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Biomedical subjects

A M Lawson

Publications and source records attributed to A M Lawson.

At least 19 recordsLinked to original sources
PubMed2003

Normalisation of gait data in children.

The comparative effect of semi-dimensional (SD) and non-dimensional (ND) normalisation on the results of a longitudinal study of gait in 5-12-year old children was investigated. The use of both height and leg length in the normalisation was examined. Only ND analysis could be used to identify subjects with the same accelerations. ND analysis of the children's gait indicated that there was little change in the combination of step length and cadence used to achieve a particular velocity between 5 and 12. The first peak and mid-stance trough values of the vertical component of ground reaction force did not change with age. We recommend the use of ND normalisation rather that SD to allow comparisons between individuals of differing size and mass.

Acceleration↗
PubMed2001

NMR studies of mannitol-terminating oligosaccharides derived by reductive alkaline hydrolysis from brain glycoproteins.

Interest in the characterisation of O-mannosyl glycan structures has been stimulated following the identification of mannitol-terminating oligosaccharides among the chains released from mammalian proteins in nervous and muscle tissues, and by the discovery of a putative human O-mannosyl transferase. Several mass spectrometry methods have been applied to structure elucidation particularly when low amounts of oligosaccharide are available for analysis. However, when sufficient amounts are available, a combination of through-bond homo- and heteronuclear, and of through-space homonuclear NMR experiments permit the complete identification of these oligosaccharide sequences. We describe here the assignment of 1H and 13C NMR chemical shifts from such experiments for four mannitol-terminating oligosaccharide alditols, GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)[Fucalpha-(1-->3)]GlcNAcbeta-(1-->2)Manol and NeuAcalpha-(2-->3)Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, that were released from brain glycopeptides by alkaline borohydride treatment.

Animals↗
PubMed2001

Negative-ion electrospray mass spectrometry of neutral underivatized oligosaccharides.

Negative-ion electrospray mass spectrometry (ES-MS) with collision-induced dissociation (CID) and MS/MS scanning on a quadrupole-orthoganal time-of-flight instrument provide a sensitive means for structural analysis of neutral underivatized oligosaccharides. Molecular mass information can be readily obtained from the dominant [M - H]- ions in the ES mass spectrum formed with subnanomole amounts of oligosaccharides, and similar sensitivity is available from CID-MS/MS to give structural details. The CID spectra of 14 oligosaccharides demonstrated that sequence and partial linkage information can be derived and isomeric structures can be differentiated. Series of C-type fragment ions give sequence information while the double glycosidic D-type cleavage of a 3-linked GlcNAc or Glc and the saccharide ring fragmentation of the 0,2A-type from 4-linked GlcNAc or Glc can provide partial linkage information. The distinctive D- and A-cleavages are important for differentiation of oligosaccharide type 1 and type 2 chains and to define the blood group H, Le(a), Le(x), Le(b), and Le(y) determinants carried by their fucosylated analogues.

Carbohydrate Sequence↗
PubMed2001

10E4 antigen of Scrapie lesions contains an unusual nonsulfated heparan motif.

The carbohydrate antigen on heparan sulfate recognized by monoclonal antibody 10E4 is uniquely codistributed with the abnormal prion protein, PrP(Sc), even in the earliest detectable brain lesions of scrapie-infected mice. Determining the chemical structure of 10E4 antigen is, therefore, an important aspect of structure elucidation of scrapie lesions, and a prerequisite for designing experiments to understand its role in scrapie pathogenesis. Toward this aim, we have examined preparations of heparan sulfate, with differing sulfate contents, for binding by 10E4 antibody. The highest antigenicity was observed in a preparation (HS-1) with the lowest sulfate content. HS-1 was partially depolymerized with heparin lyase III, and oligosaccharide fragments examined for 10E4 antigen expression by the neoglycolipid technology. An antigen-positive and two antigen-negative tetrasaccharides were isolated and examined by electrospray mass spectrometry. The antigen-positive tetrasaccharide sequence on heparan sulfate was thus deduced to contain a unique unsulfated motif that includes an N-unsubstituted glucosamine in the sequence, UA-GlcN-UA-GlcNAc. Antibody binding experiments with neoglycolipids prepared from a series of heparin/heparan sulfate disaccharides, and the trisaccharide derived from the antigen-positive tetrasaccharide after removal of the terminal hexuronic acid, show that both the penultimate glucosamine and the outer nonsulfated hexuronic acid are important for 10E4 antigenicity.

Animals↗
PubMed2001

Inhibition of adhesion of Plasmodium falciparum-infected erythrocytes by structurally defined hyaluronic acid dodecasaccharides.

