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M A Ferguson

Publications and source records attributed to M A Ferguson.

At least 127 records · Page 7Linked to original sources
PubMed1994

Glycosyl-phosphatidylinositol molecules of the parasite and the host.

The glycosyl-phosphatidylinositol (GPI) protein-membrane anchors are ubiquitous among the eukaryotes. However, while mammalian cells typically express in the order of 100 thousand copies of GPI-anchor per cell, the parasitic protozoa, particularly the kinetoplastids, express up to 10-20 million copies of GPI-anchor and/or GPI-related glycolipids per cell. Thus GPI-family members dominate the cell surface molecular architecture of these organisms. In several cases, GPI-anchored proteins, such as the variant surface glycoprotein (VSG) of the African trypanosomes, or GPI-related glycolipids, such as the lipophosphoglycan (LPG) of the Leishmania, are known to be essential for parasite survival and infectivity. The highly elevated levels and specialised nature of GPI metabolism in the kinetoplastid parasites suggest that the GPI biosynthetic pathways might be good targets for the development of chemotherapeutic agents. This article introduces the range of GPI structures found in protozoan parasites, and their mammalian hosts, and discusses some aspects of GPI biosynthesis.

Animals↗
PubMed1994

Comparison of measures of frequency resolution and recruitment in patients undergoing neuro-otological investigation.

Loudness recruitment and reduced frequency resolution both occur in cochlear types of hearing loss. One theory of loudness recruitment suggests that, as intensity is coded partly by spread of excitation across the nerve fibre array, recruitment is a direct consequence of the broad spread of excitation associated with poor frequency resolution. The present study investigated the relationship between these two quantities. The study involved a simple measure of frequency resolution (three-point psychoacoustical tuning curve, PTC) and conventional measures of recruitment obtained from patients undergoing neuro-otological investigation. Results from 376 ears of 226 patients without any material conductive impairment are presented. Measures of recruitment included the alternate binaural loudness balance test (ABLB) and estimates of dynamic range given by the sensation levels of the uncomfortable loudness level and the acoustic reflex threshold. Once covariation with hearing threshold level had been accounted for, no clear relationship emerged between frequency resolution and any of the measures of recruitment. This finding does not support the notion that frequency resolution and recruitment are specifically related. Rather, PTCs provide information complementary to measures of recruitment.

Adolescent↗
PubMed1994

The role of glycolipid C in the GPI biosynthetic pathway in Trypanosoma brucei bloodstream forms.

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in Trypanosoma brucei bloodstream forms includes the formation of glycolipid C. This molecule is the inositol-acylated form of the GPI anchor precursor, glycolipid A. There is no evidence for the transfer of glycolipid C to protein in vivo and the role of glycolipid C is unclear. In this paper we show that glycolipid C is not synthesised in the presence of phenylmethylsulphonyl fluoride (PMSF) and that glycolipid C is not an obligatory intermediate on the pathway to the formation of glycolipid A. Using pulse-chase experiments we show that glycolipid A and glycolipid C are in a dynamic equilibrium and we suggest that only the forward reaction (glycolipid A conversion to glycolipid C) is inhibited by PMSF.

Animals↗
PubMed1994

The developmental regulation and biosynthesis of GPI-related structures in Leishmania parasites.

Most macromolecules on the surface of Leishmania parasites, including the major surface proteins and a complex lipophosphoglycan (LPG) are anchored to the plasma membrane via GPI glycolipids. Free glycoinositol-phospholipids (GIPLs) which are not linked to protein or phosphoglycan are also abundant in the plasma membrane. From structural and metabolic labeling studies it is proposed that most Leishmania species express three distinct pathways of GPI biosynthesis. Some of these pathways (i.e. those involved in the protein and LPG anchor biosynthesis) are down-regulated during the differentiation of the insect (promastigote) stage to the mammalian (amastigote) stage. In contrast, the GIPLs are expressed in high copy number in both developmental stages. Based on analysis of the lipid moieties of the different GPI species it is possible that the pathways of GPI anchor and GIPL biosynthesis are located in different subcellular compartments. The relative flux through the GIPL and LPG biosynthetic pathways has been examined in L. major promastigotes. These studies showed that while the rate of synthesis of the GIPLs and LPG is similar, LPG is shed more rapidly from the plasma membrane and has a higher turnover. The possible metabolic relationship between the GIPL and LPG biosynthetic pathways is discussed.

