Inhibition of the Plasma Glycosylphosphatidylinositol-Specific Phospholipase D by Synthetic Analogs of Lipid A and Phosphatidic Acid

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), a plasma enzyme with extensive sequence similarity to integrin α subunits, is inhibited by micromolar concentrations of lipid A, phosphatidic acid (PA) and lysophosphatidic acid (M. G. Low and K.-S. Huang, J. Biol. Chem. 268, 8480–8490, 1993). In this study we have explored the mechanism of inhibition using synthetic analogs of lipid A, and PA. Monosaccharide analogs of lipid A, which varied in the number and position of the phosphate groups, the type of acyl group, and its linkage to the glucosamine ring, were tested for their ability to inhibit GPI-PLD. A compound (SDZ 880.431) containing 3-aza-glucosamine 1,4-diphosphate as the polar headgroup was identified which had a potency (IC50 ∼1 μM) similar to natural lipid A preparations. Removal of either phosphate residue increased the IC50 markedly. Analogs of PA such as (7-nitro-2-1,3-benzoxadiazo-4-yl)amino-PA, ceramide 1-phosphate, and hexadecyl phosphate had ∼IC50 values ranging from 1 to 5 μM, indicating that considerable variation in the structure of the hydrophobic groups was permissible. Inhibition of GPI-PLD by long-chain PA could not be blocked by high concentrations of glycerol 1-phosphate or dibutyryl PA. These results indicate that the hydrophobic groups do not have a passive role in inhibition but are directly involved in the binding interaction with GPI-PLD. We propose that this diverse group of inhibitors all bind to a common site on GPI-PLD, the central hydrophobic cavity predicted by the β-propeller model for integrin α subunits and GPI-PLD.