those loaded with saturated fatty acids were addi- tionally observed in SA113 cells grown in both LB and BHI medium, and are indicated by asterisks in Fig. 3A. These peaks would be due to the presence of an unsatu- rated fatty acid in the triacylated lipopeptides. Table 2. Calculated and observed masses of triacylated N-terminal lipopeptides of SitC and SA2202 isolated from exponentially grow- ing S. aureus SA113 cells grown in Luria-Bertani medium. Modified peptide Calculated [M + H] + Observed m ⁄ z D (ppm) C46 + CGTGGK a 1282.93 1282.91 )15.6 C47 1296.94 1296.98 30.8 C48 1310.96 1310.99 22.9 C49 1324.98 1325.02 30.2 C50 1338.99 1339.03 29.9 C51 1353.01 1353.05 29.6 C52 1367.02 1367.05 21.9 C53 1381.04 1381.11 50.7 C45 + CGNNSSK b 1455.97 1456.01 23.1 C46 1469.99 1470.04 30.6 C47 1484.01 1484.06 33.3 C48 1498.02 1498.07 31.2 C49 1512.04 1512.09 37.1 C50 1526.05 1526.12 43.5 C51 1540.07 1540.12 34.9 C52 1554.08 1554.16 48.3 C53 1568.10 1568.12 15.6 C54 1582.12 1582.16 27.7 a SitC, b SA2202. 200 400 600 800 1000 1200 A B H + C O H + O CR 3 CH 2 S CH 2 CHO CH 2 O R 2 R 1 H peptide NCH NCH O CR 3 CH 2 S CH 2 CHO R 2 OCH 2 R 1 H peptide + H 2 O 2 Energy C17 C17 C15 1339.0 * [M+H] + [M+H] + y2 y3 y4 y5 y1 y°1 Relative intensity * m/z B NC O CR 3 CH 2 H peptide H + N-acyl-dehydroalanyl peptide ion S CH 2 CH O R 2 OCH 2 R 1 HO 2,3-diacyloxypropane sulfenic acid Neutral loss 200 400 600 800 1000 1200 1355.0 y2 y3 y4 y5 C15 C18 C16 C19 C20 dAGTGGK-NH 3 dAGTGGK Relative intensity Fig. 4. Staphylococcus aureus SA113 cells also have the N-acylated triacyl-forms of SitC lipoprotein. (A) SitC lipoprotein of exponentially growing S. aureus SA113 cells in BHI medium was prepared as in Fig. 1A. Among the observed N-terminal lipopeptides of SitC similar to those described in Table 2 (LB medium) a signal at m ⁄ z 1339.0 corresponding to C50-triacylated lipopeptides was analyzed by MALDI-TOF MS ⁄ MS. Asterisks indicate molecular ion peaks generated by the neutral loss of diacylthioglycerol. (B) Lipopeptides used in (A) were oxi- dized by spotting hydrogen peroxide solution on to the sample–matrix co-crystal before MALDI-TOF MS. MALDI-TOF MS ⁄ MS spectrum of the on-target oxidized C50-triacylated lipopeptide at m ⁄ z 1355.0 is shown. Signals marked with C15 to C20 indicate the N-acylated dehydro- alanyl peptide ions generated by neutral loss of 2,3-diacyloxypropane-1-sulfenic acid having different length fatty acids, as indicated by carbon numbers. Peaks designated dAGTGGK or dAGTGGK-NH 3 correspond to the dehydroalanyl peptide ion or its de-ammonium ion, respectively, which are secondarily generated from N-acyl-dehydroalanyl peptide ions by losing fatty acid. (C) Reaction scheme of H 2 O 2 oxidation and collision-induced dissociation. Incorporated oxygen, shown in bold, adds 16 Da to lipopeptides, such as from m ⁄ z 1339.0 (A) to m ⁄ z 1355.0 (B). The rectangle indicates dehydroalanine. R1, R2 and R3 indicate a hydrogen or acyl group. M. Asanuma et al. Triacylated lipoproteins in S. aureus FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 721 To prove the N-acylated structure of SitC from other than the RN4220 strain, N-terminal lipopeptide from the SA113 strain grown in BHI medium was ana- lyzed by MS ⁄ MS. Figure 4A shows the MS ⁄ MS spectrum of the peak at m ⁄ z 1339.0, corresponding to triacylated lipopeptide in which the sum of the carbon number for three fatty acids is 50. The y-series ions detected in the spectrum are essentially the same as those from the SitC lipopeptide from RN4220 (Fig. 1D), confirming that the peptide moiety of N-ter- minus is identical between the two strains. Weak peaks around m ⁄ z 750 corresponding to N-acyl-dehydroala- nyl peptide