• Journal of Applied Biological Chemistry
• /
• v.58 no.1
• /
• pp.9-11
• /
• 2015

Tissue extracts were prepared from liquid-cultured Arabidopsis and reacted with UDP-[$^3H$]-GlcNAc. Phospholipid fractions were then extracted by butanol partitioning. Consecutive thin-layer chromatography identified two glycolipids sensitive to PI-specific phospholipase C, known as early intermediates in glycosylphosphatidylinositol anchor biosynthesis; phosphatidylinositol N-acetylglucosamine and phosphatidylinositol glucosamine.

• Archives of Pharmacal Research
• /
• v.25 no.1
• /
• pp.80-85
• /
• 2002

The incubation of porcine renal proximal tubules (PTs) resulted in the release of the Glycosylphosphatidylinositol (GPI)-anchored renal dipeptidase (RDPase, EC 3. 4. 13. 19) from the membrane after a lag period of approximately 6 hours. This spontaneous release of RDPase from the membrane was inhibited by antibiotics. When the incubation supernatant was added back to fresh PTs, both the antibiotic inhibition of RDPase release and the lag period disappeared. The released RDPase reacted with an anti-cross reacting determinant antibody indicating the presence of the Ins (1, 2-cyc)P. These results suggest that bacteria in the PTs, when incubated, grow find Secrete a phosphatidylinmsitol-specific phospholipase C (PIPLC). This enzyme then hydrolyses the GPI-anchored RDPase and is transferable. RDPase was purified following its release from the membrane by this simple and inexpensive method which may also be applied to other GPI-anchored proteins.

• Molecules and Cells
• /
• v.24 no.2
• /
• pp.294-300
• /
• 2007

The mammalian glycosylphosphatidylinositol (GPI) anchor consists of three mannoses attached to acylated GlcN-(acyl)PI to form $Man_3$-GlcN-(acyl)PI. The first of the three mannose groups is attached to an intermediate to generate Man-GlcN-(acyl)PI by the first mannosyltransferase (GPI-MT-I). Mammalian and protozoan GPI-MT-I have different substrate specificities. PIG-M encodes the mammalial GPI-MT-I which has 423 amino acids and multiple transmembrane domains. In this work we cloned PIG-M homologues from humans, Plasmodium falciparum (PfPIG-M), and Saccharomyces cerevisiae (GPI14), to test whether they could complement GPI-MT-I-deficient mammalian cells, since this biosynthetic step is likely to be a good target for selective screening of inhibitors against many pathogenic organisms. PfPIG-M partially restored cell surface expression of the GPI-anchored protein CD59 in PIG-M deficient mammalian cells, and first mannose transfer activity in vitro; however, this was not the case for GPI14.

• Proceedings of the PSK Conference
• /
• 2003.04a
• /
• pp.222.2-223
• /
• 2003

Renal dipeptidase (RDPase, EC 3.4.13.19), an ectoenzyme of renal proximal tubules, is covalently bound to outer leaflet of lipid bilayer via glycosylphosphatidylinositol (GPI)-anchor. Chitin is a major component of the shells of crustacea such as crab, shrimp and crawfish. This study was conducted to examine the effect of chitosan on RDPase release from renal proximal tubules. Nitric oxide (NO), highly reactive free radical, inhibits the release of RDPase from porcine proximal tubules. (omitted)

• Proceedings of the PSK Conference
• /
• 2002.10a
• /
• pp.319.2-319.2
• /
• 2002

Chitin is a major component of the shells of crustacea such as crab. shrimp and crawfish. Renal dipeptidase (RDPase. EC 3.4.13.19), an ectoenzyme of renal proximal tubules. is covalently bound to outer leaflet of lipid bilayer via glycosylphosphatidylinositol (GPI)-anchor. The biological role of RDPase was suggested as the hydrolysis of dipeptide into free-amino acids before renal reabsorption. The underlying biochemical mechanism of decreased RDPase release was suggested as nitric oxide (NO) production. (omitted)

• BMB Reports
• /
• v.37 no.3
• /
• pp.362-369
• /
• 2004

The human folate receptor (hFR) is a glycosylphosphatidylinositol (GPI) linked plasma membrane protein that mediates delivery of folates into cells. We studied the sorting of the hFR using transfection of the hFR cDNA into MDCK cells. MDCK cells are polarized epithelial cells that preferentially sort GPI-linked proteins to their apical membrane. Unlike other GPI-tailed proteins, we found that in MDCK cells, hFR is functional on both the apical and basolateral surfaces. We verified that the same hFR cDNA that transfected into CHO cells produces the hFR protein that is GPI-linked. We also measured the hFR expression on the plasma membrane of type III paroxysmal nocturnal hemoglobinuria (PNH) human erythrocytes. PNH is a disease that is characterized by the inability of cells to express membrane proteins requiring a GPI anchor. Despite this defect, and different from other GPI-tailed proteins, we found similar levels of hFR in normal and type III PNH human erythrocytes. The results suggest the hypothesis that there may be multiple mechanisms for targeting hFR to the plasma membrane.