Phosphatidylinositol‐glycan‐specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high‐density lipoproteins

Abstract

Phosphatidylinositol (Ptd1ns)-glycan-specific phospholipase D was purified from bovine and human serum by phase separation in Triton X-114 and by chromatography on DEAE-cellulose, octylSepharose, concanavalin-A -Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200-fold with a recovery of 3-5%. Bovine serum contained about 40 pgglml of PtdIns-glycan-specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns-glycan-specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X-100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X-100. In the absence of Triton X-100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high-density lipoprotein fraction. PtdIns-glycanspecific phospholipase D from bovine and human serum contained 27 and 28 N-acetylglucosamine residues, respectively. Treatment with N-glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns-glycan-specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.

Shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins

“…Despite its high concentration in mammalian serum [214] and relatively wellcharacterized molecular biology [215] and biochemistry [216] the physiological role of GPI-PLD is not clear. In serum, GPI-PLD is associated with HDL and is apparently not active [217]. Initial reports indicated that GPI-PLD was active against GPI-anchored proteins only in the presence of detergent, and was not able to cleave the anchors of proteins in a native membrane context [218].…”

Investigation of an albumin‐enriched fraction of human serum and its albuminome

“…Twenty-six of the 35 albumin-associated proteins have been previously reported to be potential biomarkers. Of these, nine were observed only in the albumin-enriched fraction, not the albumin-depleted fraction (compare with [44]). Proteins in this category are alpha-2HS-glycoprotein, apolipoprotein AI, ceruloplasmin, interalpha trypsin inhibitor H4, kininogen, apolipoprotein CIII, fibrinogen, retinol-binding protein, and beta thromboglobulin.…”

Glycosyl‐phosphatidylinositol‐specific phospholipase D

“…In mammals, the only purified and well characterized GPIspecific phospholipase is of the D-type (GPI-PLD) producing phosphatidic acid as one of the products. GPI-PLD was originally discovered in serum [2,3] and later in whole brain [4], neurons [5], and cerebrospinal fluid [6], in the islets of Langerhans [7], liver [8,9], liver mast cells [lo], and milk [6]. GPI-PLD has been purified and characterized from serum [6,1 l-131, and its primary sequence is known from bovine liver cDNA [S].…”