ions generated by the neutral losses of dia- cylthioglycerol moieties, the hallmark of N-acylation, were also detected in the spectrum (Fig. 4A). Because these characteristic peaks are usually weak, and some- times not detectable, we developed a method that can sensitively detect the neutral losses. Figure 4B shows MS ⁄ MS spectrum of the lipopeptide of SitC on-target oxidized with H 2 O 2 , in which intense peaks between m ⁄ z 712 and 782 were obtained. The increase in N-acylated dehydroalanyl peptide ions is explained by the fact that oxidized lipoproteins undergo facile neu- tral loss of 2,3-diacyloxypropane-1-sulfenic acid in MS ⁄ MS (or MALDI-MS) (Fig. 4C). The reaction mechanism should be similar to the neutral loss of methane sulfenic acid from methionine sulfoxide by MS ⁄ MS [29]. The result shown in Fig. 4B clearly dem- onstrates that a saturated fatty acid (from C15 to C20) is linked at the a-amino group of SitC. In addition to these S. aureus strains, analysis of SitC purified from S. epidermidis [26] gives a series of 14-Da interval peaks between m ⁄ z 2512 and 2597, cor- responding to the N-terminal lipopeptides of the triacy- lated SitC (Fig. 5A). Of these, the peak at m ⁄ z 2568.5, corresponding to triacylated CGNHSNHEHHSHEGK (the sum of the carbon number for three fatty acids is 53), was analyzed by MALDI-TOF MS ⁄ MS. The MS ⁄ MS spectrum shows the characteristic N-acyl dehyroalanyl peptide ions with C17 to C20 saturated fatty acid (Fig. 5B), and its elucidated structure strongly indicates that the S. epidermidis SitC lipopro- tein is also in the N-acylated triacyl form (Fig. 5C). Characterization of other lipoproteins of S. aureus Because SA2202 lipoprotein in S. aureus SA113 strain was reported to be diacylated with two palmitic acids rather than with different length fatty acids [11], we examined whether SA2202 in the SA113 strain was triacylated and also examined the composition of the fatty acids. Using the methods described above for 2568.49 2596.522554.45 2582.51 2540.44 2526.41 2512.41 2520 2540 2560 2580 2600 2620 Relative intensity C53 C52 C51 C50 C49 C54 C55 A m/z m/z y5 y6 y7 y8 y9 y10 y11 y12 y14 C18 C17 C19 C20 [M+H] + 2568.4 500 1000 1500 2000 2500 Relative intensity B C R 1 R 2 GN H N HE H H SHEGK y O N CH CH 2 CH S CH 2 CH 2 O O n = 20: 1968.2 19: 1954.5 18: 1940.3 17: 1926.3 R 3 H C R 1 : C l H 2l-1 O R 2 : C m H 2m-1 O R 3 : C n H 2n-1 O 1606.5 1435.5 1299.7 1212.2 1097.9 959.9 831.2 694.3 557.0 S Fig. 5. N-Acylated triacyl structure of S. epidermidis SitC. (A) MALDI-TOF mass spectrum of an organic phase of in-gel tryptic digest of SitC, isolated through TX114 phase extraction from expo- nential-growth phase S. epidermidis ATCC12228 cells in LB med- ium. The sum of carbon numbers for three fatty acids is labeled at the peaks corresponding to the monoisotopic mass values of the triacylated N-terminal lipopeptides of SitC (CGNHSNHEHHSHEGK). (B) MALDI-TOF MS ⁄ MS spectrum of the precursor ion of m ⁄ z 2568.5 (C53) shown in (A). Signals marked C17 to C20 indi- cate the N-acylated dehydroalanyl peptide ions generated by neutral loss and with different length saturated fatty acids with the indicated carbon numbers. (C) The elucidated structure of C53-tria- cylated N-terminal lipopeptides of S. epidermidis SitC with assign- ments of the observed fragment ions in panel B. The l, m and n are positive integers and l + m + n = 53. Triacylated lipoproteins in S. aureus M. Asanuma et al. 722 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS SitC in RN4220 strain, lipopeptides of SA2202 from the SA113 strain were prepared and analyzed by MALDI-TOF MS. As shown in Table 2, characteristic peaks corresponding to triacyl N-terminal