Alpha 1-4GlcN Research Articles

Conformation of heparin pentasaccharide bound to antithrombin III.

The interaction, in aqueous solution, of the synthetic pentasaccharide AGA*IA(M) (GlcN,6-SO(3)alpha 1-4GlcA beta 1-4GlcN,3,6-SO(3)alpha 1-4IdoA,2-SO(3)alpha 1-4GlcN,6-SO(3)alpha OMe; where GlcN,6-SO(3) is 2-deoxy-2-sulphamino-alpha-D-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO(3) is L-iduronic acid 2-sulphate), which exactly reproduces the structure of the specific binding sequence of heparin and heparan sulphate for antithrombin III, has been studied by NMR. In the presence of antithrombin there were marked changes in the chemical shifts and nuclear Overhauser effects (NOEs), compared with the free state. On the basis of the optimized geometry of the pentasaccharide the transferred NOEs were interpreted with full relaxation and conformational exchange matrix analysis. An analysis of the three-dimensional structures of the pentasaccharide in the free state, and in the complex, revealed the binding to be accompanied by dihedral angle variation at the A-G and I-A(M) (where G, I, A and A(M) are beta-d-glucuronic acid, 2-O-sulphated alpha-L-iduronic acid, N,6-O-sulphated alpha-D-glucosamine and the alpha-methyl-glycoside of A respectively) glycosidic linkages. Evidence is also provided that the protein drives the conformation of the 2-O-sulphated iduronic acid residue towards the skewed (2)S(0) form.

Structural studies of octasaccharides derived from the low-sulfated repeating disaccharide region and octasaccharide serines derived from the protein linkage region of porcine intestinal heparin.

Four octasaccharide serines and three octasaccharides were isolated after heparinase treatment of porcine intestinal heparin. Their structures were characterized by enzymatic digestion in conjunction with HPLC and 500 MHz 1H NMR spectroscopy. Three of the four octasaccharide serines were structurally identical with those isolated previously, whereas one has the unreported structure DeltaHexA(2-sulfate)alpha1-4GlcN(N-sulfate)alpha1-4GlcAbe ta1-4GlcNAca lpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta 1-O-Ser (DeltaHexA, GlcN, IdceA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, respectively). The other three octasaccharides were isolated for the first time as discrete structures and shared the common core hexasulfated sequence DeltaHexA(2-sulfate)alpha1-4GlcN(N-sulfate)alpha1-4IdceAa lpha1-4GlcNA calpha1-4GlcAbeta1-4GlcN(N-sulfate)alpha1-4IdceA (2-sulfate)alpha1-4Gl cN(N,6-disulfate) with one or two additional sulfate groups. The octasaccharides which were derived from the low-sulfated repeating disaccharide region of heparin contained the common trisaccharide sequence -4IdceAalpha1-4GlcNAcalpha1-4GlcAbeta1- [Yamada, S., Yamane, Y., Tsuda, H., Yoshida, K., and Sugahara, K. (1998) J. Biol. Chem. 273, 1863-1871], suggesting the programmed biosynthesis of heparin. These octasaccharides are the largest oligosaccharides isolated so far from the low-sulfated irregular region of heparin. Since oligosaccharides larger than a pentasaccharide appear to potentially exhibit binding activities toward growth factors or other functional proteins, they will be useful for investigating the structural requirement for molecular interactions between heparin and/or heparan sulfate and biologically active proteins. During the course of the present structural studies, we evaluated the NMR data accumulated thus far on heparin oligosaccharides and found several interesting rules on chemical shifts of proton signals affected by the neighboring sugar residues and their sulfation, which will be in turn useful for determining structures of unknown heparin and/or heparan sulfate oligosaccharides based on the proton resonances.

