Post-translational modification regulates prostaglandin D2 synthase apoptotic activity: characterization by site-directed mutagenesis.
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.
Ragolia
Louis
L
Vascular Biology Laboratory, Winthrop-University Hospital, Mineola, NY 11501, USA. LRagolia@winthrop.org
Hall
Christopher E
CE
Palaia
Thomas
T
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D006943:Hyperglycemia / Q000201:enzymology; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D054834:Lipocalins; D008297:Male; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar
D007527:Isoenzymes; D054834:Lipocalins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D005947:Glucose