GLYCAN REQUIREMENTS OF GLYCOSYLPHOSPHATIDYLINOSITOL PHOSPHOLIPASE-C FROM TRYPANOSOMA-BRUCEI - GLUCOSAMINYLINOSITOL DERIVATIVES INHIBIT PHOSPHATIDYLINOSITOL PHOSPHOLIPASE-C

Glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosom a brucei and phosphatidylinositol phospholipase C (PI-PLC) from Bacill us sp. both cleave glycosylphosphatidylinositols (GPIs), However, phos phatidylinositol, which is efficiently cleaved by PI-PLC, is a very po or substrate for GPI-PLC, We examined GPI-PLC substrate requirements u sing glycoinositol analogs of GPI components as potential inhibitors. Glucosaminyl(alpha 1-->6)-D-myo-inositol (GlcN(alpha 1-->6)Ins), GlcN( alpha 1-->6)Ins 1,2-cyclic phosphate, GlcN(alpha 1-->6)-2-deoxy-Ins, a nd GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate inhibited GPI-PLC. GlcN( alpha 1-->6)Ins was as effective as Man(alpha 1-->4)GlcN((alpha 1-->6) Ins; we surmise that GlcN(alpha 1-->6)Ins is the crucial glycan motif for GPI-PLC recognition. Inhibition by GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate suggests product inhibition since GPIs cleaved by GPI-PLC p ossess a GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate at the terminus of the residual glycan. The effectiveness of GlcN(alpha 1-->6)-2-deoxy-In s indicates that the D-myo-inositol (Ins) 2-hydroxyl is not required f or substrate recognition, although it is probably essential for cataly sis. GlcN(alpha 1-->6)-2-deoxy-L-myo-inositol, unlike GlcN(alpha 1-->6 )2-deoxy-Ins, had no effect on GPI-PLC; hence, GPI-PLC can distinguish between the two enantiomers of Ins, Surprisingly, GlcN(alpha 1-->6)In s 1,2-cyclic phosphate was not a potent inhibitor of Bacillus cereus P I-PLC, and GlcN(alpha 1-->6)Ins had no effect on the enzyme. However, both GlcN(alpha 1-->6)Ins 1-phosphate and GlcN(alpha 1-->6)Ins 1-dodec yl phosphonate were competitive inhibitors of PI-PLC. These observatio ns suggest an important role for a phosphoryl group at the Ins 1-posit ion in PI-PLC recognition of GPIs. Other studies indicate that abstrac tion of a proton from the Ins 2-hydroxyl is not an early event in PI-P LC cleavage of GPIs. Furthermore, both GlcN(alpha 1-->6)-2-deoxy-Ins 1 -phosphate and GlcN(alpha 1-->6)-2-deoxy-L-myo-inositol inhibited PI-P LC without affecting GPI-PLC. Last, the aminoglycoside G418 stimulated PI-PLC, but had no effect on GPI-PLC. Thus, these enzymes represent m echanistic subclasses of GPI phospholipases C, distinguishable by thei r sensitivity to GlcN(alpha 1-->6)Ins derivatives and aminoglycosides, Possible allosteric regulation of PI-PLC by GlcN(alpha 1-->6)Ins anal ogs is discussed.