Modulation of the cleavage of glycosylphosphatidylinositol-anchored proteins by specific bacterial phospholipases

1996 ◽  
Vol 74 (5) ◽  
pp. 701-713 ◽  
Author(s):  
Frances J. Sharom ◽  
Gary L. McNeil ◽  
John R. Glover ◽  
Sandra Seier
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Catalytic Activity ◽  
Soluble Form ◽  
Gpi Anchor ◽  
Net Charge ◽  
Optimum Ph ◽  
Host Plasma Membrane ◽  
And Staphylococcus Aureus ◽  
Native Plasma

Many enzymes are tethered to the extracellular face of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. These proteins can be released in soluble form by the action of GPI-specific phospholipases. Little is currently known about the factors modulating this release. We investigated the effects of several experimental variables on the cleavage of the GPI-anchored proteins 5′-nucleotidase, acetylcholinesterase, and alkaline phosphatase by phospholipases from Bacillus thuringiensis and Staphylococcus aureus. Phospholipase activity was not inhibited by isotonic salt and was relatively unaffected by buffer type and concentration. In both cases, the optimum pH for cleavage was ~ 6.5. Over 80% of 5′-nucleotidase activity present in the lymphocyte plasma membrane was cleaved by the B. thuringiensis enzyme, and the initial rate of release was linear with phospholipase concentration. All three GPI-anchored proteins were released from lymphocyte plasma membrane at comparable phospholipase concentrations, suggesting that they have similar anchor structures. The catalytic activity of 5′-nucleotidase appeared to increase following conversion to the soluble form. The relative surface charge of the host plasma membrane modulated catalytic activity towards GPI-anchored proteins, depending on the net charge of the phospholipase. Studies on purified lymphocyte 5′-nucleotidase reconstituted into bilayers of dimyristoylphosphatidylcholine indicated that the efficiency of phospholipase cleavage was 12- to 50-fold lower when compared with the native plasma membrane. The ability of the phospholipase to cleave the GPI anchor was further reduced when the bilayer was in the gel phase.Key words: glycosylphosphatidylinositol anchor, phospholipase C, 5′-nucleotidase, acetylcholinesterase, alkaline phosphatase.

scholarly journals Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5′-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer

1998 ◽  
Vol 332 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Marty T. LEHTO ◽  
Frances J. SHAROM
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Lipid Bilayer ◽  
Hydrolytic Enzymes ◽  
Detergent Solution ◽  
Gpi Anchor ◽  
Rich Mixture ◽  
Catalytic Activation ◽  
Dimyristoyl Phosphatidylcholine ◽  
Native Plasma

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5´-nucleotidase (5´-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5´-nucleotidase all obeyed Michaelis–Menten kinetics, with a Km for 5´-AMP in the range 11–16 µM. The GPI anchor was removed from essentially all 5´-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5´-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5´-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5´-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5´-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.

scholarly journals A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin.

1994 ◽  
Vol 14 (7) ◽  
pp. 4825-4833 ◽  
Author(s):  
C F Lu ◽  
J Kurjan ◽  
P N Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Soluble Form ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Experimental Support ◽  
Cell Biol ◽  
A Cell

Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall.

Processing of Hemojuvelin Requires Retrograde Trafficking to the Golgi in HepG2 Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3845-3845
Author(s):  
An-Sheng Zhang ◽  
Julia Julia Maxson ◽  
Caroline A Enns
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Hepg2 Cells ◽  
Iron Homeostasis ◽  
Bmp Signaling ◽  
Soluble Form ◽  
Gpi Anchor ◽  
Retrograde Trafficking ◽  
Hepcidin Expression ◽  
Bone Morphogenic Proteins

Abstract Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with the cells through a GPI-anchor or released as a soluble form. The cellular form acts as a co-receptor for bone morphogenic proteins (BMPs) and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes BMP signaling and suppresses hepcidin expression. Secretion of HJV requires binding to the transmembrane receptor neogenin. In this study we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, had complex oligosaccharides and could be derived from the pool of HJV at the plasma membrane. Neogenin did not play a role in HJV trafficking to the cell surface but was necessary for secretion of HJV, suggesting that it could be involved in either retrograde trafficking of HJV or in cleavage leading to secretion.

A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin

1994 ◽  
Vol 14 (7) ◽  
pp. 4825-4833
Author(s):  
C F Lu ◽  
J Kurjan ◽  
P N Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Soluble Form ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Experimental Support ◽  
Cell Biol ◽  
A Cell

Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall.

scholarly journals Electrophoretic characterization of hepatic alkaline phosphatase released by phosphatidylinositol-specific phospholipase C. A comparison with liver membrane and serum-soluble forms

1985 ◽  
Vol 227 (1) ◽  
pp. 183-189 ◽  
Author(s):  
T Kominami ◽  
A Miki ◽  
Y Ikehara
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Phospholipase C ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Polyacrylamide Gel ◽  
Soluble Form ◽  
Enzyme Preparations

Alkaline phosphatase was solubilized from plasma membrane of rat liver with butanol-ol, bile acids or sodium deoxycholate, and electrophoretically compared with a soluble form in serum which was derived from the liver. The three enzyme preparations from the plasma membrane migrated at the same position on polyacrylamide-gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulphate. The mobility of them, however, was distinctly different from that of the serum-soluble form of the liver-derived alkaline phosphatase. On the other hand, phosphatidylinositol-specific phospholipase C isolated from Bacillus cereus was used to release alkaline phosphatase from plasma membrane. The released alkaline phosphatase was demonstrated to have the same mobility as the serum-soluble form on polyacrylamide-gel electrophoresis in the presence or absence of detergents. The phospholipase C also converted the butan-1-ol-extracted membrane form into the serum-soluble form. The results suggest that release of alkaline phosphatase from the liver into serum is not simply caused by a detergent effect of bile salts, but involves an enzymic hydrolysis of phosphatidylinositol, with which alkaline phosphatase may strongly interact in the membrane.

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

1995 ◽  
Vol 8 (9) ◽  
pp. 865-871 ◽  
Author(s):  
Chris G. Dealwis ◽  
Liqing Chen ◽  
Catherine Brennan ◽  
Wlodek Mandecki ◽  
Cele Abad-Zapatero
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Catalytic Activity ◽  
Magnesium Binding

Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans.

1976 ◽  
Vol 22 (7) ◽  
pp. 972-976 ◽  
Author(s):  
H Van Belle
Keyword(s):  
Alkaline Phosphatase ◽  
Substrate Concentration ◽  
Human Liver ◽  
Alkaline Phosphatases ◽  
Mechanism Of Inhibition ◽  
Optimum Ph ◽  
Alkaline Phosphatase Isoenzymes

Abstract I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.

scholarly journals Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C

1987 ◽  
Vol 241 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Y Ikehara ◽  
Y Hayashi ◽  
S Ogata ◽  
A Miki ◽  
T Kominami
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Soluble Form ◽  
Microsomal Membranes ◽  
Hydrophobic Nature ◽  
Rat Hepatoma

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.

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