Midportion antibodies stimulate glycosylphosphatidylinositol-specific phospholipase D activity.

Limited information is known regarding the regulation, structural features, and functional domains of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD, EC 3. 1.4.50). Previous studies demonstrated that trypsin cleavage of GPI-PLD at or near Arg325 and/or Arg589 in bovine serum GPI-PLD was associated with an increase in enzymatic activity. Since the Arg325 is predicted to be in a region between the catalytic domain and predicted beta-propeller structure in the C-terminal portion of GPI-PLD (T. A. Springer, Proc. Natl. Acad. Sci. USA 94, 65-72, 1997), we hypothesized that this connecting region is important for catalytic activity. Trypsin cleavage of human serum GPI-PLD, which has an Arg325 but lacks the Arg589 present in bovine serum GPI-PLD, also increased GPI-PLD activity. Peptide-specific antibodies to residues 275-296 (anti-GPI-PLD(275)) and a monoclonal antibody, 191, with an epitope encompassing Arg325, also stimulated GPI-PLD activity. Pretreating human GPI-PLD with trypsin demonstrated that anti-GPI-PLD(275) only stimulated the activity of intact GPI-PLD. These results suggest that trypsin activation and anti-GPI-PPLD(275) may have similar effects on GPI-PLD. Consistent with this is the observation that both manipulations decreased the affinity of GPI-PLD for mixed micelle substrates. These results indicate that the midportion region of GPI-PLD is important in regulating enzymatic activity.