A Major Common Trisulfated Hexasaccharide Core Sequence, Hexuronic Acid(2-Sulfate)-Glucosamine(N-Sulfate)-Iduronic Acid-N-Acetylglucosamine-Glucuronic Acid-Glucosamine(N-Sulfate), Isolated from the Low Sulfated Irregular Region of Porcine Intestinal Heparin

The major structure of the low sulfated irregular region of porcine intestinal heparin was investigated by characterizing the hexasaccharide fraction prepared by extensive digestion of the highly sulfated region with Flavobacterium heparinase and subsequent size fractionation by gel chromatography. Structures of a tetrasaccharide, a pentasaccharide, and eight hexasaccharide components in this fraction, which accounted for approximately 19% (w/w) of the starting heparin representing the major oligosaccharide fraction derived from the irregular region, were determined by chemical and enzymatic analyses as well as 1H NMR spectroscopy. Five compounds including one penta- and four hexasaccharides had hitherto unreported structures. The structure of the pentasaccharide with a glucuronic acid at the reducing terminus was assumed to be derived from the reducing terminus of a heparin glycosaminoglycan chain and may represent the reducing terminus exposed by a tissue endo-beta-glucuronidase involved in the intracellular post-synthetic fragmentation of macromolecular heparin. Eight out of the 10 isolated oligosaccharides shared the trisaccharide sequence, -4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-, and its reverse sequence, -4GlcA beta 1-4GlcNAc alpha 1-4IdceA alpha 1-, was not found. The latter has not been reported to date for heparin/heparan sulfate, indicating the substrate specificity of the D-glucuronyl C-5 epimerase. Furthermore, seven hexasaccharides shared the common trisulfated hexasaccharide core sequence delta HexA(2-sulfate)alpha 1-4GlcN(N-sulfate)alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcN(N-sulfate) which contained the above trisaccharide sequence (delta HexA, IdceA, GlcN, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, D-glucosamine, and D-glucuronic acid, respectively) and additional sulfate groups. The specificity of the heparinase used for preparation of the oligosaccharides indicates the occurrence of the common pentasulfated octasaccharide core sequence, -4GlcN(N-sulfate)alpha 1-4HexA(2-sulfate)1-4GlcN(N-sulfate) alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4 GlcN(N-sulfate)alpha 1-4HexA(2-sulfate)1-, where the central hexasaccharide is flanked by GlcN(N-sulfate) and HexA(2-sulfate) on the nonreducing and reducing sides, respectively. The revealed common sequence constituted a low sulfated trisaccharide representing the irregular region sandwiched by highly sulfated regions and should reflect the control mechanism of heparin biosynthesis.

Structures of Five Sulfated Hexasaccharides Prepared from Porcine Intestinal Heparin Using Bacterial Heparinase: STRUCTURAL VARIANTS WITH APPARENT BIOSYNTHETIC PRECURSOR-PRODUCT RELATIONSHIPS FOR THE ANTITHROMBIN III-BINDING SITE

Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G. (1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.

The Lipid Structure of the Glycosylphosphatidylinositol-anchored Mucin-like Sialic Acid Acceptors of Trypanosoma cruzi Changes during Parasite Differentiation from Epimastigotes to Infective Metacyclic Trypomastigote Forms

The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (approximately 70%) inositol phosphoceramides, consisting of a C18:0 sphinganine long chain base and mainly C24:0 and C16:0 fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Man alpha 1-2Man alpha 1-2Man alpha 1- 6Man alpha 1-4GlcN. The glycans are substituted at the GlcN residue and at the third alpha Man distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by beta-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gonçalves, L. P. B., Wait, R., Travassos, L. R., and Mendoça-Previato, L. (1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.

The glycoinositol-phospholipids of Phytomonas.