lipopeptides of SA2202 with a total of 45 to 54 carbon numbers of three saturated fatty acids were detected in the MALDI-TOF mass spectrum of the in-gel tryptic digest itself and its organic phase extract, but diacyl lipopeptide signals were not. Like SitC, SA2202 pro- tein in the SA113 strain was modified, mainly with shorter fatty acids than in RN4220 (Tables 2 and 3) and peaks containing unsaturated fatty acids were also observed (data not shown). Figure 6A,B shows the MS ⁄ MS spectrum and the elucidated structure of the most abundant peak, corresponding to the oxidized C50-triacyl lipopeptides. The spectrum clearly showed the characteristic N-acyl-dehydroalanyl peptide ions with C15 to C20 saturated fatty acid, suggesting that the SA2202 lipoprotein in SA113 is the N-acylated triacyl form with different length fatty acids. We then asked whether other lipoproteins in the RN4220 strain were N-acylated. To address this, we searched for other lipoproteins in the TX114 phase of S. aureus RN4220. LC-MS ⁄ MS of the in-gel tryptic Table 3. Calculated and observed masses of the triacylated N- terminal lipopeptides of SA2202, SA0739, SA0771, SA2074, and SA2158 proteins isolated from exponentially growing S. aureus RN4220 cells grown in LB medium. Modified peptide Calculated [M+H] + Observed m ⁄ z D (ppm) C48 + CGNNSSK a 1498.02 1497.94 )53.2 C49 1512.04 1511.94 )63.0 C50 1526.05 1525.97 )53.0 C51 1540.07 1539.98 )56.2 C52 1554.08 1554.00 )52.8 C53 1568.10 1568.01 )55.9 C54 1582.11 1582.03 )52.6 C55 1596.13 1596.05 )49.4 C44 + CGHHQDSAK b 1715.08 1715.01 )42.8 C45 1729.10 1729.04 )33.9 C46 1743.11 1743.11 )1.4 C47 1757.13 1757.16 17.2 C48 1771.14 1771.14 1.3 C49 1785.16 1785.15 )5.6 C50 1799.17 1799.17 )1.2 C51 1813.19 1813.17 )11.7 C52 1827.20 1827.17 )19.1 C47 + CGNGNK c 1366.96 1366.95 )8.4 C48 1380.98 1380.98 2.1 C49 1394.99 1394.95 )30.6 C50 1409.01 1408.98 )20.1 C51 1423.02 1422.95 )51.9 C52 1437.04 1436.96 )55.3 C53 1451.06 1450.98 )51.7 C54 1465.07 1464.97 )68.7 C55 1479.09 1478.97 )78.6 C49 + CSNSNDNNESK d 2014.20 2014.15 )25.5 C50 2028.22 2028.16 )28.0 C51 2042.23 2042.22 )6.1 C52 2056.25 2056.24 )3.9 C53 2070.26 2070.24 )11.4 C54 2084.28 2084.27 )4.5 C55 2098.29 2098.26 )16.6 C45 + CGQDSDQQK e 1755.08 1755.11 16.6 C46 1769.10 1769.14 20.8 C47 1783.12 1783.13 8.5 C48 1797.13 1797.16 13.3 C49 1811.15 1811.15 0.5 C50 1825.16 1825.19 12.6 C51 1839.18 1839.17 )3.9 C52 1853.19 1853.18 )5.1 a SA2202, b SA0739, c SA0771, d SA2074, e SA2158. R 1 S CH 2 CHO OCH 2 R 2 C17 1542.0 [M+H] + y2 y3 y4 y5 C15 C18 C16 C19 C20 y6 200 600 1000 1400 m/z Relative intensity R 1 : C l H 2l-1 O R 2 : C m H 2m-1 O R 3 : C n H 2n-1 O O N CH O C CH 2 S H R 3 G N N S S K 234.0321.1435.1 y 549.1606.1 n = 20: 969.5 19: 955.5 18: 941.5 17: 927.5 16: 913.5 15: 899.5 O A B Fig. 6. Lipoprotein SA2202 of S. aureus SA113 shows N-acylated triacyl-forms. (A) MALDI-TOF MS ⁄ MS was carried out for N-termi- nal lipopeptides of SA2202 lipoprotein from exponentially growing SA113 cells as described in Fig. 4B. Precursor peaks used were molecular ions of the on-target oxidized C50-triacylated lipopeptide at m ⁄ z 1542.0. Signals marked with C15 to C20 indicate the N-acyl- ated dehydroalanyl peptide ions that were generated by neutral loss and had different length fatty acids with the indicated carbon num- bers. (B) Elucidated structures of the oxidized N-terminal triacyl lipo- peptides. R1, R2 and R3 indicate acyl group. The l, m and n are positive integers and l + m + n = 50. The value of n was deter- mined to be from 15 to 20 (A). Calculated mass values of the N-ter- minal triacyl lipopeptides of SA2202 are found in Table 2. M. Asanuma et al. Triacylated lipoproteins in S. aureus FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 723 digest of a minor band enabled us to identify five other lipoproteins, SA0739, SA0771, SA2074 (ModA), SA2158 and SA2202. To concentrate these lipoproteins, the Zn-stained lipoproteins on the acrylamide gels were collected and then eluted from gel pieces by simple dif- fusion, concentrated and resubjected to SDS ⁄ PAGE. The concentrated protein band was then in-gel digested. Using the methods described above for SitC, the result- ing lipopeptides were extracted by chloroform ⁄ metha- nol and analyzed by MALDI-TOF MS. Table 3 shows the observed and calculated masses of these lipopeptide peaks in each lipoprotein. A series of these peaks at an interval of m ⁄ z 14 in each protein corresponded to the calculated molecular mass values of triacylated N-terminal lipopeptides of each. In contrast, mass signals corresponding to diacylated lipopeptides were not observed (data not shown). Therefore, these five lipoproteins are suggested to be mainly triacylated. N-Acylation of these triacylated lipoproteins from RN4220 was further demonstrated by MALDI-TOF MS ⁄ MS (Fig. 7). Regarding SA2202 protein, Fig. 7A shows the MS ⁄ MS spectrum of the most abundant peak at m ⁄ z 1553.9, corresponding to the N-terminal tria- cylated CGNNSSK lipopeptide (the sum of the carbon number for three fatty acids was 52; see Table 3). The oxidized lipopeptides were also analyzed by MS ⁄ MS (shown as an inset in Fig. 7A). Both spectra represented the characteristic N-acyl-dehydroalanyl peptide ions due to neutral loss, whose signals were enhanced in the oxidized lipopeptides. The fatty acid linked to the a-amino group was a saturated fatty acid with a length of C16 to C20. Likewise, two lipoproteins (SA0739 and SA0771) were also successfully determined to be N-acyl- ated due to the detection of the N-acyl-dehydroalanyl peptides caused by the neutral losses using the oxidized lipopeptides (Fig. 7B,C). Figure 7D shows the MS ⁄ MS spectrum of the triacylated N-terminal lipopeptide of SA2074, which presents relatively week but significant peaks of the N-acyl-dehydroalanyl peptide ions (C15– C20) generated by the neutral loss of diacylthioglycerol, indicating N-acylation of the lipopeptide. An MS ⁄ MS spectrum of the triacylated lipopeptides of SA2158 pro- tein did not show significant signals because of the low intensity of the lipopeptide peaks. Discussion This study presents, for the first time, the structure of N-terminal lipids of native S. aureus lipoproteins. Here, we provide solid structural evidence for N-acylated triacyl forms of SitC and four other lipoproteins in S. aureus RN4220 using intensive MS-based analysis, combined with LPL or H 2 O 2 treatment. The triacylated lipoproteins were confirmed in other three S. aureus strains including SA113 and in one S. epidermidis strain, strongly suggesting that lipoproteins of S. aureus are mainly N-acylated triacyl forms. Lipoproteins in Firmicutes were thought to be diacylated because of the absence of E. coli-type Lnt in these bacteria [22]; the hypothesis was that immune cells could discrimi- nate between Gram-negative and Gram-positive bacte- ria by the ability of TLR2 to form heterodimers with 200 400 600 800 1000 1200 1400 m/z C B A HQ HQD y5 y4 y6 y7 y8 b5 b6 [M+H] + 1827.12 200 400 600 800 1000 1200 1400 1600 1800 Relative intensity C17 C18 1160 1200 1240 C15 C16 C19 C20 [M+H] + y3 y4 y6 y5 y2 y1 1553.9 C17 C18 900 940 980 C16 C19 C20 200 400 600 800 1000 1200 1400 Relative intensity C [M+H] + y2 y4 y5 y6 y7 y8 y10 y9 2043.37 200 400 600 800 1000 1200 1400 1600 1800 2000 Relative intensity [M+H] + y5 y4 y3 y2 y°1 1437.10 200 400 600 800 1000 1200 1400 Relative intensity m/z 800 840 880 