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Biomedical subjects

M A Ferguson

Publications and source records attributed to M A Ferguson.

At least 109 records · Page 6Linked to original sources
PubMed1995

Hydrophobic glycosides of N-acetylglucosamine can act as primers for polylactosamine synthesis and can affect glycolipid synthesis in vivo.

Several hydrophobic glycosides of N-acetylglucosamine (GlcNAc) served as primers for polylactosamine synthesis when added to Chinese hamster ovary (CHO) cells. The modified glycosides, containing one to six lactosamine repeats in linear array, were sialylated and secreted into the culture medium. The relative efficiencies of the glycosides to serve as primers were dependent on the nature of the aglycone and on the anomeric configuration of the GlcNAc residue. The same compounds were tested for their effects on glycolipid synthesis in CHO cells. All of the beta-glycosides significantly inhibited the synthesis of the lactoseries glycolipid GM3 whereas the alpha-glycoside was inactive. The compound GlcNAc alpha 1-O-benzyl- was the most efficient primer of polylactosamine synthesis and had no effect on glycolipid synthesis. This compound may have potential for the assay of the polylactosamine synthetic capacity of living cells.

Acetylglucosamine↗
PubMed1995

Primary structure of CD52.

The CD52 antigen was extracted from human spleens with organic solvents and purified by immunoaffinity and reverse-phase chromatography. The latter step resolved two CD52 species, called CD52-I and CD52-II. Both species were found to contain similar N-linked oligosaccharides and glycosylphosphatidylinositol (GPI) anchor glycans. The N-linked oligosaccharides were characterized by methylation linkage analysis and, following exhaustive neuraminidase and endo-beta-galactosidase digestion, by the reagent array analysis method. The results showed that the single CD52 N-glycosylation site is occupied by large sialylated, polylactosamine-containing, core-fucosylated tetraantennary oligosaccharides. The locations of the phosphoryl substituents on the GPI anchor glycan were determined using a new and sensitive method based upon partial acid hydrolysis of the GPI glycan. The difference between CD52-I and CD52-II was in the phosphatidylinositol (PI) moieties of the GPI anchors. The phosphatidylinositol-specific phospholipase C-sensitive CD52-I contained exclusively distearoyl-PI, while the PI-phospholipase C-resistant CD52-II contained predominantly a palmitoylated stearoyl-arachidonoyl-PI, as judged by electrospray ionization mass spectrometry. Tandem mass spectrometric studies indicated that the palmitoyl residue of the CD52-II anchor is attached to the 2-position of the myo-inositol ring. Both the CD52-I and CD52-II PI structures are unusual for GPI anchors and the possible significance of this is discussed. The alkali-lability of the CD52 epitope recognized by the Campath-1H monoclonal antibody was studied. The data suggest that the alkali-labile hydroxyester-linked fatty acids of the GPI anchor are necessary for antibody binding.

Antigens, CD↗
PubMed1995

The cation-independent mannose-6-phosphate receptor binds to soluble GPI-linked proteins via mannose-6-phosphate.

The cation-independent mannose-6-phosphate/insulin-like growth factor II receptor has been observed to bind to soluble forms of glycosyl-phosphatidylinositol-linked molecules, one of mammalian origin (rat Thy-1) and two of protozoan origins. Of the two phosphate groups found on the soluble forms of the protozoan glycosyl-phosphatidylinositol-linked molecules: (i) the internal mannose-6-phosphate diester (which forms a part of the ethanolamine bridge) and (ii) the inositol-1,2 cyclic phosphate group (which arises after cleavage of the membrane associated form with phosphatidylinositol-specific phospholipase C), only the former appears to be recognized by the mannose-6-phosphate/insulin-like growth factor II receptor, as mild acid hydrolysis which destroys the latter has been observed not to affect the receptor binding site.

Animals↗
PubMed1995

Progression of neuropathy in peripheral arterial disease.

Atherosclerotic peripheral arterial disease (PAD) can cause muscle denervation, but whether it is associated with more severe peripheral nerve disease is not clear. Equally unclear is the effect of exercise training on the neuromuscular aspects of this disease. We performed serial electrophysiologic studies (nerve conduction studies and quantitative electromyography) and muscle strength assessment on 16 patients with moderately severe PAD. Seven of the patients were assigned to a natural history (control) group and 9 to an exercise training group. Over study periods ranging from 3 to 23 months, 6 of 7 patients in the control group and 8 of 9 patients in the exercise training group showed progression of multifocal neuropathic disease in their ischemic legs. For equivalent lengths of time, the degree of neuropathic progression was not significantly different between the control and exercise training groups. There was a significant decline in muscle strength for the control group but not for the exercise training group. These results demonstrate that PAD can be associated with the development of a multifocal predominantly motor neuropathy, which is most likely ischemic in etiology. Furthermore, exercise training does not pose an additional risk for the development of neuropathy in patients with moderately severe PAD.