Animals↗
PubMed1994

Novel GPI structures of the membrane anchor of acetylcholinesterase from the electric organ of Torpedo californica.

The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 Man alpha 1-4 GlcN alpha 1-6myo-inositol, and Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 (GalNAc beta 1-4) Man alpha 1-4 GlcN 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.

Acetylcholinesterase↗
PubMed1994

GPI-anchored glycoconjugates from Trypanosoma cruzi trypomastigotes are recognized by lytic anti-alpha-galactosyl antibodies isolated from patients with chronic Chagas' disease.

The target molecules on the cell surface of Trypanosoma cruzi trypomastigotes reacting with lytic anti-alpha-galactosyl antibodies from chronic patients with Chagas' disease (Ch anti-Gal) have been purified by solvent extraction and identified as glycoconjugates migrating in the 74-96-kDa range (F2 antigen) and in the 120-200-kDa range (F3 antigen) on SDS-PAGE. The F3 antigen was tested for binding to Ch and normal human serum (NHS) anti-Gal and to MoAb 3C9. We observed that Ch anti-Gal and MoAb 3C9, but not NHS anti-Gal, bind strongly to the trypomastigote glycoconjugates. These antibodies, however, did not compete with each other for binding to F3 molecules, indicating that they are recognizing different epitopes. Binding of Ch anti-Gal to F3 antigen is abolished by treatment of these molecules with alpha- but not beta-galactosidase. Binding of 3C9 MoAb is abolished by treatment of F3 with sialidase. F2/F3 antigens absorbed Ch anti-Gal as well as lytic antibodies from total chagasic sera. These antigens also specifically discriminate between the serum reactivity of patients with active Chagas' disease and those of sera from cured patients, drug-treated patients with dissociated serology (positive conventional serology, negative trypanolytic activity), healthy individuals, and patients with several other infectious diseases. We also observed that F2/F3 antigens are anchored to the parasite membrane via glycosylphosphatidylinositol (GPI). The alpha-galactosyl epitopes recognized by Ch anti-Gal are present in a series of O-linked oligosaccharide chains in the mucin-like glycoprotein component of the complex.

Animals↗
PubMed1993

Structures of glycosylphosphatidylinositol membrane anchors from Saccharomyces cerevisiae.

Metabolic labeling studies suggest that Saccharomyces cerevisiae contains many glycoproteins that are anchored in the lipid bilayer by glycosylphosphatidylinositol membrane anchors. Membrane anchors were purified from a crude yeast membrane protein fraction and analyzed by two-dimensional 1H-1H NMR, fast atom bombardment-mass spectrometry, compositional and methylation linkage analyses, as well as chemical and enzymatic modifications. The yeast glycosylphosphatidylinositol anchors consist of the following structures: ethanolamine-PO4-6(R-2)Man alpha 1-2Man alpha 1-6Man alpha 1-4Glc-NH2 alpha 1-6myo-inositol-1-PO4-lipid, where R is mainly Man alpha 1- (80%) with some Man alpha 1-2Man alpha 1- (15%) and Man alpha 1-3Man alpha 1- (5%). The core region of the yeast anchors (ethanolamine-PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2 alpha 1-6myo-inositol-1-PO4) is identical to the conserved core region found in glycosylphosphatidylinositol anchors from protozoa and mammals. The lipid moieties of the total yeast glycosylphosphatidylinositol anchors are mainly ceramides, consisting mostly of C18:0 phytosphingosine and C26:0 fatty acid. However, the lipid moiety of the glycosylphosphatidylinositol anchor of the purified ggp125 protein is a lyso- or diacylglycerol, containing C26:0 fatty acids. This suggests that yeast adds different lipid components to the glycosylphosphatidylinositol anchors of different proteins.

