Sep 5, 1990 - Nystrom, S., K.-E. Johansson, and A. Wieslander. 1986. Selec- tive acylation of membrane proteins in Acholeplasma laidlawii. Eur. J. Biochem.
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0019-9567/91/030781-04$02.00/0 Copyright © 1991, American Society for Microbiology
Palmitoylated Proteins in Ureaplasma urealyticum D. THIRKELL,* A. D. MYLES, AND W. C. RUSSELL
Department of Biochemistry and Microbiology, University of St. Andrews, Irvine Building, North Street, St. Andrews, Fife KY16 9AL, Scotland Received 5 September 1990/Accepted 1 December 1990
After incubation of Ureaplasma urealyticum serotype 8 in the presence of 3H-labeled palmitic acid, about 25 acylated proteins were detected by electrophoresis and fluorography. Of these, at least six were shown to be antigenic by immunoprecipitation of solubilized palmitate-labeled cells with a homologous polyclonal serum. These six included the serotype 8-specific surface-expressed 96-kDa antigen. After phase partition of palmitate-labeled cells with Triton X-114, all but six acylated proteins partitioned entirely into the detergent phase. The others, including the 96-kDa antigen, partitioned preferentially into the detergent phase and were apparently amphipathic. These results are consistent with the acylated proteins being mainly membrane associated.
Ureaplasma urealyticum is the mycoplasma most commonly isolated from the human urogenital tract, where it may be a commensal organism or a pathogen (14). Because they are wall-less, membrane components, particularly membrane proteins, must fulfill important functions. Information on individual membrane proteins is sparse, but the possibility exists that some may be modified by acylation. Fatty acid acylation of eukaryotic cell proteins is well documented and was reviewed by Magee and Schlesinger (9). Prokaryotic membranes of some mycoplasmas have been shown to contain a larger fraction of acylated proteins than other bacteria or eukaryotic cells (18), and such modified proteins have been reported in a number of members of the class Mollicutes (2, 4, 5, 11, 19). From work with chicken embryo fibroblasts, it was shown that while several surface-oriented glycoproteins were palmitoylated, proteins which were myristylated appeared to be internal (8). The evidence also suggested that palmitic acid is probably linked to protein by thioester bonds to cysteine side chains, whereas myristic acid is probably linked to protein through amide bonds. With the assumption that there is preferential acylation of membrane proteins with palmitic acid, this investigation was undertaken to determine whether there was any evidence of palmitoylation of proteins in U. urealyticum and, if so, whether any such proteins were antigenic.
(CCU)/ml (the determination of CCU has been described elsewhere [16]). Incubation was carried out at 37°C until the pH of the medium reached 7. 1, after which labeled cells were pelleted by centrifugation (20,000 x g, 20 min, 4°C) and washed once with phosphate-buffered saline (PBS) (7), and the final pellet was suspended in 500 RI of PBS. This preparation was used immediately or stored at -20°C. SDS-PAGE and fluorography. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously (15) with 15% (wt/vol) polyacrylamide slab gels. All samples for electrophoresis were boiled (2 min) after addition of an equal volume of a solution containing 5 M urea, 2% (wt/vol) SDS, 3.5 M f3-mercaptoethanol, and 0.1% (wt/vol) bromophenol blue (denaturing mix). For fluorography, electrophoresed gels were fixed in 10% (vol/vol) acetic acid-40% (vol/vol) methanol-50% (vol/ vol) distilled water (20 min), washed in 50 ml of dimethyl sulfoxide (three times for 30 min each), and then soaked in 22% (vol/vol) 2.5-dimethyloxazole in dimethyl sulfoxide (3 h). Gels were then placed under running cold water (30 min), dried under vacuum (40°C, 2 h), and exposed to X-ray film (Kodak fast film) at -70°C for up to 3 months. Otherwise, gels were stained with silver stain (Bio-Rad) according to the supplier's instructions. Apparent molecular masses of labeled proteins were determined, where appropriate, by comparison with protein standards of known molecular mass (Bethesda Research Laboratories Inc.). Phase partitioning of labeled cells with Triton X-114. The method of Bordier (1) was modified to include centrifugation stages (13). Sonicated cell suspension (0.2 to 1.0 mg of protein per ml) was prepared in 200 p1 of buffer B (10 mM Tris hydrochloride [pH 7.4], 150 mM NaCl, 1% [vol/vol] Triton X-114 [Sigma]) and maintained on ice (10 min). Residual particulate material was removed by centrifugation (2,500 x g, 3 min), and the supernatant was overlaid on 200 RI of buffer B containing 6% (wt/vol) sucrose and 0.1% (vol/vol) Triton X-114 in an Eppendorf tube. After incubation (30°C, 3 min) and centrifugation (300 x g, 3 min), a small oily droplet pelleted; the upper aqueous supernatant was removed and made to 0.5% (vol/vol) Triton X-114, and the phase partition was repeated. The final aqueous phase was made 2% (vol/vol) Triton X-114; after phase separation, the resultant detergent phase was discarded. With buffer and Triton X-114, both final detergent and aqueous phases were