Aged↗
PubMed1995

Characterization of the lipid moiety of the glycosylphosphatidylinositol anchor of Trypanosoma cruzi 1G7-antigen.

The 90-kDa stage-specific 1G7-antigen has been implicated in the invasion of host cells by the metacyclic forms of Trypanosoma cruzi. The antigen is attached to the plasma membrane via glycosylphosphatidylinositol, the partial structure of which was the first to be determined for a protein of this parasite. In this study, the complete structure of the lipid component of the anchor was determined by electrospray mass spectrometry, gas chromatography mass spectrometry, phospholipase sensitivity and high-performance thin-layer chromatography of the diaradylglycerol components after benzoylation. These analyses showed that the lipid moiety of 1G7-antigen is composed essentially of 1-O-hexadecyl-2-O-hexadecanoyl-phosphatidylinositol and 1-O-hexadecyl-2-O-octadecanoyl-phosphatidylinositol. The high sensitivity of the electrospray mass spectrometric analysis unexpectedly revealed the presence of a small proportion of putative inositol-phosphoceramide structures, and confirmed the absence of inositol-acylated species. An interesting finding was that the biosynthetic incorporation of [3H]palmitate labelled solely the acyl position, and not the 1-O-alkyl chain in the 1G7-antigen anchor.

Animals↗
PubMed1994

Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi.

Sera of patients with chronic Chagas' disease (American trypanosomiasis) contain elevated levels of anti-alpha-galactosyl antibodies that are lytic to Trypanosoma cruzi. The T. cruzi trypomastigote F2/3 antigen complex recognized by these antibodies runs as a broad smear on SDS/PAGE [Almeida, Krautz, Krettli and Travassos (1993) J. Clin. Lab. Anal. 7, 307-316]. Treatment of T. cruzi trypomastigote cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished most of their reactivity to chronic Chagas'-disease ((Chagasic, Ch) anti-alpha-galactosyl antibodies (anti-Gal). The F2/3 antigen complex, purified by solvent extraction and hydrophobic-interaction chromatography, contained 60% carbohydrate by weight and substantial amounts of Thr, Ser, Glx, Asx, Gly, Ala and Pro, but relatively few hydrophobic amino acids. The presence of myoinositol, ethanolamine and 1-O-hexadecylglycerol suggested the presence of glycosyl-phosphatidylinositol membrane anchors. This was confirmed by PI-PLC treatment, which rendered the F2/3 molecules hydrophilic and reactive to anti-(cross-reacting determinant) antibodies. The majority of the GlcNAc content of the F2/3 antigens was found at the reducing termini of oligosaccharides in O-glycosidic linkage to Thr residues. These O-linked oligosaccharides could be released by beta-elimination and by mild hydrazinolysis. The smallest released oligosaccharitol that was reactive with the Ch anti-Gal was Gal alpha 1-3Gal beta 1-4GlcNAcol (where GlcNAcol is N-acetyl-glucosaminitol). Several other Gal-containing oligosaccharitols were observed, most of which were branched and contained 4,6-di-O-substituted GlcNAcol at their reducing termini. About half of the total released oligosaccharitols could bind to immobilized Ch anti-Gal, but none of them bound to the anti-Gal isolated from normal human sera. These data suggest that the specificities of the Ch anti-Gal are quite different from the natural anti-Gal isolated from normal human sera. Therefore, these novel T. cruzi O-linked oligosaccharides are highly immunogenic under the conditions of natural infection and are the targets for lytic Ch anti-Gal.

Animals↗
PubMed1994

Glycoinositol-phospholipid profiles of four serotypically distinct Old World Leishmania strains.