The Phytomonas spp. are trypanosomatid parasites of plants. A polar glycolipid fraction of a Phytomonas sp., isolated from the plant Euphorbia characias and grown in culture, was fractionated into four major glycolipid species (Phy 1-4). The glycolipids were analysed by chemical and enzymic modifications, composition and methylation analyses, electrospray mass spectrometry and microsequencing after HNO2 deamination and NaB3H4 reduction. The water-soluble headgroup of the Phy2 glycolipid was also analysed by 1H NMR. All four glycolipids were shown to be glycoinositol-phospholipids (GIPLs) with phosphatidylinositol (PI) moieties containing the fully saturated alkylacylglycerol lipids 1-O-hexadecyl-2-O-palmitoylglycerol and 1-O-hexadecyl-2-O-stearoylglycerol. The structures of the Phy 1-4 GIPLs are: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI, Glc alpha 1-2(NH2-CH2CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI, [formula: see text] Glc alpha 1-2(NH2CH2CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4(NH2-CH2CH2-HPO4-)GlcN alpha 1-6PI [formula: see text] and Glc alpha 1-2Glc alpha 1-2(NH2CH2-CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4(NH2CH2CH2-HPO4-)-GlcN alpha 1-6PI. [formula: see text] The Phytomonas GIPLs represent a novel series of structures. This is the first description of the chemical structure of cell-surface molecules of this plant pathogen. The Phytomonas GIPLs are compared with those of other trypanosomatid parasites and are discussed with respect to trypanosomatid phylogenetic relationships.

Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase: COMPREHENSIVE STRUCTURAL STUDIES ON THE PORCINE ANCHOR AND INTERSPECIES COMPARISON OF THE GLYCAN CORE STRUCTURES

The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal membrane dipeptidase (EC 3.4.13.19) were determined following deamination and reduction by a combination of liquid chromatography, exoglycosidase digestions, and methylation analysis. The glycan core was found to exhibit microheterogeneity with three structures observed for the porcine GPI anchor: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN (29% of the total population), Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN (33%), and Man alpha 1-2Man alpha 1-6(Gal beta 1-3GalNAc beta 1-4)Man alpha 1-4GlcN (38%). The same glycan core structures were also found in the human anchor but in slightly different proportions (25, 52, and 17%, respectively). Additionally, a small amount (6%) of the second structure with an extra mannose alpha (1-2)-linked to the non-reducing terminal mannose was also observed in the human membrane dipeptidase GPI anchor. A small proportion (maximally 9%) of the porcine GPI anchor structures was found to contain sialic acid, probably linked to the GalNAc residue. The porcine GPI anchor was found to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion electrospray-mass spectrometry revealed the presence of exclusively diacyl-phosphatidylinositol (predominantly distearoyl-phosphatidylinositol with a minor amount of stearoyl-palmitoyl-phosphatidylinositol) in the porcine membrane dipeptidase anchor. Porcine membrane dipeptidase was digested with trypsin and the C-terminal peptide attached to the GPI anchor isolated by removal of the other tryptic peptides on anhydrotrypsin-Sepharose. The sequence of this peptide was determined as Thr-Asn-Tyr-Gly-Tyr-Ser, thereby identifying the site of attachment of the GPI anchor as Ser368. This work represents a comprehensive study of the GPI anchor structure of porcine membrane dipeptidase and the first interspecies comparison of mammalian GPI anchor structures on the same protein.

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 hydrophobic mannoside Manα1-6Manα1-S-(CH2)7-CH3 acts as an acceptor for the UDP-Gal:glycosylphosphatidylinositol anchor α1,3-galactosyltransferase of Trypanosoma brucei

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. This anchor contains the core sequence ethanolamine-PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol, which is conserved in all GPI anchors, and a unique alpha Gal side chain attached to the 3-position of the alpha Man residue adjacent to the alpha GlcN residue. Here we report that trypanosome membranes can catalyse the transfer of Gal from UDP-Gal to the hydrophobic thioglycoside Man alpha 1-6Man alpha 1-S-(CH2)7-CH3. Characterization of the galactosylated products by electrospray mass spectrometry, exoglycosidase digestion and periodate-oxidation studies revealed that the major product was Man alpha 1-6(Gal alpha 1-3)Man alpha 1-S-(CH2)7-CH3. The similarity of this product to part of the mature VSG GPI anchor suggests that the thioglycoside is able to act as an acceptor for the trypanosome-specific UDP-Gal-GPI anchor alpha 1,3-galactosyltransferase.

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.