C16 C17 C18 C19 C20 14401400 1480 C17 C16 C18 C19 C15 C20 D Fig. 7. Other N-acylated triacyl-lipoproteins of S. aureus RN4220 cells. MALDI-TOF MS ⁄ MS spectrum of N-terminal lipopeptides of SA2202 protein with C52 (A), SA0739 protein with C52 (B), SA0771 protein with C52 (C) or SA2074 protein with C51 (D) prepared from exponential-growth phase S. aureus RN4220 cells in LB as SitC of Fig. 1A. The mass of the precursor ion for each is described in Table 3. The inset in each panel is the MALDI-TOF MS ⁄ MS spectrum of on-target oxidized lipopeptides for each (A–C) or the magnified view (D). N-Acyl-dehydroalanyl peptide ions gener- ated by the neutral loss of 2,3-diacyloxypropane-1-sulfenic acid or diacylthioglycerol were observed and are indicated by C15 to C20 in the insets. Peaks designated with HQ and HQD in (B) are internal fragment ions. Triacylated lipoproteins in S. aureus M. Asanuma et al. 724 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS TLR1 or TLR6, in response to triacyl lipopeptides or diacyl lipopeptides, respectively [30]. However, our study provides some clear evidence that these predic- tions may need to be reconsidered. Contrary to our results, Hashimoto’s group reported that the N-terminus of SA2202 lipoprotein in S. aureus SA113 was only an S-dipalmitoylglyceryl cysteine form in S. aureus SA113 [11]. Discrepancies with our results are likely due to differences in cell-culture, lipoprotein preparation or analysis methods, because the SA113 strain we used was received from Hashimoto’s group. Differences in method resulted in several hundred-fold differences in the recovery of lipoprotein for structural analysis; their previous overall yield was only 1.6 lgof each lipoprotein per l-culture [11], whereas we obtained several hundred micrograms of SitC in our TX114 phase per l-culture (data not shown). In addition, we developed the analytical method including in-gel diges- tion and organic solvent extraction in the presence of 0.1% n-decyl-b-D-glucopyranoside (see Experimental procedures), which prevents the loss of very hydropho- bic lipopeptides. As a consequence, we detected a series of peaks modified with different length saturated and unsaturated fatty acids. Because phospholipids in bac- teria have various length fatty acids and Lgt transfers a diacylglycerol moiety from phospholipids to the lipo- protein precursor, it is reasonable to conclude that lipoproteins are modified with different length fatty acids. Therefore, detection of only the dipalmitoyl- glyceryl form in SA2202 [11] indicates insufficient sensi- tivity to detect heterogeneity of the acylation in their analysis. Further, we showed the predominance of tria- cylated SitC in cell lysate (Fig. 2). Therefore, we believe that we have obtained unbiased results, and that the major form of lipoproteins in S. aureus is the triacylat- ed one. Recently, a similar procedure with us allowed the detection of triacylated lipopeptides of LppX lipo- protein from M. smegmatis [9]. However, our results do not rule out the existence of diacylated lipoproteins in the bacterium. Because they are intermediate forms during biosynthesis of the triacylated lipoproteins, they are likely to be a minor component under our condi- tions. In fact, in addition to mass signals of the tria- cylated form, relatively weak signals corresponding to diacylated lipopeptides of SitC with various fatty acids were also detected from high-temperature culture (data not shown), suggesting that the degree of N-acylation may depend on bacterial growth conditions. Our results also suggest that unsaturated fatty acid was incorporated in lipoproteins of S. aureus SA113 strain. Although further analysis to determine