MATERIALS AND METHODS Ureaplasma strain. U. urealyticum serotype 8 (T960) was a gift from D. Taylor-Robinson (Clinical Research Centre,
Harrow, U.K.). Labeling of U. urealyticum with palmitic acid. Labeling was carried out as follows. To 100 ml of medium (94.5 ml of PPLO broth [Difco Laboratories], 1.7 ml of horse serum [Northumbria Biologicals Ltd.], 3.4 ml of freshly prepared yeast extract, incorporating 0.1% [wt/vol] phenol red, 103 IU of penicillin G [Glaxo] per ml, and 20 mM HEPES [4-(2hydroxyethyl)-l-piperazine-ethanesulfonic acid; Sigma] at a starting pH of 6.0), 500 R1 of 9.10 (n)-[3H]palmitic acid was added, and the medium was inoculated with 100 ,ul of a U. urealyticum culture containing 106 color change units *
Corresponding author. 781
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made to the same volume (200 RI) and to 1% (vol/vol) Triton X-114. Both the final detergent and aqueous phases were subjected to SDS-PAGE and fluorography. Antibody preparation. The production of a polyclonal serum against U. urealyticum serotype 8 (12), of a polyclonal serum against Mycoplasma ovipneumoniae (956/2) (17), and of monoclonal antibodies (MAbs) against both U. urealyticum serotype 8 and purified Ureaplasma urease (16) has already been described. The MAbs used in this study were UU8/17 (antiurease), UU8/29 (recognizes a serotype 8-specific 96-kDa antigen), and UU8/39 (recognizes two antigens of 16 and 17 kDa). Immunoprecipitation of [3lHpalmitate-labeled cells. The method described previously (17) was used with MAbs UU8/17, UU8/29, and UU8/39, with homologous polyclonal serum (raised against killed whole cells), and, as a negative control, with polyclonal serum against M. ovipneumoniae. Basically, labeled cells were sonicated on ice in immunoprecipitation buffer (10 mM Tris-HCI [pH 7.2], 5 mM EDTA, 0.5% [vol/vol] Nonidet P-40 [Sigma], 0.65 M NaCl, 0.1% [wt/vol] NaN3, 1 mM phenylmethylsulfonyl fluoride). Solubilized labeled antigens were obtained by centrifugation (1,200 x g, 10 min, 4°C). Immune complexes were formed by incubating 50-,u aliquots with 5 pA of undiluted appropriate ascitic fluid or with a volume of polyclonal serum shown (by dot-blot staining on a nitrocellulose sheet with naphthalene black) to contain approximately the same amount of protein, for 1 h on ice. Immune complexes were isolated on an excess of a fixed suspension (20 ,ul of a 10% [wt/vol] suspension per RI of antibody) of Cowan strain A Staphylococcus aureus by centrifugation (1,200 x g, 10 min, 4°C), resuspended in 1 ml of immunoprecipitation buffer containing 10% (wt/vol) sucrose, and centrifuged as above. Resuspension of the pellet in this buffer was repeated three times, after which the final pellet was suspended in 100 RI of denaturing mix and boiled (3 min). After further centrifugation (as above), the supernatants were subjected to SDS-PAGE and fluorography. RESULTS AND DISCUSSION Uptake of [3lHlpalmitic acid by U. urealyticum. Preliminary experiments had shown that a significant level of incorporation of [3H]palmitic acid into ureaplasmal acylated proteins was difficult to achieve even when the labeled palmitate was present for the duration of the incubation. Labeling was detectable only on very long exposure fluorograms when exponential-phase cells, suspended in 3 ml of PBS with 0.05% (vol/vol) horse serum, were exposed to 500 ,uCi of the labeled palmitate. It has been reported (6) that the fatty acids in Mycoplasma capricolum growth medium are primarily used for phospholipid biosynthesis. If a similar situation exists in ureaplasmal growth, then the size of the available palmitate pool in growth medium, together with presumably only a very small proportion of the available palmitate being used for acylation of membrane proteins, would explain why long exposure of fluorograms was necessary. Because of the high specific activity of labeled palmitate required and the cost of the investigation, we were restricted to small 100-ml cultures to achieve the results presented, and even then fluorograms required up to 3 months of exposure at -70°C. Such cultures yield a relatively small cell pellet and precluded a parallel study with labeled membrane preparations. SDS-PAGE and fluorography. After electrophoresis, the fluorogram of labeled cells, obtained after 3 months of exposure of the X-ray film, produced the pattern shown in Fig. 1 (lane A). About 25 discrete bands were labeled, and
A
C
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kDa 126-
.:.