Isolation of the porcine heparin tetrasaccharides with glucuronate 2-O-sulfate.: HEPARINASE CLEAVES GLUCURONATE 2-O-SULFATE-CONTAINING DISACCHARIDES IN HIGHLY SULFATED BLOCKS IN HEPARIN

Eleven tetrasaccharides were isolated from the repeating disaccharide region of porcine intestinal heparin after strong digestion with Flavobacterium heparinase. Their structures were determined by composition analysis, enzymatic analysis, and 1H NMR spectroscopy. Nine of them have the common tetrasaccharide backbone, delta HexA alpha 1-4GlcN alpha 1-4IdoA alpha 1-4GlcN, where delta HexA and IdoA represent 4,5-unsaturated hexuronic acid and L-iduronic acid, respectively, and their structural variations are based upon the positions of sulfate groups. The nine compounds include one hexasulfated, three pentasulfated and five tetrasulfated compounds, and four of them have not been isolated previously as discrete structures. The other two of the 11 tetrasaccharides have the following hitherto unreported structures with novel glucuronate 2-O-sulfate at the internal position: delta HexA(2-sulfate) alpha 1- 4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N-sulfate) and delta HexA(2-sulfate) alpha 1-4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N,6-disulfate). Thus, 2-O-sulfated glucuronate in the highly sulfated tetrasaccharide structures typical of heparin has been demonstrated. The former and the latter tetrasaccharides account for 0.31 and 0.32% (w/w) of the starting heparin, respectively. Their yield, however, is an underestimation, since these tetrasaccharide structures in longer sequences will be degraded by heparinase. Although the latter tetrasaccharide described above was unexpectedly cleaved by heparinase into two disaccharide units, the former was not degraded by the enzyme most likely due to the lack of the 6-O-sulfate group on the GlcN residue at the reducing terminus. The results indicate its capability of catalyzing both anti and syn elimination, a property shared by heparitinases I and II and chondroitinase ABC. Both tetrasaccharides were degraded into disaccharides by heparitinase II. Therefore, it is necessary to reevaluate the disaccharide composition of heparin/heparan sulfate or oligosaccharide structures, which were previously determined after heparinase or heparitinase II digestion. It is no longer possible to conclude that the 2-O-sulfated unsaturated uronic acid residues obtained from heparin/heparan sulfate by lyase digestions are always derived from iduronate 2-O-sulfate residues in the original polymer. It is quite possible that the novel glucuronate 2-O-sulfate structure in the highly sulfated region of heparin is involved in some of the biological activities of heparin.

Structure of the glycosylphosphatidylinositol membrane anchor of human placental alkaline phosphatase

The glycosylphosphatidylinositol membrane anchor of human placental alkaline phosphatase was isolated by exhaustive proteolysis followed by hydrophobic interaction chromatography. The resulting glycosylphosphatidylinositol-peptide was subjected to compositional analysis and chemical and enzymic modifications. The neutral-glycan fraction, prepared by dephosphorylation followed by HNO2 deamination and reduction, was sequenced using exoglycosidases and acetolysis. The phosphatidylinositol moiety was analysed by fast-atom bombardment mass spectrometry and gas chromatography-mass spectrometry. Taken together the data suggest the structure, Thr-Asp-ethanolamine-PO4-Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN-(sn-1-O- alkyl-2-O-acylglycerol-3-PO4-1-myo-D-inositol), which contains an additional ethanolamine phosphate group at an unknown position.

Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates.

We prepared a series of oligosaccharides from porcine intestinal heparin after extensive digestion with a mixture of Flavobacterium heparinase as well as heparitinases I and II. Previously, we reported the structures of the two glycoserines derived from the carbohydrate-protein linkage region [Sugahara et al., J. Biol. Chem., 267, 1528-1533 (1992)] and three tetrasaccharides derived from the antithrombin III-binding site [Yamada et al., J. Biol. Chem., 268, 4780-4787 (1993)]. In this study, we determined the structures of 10 other tetrasaccharides and a trisaccharide by enzymatic digestion, fast atom bombardment mass spectrometry and 500-MHz 1H NMR spectroscopy. These tetrasaccharides share the common disulphated structure, delta HexA alpha 1-4GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate)alpha 1-4GlcN (where HexA is hexuronic acid and IdoA is L-iduronic acid), and their structural variations are based upon the positions of additional sulphate groups. Eight among the 10 have never been isolated as discrete structures. The structure of the trisaccharide is GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate) alpha 1-4GlcN(N,6-disulphate) and is derived from the non-reducing terminus of heparin chains. This structure may represent the terminus of a biosynthetically formed native heparin chain or a newly formed non-reducing terminus exposed by a tissue endo-beta-glucuronidase which may be involved in the intracellular post-synthetic fragmentation of macromolecular heparin. The 11 structures characterized in the present study and 6 additional tetrasaccharides were used to investigate the substrate specificities of heparinase, as well as heparitinases I and II. The results indicate that modification of the adjacent glucosamine on the reducing side of the disaccharide cleavage site influences the enzymatic action of the lyases, whereas the adjacent uronic acid on the non-reducing side is not recognized by these enzymes.

