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*
Department of Oral Bacteriology, Hokkaido University School of Dentistry, Sapporo, Japan; and
Department of Bio-material Chemistry, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by a monocytic cell
line, THP-1, and ICAM-1 expression and IL-6 production by human
gingival fibroblasts were detected in plural membrane lipoproteins of
Mycoplasma salivarium. Although SDS-PAGE of the
lipoproteins digested by proteinase K did not reveal any protein bands
with molecular masses higher than approximately10 kDa, these activities
were detected in the front of the gel. A lipoprotein with a molecular
mass of 44 kDa (Lp44) was purified. Proteinase K did not affect the
ICAM-1 expression-inducing activity of Lp44, but lipoprotein lipase
abrogated the activity. These results suggested that the proteinase
K-resistant and low molecular mass entity, possibly the N-terminal
lipid moiety, played a key role in the expression of the activity. The
N-terminal lipid moiety of Lp44 was purified from Lp44 digested with
proteinase K by HPLC. Judging from the structure of microbial
lipopeptides as well as the amino acid sequence and
infrared spectrum of Lp44, the structure of the N-terminal lipid moiety
of Lp44 was speculated to be S-(2,
3-bisacyloxypropyl)-cysteine-GDPKHPKSFTEWV-. Its analogue,
S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF, was
synthesized. The lipopeptide was similar to the N-terminal lipid moiety
of Lp44 in the infrared spectrum and the ICAM-1 expression-inducing
activity. Thus, this study suggested that the active entity of Lp44 was
its N-terminal lipopeptide moiety, the structure of which was very
similar to S-(2,
3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, it was found that lipoproteins in the cell membranes of Mycoplasma salivarium triggered the transcription of ICAM-1 mRNA in normal human gingival fibroblasts (HGF) and induced its cell surface expression by a mechanism distinct from that of Escherichia coli LPS (4). In addition, the lipid moiety of the lipoproteins was suggested to play a key role in the expression of the activity (4). M. salivarium is a member of oral microbial flora and inhabits preferentially in gingival sulci and is suspected to play an etiological role in some cases of oral infections including periodontal diseases (7, 8, 9). Chronic periodontal diseases are characterized by dense infiltrations of lymphocytes and macrophages in the connective tissue (10). Cell adhesion molecules are involved in the infiltration of activated leukocytes in inflammatory sites. Evidence for the importance of ICAM-1 in periodontal diseases has been accumulated (11, 12, 13, 14). Therefore, we are very much interested in the ICAM-1-inducing activity of the organism.
In this study, it was found that plural membrane-bound lipoproteins of M. salivarium were capable of inducing the ICAM-1 expression on the cell surface of HGF, and their active entities were N-terminal lipopeptide moieties. Furthermore, the membrane-bound lipoprotein of M. salivarium with a molecular mass of 44 kDa was characterized, and the putative structure of its N-terminal lipopeptide was partially defined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-cysteine (Pam3-cysteine) was purchased from Bachem AG (Bubendorf, Switzerland); and mAb to ICAM-1 (HA58) used for Cell-ELISA from PharMingen (San Diego, CA). All of the other chemicals were obtained from commercial sources and were of analytical or reagent grade.
Organism and culture conditions
M. salivarium ATCC 23064 was grown in pleuropneumonia-like organisms broth (Difco Laboratories, Detroit, MI) supplemented with 10% (v/v) horse serum (Life Technologies, Grand Island, NY), 1% (w/v) yeast extract (Difco), 1% (w/v) L-arginine-hydrochloride, 0.002% (w/v) phenol red, and penicillin G (1000 U/ml). When there was a rise in pH of 1 U, the cells were harvested by centrifuging the cultures at 15,000 x g for 15 min, washed three times with sterile PBS, and suspended in it.
Preparation of lipoproteins by the Triton X-114 phase separation
M. salivarium cells were treated with Triton X-114 to extract lipoproteins (TXLP) according to the method described previously (3). TXLPs in the Triton X-114 phase were precipitated by methanol and used for stimulation after being suspended in sterile PBS by light sonication. Protein concentration was determined by the method of Dully and Grieve (15).