9662 55 50 4239 33 31 -
24-
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.4.4
FIG. 1. [3H]palmitate-labeled U. urealyticum serotype 8. Lanes: A, fluorogram of labeled cells after SDS-PAGE (3-month exposure of X-ray film); B, molecular mass markers (shown in kilodaltons); C, polypeptides visualized with silver stain after SDS-PAGE. All lanes, 15% (wt/vol) polyacrylamide gel.
those most intensively labeled had apparent molecular masses of 126, 104, 96, 63, 55, 50, 42, 39, 33, 31, 24, and 16 kDa. This is the first demonstration of acylated proteins in U. urealyticum, and the number of molecular species demonstrated (ca. 25) is similar to the number of acylated membrane proteins in M. capricolum (4) and greater than the 20 reported for M. hyorhinis (3) and the 4 reported for M. hyopneumoniae (19). A significantly different pattern was obtained after silver staining of labeled cells (Fig. 1, lane C), suggesting that the labeling pattern is of acylated proteins rather than the result of anabolic reactions with degradative products of the label. Furthermore, it was significant that the derived molecular masses of the acylated proteins corresponded to those which may be ascribed to silver-stained polypeptides after SDS-PAGE of unlabeled cells (10). However, further work is required to correlate beyond question the acylated proteins with bands of approximately similar mass as seen above. (It should be noted that metabolic labeling with labeled amino acids is equally difficult to achieve and was not attempted in these studies.) Phase/partition of labeled cells with Triton X-114. The final detergent and aqueous phases were subjected to SDS-PAGE and fluorography (Fig. 2A, lanes A and B, respectively). About 20 labeled bands were sufficiently hydrophobic to partition entirely into the detergent phase; this is consistent
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2a
2b
A
B
A
B
c
D
E
kDa _97,~974 -68.0 go
-43.0
-25.7
-18.4 -14.3
FIG. 2. (a) Phase partition of [3H]palmitate-labeled cells with Triton X-114. Lanes: A; fluorogram of the final detergent phase after SDS-PAGE; B; fluorogram of the final aqueous phase after SDSPAGE. (b) Immunoprecipitations with [3H]palmitate-labeled cells. Lanes: A, MAb UU8/29 (anti-96-kDa antigen); B, MAb UU8/39 (anti-16- and anti-17-kDa antigens); C, MAb UU8/17 (antiurease); D, homologous polyclonal serum; E, heterologous polyclonal serum (done on a separate gel). All lanes; 15% (wt/vol) polyacrylamide gel; X-ray film exposed for 3 months.
solubilized palmitate-labeled cells, immunoprecipitation was achieved with the anti-96-kDa protein MAb UU8/29 (Fig. 2b, lane A) but not with either MAb UU8/39 (anti-16- and anti-17-kDa antigens) or MAb UU8/17 (antiurease) (Fig. 2b, lanes B and C). It appears, therefore, that the serotype 8-specific surface-expressed 96-kDa antigen is acylated whereas the 16- and 17-kDa surface-expressed antigens are not. A labeled band with an apparent molecular mass of ca. 16 kDa can be discerned in Fig. 1 (lane A) from the primary labeling experiment, but it is apparently not the 16-kDa antigen which is recognized by MAb UU8/39. While the function(s) of acylated proteins has yet to be demonstrated, it has been suggested (2, 19) that they may be a powerful mechanism for generating antigenic diversity and for mediating immune responses affecting the growth and survival of the organisms. It is therefore significant that the serotype 8-specific surface-expressed 96-kDa antigen was acylated. It has also been suggested (2) that lipid modification of membrane proteins may play a role in dictating host interactions and so play a role in pathogenicity. ACKNOWLEDGMENT This work was supported by a grant from the British Technology Group.
