Binding of glycoglycerolipid derived from membranes of Acholeplasma laidlawii PG8 and synthetic analogues to lymphoid cells

Saori Toujima1, Koichi Kuwano1, Ye Zhang1, Naoyuki Fujimoto1, Masahiro Hirama2, Tohru Oishi2, Sumiko Fukuda2, Yoko Nagumo2, Hiroto Imai2, Tsukasa Kikuchi2 and Sumio Arai1

Department of Bacteriology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan1
Department of Chemistry, Graduate School of Science, Tohoku University Aoba, Aramaki aza, Aoba-ku, Sendai 980-8578, Japan2

Author for correspondence: Sumio Arai. Tel: +81 942 31 7548. Fax: +81 942 31 0343. e-mail: sumiyuku{at}mx2.tiki.ne.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A component that binds to human lymphoid cells was isolated from the membranes of Acholeplasma laidlawii PG8. The component was extracted using the Bligh–Dyer method and purified using a silica-gel column and TLC. The active component was identified as 3-O-[2'-O-({alpha}-D-glucopyranosyl)- 6'-O-acyl-{alpha}-D-glucopyranosyl]-1,2-di-O- acyl-sn-glycerol (GAGDG) using 1H- and 13C-NMR and GC-MS. The compositions of the major saturated fatty acids were nC 14 (17·8%), isoC14 (10·7%) and nC 16 (34·9%) as determined by GC-MS. The amounts of unsaturated species were less than 10% of those of the corresponding saturated acids. GAGDGs which have three tetradecanoyl groups were synthesized. These synthetic GAGDGs, as well as GAGDGs derived from A. laidlawii membranes, had a high binding affinity for MOLT-4 and HUT-78 (human T cell lines), Raji (a B cell line), HL-60 (a monoblastoid cell line) and primary cultured human T cells. The binding affinities of GAGDGs with an isoC14 acyl group was higher than those with nC14 and nC16 acyl groups. The binding to lymphoid cells reveals a novel biological activity of GAGDGs.

Keywords: Acholeplasma laidlawii, adherence, glycoglycerolipids, lymphoid cells

Abbreviations: GAGDG, 3-O-[2'-O-({alpha}-D-glucopyranosyl)- 6'-O-acyl-{alpha}-D-glucopyranosyl]-1,2-di-O- acyl-sn-glycerol


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The first stage in many bacterial infections is adherence and formation of microcolonies on mucous membranes. Various components on the outer surface of bacteria, such as protein, polysaccharides and teichoic acid, are known to contribute to adherence (Espersen & Clemmensen, 1982 ; van Houte 1983 ; Elleman et al., 1986 ; Courtney et al., 1986 ). However, ß-lactam antibiotics, which inhibit cell wall synthesis, can induce the formation of L-form variants (Allan et al. , 1993 ). Glycoglycerolipids are found not only in the cell membranes of mycoplasmas, but also in those of Gram-positive and Gram-negative bacteria (Kates, 1990 ). Although there have been many investigations into the role of microbial glycoglycerolipids in the membrane physiology of prokaryotic cells (Shaw, 1970 ; Silvius et al., 1980 ; Boggs 1987 ), we have no knowledge of interactions of glycoglycerolipids with eukaryotic cells. It is important in investigating the pathogenesis of bacteria lacking cell walls that such factors which may influence attachment to host cells be studied.

The aim of the work described in this paper was to examine the capacity of glycoglycerolipids isolated from the membranes of Acholeplasma laidlawii, and of chemically synthesized glycoglycerolipids, to bind to human lymphoid cells.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells.
Two human T-cell lines, MOLT-4 and HUT-78, a human B cell line, Raji, and a human monoblastic cell line, HL60, were maintained in exponential growth in RPMI1640 medium containing 10% foetal calf serum (FCS; Mitsubishi Chemical), 2 mM L-glutamine, 100 µg penicillin G ml-1 and 100 µg streptomycin ml-1 (RPMI/FCS). To exclude the possibility of mycoplasma infections in the cell cultures, these cell suspensions were inoculated onto Hayflick’s mycoplasma agar medium once a week.

Peripheral mononuclear cells from healthy donors were isolated using Lymphoprep (Nycomed Pharma AS Diagnostics) as described previously (Arai et al., 1983 ). Five million cells ml -1 were incubated in a Petri dish at 37 °C for 1 h to remove adherent cells. Nonadherent cells were collected and incubated with M-450 CD4 Dynabeads (Dynal) for 3 h at 37 °C, and CD4+ cells were then collected according to the manufacturer’s instructions. The purified CD4+ cells were cultured in RPMI1640 with 12% FCS, 100 units human recombinant interleukin-2 (Becton Dickinson Labware) ml-1 and 100 units of human recombinant interleukin-4 (Genzyme Diagnostics) ml-1 in a 24-well cell culture plate (Costar) coated with anti-human CD3 antibody (Serotec).