Preparation of lipoproteins by using
n-octyl-
-glucopyranoside
Cell membranes of M. salivarium were prepared
according to the method described previously (16). The
lipoproteins were extracted from the cell membranes by using
n-octyl--glucopyranoside (OG) according to the method of
Mühlradt et al. (5) modified slightly. Briefly, a
10-ml volume of cell membrane suspensions (10 mg of protein/ml) was
treated twice with 10 ml of chloroform-methanol (2:1, v/v) at room
temperature. The delipidated interphase was freed of the organic
solvents in vacuo at 37°C and lyophilized to remove water. The
lyophilized material was suspended in 50 mM OG in PBS, sonicated,
treated for 6 min in boiling water, and centrifuged at 20,000 x
g for 30 min. The supernatant was collected, filtrated
through a 0.45-µm-pore-size filter, and used as lipoproteins
(OGLP).
ICAM-1 expression on HGF and cytokine production by THP-1 cells
HGF prepared and used in the previous study (4) were cultured in DMEM (Life Technologies) containing 10% (v/v) FBS (Life Technologies), penicillin G (100 U/ml), and streptomycin (100 µg/ml) in plastic culture dishes. In this study, HGF between passages 6 and 8 were used.
The activity to induce the expression of ICAM-1 on the cell surface of HGF was measured by the Cell-ELISA according to the method of Hayashi et al. (17). Briefly, 104 of HGF were seeded into a 96-well flat-bottom microplate. After HGF reached confluence, the cells were stimulated. The culture supernatant was collected and examined for IL-6 production by using a TiterZyme ELISA kit (PerSeptive Diagnostics, Cambridge, MA). The cells were fixed with 3% (w/v) paraformaldehyde in PBS supplemented with 8% (w/v) saccharose. Nonspecific binding was blocked by the addition of PBS containing 10% (v/v) horse serum. The cells were reacted with anti-ICAM-1 mAb (HA58) and then with peroxidase-conjugated goat anti-mouse IgG Ab. Peroxidase activity was measured by the addition of tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and stopped by the addition of equal volume of 1.8 M sulfuric acid. The OD at 450 nm was measured by using a microplate reader.
THP-1 cells, a human myelomonocytic cell line, were obtained from
Health Science Research Resources Banks (Osaka, Japan) and cultured in
RPMI 1640 culture medium (Life Technologies) containing 10% FBS,
penicillin G (100 U/ml), and streptomycin (100 µg/ml). After a 0.2-ml
vol of cell suspensions of THP-1 (5 x 105
cells) in each well of a 96-well tissue culture plate was incubated at
37°C for 15 h with OGLP (40 µg of protein) in the culture
medium supplemented with 1% (v/v) human serum, the culture supernatant
was collected by centrifugation at 400 x g for 10 min.
TNF- in the supernatant was determined by using a TiterZyme kit
(PerSeptive).
Identification of lipoproteins responsible for the ICAM-1-inducing activity by the monocyte Western blotting
Lipoproteins in TXLP or OGLP responsible for the ICAM-1-inducing activity were determined by the monocyte Western blotting described previously (18). SDS-PAGE of TXLP or OGLP was performed in 10% gel according to the method of Laemmli (19). Lipoproteins separated were transferred to 0.45-µm-pore-size nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The membranes were cut into a 4-mm strip and dissolved in 1 ml of DMSO. The Ag-coated particles were formed by the dropwise addition of 3 ml of 50 mM sodium carbonate buffer (pH 9.6) into the melted membrane, washed three times with PBS, and incubated with HGF confluent culture. The ICAM-1 expression was measured by the Cell-ELISA described above.
Purification of Lp44 (a lipoprotein with a molecular mass of 44 kDa) from SDS-PAGE gels
SDS-PAGE of OGLP was performed in 10% gel according to the method of Laemmli et al. (19). Lp44 was extracted from the gels according to the modified method of Hager and Burgess (20). Briefly, the gels were rinsed with distilled water (DW) and stained with ice-cold 0.25 M KCl and 1 mM DTT. Gel pieces containing Lp44 were cut out and soaked for 15 min in DW and 1 mM DTT. The gel pieces were crushed in 50 mM Tris-HCl buffer (pH 7.9) containing 0.1 mM EDTA, 5 mM DTT, BSA (0.1 mg/ml), and 0.15 M NaCl, incubated at 25°C for 2 h with agitation, and centrifuged at 2000 x g to pellet the crumbled gel. Lp44 was precipitated by adding 4 volumes of cold acetone prechilled at -20°C to the supernatant. The precipitate was dissolved in 6 M guanidium hydrochloride and dialyzed against DW. The resulting white aggregate was suspended in 20 mM OG in PBS by light sonication and incubated at 50°C for 30 min.