the modification site(s) and molecular species of the unsat- urated fatty acid in bacterial lipoproteins is required, its roles in ligand recognition and receptor activation for TLR2 are curious. In addition to our studies, several reports provide indirect evidence of triacylated lipoprotein(s) in Firmi- cutes [13,14] and Mollicutes [16,17]. Although Firmi- cutes do not have an E. coli Lnt homolog [5–8], our results strongly suggest that S. aureus and also S. epide- rmidis have an unidentified enzyme which can catalyze the N-acylation of diacylated lipoproteins with a satu- rated fatty acid, whose structure is distinct from E. coli and M. smegmatis Lnt. N-Acylation of lipoproteins in E. coli is characterized as being required for lipoprotein localization machinery LolCDE-dependent release of outer membrane-specific lipoproteins from the inner membrane [3], and deficiency of Lnt is known to cause mislocation of lipoproteins [31]. Identification of the unidentified N-acylation enzyme in S. aureus should facilitate understanding of the biological significance of the lipoprotein N-acylation. Experimental procedures Bacterial strains, plasmids, and culture conditions The S. aureus SA113 strain was a gift from Dr M. Hashim- oto (Kagoshima University, Japan). The methicillin-resistant S. aureus MW2 strain and methicillin-sensitive S. aureus MSSA476 strain were obtained from the Network on Anti- microbial Resistance in Staphylococcus aureus (Chantilly, VA). The Staphylococcus epidermidis ATCC12228 strain was obtained from the Korean Culture Center of Microorgan- isms (Seoul, Korea). These S. aureus and S. epidermidis strains were grown in LB or BHI medium at 37 °C until late exponential-growth phase, as described previously [15]. Purification of lipoproteins by TX114 phase partitioning Lipoproteins were obtained using the TX114 phase-parti- tioning method, as described previously [15]. Briefly, the harvested S. aureus cells were disrupted with glass beads and centrifuged at 2000 g for 10 min to remove the glass beads and unbroken cells, and the resulting supernatant was further centrifuged at 20 000 g for 10 min to remove the peptidoglycan fraction. The obtained supernatant was used as a cell lysate, or was supplemented with TX114 to a final concentration of 2%, and then incubated at 4 °C for 1 h. The mixture was subsequently incubated at 37 °C for 10 min for phase separation. After centrifugation at 10 000 g for 10 min at 25 °C, the upper aqueous phase was removed and was replaced with the same volume of a TBSE solution (20 mm Tris ⁄ HCl, pH 8, 130 m m NaCl and 5mm EDTA). This procedure was repeated twice. The final M. Asanuma et al. Triacylated lipoproteins in S. aureus FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 725 [...]... protein identification used here were from S aureus strain N315 strain [7] Acknowledgements We thank Dr Masahito Hashimoto of Kagoshima University for providing S aureus SA113 strain and Dr Youngnim Choi of Seoul National University for help with analysis of TLR2-expressing CHO We also thank Mr Daisuke Higo of Thermo-Fischer Scientific and Dr Ryo Taguchi of University of Tokyo for help with MALDI-IT MS ⁄ MS... 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MALDI-TOF ⁄ TOF instrument (Ultraflex; Bruker Daltonics) using CHCA or 2, 5-dihydroxybenzoic acid as matrix Fragments of tryptic lipopeptides observed in MALDI MS ⁄ MS spectrum are mainly C-terminus-containing sequence ions (y-type) and less intense N-terminuscontaining ions (b-type) because positive charge tends to localize at C-terminal lysine or arginine of the peptides 726 On-target oxidation of lipopeptides... 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