Cell binding assay.
Extracts from the membranes of A. laidlawii and synthetic 3-O-[2'-O-({alpha}-D-glucopyranosyl)- 6'-O-acyl-{alpha}-D-glucopyranosyl]-1,2-di-O-acyl-sn-glycerols (GAGDGs) were dissolved in chloroform and 50 µl samples were spread on individual 10 mm diameter coverglasses (Matsunami) to make thin films of GAGDGs. To avoid nonspecific adherence of cells, each coverglass was immersed in RPMI/FCS medium. After 1 h, the medium was removed by suction and replaced with serum-free tissue culture medium (Cosmo medium; Cosmo Bio). Cells were suspended in Cosmo medium at a concentration of 4x105 cells ml -1 and 0·5 ml of the suspension was added to each well of a 24-well cell culture plate with a GAGDG-coated coverglass. The plate was then incubated at 37 °C in a CO2 incubator for 4 h. The coverglass was then removed with forceps and gently dipped twice in warmed PBS (137 mM NaCl, 2·68 mM KCl, 1·47 mM KH2PO4 , 8·1 mM Na2HPO4). The adherent cells on the coverglass were scraped off with a rubber policeman. The cell suspensions from five coverglasses were centrifuged at 170 g for 5 min and the cells were resuspended in 0·2 ml RPMI/FCS. The cells were counted with a haemocytometer. For detection of adherence to A. laidlawii, 0·1 ml of a 1x107 c.f.u. ml -1 suspension of A. laidlawii was inoculated into each well of a cell culture plate and incubated in 1 ml RPMI/FCS medium for 3 d at 37 °C in a CO2 incubator. The medium was then removed and suspensions of eukaryotic cells in Cosmo medium were added to different wells. The adherent cells were counted as described above. An alternative method for estimation of cell numbers was also employed. Briefly, after removing nonadherent cells, residual cells on the coverglasses were incubated for 4 h at 37 °C in a CO2 incubator in the presence of 1 µCi (37 kBq) [6-3H]thymidine [specific activity 16·3 Ci (603 GBq) mmol-1 ; Dupont NEN]. The cells were lysed, DNA was precipitated onto glass filters and the amount of [6-3H]thymidine incorporation was determined. Both methods for estimation of numbers of binding cells were used in five independent experiments.

Fractionation of A. laidlawii membranes.
Membranes of A. laidlawii PG8 ATCC 23206 were isolated by osmotic lysis as described previously (Iyama et al., 1996 ). The membranes of A. laidlawii were extracted twice using the Bligh–Dyer method with 40 vols water/chloroform/methanol (0·8:2:1, by vol.). Extracted lipids were dried on a rotary evaporator. Fifty milligrams of the lipid was resuspended in chloroform for application to a column of 10 g activated silica gel (Silica gel 60; Merck) prepared in chloroform. Sequential elution of the lipids from the column was performed with a discontinuous gradient starting with 400 ml of chloroform and followed by 400 ml each of the following mixtures of chloroform/methanol (v/v): 95:5, 90:10, 80:20, 70:30, 60:40, and finally 400 ml methanol. The fraction showing binding activity was further purified by TLC (Silica gel 60, 1·2 mm in thickness) using chloroform/methanol/acetic acid (85:15:0·5, by vol.).

Structure determination by NMR and GC-MS.
The structure of a component of the membranes of A. laidlawii with binding activity was determined by 13C-NMR, 1 H-NMR and GC-MS analysis. Detailed one- and two-dimensional NMR studies of +39 °C (c 0·1, CHCl3)} by 1H-1H DQFCOSY, HOHAHA ({tau}m=60 ms) and HMQC experiments with a JEOL {alpha}-500 spectrometer in CHCl3-CH3OD (1:1, v/v) allowed sequential assignments of proton and carbon resonances. Molecular ion peaks of the fatty acid methyl esters from lipid X by GC- MS indicated that the fatty acyl groups of the lipid were a mixture of straight- and branched-chain fatty acids. The lipids (0·7 mg in 0·5 ml methanol) were treated with 15 µl 140 mM sodium methoxide in methanol and the resulting fatty acid methyl esters were analysed by GC-MS equipped with a 25 m PEGM capillary column and electron ionization-MS spectrometry (Huang & Anderson, 1995 ).