Carbohydrate Conformation↗
PubMed1993

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.

Acetylcholinesterase↗
PubMed1993

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.

Animals↗
PubMed1993

Characterization of glycoinositol phospholipids in the amastigote stage of the protozoan parasite Leishmania major.

The major macromolecules on the surface of the parasitic protozoan Leishmania major appear to be down-regulated during transformation of the parasite from an insect-dwelling promastigote stage to an intracellular amastigote stage that invades mammalian macrophages. In contrast, the major parasite glycolipids, the glycoinositol phospholipids (GIPLs), are shown here to be expressed at near-constant levels in both developmental stages. The structures of the GIPLs from tissue-derived amastigotes have been determined by h.p.l.c. analysis of the deaminated and reduced glycan head groups, and by chemical and enzymic sequencing. The deduced structures appear to form a complete biosynthetic series, ranging from Man alpha 1-4GlcN-phosphatidylinositol (PI) to Gal alpha 1-3Galf beta 1-3Man alpha 1-3Man alpha 1-4GlcN-PI (GIPL-2). A small proportion of GIPL-2 was further extended by addition of a Gal residue in either alpha 1-6 or beta 1-3 linkage. From g.c.-m.s. analysis and mild base treatment, all the GIPLs were shown to contain either alkylacylglycerol or lyso-alkylglycerol lipid moieties, where the alkyl chains were predominantly C18:0, with lower levels of C20:0, C22:0 and C24:0. L. major amastigotes also contained at least two PI-specific phospholipase C-resistant glycolipids which are absent from promastigotes. These neutral glycolipids were resistant to both mild acid and mild base hydrolysis, contained terminal beta-Gal residues and were not lost during extensive purification of amastigotes from host cell membranes. It is likely that these glycolipids are glycosphingolipids acquired from the mammalian host. The GIPL profile of L. major amastigotes is compared with the profiles found in L. major promastigotes and L. donovani amastigotes.

Animals↗
PubMed1993

Post-translational modifications of the Dictyostelium discoideum glycoprotein PsA. Glycosylphosphatidylinositol membrane anchor and composition of O-linked oligosaccharides.

Prespore-specific antigen (PsA) is a cell-surface glycoprotein isolated from Dictyostelium discoideum, which is post-translationally modified by addition of carbohydrate to threonine residues of the carboxy-terminal peptide domain, and a glycosylphosphatidylinositol (GPI) anchor which attaches the glycoprotein to the cell membrane. The GPI anchor was isolated by proteolytic cleavage of the protein, and the structure of the lipid and glycan portions of the anchor were determined. The lipid moiety of the anchor is an inositolphosphoceramide which contains C18:0 phytosphingosine as a long chain base, and a mixture of fatty acids with a C18:1 mono-unsaturated fatty acid as the major component. The purified GPI anchor was susceptible to digestion by a bacterial phosphatidylinositol-specific phospholipase-C enzyme. The glycan of the GPI anchor consisted of two molecular species present in the ratio 55:45, the structures of which were determined by exoglycosidase sequencing and found to be Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2 and Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine in both structures is glycosidically linked to the inositol ring of the inositolphosphoceramide. The GPI glycan structures are consistent with the conserved core structure of all characterised GPI anchors, and the structure of the D. discoideum GPI moiety has features in common with structures from yeast, protozoa and higher eukaryotes. Compositional analysis of the carbohydrate attached to threonine residues in the carboxy-terminal peptide domain is also presented. The oligosaccharides bind to wheat germ agglutinin, and contain glucosamine and fucose as the major constituents.

Amino Acid Sequence↗
PubMed1993

Structure of the CAMPATH-1 antigen, a glycosylphosphatidylinositol-anchored glycoprotein which is an exceptionally good target for complement lysis.