Structural studies on the oligosaccharides isolated from bovine kidney heparan sulphate and characterization of bacterial heparitinases used as substrates.

We prepared a series of oligosaccharides from commercial bovine kidney heparan sulphate after limited digestion with heparitinase I from Flavobacterium heparinum, and determined the structures of eight tetrasaccharides and a hexasaccharide by enzymatic analysis, fast atom bombardment mass spectrometry and 500 MHz 1H NMR spectroscopy. The tetrasaccharides share the common core structure delta 4,5HexA alpha 1-4GlcN alpha 1-4HexA1-4GlcN (where delta 4,5HexA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid and HexA is hexuronic acid), with zero, one or two sulphate groups. Seven of them contain non-sulphated glucuronic or iduronic acid, and the other, 2-O-sulphated iduronic acid at the internal position. Although they contain ordinary structures which should be widely distributed in the relatively low-sulphated region of heparan sulphate, five of the tetrasaccharides were isolated for the first time as discrete structures. The structure of the hexasaccharide was determined as delta 4,5HexA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta-1-4 Xyl and is derived from the carbohydrate-protein linkage region of the heparan sulphate chains. The hexasaccharide seems to have been released by the alkaline treatment used to prepare the heparan sulphate. The Gal residues were non-sulphated as are those in the porcine intestinal heparin chains, but in contrast to the sulphated Gal structures previously demonstrated in the carbohydrate-protein linkage region of chondroitin sulphate chains. These oligosaccharides were used to investigate the substrate specificity of heparitinases I and II from F. heparinum. The results revealed that heparitinase I cleaves hexosaminidic bonds linked to non-sulphated glucuronic or iduronic acid residues. The glucosaminidic linkage of the hexasaccharide was sensitive to heparitinase I, but resistant to heparitinase II, demonstrating the differential specificity of these enzymes towards the carbohydrate-protein linkage region.

Structure of the glycosyl-phosphatidylinositol membrane anchor of acetylcholinesterase from the electric organ of the electric-fish, Torpedo californica

The structure of the glycan moiety of the glycosyl-phosphatidylinositol (GPI) membrane anchor from Torpedo californica (electric fish) electric-organ acetylcholinesterase was solved using n.m.r., methylation analysis and chemical and enzymic micro-sequencing. Two structures were found to be present: Glc alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol and Glc alpha 1-2Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.

The mechanism of inhibition of glycosylphosphatidylinositol anchor biosynthesis in Trypanosoma brucei by mannosamine.