Glycoinositol-phospholipids (GIPLs) are the major glycolipid class and prominant surface antigens of leishmanial parasites. The GIPLs from four serologically distinct Old World strains of Leishmania were characterized to determine inter- and intra-specific differences in these glycolipids. These studies showed that: (1) the major GIPLs of Leishmania topica (LRC-L36) and Leishmania aethiopica (LRC-L495) belong to the alpha-mannose-terminating GIPL series (iM2, iM3 and iM4) that are structurally related to the glycosyl-phosphatidylinositol anchors of both the surface proteins and the abundant lipophosphoglycan (LPG). In contrast, the GIPLs from two Leishmania major strains (LRC-L456 and LRC-L580) belong to the alpha-galactose-terminating GIPL series (GIPL-1, -2 and -3) that are more structurally related to the LPG anchor; (2) the GIPL profiles of the L. major strains differed in that a significant proportion of the GIPL-2 and -3 species (approximately 40% and 80%, respectively) in LRC-L580 are substituted with a glucose-1-PO4 residue, while this type of substitution was not detected in LRC-L456; and (3) all the GIPLs contained either an alkylacyl- or a lysoalkyl-phosphatidylinositol lipid moiety. However, the alkyl chain compositions of different GIPLs within the same strain was variable. In L. major, the major GIPL species contained alkylacylglycerols with predominantly C18:0 and C24:0 alkyl chains, whereas the glucose-1-PO4-substituted GIPLs contained exclusively lysoalkylglycerols with C24:0 alkyl chains. In L. tropica, the major GIPL, iM2, contained predominantly C24:0 alkyl chains whereas the structurally related iM3 and iM4 GIPLs in this strain contained predominantly C18:0 alkyl chains. In L. aethiopica all the GIPLs (iM2, iM3, iM4) contained C18:0 alkyl chains. These data suggest that the synthesis of the GIPLs may occur in more than one subcellular compartment. The possibility that species-specific differences in the predominantly surface glycan structures may modulate the interaction of the parasite with the insect and mammalian hosts is discussed.

Animals↗
PubMed1994

O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans.

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyltransferases to utilize peptide-linked versus glycolipid-linked acceptors.

Acid Phosphatase↗
PubMed1994

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.

Alkaline Phosphatase↗
PubMed1994

Hydrophilic-interaction chromatography of complex carbohydrates.

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

Animals↗
PubMed1994

The effects of phenylmethylsulfonyl fluoride on inositol-acylation and fatty acid remodeling in African trypanosomes.

Phenylmethylsulfonyl fluoride (PMSF) has been shown to inhibit the addition of ethanolamine phosphate to glycosylphosphatidylinositol (GPI) intermediates in Trypanosoma brucei (Masterson, W. J., and Ferguson, M. A. J. (1991) EMBO J. 10, 2041-2045). Here we show that the Man3-GlcN-PI intermediate that accumulates in the presence of PMSF can undergo fatty acid remodeling, suggesting that the fatty acid remodeling enzymes are not specific for ethanolamine phosphate-containing GPI intermediates. We also show that PMSF inhibits the acylation of the inositol residue of GPI intermediates in bloodstream form T. brucei. Pulse-chase experiments demonstrate that glycolipid C (ethanolamine-PO4-Man3-GlcN-(acyl)PI) is not an obligatory precursor of glycolipid A (ethanolamine-PO4-Man3-GlcN-PI) and that glycolipid C can be converted to glycolipid A. These data suggest a model where glycolipid C is the terminal product of the GPI biosynthetic pathway, in dynamic equilibrium with glycolipid A. The inhibition of ethanolamine phosphate addition and inositol acylation by PMSF was also observed for procyclic forms of T. brucei but not for mammalian HeLa cells. These results suggest differences between the relevant parasite and mammalian enzymes.

Acylation↗
PubMed1994

Characterization of GDP-alpha-D-arabinopyranose, the precursor of D-Arap in Leishmania major lipophosphoglycan.

Protozoan parasites of the genus Leishmania synthesize a complex lipophosphoglycan (LPG), which is the major cell surface macromolecule. The LPG from Leishmania major contains beta-D-Arap-terminating side chains that are involved in regulating the attachment of the parasite to the midgut epithelium of its insect vector. An arabinose sugar nucleotide donor was identified in soluble extracts of L. major promastigotes. This sugar nucleotide was biosynthetically labeled with D-[2-3H]Glc and with D-[5-3H]Ara. The labeled sugar nucleotide generated [3H]Ara and GDP after mild acid hydrolysis. The absolute configuration of the arabinose was determined after reduction and acylation with a pure enantiomer of Mosher's acid chloride. The pyranose ring configuration was inferred from the ability of GDP-Ara to form borate complexes, and the anomeric configuration was deduced from the results of mild base hydrolysis experiments. Taken together these data suggest that the sugar nucleotide has the structure GDP-alpha-D-Arap. This sugar nucleotide has not been previously described from natural sources and may be unique to trypanosomatid protozoan parasites. Ara-1-PO4 and GDP-Ara were the only soluble metabolites labeled with [3H]Ara, and pulse-chase experiment data are consistent with them being precursors of the arabinosyl residues of LPG.

