Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

Synthetic analogues of D-GlcNalpha1-6D-myo-inositol-1-HPO(4)-3(sn-1, 2-diacylglycerol) (GlcN-PI), with the 2-position of the inositol residue substituted with an O-octyl ether [D-GlcNalpha1-6D-(2-O-octyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-octyl) PI] or O-hexadecyl ether [D-GlcNalpha1-6D-(2-O-hexadecyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell-free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N-acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell-free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell-free system. GlcN-(2-O-hexadecyl)PI was shown to inhibit the first alpha-mannosyltransferase of the trypanosomal GPI pathway. The N-acetylated derivative GlcNAc-(2-O-octyl)PI is a substrate for the trypanosomal GlcNAc-PI de-N-acetylase and this compound, like GlcN-(2-O-octyl)PI, is processed predominantly to Man(2)GlcN-(2-O-octyl)PI by the T.brucei cell-free system. Both GlcN-(2-O-octyl)PI and GlcNAc(2-O-octyl)PI also inhibit inositol acylation of Man(1-3)GlcN-PI and, consequently, the addition of the ethanolamine phosphate bridge in the T.brucei cell-free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway-specific inhibitors.     

Cloning of Trypanosoma brucei and Leishmania major genes encoding the GlcNAc-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol biosynthesis that is essential to the African sleeping sickness parasite

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol (GlcNAc-PI) to d-GlcNalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol by GlcNAc-PI de-N-acetylase. The genes encoding this activity are PIG-L and GPI12 in mammals and yeast, respectively. Fragments of putative GlcNAc-PI de-N-acetylase genes from Trypanosoma brucei and Leishmania major were identified in the respective genome project data bases. The full-length genes TbGPI12 and LmGPI12 were subsequently cloned, sequenced, and shown to complement a PIG-L-deficient Chinese hamster ovary cell line and restore surface expression of GPI-anchored proteins. A tetracycline-inducible bloodstream form T. brucei TbGPI12 conditional null mutant cell line was created and analyzed under nonpermissive conditions. TbGPI12 mRNA levels were reduced to undetectable levels within 8 h of tetracycline removal, and the cells died after 3-4 days. This demonstrates that TbGPI12 is an essential gene for the tsetse-transmitted parasite that causes Nagana in cattle and African sleeping sickness in humans. It also validates GlcNAc-PI de-N-acetylase as a potential drug target against these diseases. Washed parasite membranes were prepared from the conditional null mutant parasites after 48 h without tetracycline. These membranes were shown to be greatly reduced in GlcNAc-PI de-N-acetylase activity, but they retained their ability to make GlcNAc-PI and to process d-GlcNalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol to later glycosylphosphatidylinositol intermediates. These results suggest that the stabilities of other glycosylphosphatidylinositol pathway enzymes are not dependent on GlcNAc-PI de-N-acetylase levels.     

Substrate specificity of the Plasmodium falciparum glycosylphosphatidylinositol biosynthetic pathway and inhibition by species-specific suicide substrates

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcN(alpha)1-6-D-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de-N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN-[L]-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or betaGlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor [Smith, T. K., et al. (2001) EMBO J. 20, 3322-3332], GalNCONH(2)-PI and GlcNCONH(2)-beta-PI were designed and found to be potent (IC(50) approximately 0.2 microM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs.     

Differences between the trypanosomal and human GlcNAc-PI de-N-acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis

De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc-PI) is the second step of glycosylphosphatidylino-sitol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. In this article, we used a direct de-N-acetylase assay, based on the release of [14C]acetate from synthetic GlcN[14C]Ac-PI and analogues thereof, and an indirect assay, based on the mannosylation of GlcNAc-PI analogues, to study the substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells. The HeLa enzyme was found to be more fastidious than the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was unable to act on a GlcNAc-PI analogue containing 2-O-octyl-d- myo -inositol or on the GlcNAc-PI diastereoisomer containing l- myo -inositol (GlcNAc-P(l)I). These results suggest thatselective inhibition of the trypanosomal de-N-acetylase may be possible and that this enzyme should be considered as a possible therapeutic target. The lack of strict stereospecificity of the trypanosomal de-N-acetylase for the d- myo -inositol component was also seen for the trypanosomal GPI alpha-manno-syltransferases when GlcNAc-P(l)I was added to the trypanosome cell-free system, but not when GlcN-P(l)I was used. In an attempt to rationalize these data, we modeled the structure and dynamics of d-GlcNAcalpha1-6d- myo -inositol-1-HPO4-( sn )-3-glycerol and its diastereoisomer d-GlcNAcalpha1-6l- myo -inositol-1-HPO4-( sn )-3-glycerol. These studies indicate that the latter compound visits two energy minima, one of which resembles the low-energy conformer of former compound. Thus, it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(l)I when it occupies a GlcNAc-PI-likeconformation and that GlcN-P(l)I emerging from the de-N-acetylase may be channeled to the alpha-mannosyltransferases in this conformation.     

Parasite-specific inhibition of the glycosylphosphatidylinositol biosynthetic pathway by stereoisomeric substrate analogues

The natural substrate for the first alpha-D-mannosyltransferase of glycosylphosphatidylinositol biosynthesis in the protozoan parasite Trypanosoma brucei is D-GlcNalpha1-6-D-myo-inositol-1-P-sn-1, 2-diacylglycerol. Here we show that a diastereoisomer, D-GlcNalpha1-6-L-myo-inositol-1-P-sn-1,2-diacylglycerol, is an inhibitor of this enzyme in a trypanosomal cell-free system. Tests with other L-myo-inositol-containing compounds revealed that L-myo-inositol-1-phosphate is the principal inhibitory component and that methylation of the 2-OH group of the L-myo-inositol residue abolishes any inhibition. Comparisons between the natural substrate and the inhibitors suggested that the inhibitors bind to the first alpha-D-mannosyltransferase by means of charge interactions with the 1-phosphate group and/or hydrogen bonds involving the 3-, 4-, and 5-OH groups of the L-myo-inositol residue, which are predicted to occupy orientations identical to those of the 1-phosphate and 5-, 4-, and 3-OH groups, respectively, of the D-myo-inositol residue of the natural substrate. However, additional experiments indicated that the 4-OH group of the D-myo-inositol residue is unlikely to be involved in substrate recognition. None of the L-myo-inositol-containing compounds that inhibited glycosylphosphatidylinositol (GPI) biosynthesis in a parasite cell-free system had any effect on GPI biosynthesis in a comparable human (HeLa) cell-free system, suggesting that other related parasite-specific inhibitors of this essential pathway might be developed.     

Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors.

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.     

The N-acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of glycosylphosphatidylinositol biosynthesis is a zinc metalloenzyme

The de-N-acetylation of N-acetyl-D-glucosaminylphosphatidylinositol (GlcNAc-PI) is the second step of mammalian and trypanosomal glycosylphosphatidylinositol biosynthesis. Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei, the causative agent of African sleeping sickness, and GlcNAc-PI de-N-acetylase has previously been validated as a drug target. Inhibition of the trypanosome cell-free system and recombinant rat GlcNAc-PI de-N-acetylase by divalent metal cation chelators demonstrates that a tightly bound divalent metal cation is essential for activity. Reconstitution of metal-free GlcNAc-PI de-N-acetylase with divalent metal cations restores activity in the order Zn(2+) > Cu(2+) > Ni(2+) > Co(2+) > Mg(2+). Site-directed mutagenesis and homology modeling were used to identify active site residues and postulate a mechanism of action. The characterization of GlcNAc-PI de-N-acetylase as a zinc metalloenzyme will facilitate the rational design of anti-protozoan parasite drugs.     

Further probing of the substrate specificities and inhibition of enzymes involved at an early stage of glycosylphosphatidylinositol (GPI) biosynthesis

1-D-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1-O-hexadecyl-myo-inositol (14), 1-D-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-(octadecyl phosphate) (18), 1-D-6-O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (24), 1-D-6-O-(2-amino-2-deoxy-alpha-D-mannopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (30) and the corresponding 2-amino-2-deoxy-alpha-D-galactopyranosyl analogue 36 have been prepared and tested in cell-free assays as substrate analogues/inhibitors of alpha-(1 --> 4)-D-mannosyltransferases that are active early on in the glycosylphosphatidylinositol (GPI) biosynthetic pathways of Trypanosoma brucei and HeLa (human) cells. The corresponding N-acetyl derivatives of these compounds were similarly tested as candidate substrate analogues/inhibitors of the N-deacetylases present in both systems. Following on from an early study, 1-L-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-2-O-methyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (44) was prepared and tested as an inhibitor of the trypanosomal alpha-(1 --> 4)-D-mannosyltransferase. A brief summary of the biological evaluation of the various analogues is provided.     

Synthesis of some second-generation substrate analogues of early intermediates in the biosynthetic pathway of glycosylphosphatidylinositol membrane anchors

1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-2-O-octyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (23) and the corresponding 2-O-hexadecyl-D-myo-inositol compound 24 have been prepared as substrate analogues of an early intermediate in the biosynthetic pathway of glycosylphosphatidylinositol (GPI) membrane anchors. 1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-octyl-sn-glycerol 3-phosphate) has also been prepared as a substrate analogue. Biological evaluation of the analogues 23 and 24 revealed that they are neither substrates nor inhibitors of GPI biosynthetic enzymes in the human (HeLa) cell-free system but are potent inhibitors at different stages of GPI biosynthesis in the Trypanosoma brucei cell-free system.     

The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei.

The compound diisopropylfluorophosphate (DFP) selectively inhibits an inositol deacylase activity in living trypanosomes that, together with the previously described phenylmethylsulfonyl fluoride (PMSF)-sensitive inositol acyltransferase, maintains a dynamic equilibrium between the glycosylphosphatidylinositol (GPI) anchor precursor, glycolipid A [NH2(CH2)2PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-sn-1,2-dimyristoylglycerol], and its inositol acylated form, glycolipid C. Experiments using DFP in living trypanosomes and a trypanosome cell-free system suggest that earlier GPI intermediates are also in equilibrium between their inositol acylated and nonacylated forms. However, unlike mammalian and yeast cells, bloodstream form trypanosomes do not appear to produce an inositol acylated form of glucosaminylphosphatidylinositol (GlcN-PI). A specific function of inositol acylation in trypanosomes may be to enhance the efficiency of ethanolamine phosphate addition to the Man3GlcN-(acyl)PI intermediate. Inositol deacylation appears to be a prerequisite for fatty acid remodelling of GPI intermediates that leads to the exclusive presence of myristic acid in glycolipid A and, ultimately, in the variant surface glycoprotein (VSG). In the presence of DFP, the de novo synthesis of GPI precursors cannot proceed beyond glycolipid C' (the unremodelled version of glycolipid C) and lyso-glycolipid C'. Under these conditions glycolipid C'-type GPI anchors appear on newly synthesized VSG molecules. However, the efficiencies of both anchor addition to VSG and N-glycosylation of VSG were significantly reduced. A modified model of the GPI biosynthetic pathway in bloodstream form African trypanosomes incorporating these findings is presented.     

The synthesis of some deoxygenated analogues of early intermediates in the biosynthesis of glycosylphosphatidylinositol (GPI) membrane anchors

Syntheses are described of 2-azido-4,6-di-O-benzyl-2,3-dideoxy-d-ribo-hexopyranosyl fluoride, 6-O-acetyl-2-azido-3-O-benzyl-2,4-dideoxy-d-xylo-hexopyranosyl fluoride and 2-azido-3,4-di-O-benzyl-2,6-dideoxy-d-glucopyranosyl fluoride. These glycosyl donors were coupled with the acceptor 1d-2,3,4,5-tetra-O-benzyl-1-O-(4-methoxybenzyl)-myo-inositol and the α-coupled products were transformed into α-d-3dGlcpN-PI, α-d-4dGlcpN-PI and α-d-6dGlcpN-PI by way of the H-phosphonate route. Brief mention is made of the biological evaluation of these deoxy-sugar analogues and their N-acetylated forms as candidate substrate/inhibitors of the N-deacetylase and α-(1→4)-d-mannosyltransferase activities present in trypanosomal and HeLa (human) cell-free system.     

Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme in the glycosylphosphatidylinositol biosynthetic pathway

The second step in glycosylphosphatidylinositol biosynthesis is the de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) catalyzed by N-acetylglucosaminylphosphatidylinositol deacetylase (PIG-L). Previous studies of mouse thymoma cells showed that GlcNAc-PI de-N-acetylase activity is localized to the endoplasmic reticulum (ER) but enriched in a mitochondria-associated ER membrane (MAM) domain. Because PIG-L has no readily identifiable ER sorting determinants, we were interested in learning how PIG-L is localized to the ER and possibly enriched in MAM. We used HeLa cells transiently or stably expressing epitope-tagged PIG-L variants or chimeric constructs composed of elements of PIG-L fused to Tac antigen, a cell surface protein. We first analyzed the subcellular distribution of PIG-L and Glc-NAc-PI-de-N-acetylase activity and then studied the localization of Tac-PIG-L chimeras to identify sequence elements in PIG-L responsible for its subcellular localization. We show that human PIG-L is a type I membrane protein with a large cytoplasmic domain and that, unlike the result with mouse thymoma cells, both PIG-L and GlcNAc-PI-de-N-acetylase activity are uniformly distributed between ER and MAM in HeLa cells. Analyses of a series of Tac-PIG-L chimeras indicated that PIG-L contains two ER localization signals, an independent retention signal located between residues 60 and 88 of its cytoplasmic domain and another weak signal in the luminal and transmembrane domains that functions autonomously in the presence of membrane proximal residues of the cytoplasmic domain that themselves lack any retention information. We conclude that PIG-L, like a number of other ER membrane proteins, is retained in the ER through a multi-component localization signal rather than a discrete sorting motif.     

Synthesis of 1-D-6-O-[2-(N-hydroxyaminocarbonyl)amino-2-deoxy-alpha-D-glucopyranosyl]-myo-inositol 1-(n-octadecyl phosphate): a potential metalloenzyme inhibitor of glycosylphosphatidylinositol biosynthesis

1-D-6-O-[2-(N-hydroxyaminocarbonyl)amino-2-deoxy-alpha-D-glucopyranosyl]-myo-inositol 1-(n-octadecyl phosphate) was prepared to probe the reaction mechanism of the putative zinc-dependent metalloenzyme 2-acetamido-2-deoxy-alpha-D-glucopyranosyl-(1-->6)-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol biosynthesis.     

Glycolipid transfer protein from pig brain transfers glycolipids with beta-linked sugars but not with alpha-linked sugars at the sugar-lipid linkage

The glycolipid transfer protein purified from pig brain facilitates the transfer of various glycosphingolipids and glyceroglycolipids (Yamada, K., Abe, A. and Sasaki, T. (1985) J. Biol. Chem. 260, 4615-4621). In this paper, the transfer of Man beta 1----4Glc beta 1-Cer and Man alpha 1----4Man beta 1-Cer isolated from a bivalve, Corbicula japonica, the transfer of 3-[Glc alpha 1-]-sn-1,2-diacylglycerol and 3-[Glc alpha 1----2Glc alpha 1-]-sn-1,2-diacylglycerol prepared from Streptococcus lactis, and the transfer of 3-[Glc beta 1-]-rac-1,2-dipalmitylglycerol have been investigated. The transfer of these lipids from liposomes to mitochondria was assayed by the decrease of these lipids in the donor liposomes. These lipids were determined by chromatographic isolation of the lipids, acid hydrolysis of the isolated lipids, and subsequent determination of glucose in the hydrolysate. The glycolipid transfer protein facilitated the transfer of ManGlcCer and ManManGlcCer. The transfer protein did not facilitate the transfer of Glc alpha-diacylglycerol or Glc alpha Glc alpha-diacylglycerol. However, the transfer of Glc beta-dipalmitylglycerol was facilitated by the protein. These results strongly suggest that the glycolipid transfer protein has the specificity to the presence of beta-linked glucose or galactose directly linked to either ceramide or diacylglycerol.     

The surface glycoconjugates of trypanosomatid parasites.

Insect-transmitted protozoan parasites of the order Kinetoplastida, suborder Trypanosomatina, include Trypanosoma brucei (aetiological agent of African sleeping sickness), Trypanosoma cruzi (aetiological agent of Chagas'' disease in South and Central America) and Leishmania spp. (aetiological agents of a variety of diseases throughout the tropics and sub-tropics). The structures of the most abundant cell-surface molecules of these organisms is reviewed and correlated with the different modes of parasitism of the three groups of parasites. The major surface molecules are all glycosylphosphatidylinositol (GPI)-anchored glycoproteins, such as the variant surface glycoproteins of T. brucei and the surface mucins of T. cruzi, or complex glycophospholipids, such as the lipophosphoglycans and glycoinositolphospholipids of the leishmanias. Significantly, all of the aforementioned structures share a motif of Man alpha 1-4GlcN alpha 1-6-myo-inositol-1-HPO4-lipid and can therefore be considered to be members of a GPI superfamily.     

Raft-like membrane domains contain enzymatic activities involved in the synthesis of mammalian glycosylphosphatidylinositol anchor intermediates

The synthesis of the glycosylphosphatidylinositol (GPI) anchor occurs in different compartments within the ER. We have previously shown that GPI anchor intermediates including GlcNAc-PI and GlcN-(acyl)PI are present in Triton insoluble membranes (TIMs), believed to be derived from lipid rafts. The present study was initiated to determine if GPI anchor intermediates move to raft-like domains after their synthesis or if these domains represent another ER compartment for GPI anchor synthesis. We determined that in transfected cells Pig-Ap and Pig-Lp, two proteins involved in the synthesis of GlcNAc-PI and GlcN-PI, respectively, are present in TIMs. In addition, we detected GlcNAc-PI synthase, GlcNAc-PI deacetylase, and GlcN-PI acyltransferase activities in TIMs isolated from untransfected cells. These results lend support to the possibility of additional GPI biosynthetic compartments in the ER and to the notion that GPI anchor intermediates produced in and outside raft-like domains may have a different fate.     

Structure and dynamics of the conserved protein GPI anchor core inserted into detergent micelles

A suitable approach which combines nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have been used to study the structure and the dynamics of the glycosylphosphatidylinositol (GPI) anchor Manalphal-2Manalpha1-6Manalphal -4GlcNalpha1-6myo-inositol-1-OPO(3)-sn-1,2-dimyristoylglycerol (1) incorporated into dodecylphosphatidylcholine (DPC) micelles. The results have been compared to those previously obtained for the products obtainable from (1) after phospholipase cleavage, in aqueous solution. Relaxation and diffusion NMR experiments were used to establish the formation of stable aggregates and the insertion of (1) into the micelles. MD calculations were performed including explicit water, sodium and chloride ions and using the Particle Mesh Ewald approach for the evaluation of the electrostatic energy term. The MD predicted three dimensional structure and dynamics were substantiated by nuclear overhauser effect (NOE) measurements and relaxation data. The pseudopentasaccharide structure, which was not affected by incorporation of (1) into the micelle, showed a complex dynamic behaviour with a faster relative motion at the terminal mannopyranose unit and decreased mobility close to the micelle. This motion may be better described as an oscillation relative to the membrane rather than a folding event.     

Structure of polymerizable lipid bilayers. V. Synthesis, bilayer structure and properties of diacetylenic ether and ester lipids.

Four diacetylenic phosphatidylcholines (PC's) have been synthesized and the structures of bilayers of these lipids have been determined at low resolution by low-angle X-ray diffraction. The PC's all have 18-carbon chains but differ with respect to the ether/ester linkage at the sn-1 and sn-2 positions and the relative position of the diacetylene moiety: diester-PC (1): 1,2-bis(octadeca-4',6'-diynoyl)-sn-glycero-3-phosphocholine diester-PC (2): 1-(octadeca-4',6'-diynoyl)-2-(octadeca-5',7'-diynoy l)-sn- glycero-3-phosphocholine diester-PC (3): 1,2-bis(octadeca-8',10'-diynoyl)-sn-glycerol-3-phosphocholin e diether-PC (4): 1-O-(octadeca-4',6'-diynyl)-2-O-(octadeca-5",7"-din yl)-sn- glycero-3-phosphocholine Only (1) exhibits the typical bilayer profile, whereas (2), (3) and (4) show evidence of interdigitation and/or significant disorder. Only (1) polymerized effectively upon illumination with 254 nm light, turning deep blue in seconds, indicating the formation of long, well-ordered polydiacetylenic structures. Liposomes of these derivatives were tested for permeability by osmotic swelling. Polymerized liposomes of (1) underwent osmotic swelling with urea, glycerol, and acetamide more rapidly than did liposomes of stearoyl-oleoyl-PC, but the initial rates of osmotic swelling of polymerized liposomes of (1) were 3-10-times lower than those of unpolymerized liposomes of (1). Blue polymerized multilayer samples of (1) exhibited an irreversible thermochromic transition to red at approx. 40 degrees C. Differential scanning calorimetry with liposome suspensions of (1) revealed an endotherm at 28.3 degrees C with a transition enthalpy of 40 J/g. PC (1) is a potentially useful diacetylenic lipid which exhibits facile, complete polymerization and a bilayer thickness comparable to that of biomembrane lipids.     

Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues.

Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.     

N-Acetylglucosaminyltransferase IX acts on the GlcNAc beta 1,2-Man alpha 1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan

Mammals contain O-linked mannose residues with 2-mono- and 2,6-di-substitutions by GlcNAc in brain glycoproteins. It has been demonstrated that the transfer of GlcNAc to the 2-OH position of the mannose residue is catalyzed by the enzyme, protein O-mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific beta1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcbeta1,2-Manalpha on both the alpha1,3- and alpha1,6-linked mannose arms in the core structure of N-glycan (Inamori, K., Endo, T., Ide, Y., Fujii, S., Gu, J., Honke, K., and Taniguchi, N. (2003) J. Biol. Chem. 278, 43102-43109). Here we examined the issue of whether GnT-IX is able to act on the same sequence of the GlcNAcbeta1,2-Manalpha in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manalpha1-Ser, GlcNAcbeta1,2-Manalpha1-Ser, and Galbeta1,4-GlcNAcbeta1,2-Manalpha1-Ser as acceptor substrates, the findings show that (14)C-labeled GlcNAc was incorporated only into GlcNAcbeta1,2-Manalpha1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcbeta1,2-Manalpha1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1-Ser-PAES by mass spectrometry and (1)H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH(2) by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel beta1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.