Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 May 15;332(Pt 1):101–109. doi: 10.1042/bj3320101

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

M T Lehto 1, F J Sharom 1
PMCID: PMC1219457  PMID: 9576857

Abstract

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.

Full Text

The Full Text of this article is available as a PDF (502.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barboni E., Rivero B. P., George A. J., Martin S. R., Renoup D. V., Hounsell E. F., Barber P. C., Morris R. J. The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein. J Cell Sci. 1995 Feb;108(Pt 2):487–497. doi: 10.1242/jcs.108.2.487. [DOI] [PubMed] [Google Scholar]
  2. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Brasitus T. A., Schachter D. Lipid dynamics and lipid-protein interactions in rat enterocyte basolateral and microvillus membranes. Biochemistry. 1980 Jun 10;19(12):2763–2769. doi: 10.1021/bi00553a035. [DOI] [PubMed] [Google Scholar]
  5. Brewis I. A., Turner A. J., Hooper N. M. Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C. Biochem J. 1994 Oct 15;303(Pt 2):633–638. doi: 10.1042/bj3030633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  7. Durbin H., Young S., Stewart L. M., Wrba F., Rowan A. J., Snary D., Bodmer W. F. An epitope on carcinoembryonic antigen defined by the clinically relevant antibody PR1A3. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4313–4317. doi: 10.1073/pnas.91.10.4313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Englund P. T. The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu Rev Biochem. 1993;62:121–138. doi: 10.1146/annurev.bi.62.070193.001005. [DOI] [PubMed] [Google Scholar]
  9. Gmachl M., Sagan S., Ketter S., Kreil G. The human sperm protein PH-20 has hyaluronidase activity. FEBS Lett. 1993 Dec 28;336(3):545–548. doi: 10.1016/0014-5793(93)80873-s. [DOI] [PubMed] [Google Scholar]
  10. Hanada K., Izawa K., Nishijima M., Akamatsu Y. Sphingolipid deficiency induces hypersensitivity of CD14, a glycosyl phosphatidylinositol-anchored protein, to phosphatidylinositol-specific phospholipase C. J Biol Chem. 1993 Jul 5;268(19):13820–13823. [PubMed] [Google Scholar]
  11. Holland J. W., Cullis P. R., Madden T. D. Poly(ethylene glycol)-lipid conjugates promote bilayer formation in mixtures of non-bilayer-forming lipids. Biochemistry. 1996 Feb 27;35(8):2610–2617. doi: 10.1021/bi951999j. [DOI] [PubMed] [Google Scholar]
  12. Hooper N. M. More than just a membrane anchor. Curr Biol. 1992 Nov;2(11):617–619. doi: 10.1016/0960-9822(92)90183-b. [DOI] [PubMed] [Google Scholar]
  13. Itami C., Taguchi R., Ikezawa H., Nakabayashi T. Release of ectoenzymes from small intestine brush border membranes of mice by phospholipases. Biosci Biotechnol Biochem. 1997 Feb;61(2):336–340. doi: 10.1271/bbb.61.336. [DOI] [PubMed] [Google Scholar]
  14. Koelsch R., Gottwald S., Lasch J. Release of GPI-anchored membrane aminopeptidase P by enzymes and detergents has some peculiarities. Biochim Biophys Acta. 1994 Feb 23;1190(1):170–172. doi: 10.1016/0005-2736(94)90047-7. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lierheimer R., Kunz B., Vogt L., Savoca R., Brodbeck U., Sonderegger P. The neuronal cell-adhesion molecule axonin-1 is specifically released by an endogenous glycosylphosphatidylinositol-specific phospholipase. Eur J Biochem. 1997 Jan 15;243(1-2):502–510. doi: 10.1111/j.1432-1033.1997.0502a.x. [DOI] [PubMed] [Google Scholar]
  17. Lisanti M. P., Rodriguez-Boulan E., Saltiel A. R. Emerging functional roles for the glycosyl-phosphatidylinositol membrane protein anchor. J Membr Biol. 1990 Jul;117(1):1–10. doi: 10.1007/BF01871561. [DOI] [PubMed] [Google Scholar]
  18. Loe D. W., Glover J. R., Head S., Sharom F. J. Solubilization, characterization, and detergent interactions of lymphocyte 5'-nucleotidase. Biochem Cell Biol. 1989 Apr-May;67(4-5):214–223. doi: 10.1139/o89-033. [DOI] [PubMed] [Google Scholar]
  19. Low M. G., Finean J. B. Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C. Biochim Biophys Acta. 1978 Apr 20;508(3):565–570. doi: 10.1016/0005-2736(78)90100-1. [DOI] [PubMed] [Google Scholar]
  20. Maeda T., Balakrishnan K., Mehdi S. Q. A simple and rapid method for the preparation of plasma membranes. Biochim Biophys Acta. 1983 May 26;731(1):115–120. doi: 10.1016/0005-2736(83)90404-2. [DOI] [PubMed] [Google Scholar]