Animals↗
PubMed1994

Partial purification and characterization of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes.

N-Acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase was solubilized from the bloodstream form of African trypanosomes using Zwittergent 3-14. The solubilized GlcNAc-PI de-N-acetylase was assayed using radiolabeled GlcNAc-PI substrates. The enzyme was partially purified about 140-fold from washed trypanosome membranes using conventional liquid chromatography. The enzyme has a Km of 1.5 microM. Replacement of the di-O-substituted D-myo-inositol of the natural GlcNAc-PI substrate by the L-myo-inositol isomer did not significantly alter the ability of the compound to act as a substrate for the de-N-acetylase, suggesting that the C-2 to C-5 hydroxyl groups of the myoinositol ring do not play a critical role in substrate recognition. A substrate analogue lacking fatty acids was a relatively poor substrate for the enzyme, indicating that the lipid component plays an important role in substrate recognition and/or presentation of the substrate to the enzyme in detergent micelles. Substrate analogues lacking the glycerophosphate component were not recognized by the enzyme, suggesting that this component is important in the substrate recognition process.

Acetylglucosamine↗
PubMed1994

Sodium channels accumulate at the tips of injured axons.

The axolemmal distribution of voltage-gated sodium channels largely determines the regions of axonal electrical excitability. Using a well-characterized anti-sodium channel antibody, we examined peripheral nerve fibers focally injured by exposure to the neurotoxic agent, potassium tellurite (K2TeO3). Immunocytochemical and radioimmunoassay data showed a focal accumulation of sodium channels within the tips of injured axons. The major increase in sodium channel concentration occurred between 7 and 11 days after toxin exposure; however, immunocytochemically, excess sodium channels persisted in several axonal endings for a much longer time. The accumulation of sodium channels at injured axonal tips may be responsible, in part, for ectopic axonal excitability and the resulting abnormal sensory phenomena (especially pain and paresthesias) which frequently complicate peripheral nerve injury in humans.

Animals↗
PubMed1994

The detection of phospholipase-resistant and -sensitive glycosyl-phosphatidylinositol membrane anchors by western blotting.

Glycosyl-phosphatidylinositol (GPI) membrane anchors are present on a large number of eukaryotic plasma membrane proteins. Some of these anchors can be cleaved with bacterial phosphatidylinositol-specific phospholipases C, and a glycosyl-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei, to reveal an epitope called the cross-reacting determinant. Other glycosyl-phosphatidylinositol anchors are resistant to the action of these enzymes prior to treatment with mild base. A simple method is described for identifying both phospholipase-sensitive and -resistant anchors using anti-cross-reacting determinant antibodies on Western blots. This procedure represents a high-sensitivity general method for the identification of GPI-anchored proteins.

Animals↗
PubMed1994

Analysis of glycosylphosphatidylinositol membrane anchors by electrospray ionization-mass spectrometry and collision induced dissociation.

The multi-component nature of glycosylphosphatidylinositol membrane anchors makes the analysis of their structure complex. Nuclear magnetic resonance spectroscopy of delipidated glycosylphosphatidylinositol-peptide fractions can supply considerable information but requires relatively large quantities of material. High-sensitivity sequencing techniques are available for the oligosaccharide portions of glycosylphosphatidylinositol anchors, but there is no simple and generally applicable technique to complement this information. In this paper we describe the application of electrospray ionization-mass spectrometry and collision induced dissociation to study intact glycosylphosphatidylinositol-peptides from a Trypanosoma brucei variant surface glycoprotein. Collision of the [M + 4H]4+ pseudomolecular ions of two glycosylphosphatidylinositol-peptide glycoforms produced easily interpretable daughter ion spectra, from which detailed information on the lipid moiety, carbohydrate sequence and site of peptide attachment could be obtained. All of the collision induced dissociation cleavage events occurred in the glycosylphosphatidylinositol portion of the glycosylphosphatidylinositol-peptide. This technique supplies complementary data to the high-sensitivity oligosaccharide sequencing procedures and should greatly assist glycosylphosphatidylinositol anchor structure-function studies, particularly when sample quantities are limiting.

Amino Acid Sequence↗
PubMed1994

What can GPI do for you?

Various functions for glycosylphosphatidylinositol (GPI) protein anchors have been described in mammalian and protozoan systems. These data suggest that some functions are common to higher and lower eukaryotes, whereas others may represent adaptations that are specifically advantageous to either unicellular or metazoan organisms. In this article, Mike Ferguson discusses the current theories of GPI function that have relevance to protozoan parasites and their mammalian hosts.

Journal Article↗