Glycan Requirements of Glycosylphosphatidylinositol Phospholipase C from Trypanosoma brucei

doi:10.1074/jbc.270.6.2517

Glycan Requirements of Glycosylphosphatidylinositol Phospholipase C from Trypanosoma brucei

GLUCOSAMINYLINOSITOL DERIVATIVES INHIBIT PHOSPHATIDYLINOSITOL PHOSPHOLIPASE C (*)

From the (1)Department of Cellular Biology, University of Georgia, Athens, Georgia 30602 and
the (2)Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901

^¶ Supported by a Burroughs Wellcome Fund New Investigator Award in Molecular Parasitology. To whom correspondence should be addressed:
Dept. of Cellular Biology, 724 Biological Sciences Bldg., University of Georgia, Athens, GA 30602.
Tel.: 706-542-3355; Fax: 706-542-4271; mensawil{at}zookeeper.zoo.uga.edu

Abstract

Glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol phospholipase C (PI-PLC) from Bacillus sp. both cleave glycosylphosphatidylinositols (GPIs). However, phosphatidylinositol, which is efficiently cleaved by PI-PLC, is a very poor substrate for GPI-PLC. We examined GPI-PLC substrate requirements using glycoinositol analogs of GPI components as potential inhibitors. Glucosaminyl(α16)-D-myo-inositol (GlcN(α16)Ins), GlcN(α16)Ins 1,2-cyclic phosphate, GlcN(α16)-2-deoxy-Ins, and GlcN(α16)Ins 1-dodecyl phosphonate inhibited GPI-PLC. GlcN(α16)Ins was as effective as Man(α14)GlcN(α16)Ins; we surmise that GlcN(α16)Ins is the crucial glycan motif for GPI-PLC recognition. Inhibition by GlcN(α16)Ins 1,2-cyclic phosphate suggests product inhibition since GPIs cleaved by GPI-PLC possess a GlcN(α16)Ins 1,2-cyclic phosphate at the terminus of the residual glycan. The effectiveness of GlcN(α16)-2-deoxy-Ins indicates that the D-myo-inositol (Ins) 2-hydroxyl is not required for substrate recognition, although it is probably essential for catalysis. GlcN(α16)-2-deoxy-L-myo-inositol, unlike GlcN(α16)-2-deoxy-Ins, had no effect on GPI-PLC; hence, GPI-PLC can distinguish between the two enantiomers of Ins. Surprisingly, GlcN(α16)Ins 1,2-cyclic phosphate was not a potent inhibitor of Bacillus cereus PI-PLC, and GlcN(α16)Ins had no effect on the enzyme. However, both GlcN(α16)Ins 1-phosphate and GlcN(α16)Ins 1-dodecyl phosphonate were competitive inhibitors of PI-PLC. These observations suggest an important role for a phosphoryl group at the Ins 1-position in PI-PLC recognition of GPIs. Other studies indicate that abstraction of a proton from the Ins 2-hydroxyl is not an early event in PI-PLC cleavage of GPIs. Furthermore, both GlcN(α16)-2-deoxy-Ins 1-phosphate and GlcN(α16)-2-deoxy-L-myo-inositol inhibited PI-PLC without affecting GPI-PLC. Last, the aminoglycoside G418 stimulated PI-PLC, but had no effect on GPI-PLC. Thus, these enzymes represent mechanistic subclasses of GPI phospholipases C, distinguishable by their sensitivity to GlcN(α16)Ins derivatives and aminoglycosides. Possible allosteric regulation of PI-PLC by GlcN(α16)Ins analogs is discussed.

Footnotes

↵^§ Supported by National Institutes of Health Predoctoral Training Grant 1-T32-AIO-7322.
↵* This work was supported in part by National Institutes of Health Grants AI 33383 (to K. M.-W.) and GM 47109 (to T.-Y. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

VSG

variant surface glycoprotein

GPI

glycosylphosphatidylinositol

EtN

ethanolamine

Ins

D-myo-inositol

GPI-PLC

glycosylphosphatidylinositol phospholipase C

GlcN(α16) Ins

glucosaminyl(α16)-D-myo-inositol

PI

phosphatidylinositol

PC

phosphatidylcholine

PS

phosphatidylserine

PG

phosphatidylglycerol

PE

phosphatidylethanolamine

PI-PLC

phosphatidylinositol phospholipase C.
↵² L. Ping-Sheng and T.-Y. Shen, manuscript in preparation.
- Received May 23, 1994.
- Revision received November 8, 1994.