The inhibition of glycosylphosphatidylinositol anchor biosynthesis by mannosamine has been described previously in the procyclic forms of Trypanosoma brucei and in mammalian cells (Lisanti, M. P., Field, M. C., Caras, I. W. J., Menon, A. K., and Rodriguez-Boulan, E. (1991) EMBO J. 10, 1969-1977). A recent report has suggested that mannosamine exerts these effects by becoming incorporated into glycosylphosphatidylinositol anchor intermediates (Pan, Y-T., Kamitani, T., Bhuvaneswaran, C., Hallaq, Y., Warren, C. D., Yeh, E. T. H., and Elbein, A. D. (1992) J. Biol. Chem. 267, 21250-21255). In this paper we have analyzed the effects of mannosamine on glycosylphosphatidylinositol anchor and variant surface glycoprotein biosynthesis in the blood-stream form of T. brucei. Trypanosomes were biosynthetically labeled with [3H]mannosamine, and [3H]glucosamine in the presence of mannosamine, and the structures of the labeled glycolipids which accumulated were determined. The main glycolipid metabolite of mannosamine was shown to be ManN-Man-GlcN-PI. A trypanosome cell-free system preloaded with this compound was significantly impaired in its ability to synthesize glycosylphosphatidylinositol anchor intermediates beyond Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI. This compound is therefore proposed to be an inhibitor of the Dol-P-Man:Man alpha 1-6Man alpha 1-4GlcNa alpha 1-6PI alpha 1-2-mannosyltransferase of the GPI biosynthetic pathway. In living trypanosomes, 4 mM mannosamine had no effect on protein synthesis but reduced the rate of formation of mature glycosylphosphatidylinositol anchor precursors by 80%. This reduction in anchor precursor synthesis was insufficient to prevent the attachment of glycosylphosphatidylinositol anchors to newly synthesized variant surface glycoprotein molecules. These data suggest that the rate of anchor precursor synthesis in the bloodstream form of T. brucei, in contrast to mammalian cells and the procyclic form of T. brucei, is in large excess of the cellular requirements for protein anchorage.

The glycoinositol phospholipids of Leishmania mexicana promastigotes. Evidence for the presence of three distinct pathways of glycolipid biosynthesis

Most macromolecules at the cell surface of parasitic protozoa of the genus Leishmania, including the major surface glycoproteins and a complex lipophosphoglycan (LPG), are attached to the plasma membrane via glycosyl-phosphatidylinositol (GPI) anchors. Free glycoinositol phospholipids (GIPLs) which are not linked to protein or phosphoglycan have also been found. In this study, we show that L. mexicana promastigotes synthesize two distinct GIPL lineages, comprising at least 10 glycolipid species. These structures were characterized using a combination of gas-liquid chromatography-mass spectrometry, methylation linkage analysis, and chemical and exoglycosidase sequencing. The major lineage contains GIPLs with the glycan structures Man alpha 1-3Man alpha 1-4GlcN (iM2), Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (iM3), and Man alpha 1-2Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (iM4), which are linked to alkylacyl-PI containing predominantly C16:0 and C18:0 fatty acids and C18:0 alkyl chains (referred to as the hybrid type GIPLs). A proportion of the iM3 and iM4 species (32 and 4%, respectively) are substituted with an ethanolamine-phosphate residue. The location of this residue on the core glucosamine residue was inferred from the results of methylation analyses and alpha-mannosidase digestion. The minor GIPL lineage contains GIPLs with the same glycan sequences as the glycolipid anchor of LPG (referred to as the type-2 GIPLs). The alkylacyl-PI or lyso-alkyl-PI lipid moieties of these GIPLs differ from those of the hybrid type GIPLs and from the main pool of alkylacyl-PI in containing significant levels of C24:0 and C26:0 alkyl chains. The most polar of these GIPLs, LPGp, has the properties expected of a biosynthetic precursor to the LPG, having the structure, [formula: see text] Finally, the GPI anchors of the major promastigote proteins were found to contain the glycan sequence Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN, and an alkylacyl-PI lipid moiety which was highly enriched for C24:0 or C26:0 alkyl chains. These data suggest that L. mexicana promastigotes contain three distinct pathways of GPI biosynthesis. The possibility that the distinct alkyl chain compositions of the different GPI glycolipids reflects the subcellular compartmentalization of different GPI biosynthetic pathways is discussed.

Refined structure of the lipophosphoglycan of Leishmania donovani.