Amino acid sequence analysis
SDS-PAGE of the Lp44 extracted was performed in 10% gels. The proteins were blotted onto Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA) and stained with Coomassie brilliant blue. The Lp44 was excised and analyzed in an automated gas-phase procise sequencer model 492 (PE Biosystems, Foster City, CA).
HPLC and infrared (IR) spectrometry
HPLC was performed on preparative Nucleosil 120-7C18 column (10 x 300 mm) (Chemco Scientific, Osaka, Japan). The fractionation was done with the program: time zero, 5% N,N'-dimethylformamide (DMF)/95% water; at 15 min, 5% DMF/95% water; at 60 min, 5% DMF/95% 2-propanol; and at 70 min, 5% DMF/95% 2-propanol. The flow rate was 1.5 ml/min. Each fraction was dried in vacuo at 60°C, dissolved in 20 mM OG in PBS, and used for stimulation of HGF. IR absorption spectrum of the dried fractions in KBr pellet was measured with Foulier transform IR spectrometer (RT-210; Horiba, Kyoto, Japan). Pam3-cysteine was used as a standard.
Synthesis of S-(2,3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF
The side chain-protected GDPKHPKSF was built up with an automated peptide synthesizer, model 433 (Applied Biosystems, Foster City, CA). Fmoc-S-(2,3-bispalmitoyloxypropyl)-cysteine (Novabiochem, Laeufelfingen, Switzerland) was manually coupled to the peptide-resin by using a solvent system of 1-hydroxy-7-azabenzotriazole-1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/CH2Cl2-DMF. The Fmoc and resin were removed from the lipopeptide by trifluoroacetic acid. The lipopeptide was extracted into 90% acetic acid and lyophilized. The lipopeptide was purified by preparative HPLC with reverse-phase C18 column (30 x 250 mm). The purity (97.9%) was confirmed by analytical HPLC with reverse-phase C18 column (4.6 x 150 mm).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lipoproteins in TXLP responsible for inducing ICAM-1 expression on
the cell surface of HGF were analyzed by the monocyte Western blotting.
The activity was detected in plural lipoproteins with molecular mass
ranges of 40–50 kDa and 60–80 kDa (Fig. 1
). Previously, we found that lipoprotein
lipase abrogated the ICAM-1 expression-inducing activity of TXLP,
whereas proteinase K does not affect the activity of TXLP
(4). Therefore, the activity of TXLP digested by
proteinase K was also examined by the monocyte Western blotting.
SDS-PAGE of TXLP digested by proteinase K did not reveal any protein
bands with molecular masses higher than approximately10 kDa, but the
activity was detected in the front of the gel (Fig. 1). This result
suggests that proteinase K-resistant entities with molecular masses
lower than approximately 10 kDa common in plural lipoproteins play a
key role in the expression of the activity.
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by THP-1 cells
(unpublished data). Therefore, it was examined whether or not TXLP
possessed the cytokine production-inducing activity. TXLP was found to
induce TNF- production by THP-1 cells or IL-6 production by HGF
(Table I). Lipoproteins involved in the
cytokine production-inducing activity were also identified by the
monocyte Western blotting. The lipoprotein profile (Fig. 2A) with IL-6
production-inducing activity was similar to that (Fig. 2B)
with TNF- production-inducing activity. Also, each of them was
similar to that (Fig. 1
) with the ICAM-1 expression-inducing activity.
The cytokine production-inducing activity of TXLP digested by
proteinase K migrated to the front of the gel (Fig. 2, A and
B) as the ICAM-1 expression-inducing activity did (Fig. 1).
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Identification of a lipoprotein with a molecular mass of 44 kDa is one of the lipoproteins responsible for the ICAM-1 expression-inducing activity
OGLP extracted from the cell membrane of M. salivarium
by using OG were examined for the ICAM-1 expression-inducing activity
by the monocyte Western blotting. Lp44 was found to possess the
activity significantly higher than the others (Fig. 3). Therefore, to characterize Lp44, the
N-terminal amino acid sequence of Lp44 was examined. However, the
N-terminal amino acid of Lp44 was not determined, because any amino
acid peak significantly higher than the others was not observed in the
amino acid profile of the first cycle of Edmann degradation (Fig. 4). Amino acids after second were easily
identified, as shown in Fig. 4. This result suggests that the amino
group of the N-terminal amino acid is free. Judging from characteristic
of Edmann degradation, the N-terminal amino acid is speculated to be
cysteine. This is supported by the previous finding that the N-terminal
amino acid of lipoproteins from prokaryotes is cysteine, the Src
homology group of which is bound to lipid (21). Therefore,
the N-terminal amino acid sequence of Lp44 was speculated to be
CGDPKHPKSFTEWVA-. It was found by homology search with GenBank
databases that the amino acid sequence of Lp44 had not been reported
previously.
