Phosphatidylinositol Hydrolysis by Trypanosoma brucei Glycosylphosphatidylinositol Phospholipase C

doi:10.1074/jbc.271.26.15533

Phosphatidylinositol Hydrolysis by Trypanosoma brucei Glycosylphosphatidylinositol Phospholipase C*

^‡ Institute of Biochemistry and Molecular Biology, University of Bern, CH-3012 Bern, Switzerland and the
^¶ Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

^§To whom correspondence should be addressed:
Inst. of Biochemistry and Molecular Biology, University of Bern, Bühlstr. 28, CH-3012 Bern, Switzerland.
Tel.: 41-31-631-4113; Fax: 41-31-631-3737; E-mail: buetikofer{at}mci.unibe.ch.

Abstract

Detergent-solubilized glycosylphosphatidylinositol (GPI)-anchored structures can be cleaved by C-type phospholipases isolated from peanuts and bloodstream cells of the African trypanosome, Trypanosoma brucei. The two enzymes differ in their reported ability to hydrolyze phosphatidylinositol (PI); while the peanut enzyme readily hydrolyzes PI in vitro, the T. brucei enzyme was reported to be virtually inactive against PI and consequently named GPI-specific phospholipase C (GPI-PLC). In this paper, we describe experiments in which we reinvestigated the substrate specificity of T. brucei GPI-PLC by incubating the purified enzyme with Triton X-100/PI-mixed micelles and by studying PI hydrolysis. We found that PI hydrolysis occurred in a detergent-dependent fashion over the range of concentrations tested (5 μM to 1 mM PI). At 5 μM PI, hydrolysis was maximal at 0.005% Triton X-100, whereas at 1 mM PI, maximal hydrolysis required 0.05% Triton X-100. Hydrolysis of both PI and GPI was strongly affected by the presence of phospholipids. Endogenous PI was hydrolyzed during osmotic and detergent lysis of trypanosomes under conditions used to obtain quantitative hydrolysis of the GPI-anchored trypanosome variant surface glycoprotein. PI hydrolysis in the lysates was inhibited by sodium p-chloromercuriphenylsulfonate but unaffected by EGTA, consistent with the proposal that hydrolysis is due to GPI-PLC. These results suggest that the function of T. brucei GPI-PLC may be to regulate PI as well as (or instead of) GPI levels.

Footnotes

↵* This work was supported in part by Swiss National Science Foundation Grant 3100-039209.93, National Institutes of Health Grant AI28858, and the University of Wisconsin. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PI-PLCs

phosphatidylinositol-specific phospholipases C

PI

phosphatidylinositol

GPI

glycosylphosphatidylinositol

GPI-PLC

glycosylphosphatidylinositol-specific phospholipase C

MES

4-morpholineethanesulfonic acid

GPI-PLD

glycosylphosphatidylinositol-specific phospholipase D

AChE

acetylcholinesterase

VSG

variant surface glycoprotein

PC

phosphatidylcholine

PS

phosphatidylserine

pCMPS

sodium p-chloromercuriphenylsulfonate

CRD

cross-reacting determinant.
↵² P. Bütikofer and U. Brodbeck, unpublished observation.
- Received January 19, 1996.
- Revision received April 4, 1996.