Immunol, Cell Biol. (1990) 68. 87-93

Antibody against Bacillus thuringiensis phosphatidylinositol-phospholipase C: Some examples of its potential uses M. A. Theveniau, P. Malapert and G. N. Rougon VRA 202 CNRS, ICB-3, Place K Hugo. Marseilles J333I. Cedex 3. France (Submitted

17 July 1989. Accepted for publication 12 October 1989.)

Summary We have purified the phosphatidylinositol-phospholipase C enzyme from the bacterium Bacillm thuringiensis. This enzyme is able to release in soluble form molecules which are anchored lo membranes via a glycan-phosphatidylinositol group. It exhibits a molecular weight of-Vl-SS kDa. We raisedpolyclonalantiseraagainst the molecule and used them in immunoblot as well asradioimmunoassays for cnzymcdetcction. This last technique should facilitate monitoringof chromatographic steps during enzyme purification. We coupled antibodies to Sepharose beads in order to remove Ihe enzyme from incubation media. This reagent also proved to be particularly useful in control experiments designed to ascertain that the observed release of molecules is due lo the action of the phosphatidylinositol-phospholipase C enzyme and not to spontaneous release or to cleavage by nonspecific hydrolases. A search for cross-reactive molecules in other bacterial strains or mammalian tissues gave negative results. This leads to the conclusion that a great diversity exists between phosphatidylinositol-phospholipascs C, even among different bacterial strains.

INTRODUCTION In the past 3 years a novel mechanism by which proteins are anchored to membranes has been elucidated. This mechanism itivolves a covalent linkage of the protein to a glycan-phosphatidylinositol >

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3 2 antibodies to Sepharose beads. Then we determined the quantity of beads necessary to totally remove a given quantity of enzyme. Titration curves shown (Table 1) gave values of 30 (aL of beads to remove 1 ^g of enzyme (60 mlU). To make sure that all the enzyme was trapped by the bead-litiked antibody we verified that remaining supernatants did not contain any further PIPLC activity (not shown). Some G-PI molecules have been demonstrated to exist in soluble forms either in culture media (16) or in sera from patients with coiorectal carcinoma (13). Thus it is conceivable to postulate the existence of endogenous PI-PLC in mammalian tissues. Enzymes with such a specificity have been reported from the parasite Trypanosoma brucei (18) as well as from rat liver (19). We made the assumption that such

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1 2 3 Volume (mLxi 0"^' Fig. 4. Comparison between (-^HJ-liposomes and radioimmunoassay for enzyme detection. PI-PLC was detected on 100 |J1- of eluate samples by ['H]-liposome technique; activity was plotted as ct/min of CHJ-inositol phosphate released (—) or by radioimmunoassay (...) on I |iL sample in 100 |JL buffer,

enzymes exhibiting a common specificity could share some sequence or conformation similarities at least at their active site. Our antisera were able partially to inhibit the activity of the enzyme (around 10%), indicating that some antibody activity was directed against the active site. Thus, we searched for cross-reactive mol-

Pi-PLC ANTIBODY

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Table 1. Quantitative determination of PI-PLC removal from hydrolysis medium by antibody linked toSepharose beads. PI-PLC added 100

0005

005

107

107

01

05

1

5

104 103 47 106 We conducted experiments as described in Methods and determined ihe B/Bm^ values. We considered that as long as this value was equivalent lo B^av (value obtained with no addition of unlabelled enzyme) the beads had [he capacity to remove all the molecules from the aiedium. The validity of our data had been shown by cheeking that the remaining supcrnaiani had no PI-P1,C aciiviiy left.

ecules using the immunoblot technique (Fig. 5). Crude antisera used at low dilution (1:200) detected some bands on each of the tissue extracts analysed (panel A). To ascertain the specificity ofthe reactions observed we eluted bound antibodies from the PI-Pl.C band as described in methods and reincubated the eluted molecules with the various extracts (panel B). Although it is clear that eluted antibodies still recognized the hlotted !i. thuringien.si.s enzyme no specific cross-reaction could be seen even with the crude preparation of 5. aureus PI-PLC

(lane 2), meaning that the bands observed with total antisera were not specific. As a control, the reverse experiment was done by eluting antibodies reacting with the S. aureus preparation (panel A. lane 2) and reincubating them with B. ihuringiensis enzyme (panel C). Here again the antibodies were still reactive with the molecules they were eluted from but did not recognize/f. ?/H;/mHi PI-PLC (lane I). Again, reaction was only seen with original band of .S', aureus enzyme (lane 2).

