Purification and Characterization of a Polyisoprenyl Phosphate Phosphatase from Pig Brain

Abstract

Microsomal fractions from pig and calf brain catalyze the enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate (Dol-P) (Sumbilla, C. A., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482). The Dol-P phosphatase (EC 3.1.3.51) has been solubilized by extracting pig brain microsomes with the nonionic detergent Nonidet P-40 and purified approximately 1,107-fold by a combination of anion exchange chromatography, polyethylene glycol fractionation, dye-ligand chromatography, and wheat germ agglutinin affinity chromatography. Treatment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetylneuraminyl residues per molecule of enzyme. When the highly purified polyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymatic dephosphorylation of Dol-P by the purified phosphatase was 1) optimal at pH 7; 2) potently inhibited by F ؊ , orthovanadate, and Zn 2؉ > Co 2؉ > Mn 2؉ but unaffected by Mg 2؉ ; 3) exhibited an approximate K m for C 95 -Dol-P of 45 M; and 4) was sensitive to N-ethylmaleimide, phenylglyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not dephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5-AMP, or p-nitrophenylphosphate, but it dephosphorylated dioleoylphosphatidic acid at initial rates similar to those determined for Dol-P. Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephosphorylation by the highly purified enzyme to N-ethylmaleimide, F ؊ , phenylglyoxal, and diethylpyrocarbonate, both substrates appear to be hydrolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase from mammalian tissues. Although the enzyme is Mg 2؉ -independent and capable of dephosphorylating Dol-P and PA, several enzymological properties distinguish this lipid phosphomonoesterase from PAP2. Dolichyl monophosphate (Dol-P)1 plays an essential role as a glycosyl carrier lipid in the assembly of N-linked oligosaccharides in eukaryotes (1, 2). In view of the evidence that modulation of Dol-P levels in the ER are one factor regulating the rate of biosynthesis of dolichol-linked oligosaccharide intermediates and protein N-glycosylation (2-11), it will be important to understand the control of the enzymes involved in Dol-P metabolism. Recent studies on the biosynthesis of Dol-P indicate that the induction of the cis-isoprenyltransferase system, catalyzing the chain elongation stage in the de novo pathway, is a key regulatory event when the rates of lipid intermediate synthesis and protein N-glycosylation increase developmentally in embryonic rat brain (10) and proliferating B lymphocytes (11). The presence of membrane-bound enzymes capable of phosphorylating dolichol and dephosphorylating Dol-P has been documented for a number of animal tissues (12). Reciprocal developmental changes in dolichol kinase and polyisoprenyl ph...

The CWH8 Gene Encodes a Dolichyl Pyrophosphate Phosphatase with a Luminally Oriented Active Site in the Endoplasmic Reticulum of Saccharomyces cerevisiae

“…The rates of hydrolysis of PA were not significantly high enough to calculate meaningful kinetic constants. This is the first report of a lipid phosphatase that hydrolyzes Dol-P-P/Dol-P but not PA (5)(6)(7)(27)(28)(29). Similarly, the level of diacylglycerol pyrophosphate phosphatase activity was not changed compared with wild type strains, in microsomes from the yeast strains overexpressing CWH8 or the cwh8 null allele mutant (data not included).…”

“…Under the same in vitro conditions, expression of CWH8 also produced an increase in Dol-P phosphatase, although considerably lower than the increase seen for Dol-P-P phosphatase (Table IV). Very significantly, the lipid phosphatase encoded by CWH8 does not hydrolyze PA in contrast to virtually all other Mg 2ϩ -independent lipid phosphatases reported previously (5)(6)(7)(27)(28)(29)(30)(31)(32)(33). Membrane fractions from the insect cells overexpressing CWH8 also did not exhibit diacylglycerol pyrophosphate phosphatase (data not included).…”

The LPP1 and DPP1 Gene Products Account for Most of the Isoprenoid Phosphate Phosphatase Activities inSaccharomyces cerevisiae

“…The existence of a relatively non-selective lipid phosphatase was also suggested by observations that Mg 2ϩ -independent dolichyl-P phosphatase activities from mammalian sources were inhibited by PA and LPA (24,25). Furthermore, a dolichyl-P phosphatase that was purified to apparent homogeneity from the particulate fraction of porcine brain used both dolichyl-P and PA with similar catalytic efficiency (26).…”

Genetic defects in dolichol metabolism

“…In contrast, bacterial CPTs produce a single end product which most often contains 11 i.u. Polyprenyl diphosphate is then dephosphorylated by mono and/or diphosphatases (Frank and Waechter 1998 ) and converted to dolichol by polyprenol reductase (Cantagrel et al 2010 ) absent from prokaryotic cells. Dolichol is further phoshorylated by a dolichol kinase (Shridas and Waechter 2006 ) and might then serve as the carrier for mannose and glucose monosaccharides further used as donors in N-glycosylation, O- and C-mannosylation and GPI anchor synthesis.…”