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Lp44 extracted from gels of SDS-PAGE was analyzed by SDS-PAGE. It
revealed one dense band with a molecular mass of 44 kDa and a faint
band with a higher molecular mass, possibly an aggregate of Lp44 (Fig. 5).
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),
suggesting that the lipid moiety, but not the proteinous moiety, is
involved in the expression of the activity. To purify the lipid moiety
of Lp44, Lp44 was digested with proteinase K and then fractionated by a
reverse-phased HPLC with a linear gradient of isopropanol. The lipid
moiety of Lp44 was eluted by about 90% isopropanol, because the ICAM-1
expression-inducing activity was detected in these
fractions (Fig. 6). These fractions were
dried in vacuo and applied to IR spectrometer to get some information
on the structure of the lipid moiety of Lp44. In the spectrometry,
Pam3-cysteine was used as a standard. The IR spectra of the lipid
moiety of Lp44 and Pam3-cysteine exhibited signals about 2900
cm-1 and 1700 cm-1, which
show the presence of fatty acid alkyl chains and typical ester bonds,
respectively (Fig. 7, A and
B). Lipid is known to be bound to Src homology group
of the N-terminal cysteine in lipoproteins from many microbes
(22). The structure of N-terminal lipid of murein
lipoproteins of E. coli is determined to be S-(2,
3-bispalmitoyloxypropyl)-N-palmitoyl-cysteine-SSNKIDELSSD-
(21). Furthermore, Mühlradt et al. (5, 6) identified a lipopeptide called MALP-2,
S-(2,3-bispalmitoyloxy-propyl)-cysteine-GNNDESNISFKEK, from
Mycoplasma fermentans which is capable of activating
macrophages/monocytes.
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). IR spectrum of FSL-1 also exhibited
signals about 2900 and 1700 cm-1 due to fatty
acid alkyl chains and the ester bond as those of the HPLC-purified
lipid moiety of Lp44 and Pam3-cysteine (Fig. 7C). In
addition, the activity of FSL-1 possessed a strong activity to induce
the ICAM-1 expression on the cell surface of HGF (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mycoplasmal active entities responsible for the activation of monocytes/macrophages have not been identified until Mühlradt et al. (5, 6) purified and characterized a 2-kDa lipopeptide, MALP-2, from M. fermentans. This study indicated that Lp44, one of plural membrane-bound lipoproteins, was an active entity capable of activating fibroblasts, and its active site was the N-terminal lipopeptide moiety, the structure of which was similar to MALP-2. Therefore, lipopeptides with the N-terminal cysteine residue bound to a diacylated glyceride residue through a thioester linkage may be a potent activator of mammalian cells with some universal cell surface receptor for them on the cell surface. The universal cell surface receptors are considered to be Toll-like receptor 2, because it has very recently been reported that inflammatory signaling by bacterial lipoproteins and MALP-2 is mediated by Toll-like receptor 2 (23, 24, 25, 26).
The relationship of the structure of N-terminal lipopeptides of
prokaryote lipoproteins and their biological activities has been
partially defined (27, 28, 29). The N-terminal structure of
E. coli murein lipoprotein, which is by far the most
frequently present lipoproteins in bacteria (22), is
S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-cysteine-SSNKIDELSSD-
(Pam3-cysteine-SSNKIDELSSD-) (21). The lipopeptides
synthesized based on Pam3-cysteine-SSNKIDELSSD- are known to activate
macrophages to induce the production of IL-1, IL-6, and TNF-
(22). A well-defined series of analogues such as
Pam3-cysteine-SSNK, Pam3-cysteine-SSN, Pam3-cysteine-SS,
Pam3-cysteine-S, and Pam3-cysteine are synthesized and examined for the
activity to stimulate B lymphocytes (27). The lipopeptides
carrying 2–5 aa exhibit strong stimulation activity comparable with
native murein lipoprotein (27). In contrast, the
lipopeptides containing only 1 aa are marginally active, suggesting
that the presence of dipeptide structure is necessary for the
expression of full biological activity (27). Lipopeptides
containing two ester-bonded palmitoyl residues exhibited more potent
mitogenic activity toward murine splenocytes than the lipopeptide
containing one ester-bonded palmitoyl residue (28).