Synthesis of GAGDGs.
Synthesis of glycoglycerolipids essentially followed a method established by van Boeckel & van Boom (1985a , b , c ).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Purification of the active component from A. laidlawii membranes
The adherence of MOLT-4 cells to coverglasses bearing colonies of A. laidlawii or glycolipids extracted from A. laidlawii using the Bligh–Dyer method was tested. The number of adherent cells was determined by both direct counting of cell numbers and [6-3H]thymidine incorporation by adherent cells. The numbers of cells binding in the presence of A. laidlawii, their membranes, the chloroform/methanol phase, the water/methanol phase and in the negative control were 1·5±0·9x104, 1·3±0·1x104, 4·5±0·5x104, 0·3±0·1x104 and less than 0·1x104 per coverglass, respectively. The amounts of [6-3H]thymidine incorporated by adherent cells in the presence of these fractions, were 2·5±0·07x104, 2·8±0·05x104, 12·9±0·09x104, 0·8±0·01x104 and 0·08±0·03x104 c.p.m., respectively. These results indicated that the binding activity was in the chloroform/methanol phase, but not in the water/methanol phase. Further purification of the active component in the chloroform/methanol phase was performed using silica gel column chromatography. As shown in Table 1, binding activity was clearly demonstrated in the fraction eluted with chloroform/methanol (90:10, v/v), but not in the other six fractions. The component showing the binding activity was further purified by TLC. Two spots of RF 0·36 and RF 0·64 were separated by development with chloroform/methanol/acetic acid (85:15:0·5, by vol.). Both spots were scraped off and eluted with chloroform/methanol (1:1, v/v). The activity was clearly found in the spot with R F 0·36.


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Table 1. Binding activity of fractions of A. laidlawii membrane extracts separated by silica-gel chromatography

 
Chemical structure of active natural components and their synthetic compounds
The chemical structure of the active component separated by TLC was determined by 1H-NMR, 13C-NMR and GC-MS. The chemical structure of synthetic compounds was also examined. As shown in Figs 1 and 2(a), the two glucose units were readily assigned by sequential large axial-axial couplings for the pyranose methine protons of H2' to H5' (3·58, 3·74, 3·31, 3·75 p.p.m.) and H2' to H5' (3·40, 3·67, 3·29, 3·85 p.p.m.). Five spin systems for the glycerol protons were also easily recognized (Fig. 2a). HMQC spectra clearly showed 13C assignments for the glucosyl and glycerol units, C-1 to C-3 (66·6, 70·8, 63·5 p.p.m., C-1' to C- 6' (97·2, 77·4, 72·6, overlapped with methanol peak, 70·7, 64·4 p.p.m.) and C-1' to C-6' (97·7, 72·8, 74·3, overlapped with methanol peak, 73·2, 62·2 p.p.m.) (Fig. 2b) . Also, 1H resonance at 0·79–0·86 (m, Me), 1·17–1·31 (m, methylene), 1·54–1·62 (6H, m, CH2CH 2CO) and 2·27–2·34 p.p.m. (6H, m, CH 2CO) (Fig. 2c) and 13C resonance at 174·3, 174·8 and 175·0 p.p.m. (Fig. 2b) indicated three fatty acids attached to the diglucosyl glycerol. Downfield shift of the five proton resonance at H-2 (5·18–5·23 p.p.m.) and H-3 (4·21, 4·35 p.p.m.) of the glycerol moiety as well as the hydroxymethyl resonance H-6' (4·21, 4·35 p.p.m.) of one glucosyl unit implied that the three acyl groups were located on the C-2, C-3 and C-6 hydroxyl groups. A ROESY spectrum ({tau}m=200 ms) showed a signal cross peak between the two sugar moieties [H-2' (3·76 p.p.m.) and H-1' (4·92 p.p.m.)]. The coupling constant of the anomeric proton H-1' was 4·0 Hz. These observations indicated that the two glucosyl units had an {alpha}-glycoside linkage between C-2' and C- 1'. Another anomeric proton H-1' (4·96 p.p.m.) also showed a small coupling constant (3·7 Hz). Therefore, the anomeric carbon (C-1') also had an {alpha}-linkage. The structure of the lipid was determined as 3-O-[2'-O -({alpha}-D-glucopyranosyl)-6'-O-{alpha}- D-glucopyranosyl-1,2-di-O-acyl-sn-glycerol (GAGDG, Fig. 1). The spectra of the natural compound were identical to the spectra of the synthetic compound except for that for the acyl groups. As shown in Table 2, the compositions of the major saturated fatty acids nC14, isoC14, nC16 were 17·8, 10·7 and 34·9%, respectively, as determined by GC-MS. The amounts of the unsaturated species were less than 10% of those of the corresponding saturated acids.



