Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity

Abstract

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal b-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn2+, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity.

Figure 1. DEPC inhibits GPI-PLD activity

(A) Concentration dependence of DEPC inhibition of GPI-PLD activity. GPI-PLD was pretreated with DEPC concentrations ranging from 0 to 50 mM for 60 min, and then GPI-PLD activity was determined as described in the Materials and methods section. (B) Time course of DEPC inhibition of GPI-PLD activity. GPI-PLD was treated with 50 mM DEPC for 0–40 min. At 30 min, GPI-PLD activity was determined with (○) and without (●) the addition of hydroxylamine as described in the Materials and methods section. Results shown are mean of duplicate determinations and are representative of three independent experiments.

Figure 2. Mutating His²⁹, His¹²⁵, His¹³³ and His¹⁵⁸ abolishes GPI-PLD activity

His²⁹, His³⁴, His⁵⁶, His⁸¹, His⁸⁸, His⁹⁸, His¹²⁵, His¹³³, His¹⁵⁸ or His²¹⁹ of mouse GPI-PLD was individually mutated to an asparagine residue as described in the Materials and methods section. COS-I cells were transiently transfected with wild-type (WT) or each mutant (5 μg of plasmid DNA) using Lipofectamine™ (20 μg). After 24 h, GPI-PLD catalytic activity was determined in the medium and cell lysate (A) or GPI-PLD mass in the medium by Western blotting (B) as described in the Materials and methods section. Absolute GPI-PLD activity in the medium and cell lysates from cells transfected with wild-type GPI-PLD was 140±118 (n=6) and 43±20 (n=6) c.p.m.·h⁻¹·(mg of protein)⁻¹ respectively. Results in (A) are from three independent experiments performed in triplicate. Results in (B) are representative of three experiments.

Figure 3. Trypsin fragmentation of GPI-PLD mutants

Conditioned media from COS-I cells transfected with wild-type (WT) GPI-PLD or one among H81N, H98N, H125N, H133N and H158N GPI-PLD were concentrated and treated with (+) or without (−) trypsin as described in the Materials and methods section. Proteins and fragments were separated by SDS/PAGE (7–15% polyacrylamide) and a C-terminal epitope of GPI-PLD was visualized by Western blotting with anti-GPI-PLD⁷⁷¹ as described in the Materials and methods section. Molecular mass standards are indicated in kDa. The 27 kDa fragment is indicated by the arrow. Results are representative of three independent experiments.

Figure 4. Phosphorylation of GPI-PLD mutants

Proteins in conditioned media from COS-I cells transfected with wild-type (WT) or GPI-PLD mutants were phosphorylated with protein kinase A and separated by SDS/PAGE (7% polyacrylamide) as described in the Materials and methods section. Phosphorylated proteins were visualized by autoradiography. GPI-PLD mass was identified by Western blotting as described in the Materials and methods section.

Figure 5. Effect of Cys substitution for His²⁹, His¹²⁵, His¹³³ and His¹⁵⁸ on GPI-PLD secretion and activity

His²⁹, His¹²⁵, His¹³³ and His¹⁵⁸ were individually mutated to Cys, expressed in COS cells and GPI-PLD activity and mass determined as described in Figure 1. Results are representative of three independent experiments.