• 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.

• Parasites, Hosts and Diseases
• /
• v.47 no.3
• /
• pp.197-204
• /
• 2009

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.

• Journal of Microbiology
• /
• v.43 no.3
• /
• pp.266-271
• /
• 2005

In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.

• 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.

• Journal of Life Science
• /
• v.10 no.1
• /
• pp.20-23
• /
• 2000

Mono-ADP-ribosylation, catalyzed by ADP-ribosyltransferases, is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to an acceptor protein. Previously, we have identified and cloned a glycosylphosphatidylinositol-linked ADP-ribosyltransferase (Yac-1) from mouse lymphoma cells. Yac-1 enzyme contains three regions (region I,II,III) similar to those found in several bacterial toxins and vertebrate ADP-ribosyltransferases. Site-directed mutagenesis was performed to verify the role of Glu 233 in region III. Mutants E233Q, E233D and E233A were inactive for ADP-ribosyltransferase activity. Thus Glu 233 in Yac-1 is essential for enzyme activity, suggesting that Glu 233 in Glu-rich motif near the carboxy terminus plays a catalytic role in ADP-ribosyltransferase activity.

• 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
• /
• 2000.04a
• /
• pp.151.3-152
• /
• 2000

• Journal of Life Science
• /
• v.8 no.1
• /
• pp.102-108
• /
• 1998

The nascent from of glycosylphosphatidylinositol (GPI)-anchored proteins possesses both amino and carboxy terminal hydrophobic signal sequences to direct processing in the endoplasmic reticulum (ER). Following cleavage of the amino-terminal signal peptide, the carboxy-terminal peptide is processed. Previously, mouse lymphocyte NDA: agrinine ADP-ribosyltransferase (Yac-1) was cloned and the deduced amino acid sequence of the Yac-1 transferase contained hydrophobic amino and carboxy termini, consistent with known signal sequences of GPI-anchored proteins. This tranferase was present on the surface of NMU (rat mammary adenocarcinoma) cells transfected with the wildtype cDNA and was released with phosphatidylinositol-specific phosphilpase C. Expression of the mutant protein, lacking the carboxy terminal hydrophobic sequence, resulted in the peoduction of soluble, secreted from of the transferase. This result shows that carboxy terminal sequence is important for GPI-attachment.

• Journal of Life Science
• /
• v.8 no.2
• /
• pp.189-196
• /
• 1998

A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various animal cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini. A similar notif was observed in the sequence of the mouse lymphocyte ADP-ribosyltransferase (Yac-s). Yac-2 has significant NAD glycohydrolase activity as well as ADP-ribosyltransferase activity. To verify the role of the Glu-rich motif in Yac-2, site-directed mutagenesis was performed. Mutants E220Q, E220A, E222A were inactive for ADP-ribosyltransferase activity. For NAD glycohydrolase activity, E220A, E222Q, and E222A were inactive. In contrast, E220Q was active as wild-type. Thus, Glu-220 and Glu-222 in Yac-2 are critical for ADP-ribosyltransferase and NAD glycohydrolase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the Yac-2 enzyme activity.

• 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)