The primary structure of the major surface glycoconjugate of Leishmania donovani parasites, a lipophosphoglycan, has been further characterized. The repeating PO4-6Galp beta 1-4Man disaccharide units, which are a salient feature of the molecule, are shown to terminate with one of several neutral structures, the most abundant of which is the branched trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The phosphosaccharide core of lipophosphoglycan, which links the disaccharide repeats to a lipid anchor, contains 2 phosphate residues. One of the core phosphates has previously been localized on O-6 of the galactosyl residue distal to the lipid anchor; the second phosphate is now shown to be on O-6 of the mannosyl residue distal to the anchor and to bear an alpha-linked glucopyranosyl residue. Also, the anomeric configuration of the unusual 3-substituted Galf residue in the phosphosaccharide core is established as beta. The complete structure of the core is thus PO4-6Galp alpha 1-6Galp alpha 1-3Galf beta 1-3[Glcp alpha 1-PO4-6]Manp alpha 1-3Manp alpha 1-4GlcN alpha 1-. This further clarification of the structure of lipophosphoglycan may prove beneficial in determining the structure-function relationships of this highly unusual glycoconjugate.

Developmental changes in the glycosylated phosphatidylinositols of Leishmania donovani. Characterization of the promastigote and amastigote glycolipids

In addition to utilizing glycosylated phosphatidylinositols (GPIs) as anchors for surface proteins, protozoan parasites of the genus Leishmania synthesize two novel classes of GPI: the polydisperse lipophosphoglycans (LPGs) and a family of low molecular weight glycoinositol phospholipids (GIPLs). We now show that LPG is expressed in high copy number (6 x 10(6) molecules/cell) in the promastigote (insect) stage of L. donovani but not in the amastigote stage, which infects mammalian macrophages. Detection of these molecules was by gas chromatography-mass spectrometric analyses and by a sensitive radiolabeling procedure. In contrast, a novel family of GIPLs was present in high copy number (approximately 10(7) molecules/cell) in both promastigote and amastigote stages of L. donovani. These glycolipids were purified and analyzed by gas chromatography-mass spectrometry, methylation analysis, and by chemical and enzymatic sequencing after deamination and NaB3H4 reduction. Promastigotes contained three major GIPLs species with the following generalized structure [formula: see text] where R = H for isoM2, Man alpha 1- for isoM3 or Man alpha 1-2Man alpha 1- for isoM4. Amastigotes contained two major GIPL species that lacked the alpha 1-3-linked mannose branch and had the linear structures Man alpha 1-6Man alpha 1-4GlcN (M2) and Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN (M3) linked to alkylacyl-PI. The 1-O-alkyl-2-acyl-PI moieties of all these species contained predominantly C18:0 alkyl chains and C16:0 or C18:0 fatty acids. Amastigotes contained, in addition, a GalNAc beta 1-3 terminating glycosphingolipid with homology to the mammalian para Forssman glycolipid. This glycolipid appeared to be a constituent of the parasite membrane but was not metabolically labeled with [3H]glucose, suggesting that it was acquired from host cells. These results suggest that LPG may not be required for amastigote survival in the mammalian host and that the GIPLs are likely to be major components on the surface membrane in both stages.

Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

In common with many other plasma membrane glycoproteins of eukaryotic origin, the promastigote surface protease (PSP) of the protozoan parasite Leishmania contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. The GPI anchor of Leishmania major PSP was purified following proteolysis of the PSP and analyzed by two-dimensional 1H-1H NMR, compositional and methylation linkage analyses, chemical and enzymatic modifications, and amino acid sequencing. From these results, the structure of the GPI-containing peptide was found to be Asp-Gly-Gly-Asn-ethanolamine-PO4-6Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-(1-alkyl-2-acyl-glycerol). The glycan structure is identical to the conserved glycan core regions of the GPI anchor of Trypanosoma brucei variant surface glycoprotein and rat brain Thy-1 antigen, supporting the notion that this portion of GPIs are highly conserved. The phosphatidylinositol moiety of the PSP anchor is unusual, containing a fully saturated, unbranched 1-O-alkyl chain (mainly C24:0) and a mixture of fully saturated unbranched 2-O-acyl chains (C12:0, C14:0, C16:0, and C18:0). This lipid composition differs significantly from those of the GPIs of T. brucei variant surface glycoprotein and mammalian erythrocyte acetylcholinesterase but is similar to that of a family of glycosylated phosphoinositides found uniquely in Leishmania.