Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization

Abstract

The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography-mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity.

Figure 1. ACE acts as a GPI anchored protein–releasing factor.

(a) A single 100-kDa band isolated by the TSK gel 3000SW column was subjected to SDS-PAGE and silver stained. (b) Immunoblotting of released PLAP from reaction with purified ACE-t, ACE-S, PI-PLC or vehicle alone (Buffer). 'Input' indicates substrate of the reaction. The size of the products released by ACE and PI-PLC was similar to that of the substrate. (c) Dose dependence of ACE-S GPIase activity. The results of experiments in which captopril was applied are also indicated. Values are mean ± s.d., n = 3.

Figure 2. Differences in GPIase and peptidase activities of ACE.

(a) Effects of metal chelating reagents on the wild-type Ace. EDTA, CyDTA or EGTA was applied to the PLAP conversion assay and the released PLAP was detected by immunoblotting (top). Bottom panels indicate effects of these reagents on the peptidase activity (activity under control condition; i.e., no reagent, was considered 1.0). Dose effect of EDTA on GPIase and peptidase activities. EDTA was applied at 0.1 mM or 1 mM (left). Effects of high dose (1 mM) CyDTA and EGTA on GPIase and peptidase activities (right). Buffer indicates no reagent applied. (b) GPIase and peptidase activities of the wild-type (WT), E414D and H413K-H417K mutants. GPIase activity is expressed as released PLAP activity (left). The dipeptidyl carboxypeptidase (Dipeptidase) activity of the same samples (right). ND, not detected. (c) Effects of metal chelating reagents on the H413K-H417K mutant. EDTA, CyDTA or EGTA was applied to the PLAP conversion assay and the released PLAP was detected by immunoblotting. Dose effect of EDTA on GPIase activity (left). EDTA was applied at 0.1 mM or 1 mM. Effects of high-dose (1 mM) CyDTA and EGTA on GPIase activity (right).

Figure 3. Shedding activity of ACE on the cell surface.

(a) Expression of EGFP-GPI was examined by FACS analysis. Purple area, ACE (−); green line, ACE (+); yellow line, PI-PLC-treated; black dot, background. (b) Expression of EGFP-GPI after ACE or PI-PLC treatment examined by fluorescence microscopy. Note that the fluorescence of Golgi complex remained the same. Magnification, ×200. (c) ACE caused shedding of various endogenous GPI-anchored proteins. The amount of ACE used was equivalent to the endogenous ACE activity. Values are mean ± s.d., n = 3. ND, not determined.

Figure 4. Characteristics of GPI cleavage by ACE.

(a) Detection of GPI anchor moiety in the released products. Autoradiography using radioactive phosphate (top left) and ethanolamine (top right); immunoblotting of GFP (bottom panels). The radioactivity of the cognate band detected by immunoblotting (arrowhead) was determined. The radioactivity per quantity of protein is indicated (amount of ACE-treated product was considered 1.0). (b) Identification and characterization of carboxy-terminal peptide by high-performance liquid chromatography–mass spectrometry. Spectra of the eluted peptides are shown. The peak at the retention time (RT) of 6.2 min was considered a carboxy-terminal peptide with the indicated modification. EtN, ethanolamine; Phs, phosphate; Man, mannose. Base peak chromatogram (top). The fraction at RT = 6.2 min is indicated (red). A full-scan spectrum at RT = 6.2 min (middle). A tandem mass spectrum of m/z-660 ions (bottom).

Figure 5. Involvement of ACE GPIase activity in sperm-egg binding.

(a) Distribution of Tesp-5 (TESP5) and Ph-20 (PH-20) in the sperm of wild-type and Ace knockout mice detected by immunoblotting. The acrosin and fertilin-β are indicated for water-soluble and detergent-soluble fraction controls, respectively. +/+, wild-type; −/−, Ace knockout. DS, detergent-soluble fraction; WS, water-soluble fraction. (b) Binding of Ace-knockout sperm to the zona pellucida after various treatments. The amount of Ace used for treatment was equivalent to the endogenous ACE activity. (c) Number of sperm bound to the egg. Values are mean ± s.e.m. ^*P < 0.001, ^**P < 0.005, compared with buffer control; P < 0.3, comparison of wild-type ACE with ACE-E414D; P < 0.5, comparison of wild-type ACE with PI-PLC; P < 0.05, comparison of PI-PLC with PI-PLC + Inositol-P (Student's t-test).