M A. THfiVENlAU ET AL.

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aturated, the similarities might reside more in conformation than in sequence. We also tested the ability of different tissuesor PI-PLC from 5. aureuslo interfere with antibody-['^^I]-labelled PI-PLC complex formation in radioimmunoassay tests. We again obtained negative results; with increasing concentrations of extracts the B/T values were always equivalent to BmaxThus, if similarities exist between different PIPLC enzymes, they are not consequential enough to be detected by our antibody probe. DISCUSSION Antibodies against a 33 kDa molecule species purified from li. ihuringiensis culture supernatant unambiguously proved that the PI-PLC activity was associated with protein of such a size. Indeed antibody linked to Sepharose beads was able to remove all enzymatic activity from the incubation medium. Monitoring of the different purification steps with both radioimmunoassay. using the antisera. and tritiated phosphatidylinositol incorporated into liposomes. and detecting enzyme activity, showed superimposable results. Thus, in subsequent purifications of PI-PLC enzyme it will be possible to use radioimmunoassay instead of liposomes. Radioimmunoassay using iodide is more sensitive, easier and faster to perform than the lipo-

some technique using tritium and it allows treatment of a large number of samples in a single batch. Considering our large supply of antiserum we will make it available to potential users. Coupling the antiserum to beads provides convenient method of removing the enzyme from hydrolysis media. An alternative would be to link the enzyme itself to the beads. However, so far we have been unsuccessful in retaining a good activity. Our search for cross-reactive molecules demonstrates a structural heterogeneity of these enzymes among bacterial PI-PLCs. In mammals several types of enzymes hydrolysing various inositol-containing compounds have been described (20). Studies with purified proteins have shown that they are immunologically unrelated and that they differ in their molecular properties. An important question regarding these different forms of PI-PLCs is how their structure relates to their functional properties, with the speculative view that their diversity may reflect unique functions in the cell. Acknowledgements We thank Dr J. Barbct for the gift of .S". aurvu.s PI-PLC. Dr T. Ballz for Trypanosoma cells. The technical help of P. de Castro in enzyme purification is gratefully acknowledged. We also thank Dr J. Marvaldi for his encouragement and scientitic adviee.

REFERENCES 1. Ferguson. M. A. J. and Williams, A. F. ! 988. Cellsurface anchoring of proteins via glyeosyl-phosphatidylinositol structures. Ann, Rev. Biochem. 57: 285-320. 2. Low. M. G. and Saltiel. A. R. 1988. Structural and functional roles of glycosyl-phosphatidylinositol in membranes. Science 239: 268-275. 3. Cross. G. A. M. 1987. Eukaryotic protein modificaiion and membrane attachment via phosphatidylinositol. CW/48: 179-181. 4. lkezawa. H., Yamanegi. M., Taguchi. R.. Miyashita. T. and Ohyabu. T. 1976. Study of phosphat idyll nositol phosphodiesterase (phospholipasc C type) of Bacillus cereus-l Purification, properties and phosphatase releasing activities. Biochem. Biophys. .Ada 450: 154-164. 5. Low. M. G, and Finean. J. B. 1978. Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C. Biocliem. Biophys. .4cia 508: 565-570. 6. Taguchi. R.. Asabi. Y. and lkezawa. H. 1980. Purification and properties of phosphatidyiinositol specific phospholipase C of Bacillus thtiringiensi.s. Biochem. Biophys. .-([70 619: 48-57.