CAMPATH-1 antibodies recognize a unique molecule on human lymphocytes and are unusually efficient at causing cell lysis with homologous complement. They have been successfully used for lymphocyte depletion in vivo in a variety of diseases. We find that the antigen is a very small glycosylphosphatidylinositol (GPI)-anchored glycoprotein with a mature peptide comprising only 12 amino acids. It can be separated into two distinct antigenic fractions which differ in their susceptibility to phosphatidylinositol-specific phospholipase C. There is one N-linked glycosylation site, but no evidence for O-glycosylation despite the presence of several serine and threonine residues. The antibodies were found to bind, albeit with a generally reduced affinity, to a proteolytic fragment containing the C-terminal tripeptide and the GPI anchor. We postulate that one of the reasons why the CAMPATH-1 antibodies are so good for cell lysis is because they bind to an epitope which is likely to be very close to the lipid bilayer.

Amino Acid Sequence↗
PubMed1993

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.

Animals↗
PubMed1993

A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor.

The procyclic acidic repetitive protein is the major cell-surface glycoprotein of the insect-dwelling procyclic forms of the Trypanosoma brucei species of African trypanosomes. The glycoprotein contains an acidic Glu-Pro repeat domain, a glycosyl-phosphatidylinositol membrane anchor and a putative asparagine glycosylation site. In this paper we describe a rapid purification scheme for this glycoprotein, using solvent extraction and hydrophobic interaction chromatography, and a partial characterization of the glycosylphosphatidylinositol membrane anchor. The carbohydrate composition of the anchor is extremely unusual; it contains on average nine GlcNAc, nine Gal, and five sialic acid residues. This is the first description of such a heavily substituted and negatively charged anchor. A comparison between the trypanosome procyclic surface and the Leishmania promastigote surface is also presented.

Acetylglucosamine↗
PubMed1993

Analysis of the neutral glycan fractions of glycosyl-phosphatidylinositols by thin-layer chromatography.

The radiolabeled neutral glycan fractions of both glycosyl-phosphatidylinositol (GPI) protein anchors and related glycolipids were analyzed by thin-layer chromatography on silica gel 60, using butanol:ethanol:water (4:3:3, v/v) or a combination of 1-propanol:acetone:water (9:6:5, v/v and 5:4:1, v/v) as solvents. Dextran acid hydrolysates were used as standards, and oligomers up to 11 glucose units could be resolved. A comparison of 18 GPI-glycan standards revealed that their migration was dependent mainly on the size of the oligosaccharide. Isomers were generally not resolved, with the exception of Man alpha 1-6Man alpha 1-4AHM and Man alpha 1-3Man alpha 1-4AHM. Structures containing galactofuranose or GalNAc were well resolved from structures containing only Galp and/or Manp. The utility of this method for the microsequencing of radiolabeled neutral glycans derived from two GPI glycolipids, using exoglycosidases and chemical treatments, is demonstrated. This method is a simple and useful complement to the existing chromatographic techniques.

Carbohydrate Sequence↗
PubMed1993

Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalyzed by trans-sialidase in metacyclic forms of Trypanosoma cruzi.

We have previously shown that 35- and 50-kDa glycoconjugates of cultured metacyclic trypomastigotes participate in the attachment of parasites to mammalian cells. Here we show that when metacyclic trypomastigotes are incubated with [3H]sialyllactose, most of the sialic acid is transferred to these 35/50-kDa molecules in a reaction catalyzed by a parasite transsialidase. The sialic acid is incorporated in oligosaccharides of about 10 glucose units in size that are released from the glycoconjugate by mild alkaline hydrolysis. Compositional analysis reveals that the 35/50-kDa molecules are highly glycosylated proteins rich in threonine, galactose, N-acetyl-glucosamine and sialic acid. These glycoproteins can be labeled in vivo with [3H]palmitate, and the labeled fatty acid is released by glycosylphosphatidylinositol specific phospholipases C. This result, associated with the fact that they contain mannose, ethanolamine, myo-inositol, and lipid, indicate that these glycoproteins are anchored to the membrane by glycosylphosphatidylinositol. During cell invasion, these molecules appear to be capped and locally released by the parasite.

Amino Acids↗