  21. McConville M. J., Ferguson M. A. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J. 1993 Sep 1;294(Pt 2):305–324. doi: 10.1042/bj2940305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Müller G., Bandlow W. Lipolytic membrane release of two phosphatidylinositol-anchored cAMP receptor proteins in yeast alters their ligand-binding parameters. Arch Biochem Biophys. 1994 Feb 1;308(2):504–514. doi: 10.1006/abbi.1994.1071. [DOI] [PubMed] [Google Scholar]
  23. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  24. Rademacher T. W., Edge C. J., Dwek R. A. Dropping anchor with the lipophosphoglycans. Curr Biol. 1991 Feb;1(1):41–42. doi: 10.1016/0960-9822(91)90123-e. [DOI] [PubMed] [Google Scholar]
  25. Resta R., Thompson L. F. T cell signalling through CD73. Cell Signal. 1997 Feb;9(2):131–139. doi: 10.1016/s0898-6568(96)00132-5. [DOI] [PubMed] [Google Scholar]
  26. Roberts W. L., Myher J. J., Kuksis A., Low M. G., Rosenberry T. L. Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C. J Biol Chem. 1988 Dec 15;263(35):18766–18775. [PubMed] [Google Scholar]
  27. Robinson P. J. Signal transduction via GPI-anchored membrane proteins. Adv Exp Med Biol. 1997;419:365–370. doi: 10.1007/978-1-4419-8632-0_48. [DOI] [PubMed] [Google Scholar]
  28. Romero G. Inositolglycans and cellular signalling. Cell Biol Int Rep. 1991 Sep;15(9):827–852. doi: 10.1016/0309-1651(91)90036-i. [DOI] [PubMed] [Google Scholar]
  29. Schroeder R., London E., Brown D. Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12130–12134. doi: 10.1073/pnas.91.25.12130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sharom F. J., Lamb M. P., Kupsh C. C., Head S. Inhibition of lymphocyte 5'-nucleotidase by lectins: effects of lectin specificity and cross-linking ability. Biochem Cell Biol. 1988 Jul;66(7):715–723. doi: 10.1139/o88-082. [DOI] [PubMed] [Google Scholar]
  31. Sharom F. J., Lorimer I., Lamb M. P. Reconstitution of lymphocyte 5'-nucleotidase in lipid bilayers: behaviour and interaction with concanavalin A. Can J Biochem Cell Biol. 1985 Oct;63(10):1049–1057. doi: 10.1139/o85-130. [DOI] [PubMed] [Google Scholar]
  32. Sharom F. J., McNeil G. L., Glover J. R., Seier S. Modulation of the cleavage of glycosylphosphatidylinositol-anchored proteins by specific bacterial phospholipases. Biochem Cell Biol. 1996;74(5):701–713. doi: 10.1139/o96-077. [DOI] [PubMed] [Google Scholar]
  33. Shukla S. D., Coleman R., Finean J. B., Michell R. H. Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C. Biochem J. 1980 Apr 1;187(1):277–280. doi: 10.1042/bj1870277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  35. Stevens V. L. Biosynthesis of glycosylphosphatidylinositol membrane anchors. Biochem J. 1995 Sep 1;310(Pt 2):361–370. doi: 10.1042/bj3100361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stochaj U., Flocke K., Mathes W., Mannherz H. G. 5'-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol-glycan. Biochem J. 1989 Aug 15;262(1):33–40. doi: 10.1042/bj2620033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Strohmeier G. R., Lencer W. I., Patapoff T. W., Thompson L. F., Carlson S. L., Moe S. J., Carnes D. K., Mrsny R. J., Madara J. L. Surface expression, polarization, and functional significance of CD73 in human intestinal epithelia. J Clin Invest. 1997 Jun 1;99(11):2588–2601. doi: 10.1172/JCI119447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Udenfriend S., Kodukula K. How glycosylphosphatidylinositol-anchored membrane proteins are made. Annu Rev Biochem. 1995;64:563–591. doi: 10.1146/annurev.bi.64.070195.003023. [DOI] [PubMed] [Google Scholar]
  39. Wang X., Jansen G., Fan J., Kohler W. J., Ross J. F., Schornagel J., Ratnam M. Variant GPI structure in relation to membrane-associated functions of a murine folate receptor. Biochemistry. 1996 Dec 17;35(50):16305–16312. doi: 10.1021/bi961098q. [DOI] [PubMed] [Google Scholar]
  40. Wong Y. W., Low M. G. Phospholipase resistance of the glycosyl-phosphatidylinositol membrane anchor on human alkaline phosphatase. Clin Chem. 1992 Dec;38(12):2517–2525. [PubMed] [Google Scholar]
  41. Zekri M., Harb J., Bernard S., Poirier G., Devaux C., Meflah K. Differences in the release of 5'-nucleotidase and alkaline phosphatase from plasma membrane of several cell types by PI-PLC. Comp Biochem Physiol B. 1989;93(3):673–679. doi: 10.1016/0305-0491(89)90394-5. [DOI] [PubMed] [Google Scholar]
  42. Zimmermann H. 5'-Nucleotidase: molecular structure and functional aspects. Biochem J. 1992 Jul 15;285(Pt 2):345–365. doi: 10.1042/bj2850345. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES