Specificities of enzymes of glycosylphosphatidylinositol biosynthesis in Trypanosoma brucei and HeLa cells

A series of synthetic analogues of d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, consisting of 22 variants of the d-GlcN or lipid components, were tested in trypanosomal and human (HeLa) cell-free systems. The assays measured the abilities of the analogues to act as substrates or inhibitors of the enzymes of glycosylphosphatidylinositol biosynthesis downstream of GlcNAc-phosphatidylinositol (GlcNAc-PI) de-N-acetylase. One compound, 4-deoxy-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, proved to be an inhibitor of both the trypanosomal and HeLa pathways, whereas 4-O-methyl-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol and the 4'-epimer, d-GalN-alpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, were neither substrates nor inhibitors. The results with other analogues showed that the 6-OH of the alpha-d-GlcN residue is not required for substrate recognition in the trypanosomal and human pathways, whereas the 3-OH group is essential for both. Parasite-specific recognition of the beta-linked analogue d-GlcN beta 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol is striking. This suggests that, like the GlcNAc-PI de-N-acetylase, the trypanosomal glycosylphosphatidylinositol alpha-mannosyltransferases, inositol acyltransferse and ethanolamine phosphate transferase, do not recognize the 2-, 3-, 4-, and 5-OH groups of the d-myo-inositol residue, whereas the human inositol acyltransferase and/or first alpha-mannosyltransferase recognizes one or more of these groups. All of the various lipid analogues tested served as substrates in both the trypanosomal and HeLa cell-free systems, suggesting that a precise lipid structure and stereochemistry are not essential for substrate recognition. However, an analogue containing a single C18:0 alkyl chain in place of sn-1,2-dipalmitoylglycerol proved to be a better substrate in the trypanosomal than in the HeLa cell-free system. These findings should have a bearing on the design of future generations of specific inhibitors of the trypanosomal glycosylphosphatidylinositol biosynthetic pathway.     

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.     

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.     

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.     

GPI-anchored proteins and free GPI glycolipids of procyclic form Trypanosoma brucei are nonessential for growth, are required for colonization of the tsetse fly, and are not the only components of the surface coat

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine-containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine-containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI-anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.     

The suppression of galactose metabolism in procylic form Trypanosoma brucei causes cessation of cell growth and alters procyclin glycoprotein structure and copy number

Galactose metabolism is essential in bloodstream form Trypanosoma brucei and is initiated by the enzyme UDP-Glc 4'-epimerase. Here, we show that the parasite epimerase is a homodimer that can interconvert UDP-Glc and UDP-Gal but not UDP-GlcNAc and UDP-GalNAc. The epimerase was localized to the glycosomes by immunofluorescence microscopy and subcellular fractionation, suggesting a novel compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T. brucei single-allele knockouts, and conditional (tetracycline-inducible) null mutants were constructed. Under non-permissive conditions, conditional null mutant cultures ceased growth after 8 days and resumed growth after 15 days. The resumption of growth coincided with constitutive re-expression epimerase mRNA. These data show that galactose metabolism is essential for cell growth in procyclic form T. brucei. The epimerase is required for glycoprotein galactosylation. The major procyclic form glycoproteins, the procyclins., were analyzed in TbGALE single-allele knockouts and in the conditional null mutant after removal of tetracycline. The procyclins contain glycosylphosphatidylinositol membrane anchors with large poly-N-acetyl-lactosamine side chains. The single allele knockouts exhibited 30% reduction in procyclin galactose content. This example of haploid insufficiency suggests that epimerase levels are close to limiting in this life cycle stage. Similar analyses of the conditional null mutant 9 days after the removal of tetracycline showed that the procyclins were virtually galactose-free and greatly reduced in size. The parasites compensated, ultimately unsuccessfully, by expressing 10-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.     

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.     

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.     

Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation.

alpha-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of alpha-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response.     

Partial structure of glutamic acid and alanine-rich protein,a major surface glycoprotein of the insect stages of Trypanosoma congolense

The tsetse fly transmitted salivarian trypanosome, Trypanosoma congolense of the subgenus Nanomonas, is the most significant of the trypanosomes with respect to the pathology of livestock in sub-Saharan Africa. Unlike the related trypanosome Trypanosoma brucei of the subgenus Trypanozoon, the major surface molecules of the insect stages of T. congolense are poorly characterized. Here, we describe the purification and structural characterization of the glutamic acid and alanine-rich protein, one of the major surface glycoproteins of T. congolense procyclic and epimastigote forms. The glycoprotein is a glycosylphosphatidylinositol-anchored molecule with a galactosylated glycosylphosphatidylinositol anchor containing an sn-1-stearoyl-2-l-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol phospholipid moiety. The 21.6-kDa polypeptide component carries two large mannose- and galactose-containing oligosaccharides linked to threonine residues via phosphodiester linkages. Mass spectrometric analyses of tryptic digests suggest that several or all of the closely related glutamic acid and alanine-rich protein genes are expressed simultaneously in a T. congolense population growing in vitro.     

The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation

A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.     

Structure of the lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum

The lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum were extracted from cytoplasmic membranes or from disintegrated bacteria with aqueous phenol and purified by gel chromatography. The lipoteichoic acid preparations contained phosphate, glycerol, galactose, glucose and fatty acids in a molar ratio of 1.0:1.0:1.3:1.2:0.3. Chemical analysis and NMR studies of the native preparations and of products from various acid and alkaline hydrolysis procedures gave evidence for the structure of two lipoteichoic acids. The lipid anchor appeared to be 3-O-(6'-(sn-glycero-1-phosphoryl)diacyl-beta-D-galactofuranosyl)-sn-1, 2-diacylglycerol. The polar part showed two structural features not previously described for lipoteichoic acids. A 1,2-(instead of the usual 1,3-) phosphodiester-linked sn-glycerol phosphate chain is only used substituted at the terminal glycerol unit with a linear polysaccharide, containing either beta(1----5)-linked D-galactofuranosyl groups or beta(1----6)-linked D-glucopyranosyl groups.     

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.     

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.     

Galactose starvation in a bloodstream form Trypanosoma brucei UDP-glucose 4'-epimerase conditional null mutant

Galactose metabolism is essential for the survival of Trypanosoma brucei, the etiological agent of African sleeping sickness. T. brucei hexose transporters are unable to transport galactose, which is instead obtained through the epimerization of UDP-glucose to UDP-galactose catalyzed by UDP-glucose 4'-epimerase (galE). Here, we have characterized the phenotype of a bloodstream form T. brucei galE conditional null mutant under nonpermissive conditions that induced galactose starvation. Cellular levels of UDP-galactose dropped rapidly upon induction of galactose starvation, reaching undetectable levels after 72 h. Analysis of extracted glycoproteins by ricin and tomato lectin blotting showed that terminal beta-d-galactose was virtually eliminated and poly-N-acetyllactosamine structures were substantially reduced. Mass spectrometric analysis of variant surface glycoprotein confirmed complete loss of galactose from the glycosylphosphatidylinositol anchor. After 96 h, cell division ceased, and electron microscopy revealed that the cells had adopted a morphologically distinct stumpy-like form, concurrent with the appearance of aberrant vesicles close to the flagellar pocket. These data demonstrate that the UDP-glucose 4'-epimerase is essential for the production of UDP-galactose required for galactosylation of glycoproteins and that galactosylation of one or more glycoproteins, most likely in the lysosomal/endosomal system, is essential for the survival of bloodstream form T. brucei.     

Deletion of the glucosidase II gene in Trypanosoma brucei reveals novel N-glycosylation mechanisms in the biosynthesis of variant surface glycoprotein

The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, and Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man(4-3)GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.     

Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.     

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.