7. Ferguson. M. A. J.. Low, M. G. and Cross, G.A. M. 1985. Glyeosyl-sn-1. 2-dimyrislylphosphatidylinositol is eovalenliy linked to Trypanosoma brucei variant surface glyeoprotein. J. Biol. Chem. 260: 14 547-14 555. 8. Low, M. G. 1981. Pbosphatidylinositol-specific phospholipase C from Slaphylococcus aureus. Meth. Enzymolll: 741-746. 9. Salaeinski. P., MeLean. C . Sykes, J.. ClementJones, V. and Lowry. P. 1981. lodination of proteins, glyeoproteins. and peptides using solid phase oxidizing agenl l,3.4.6-tctrachloro36diphenyglycoluril (iodogen). Anal. Biochem 117: 136-146. 10. Rougon. G.. Ccard, B., Van Reitschoten. J.. Jordan, B. and Barbet. J. 1984. Induction wilh a synthetic peptidc of antibodies to HLA class I Cterminal intraeytoplasmic region. Moi Immunol 21: 461-468. 11. Merril. C R., Goldman. D.. Sedman. S. A. and Ebert. M. H. 1981. Ultrasensitive stain for proteins in po!yacr\'lamide gels shows regional variation in cerebrospinal fluid proteins. Science 2\l\ 1437-1438.

PI-Pl.C ANTIBODY 12. Pierres. M..Naquet, P..Barbct.J.f/a/. 1988. Evidence thai murine hematopoietic cell subset marker J11 d is attached to glycosyl-phosphatidylinositol membrane anchor. Eur, J, Immunol 17: 1781-1785. 13. Jean, F., Malapert, P.. Rougon, G. and Barbel, J. 1988. Cell membrane but not circulating carcinoembryonic antigen is linked to a phosphatidylinosilol containing hydrophobic domain. Bio(hem. Btophys. Res. Comm 155: 794-800. 14. Fouchier. F.. Bastiani, P. Baltz. T.. Aunis. D. and Rougon, G. 1988. Glycosyl-phosphatidylinosilol is involved in the membrane attachment of proicins in granules of chromaffin cells. Biochem. J. 256: 103-108. I 5. Gcnnarini. G., Rougon, G.. Vitiello, F., Corsi, P., Di Benedetta. C. and Goridls. C. 1989. Identification and cDNA cloning of a new member of ihe L2/HNK-I family of neural surface glycoproteins. 7. Neurosci.

Res- 22: 1-12.

16. He. H. T., Finne. J. and Goridis. C 1988. Biosynthesis, membrane association, and release of NCAM-120, phosphatidylinositol-linked form of the neural cell adhesion molecule. J. Cell Biol. 105: 2489-2500.

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17. Pierrcs, M., Barbet. J.. Naquel, P. I'tal. 1987. Thy1, Thy-3 and thymocytc activating molecules (ThAM): signal transducing T cell markers by rat monoclonal antibodies raised against Pl-specific phospholipase C solubilized thymocyte surface antigens. In The T-cell Rcceplor. J. Kappler and M. Davis (eds). Alan R. Liss. Inc.. New York. pp. 293-300. 18. Bulow. R. and Overath. P. 1986. Purification and characterization of the membrane form variant surface glycoprolcin hydrolase of Trypanosoma brucei.J. Bioi Chem. 261: II 918-11 923. 19. Fox, J. A.. Soliz. N. M. and Saltid. A. R. 1987. Purification of phosphatidylinositol glycanspecific phospholipase C from liver plasma membranes: A possible target of insulin action. Proc. Nail Sci. USA 84; 2663-2667. 20. Katan. M.. Kriz, R.. Totty. N., Philip, R.. Mcldrum, E., Aldape. R., Knopf, J. and Parker. P. 1988. Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-spccific phospholipase C activities. Cell 54: 171-177.

Antibody against Bacillus thuringiensis phosphatidylinositol-phospholipase C: some examples of its potential uses.

We have purified the phosphatidylinositol-phospholipase C enzyme from the bacterium Bacillus thuringiensis. This enzyme is able to release in soluble ...
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