Post-translational modification regulates prostaglandin D2 synthase apoptotic activity: characterization by site-directed mutagenesis

Abstract

Lipocalin-type prostaglandin D(2) synthase (L-PGDS) is a highly glycosylated protein found in several body fluids. Elevated L-PGDS levels have been observed in the serum of patients with renal impairment, diabetes mellitus, and hypertension. Recently, we demonstrated the ability of L-PGDS to induce apoptosis in a variety of cell types including epithelial cells, neuronal cells, and vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the effect several site-directed mutations had on L-PGDS-induced apoptosis in order to identify potential sites of regulation. Point mutations created in a glycosylation site (Asn51), a protein kinase C phosphorylation site (Ser106), and the enzymatic active site (Cys65) all inhibited L-PGDS-induced apoptosis as determined by both terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and caspase3 activity. We also compared the L-PGDS isoforms present in GK rat serum to WKY control serum using two-dimensional gel electrophoresis and observed distinct differences which vanished after PNGase F glycolytic digestion. We conclude that post-translational modification of L-PGDS, by either glycosylation or phosphorylation, enhances its apoptotic activity and inhibits VSMC hyperproliferation and postulate that this process is altered in type 2 diabetes.

Figure 1. Two-dimensional gel electrophoresis of L-PGDS Isoforms

Protein samples (5.0 μg) were resolved by isoelectric focusing as described in Materials and Methods. After electrophoresis, the gels were probed for L-PGDS and analyzed by using enhanced chemiluminescence (Panel A). Isoforms were compared using Photoshop^® CS overlaying which highlights differences in orange (Panel B). Proteins were digested with PNGase F to remove glycosylated residues (Panel C). Molecular weights (right) and isoelectric points (below) are provided for reference.

Figure 2. Site-directed Mutagenesis of L-PGDS

Point mutations in L-PGDS were created at two prospective glycosylation sites (Gly1 and Gly2), an apparent protein kinase C phosphorylation site (PKC), and the enzymatic active site (ACT) as described in Materials and Methods. Gly1 (Asn51), Gly2 (Asn78), and the potential PKC phosphorylation site (Ser106) were all exchanged for alanine, and the ACT (Cys65) mutation was created by exchanging Cys65 with a glycine residue.

Figure 3. Effect of Mutation on L-PGDS-induced apoptosis

VSMCs were cultured as described in Materials and Methods. At approximately 100% confluency the cells, where indicated, were treated with L-PGDS (50 μg/ml) for 18h, and the apoptotic index calculated by TUNEL assay (Panel A) or Caspase3 assay (Panel B). Results are the mean ± SEM of three different experiments each performed in duplicate. Asterisks (*) represents a P<0.05 versus GK controls.

Figure 4. Effect of Mutation on L-PGDS Enzymatic Activity

VSMCs were cultured as described in Materials and Methods. At approximately 100% confluency the PG D₂ concentration of both the culture medium, and the cell lysate was determined by the conversion of PG D₂ into a stable ‘MOX’ product. Results are the mean ± SEM of three experiments performed in duplicate and expressed as μg of PG D₂ formed/ μg of protein /ml. Asterisks (*) represent a P<0.05 when compared to wildtype L-PGDS.

Figure 5. Effect of Hyperglycemia on L-PGDS-mediated Cell Proliferation

Diabetic GK VSMCs were cultured as described in Materials and Methods in the presence or absence of L-PGDS (50 μg/ml) in media containing either unmodified (5 mM) or high (20 mM) glucose. Cells were plated at a density of 1x10⁴ cells/ml in 35 mm dishes and counted using a hemocytometer after the indicated time. Results are the mean ± S.E.M. of at least three experiments, each performed in duplicate. Asterisks (*) represent a P<0.01 when compared to cells cultured in high glucose.