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with the view that the acylated proteins are membrane associated. The much smaller number (five) of labeled bands seen in the aqueous phase aligned with components present in the detergent phase at greater concentration. This suggests that some of these acylated proteins, including the serotype 8-specific 96-kDa antigen, are amphipathic, with a sufficient degree of hydrophilicity to allow better interaction, presumably of exposed regions, with a surrounding aqueous environment. The result with the 96-kDa antigen confirms previously published data (16) and, we believe, eliminates the possibility that partition of a proportion of some acylated proteins into the aqueous phase is due to fragmented polypeptides. The results with the majority of the acylated proteins in the detergent phase suggests that partition was complete. Immunoprecipitations. No immunoprecipitation was achieved with the negative control polyclonal serum raised against M. ovipneumoniae (Fig. 2b, lane E). With the homologous polyclonal serum and with solubilized palmitate-labeled cells, five of the acylated proteins were shown to be antigenic (Fig. 2b, lane D). These antigens had apparent molecular masses of 104, 96, 55, 50, and 42 kDa. These five are among the antigens which have previously been routinely detected in blotting experiments with this serum (10). The 16-, 17-, and 96-kDa antigens of serotype 8 have been shown previously to be surface expressed (16), and the urease has been shown to be cytosolic (10). By using
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REFERENCES Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256:1604-1607. Boyer, M. J., and K. S. Wise. 1989. Lipid-modified surface antigens expressing size variation within the species Mycoplasma hyorhinis. Infect. Immun. 57:245-254. Bricker, T. M., M. J. Boyer, J. Keith, R. Watson-McKown, and K. S. Wise. 1988. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis. Infect. Immun. 56:295-301. Dahl, C. E., J. S. Dahl, and K. Bloch. 1983. Proteolipid formation in Mycoplasma capricolum: influence of cholesterol on unsaturated fatty acid acylation of membrane proteins. J. Biol. Chem. 258:11814-11818. Dahl, C. E., N. C. Sacktor, and J. S. Dahl. 1985. Acylated proteins in Acholeplasma laidlawii. J. Bacteriol. 162:445-447. Dahl, J. S. 1988. Uptake of fatty acid by Mycoplasma capricolum. J. Bacteriol. 170:2022-2026. Dulbecco, R., and H. Vogt. 1954. Plaque formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med. 99:167182. Magee, A. 1., and S. A. Courtneidge. 1985. Two classes of fatty acid acylated proteins exist in eukaryotic cells. EMBO J. 4:1137-1144. Magee, A. I., and M. J. Schlesinger. 1982. Fatty acid acylation of eukaryotic cell membrane proteins. Biochim. Biophys. Acta 694:279-289.
10. Myles, A. D. 1989. Molecular characterisation of Ureaplasma urealyticum. Ph.D. thesis, University of St. Andrews, St. Andrews, Scotland. 11. Nystrom, S., K.-E. Johansson, and A. Wieslander. 1986. Selective acylation of membrane proteins in Acholeplasma laidlawii. Eur. J. Biochem. 156:85-94. 12. Precious, B. L., D. Thirkell, and W. C. Russell. 1987. Preliminary characterization of the urease and a 96 kDa surfaceexpressed polypeptide in Ureaplasma urealyticum. J. Gen. Microbiol. 133:2659-2676. 13. Reithman, H. C., M. J. Boyer, and K. S. Wise. 1987. Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing in Mycoplasma hyorhinis. Infect. Immun. 55:1094-1100. 14. Robertson, J. A. 1986. Potential virulence factors of Ureaplasma urealyticum. Pediatr. Infect. Dis. 5:S322-S324.
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15. Russell, W. C., and G. E. Blair. 1977. Polypeptide phosphorylation in adenovirus-infected cells. J. Gen. Virol. 34:19-35. 16. Thirkell, D., A. D. Myles, and W. C. Russell. 1989. Serotype 8 and serocluster-specific surface-expressed antigens of Ureaplasma urealyticum. Infect. Immun. 57:1697-1701. 17. Thirkell, D., R. K. Spooner, G. E. Jones, and W. C. Russell. 1990. Polypeptide and antigenic variability among strains of Mycoplasma ovipneumoniae demonstrated by SDS-PAGE and
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immunoblotting. Vet. Microbiol. 21:241-254. 18. Wieslander, A., P. Wallbrandt, S. Nystrom, and K.-E. Johanson. 1990. Is the (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii membranes lipid modified? Abstr. 8th Int. Cong. I.O.M., Istanbul, p. 96-97. 19. Wise, K. S., and M. F. Kim. 1987. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid. J. Bacteriol. 169:5546-5555.