Lipopeptides containing two ester-bonded palmitoyl residues and a free
N terminus exhibit more potent activity toward murine splenocytes than
the lipopeptides containing three palmitoyl residues and the
N-terminally elongated lipopeptides (29). Nonlipidated
MALP-2 and Pam3-cysteine fail to activate monocytes/macrophages
(30).
These findings suggest that both peptide and fatty acid portions of microbial lipopeptides are indispensable for the expression of their biological activities. This speculation is also supported by the present finding that Pam3-cysteine and tripalmitin did not possess the activity to induce the ICAM-1 expression on the cell surface of HGF. Furthermore, mycoplasmal lipoproteins are speculated to be a more potent activator of monocytes/macrophages or fibroblasts than bacterial lipoproteins, because the amino group of the N-terminal cysteine of mycoplasmal lipoproteins or lipopeptides like MALP-2 and the N-terminal lipid moiety of Lp44 is free, whereas that of lipoproteins of many bacteria is bounded to some fatty acid.
Lipoproteins or lipopeptides of E. coli, Treponema pallidum, and Borrelia burgdorferi activate monocytes/macrophages, and the activity resides in their N-terminal lipopeptide moieties (31, 32). As described above, lipoproteins with the structure analogous to E. coli murein lipoproteins exist in many bacterial species (22).
Taken together, it is very likely that microbial lipoproteins play important etiological roles in diseases caused by these microbes. Especially, lipoproteins of wall-less microbes such as mycoplasmas might play more important roles, because they interact directly with host cells.
M. salivarium, a member of oral microbial flora, is suspected to play some etiological role in some cases of oral infections, including periodontal diseases (7, 8, 9), but its etiological roles remain unknown. The present finding that the organism possesses membrane-bound lipoproteins capable of activating monocytes/macrophages and HGF may give a clue to clarify etiological roles in oral infections, especially periodontal disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ken-ichiro Shibata, Department of Oral Bacteriology, Hokkaido University School of Dentistry, Nishi 7, Kita 13, Kita-Ku, Sapporo 060-8586, Japan.
3 Abbreviations used in this paper: MALP-2, macrophage-activating lipopetide; DMF, N,N'-dimethylformamide; DW, distilled water; HGF, human gingival fibroblasts; IR, infrared; Lp44, a lipoprotein with a molecular mass of 44 kDa; OG, n-octyl--glucopyranoside; OGLP, lipoproteins obtained from M. salivarium cell membranes; Pam3-cysteine, S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-cysteine; TXLP, lipoproteins obtained from M. salivarium cells by Triton X-114 phase separation.
Received for publication April 24, 2000. Accepted for publication September 6, 2000.
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M. Fujita, T. Into, M. Yasuda, T. Okusawa, S. Hamahira, Y. Kuroki, A. Eto, T. Nisizawa, M. Morita, and K.-i. Shibata Involvement of Leucine Residues at Positions 107, 112, and 115 in a Leucine-Rich Repeat Motif of Human Toll-Like Receptor 2 in the Recognition of Diacylated Lipoproteins and Lipopeptides and Staphylococcus aureus Peptidoglycans J. Immunol., October 1, 2003; 171(7): 3675 - 3683. [Abstract] [Full Text] [PDF]
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T. Into, M. Fujita, T. Okusawa, A. Hasebe, M. Morita, and K.-I. Shibata Synergic Effects of Mycoplasmal Lipopeptides and Extracellular ATP on Activation of Macrophages Infect. Immun., July 1, 2002; 70(7): 3586 - 3591. [Abstract] [Full Text] [PDF]
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G. H. Waetzig, D. Seegert, P. Rosenstiel, S. Nikolaus, and S. Schreiber p38 Mitogen-Activated Protein Kinase Is Activated and Linked to TNF-{alpha} Signaling in Inflammatory Bowel Disease J. Immunol., May 15, 2002; 168(10): 5342 - 5351. [Abstract] [Full Text] [PDF]
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A. Hasebe, K.-I. Shibata, and T. Watanabe A 4.1-Kilodalton Polypeptide in the Cultural Supernatant of Mycoplasma fermentans Is One of the Substances Responsible for Induction of Interleukin-6 Production by Human Gingival Fibroblasts Infect. Immun., November 1, 2001; 69(11): 7173 - 7177. [Abstract] [Full Text] [PDF]
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