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Fig. 1. Chemical structure of glycoglycerolipid isolated from membranes of A. laidlawii.

 


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Fig. 2. Assignments for the compounds obtained by TLC (natural) and their synthetic analogues (synthesized). (a, c) 1H-NMR, 500 MHz; (b) 13C-NMR, 125 MHz.

 

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Table 2. Fatty acid composition of the lipid side chain as determined by GC-MS

 
The binding activity of synthetic GAGDGs
As shown in Fig. 3(a), the number of adherent cells was directly related to the concentration of GAGDGs. However, GAGDGs with nC 14 and nC16 had a lower binding activity compared with those with isoC14. As shown in Fig. 3(b), the adherence of cells began within 30 min and reached a maximum at 6 h.



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Fig. 3. (a) Effect of GAGDG concentration on binding to MOLT-4 cells. Cells (20x104) were incubated in a culture plate in the presence of a coverglass with synthetic GAGDGs containing isoC14 ({bullet}), nC14 ({circ}) or nC 16 ({square}), or GAGDGs of A. laidlawii membranes ({blacksquare}) for 4 h at 37 °C. Values shown are means+SD of five assays. (b) Cell attachment over time. MOLT-4 cells (20x104 ml-1) were incubated in a culture plate in the presence of a coverglass coated with 10 µg of synthetic GAGDGs containing isoC14 ({bullet}), nC14 ({circ}) or nC 16 ({square}), GAGDGs of A. laidlawii membranes ({blacksquare}) or an uncoated control ({triangleup}). Values shown are means+SD of five assays from one representative experiment.

 
Adherent cells were not observed in the absence of GAGDG. Table 3 shows one set of representative results from five experiments on the adherence of various cells to GAGDGs. The number of adherent cells was highest for Raji cells, but the difference between the other types of cells was not significant.


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Table 3. Binding of GAGDGs to various lymphoid cells

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study we have shown that glycoglycerolipids obtained from A. laidlawii membranes and synthetic glycoglycerolipids bind to both human lymphoid cell lines and human peripheral T cells. Our results provide new information on the interaction between glycoglycerolipids of prokaryotic cell membranes and eukaryotic cells.

Glycoglycerolipids are not only found in the cell membranes of mycoplasmas, but also in those of Gram-negative and Gram-positive bacteria, and their L-forms (Kates, 1990 ). However, their function in the membranes of prokaryotic cells is not clear. Several investigators have reported that glycolipids in the membranes of A. laidlawii are mono- and di-glycosyldiacylglycerol (Bhakoo et al., 1987 ; Dahlqvist et al., 1995 ; Gross & Rottem, 1979 ; Shaw 1968 ) and that they might play an important role in physiological functions such as membrane viscosity (De Kruyft et al., 1973 ; McElhaney et al., 1970 ; McElhaney, 1975 ; Rottem et al., 1973 ). Several reports suggest that the fusion of mycoplasmas with eukaryotic cells may result in the delivery of mycoplasma components into the host cells (Toole & Lowdell, 1990 ; Franzoso et al., 1992 ). Furthermore, Rottem (1980) reported that the presence of cholesterol in small unilamellar vesicles is required to allow their fusion with mycoplasmas. These findings suggest that GAGDGs, a major component of A. laidlawii membranes (Shaw, 1970 ; Silvius et al., 1980 ), might play an important role not only in the membrane fluidity of prokaryotic cells, but also in interactions between micro-organisms lacking cell walls and eukaryotic cells.

Our results strongly suggest that glycoglycerolipids in A. laidlawii membranes might participate in the adhesion of bacterial cells to eukaryotic cells. The role of glycoglycerolipids in the pathogenesis of A. laidlawii infections is unknown. The induction of conformational changes in the membranes of eukaryotic cells as well as in those of prokaryotic cells by glycoglycerolipids may be responsible for these biological activities. In this study, we have clearly demonstrated that glycoglycerolipids possess the ability to bind to certain kinds of eukaryotic cells. However, we need to clarify the initial binding sites and the significance of this phenomenon in infections.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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van Boeckel, C. A. A. & van Boom, J. H. (1985b). Synthesis of phosphatidyl-ß-glucosyl glycerol containing a dioleoyl diglyceride moiety. Application of the tetraisopropyldisiloxane-1,3-diyl (tips) protecting group in sugar chemistry, part IV. Tetrahedron 41, 4557-4565.

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Received 7 January 2000; revised 10 May 2000; accepted 23 May 2000.



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