We recently reported that Plasmodium falciparum-infected erythrocytes (IRBCs) can adhere to hyaluronic acid (HA), which appears to be a receptor, in addition to chondroitin sulfate A (CSA), for parasite sequestration in the placenta. Further investigations of the nature and specificity of this interaction indicate that HA oligosaccharide fragments competitively inhibit parasite adhesion to immobilized purified HA in a size-dependent manner, with dodecasaccharides being the minimum size for maximum inhibition. Rigorously purified and structurally defined HA dodecasaccharides, free of contamination by CSA or other glycosaminoglycans, effectively inhibited IRBC adhesion to HA but not CSA, providing compelling evidence of a specific interaction between IRBCs and HA.

Adhesiveness↗
PubMed2000

Expression in Escherichia coli, folding in vitro, and characterization of the carbohydrate recognition domain of the natural killer cell receptor NKR-P1A.

NKR-P1A is a homodimeric type II transmembrane protein of the C-type lectin family found on natural killer (NK) cells and NK-like T cells and is an activator of cytotoxicity. Toward structure determination by NMR, the recombinant carbohydrate-recognition domain (CRD) of NKR-P1A has been expressed in high-yield in Escherichia coli and folded in vitro. The purified protein behaves as a monomer in size-exclusion chromatography and is bound by the conformation-sensitive antibody, 3.2.3, indicating a folded structure. A polypeptide tag at the N-terminus is selectively cleaved from the CRD after limited trypsin digestion in further support of a compact folded structure. The disulfide bonds have been identified by peptide mapping and electrospray mass spectrometry. These are characteristic of a long form CRD. The 1D NMR spectrum of the unlabeled CRD and the 2D HSQC spectrum of the (15)N-labeled CRD are those of a folded protein. Chemical shifts of H(alpha) and NH protons indicate a considerable amount of beta-strand structure. Successful folding in the absence of Ca(2+), coupled with the lack of chemical shift changes upon addition of Ca(2+), suggests that the NKR-P1A-CRD may not be a Ca(2+)-binding protein.

Amino Acid Sequence↗
PubMed2000

Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria.

Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.

Animals↗
PubMed2000

Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery.

A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1, 2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHP-neoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequence-specific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Lea- and sialyl-Lex-active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.

Amination↗
PubMed1999

Recombinant soluble human CD69 dimer produced in Escherichia coli: reevaluation of saccharide binding.

We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein. We have expressed in Escherichia coli a new construct of the extracellular part (Q(65)-K(199)) of human CD69. We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine, linked to bovine serum albumin. Monosaccharide-binding signals are not detectable. Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan.

Amino Acid Sequence↗
PubMed1999

Core-branching pattern and sequence analysis of mannitol-terminating oligosaccharides by neoglycolipid technology.

The occurrence of mannitol-terminating oligosaccharides (2-substituted or 2,6-disubstituted) among the O-glycans released by alkaline borohydride treatment from glycoproteins of the nervous system has prompted the development of a microscale method to analyze the core-branching pattern and sequence by the neoglycolipid (NGL) technology, analogous to a method previously described for GalNAcol-terminating oligosaccharides (M. S. Stoll, E. F. Hounsell, A. M. Lawson, W. Chai, and T. Feizi, Eur. J. Biochem. 189, 499-507, 1990). The approach involves the selective cleavage at the core mannitol by mild periodate treatment and analysis of the reaction products as NGLs by in situ TLC/liquid secondary ion mass spectrometry. Oxidation conditions have been optimized using as reference compounds 2-, 3-, 4-, or 6-monosubstituted mannobi-itols, 3,6-disubstituted mannitol-terminating pentasaccharides, and 2-mono- and 2,6-disubstituted mannitol-terminating neutral and sialylated oligosaccharides isolated from brain glycopeptides. When a 2:1 molar ratio of periodate to alditol is used, the core mannitol is cleaved at the C3-C4 threo-diol bond and in the absence of a threo-diol cleavage occurs to a lesser extent at erythro-diols. Saccharide ring diols are not cleaved under these conditions, and it is also shown that the side chain of sialic acid on the oligosaccharide is largely unaffected. Substituents at 2- and 6-positions of the core mannitol can be identified, and the method is applicable to neutral and sialylated oligosaccharide alditols. Typically, the starting material is 5 nmol of oligosaccharide and 0.5-1 nmol of derivatives is applied for analysis. By this strategy, the core-branching pattern and position of sialic acid of two branched monosialylated mannitol-terminating oligosaccharide isomers have been determined.

Animals↗
PubMed1999

High prevalence of 2-mono- and 2,6-di-substituted manol-terminating sequences among O-glycans released from brain glycopeptides by reductive alkaline hydrolysis.

Di- to heptasaccharides isolated from total nondialyzable brain glycopeptides after release by alkaline borohydride treatment have been subjected to mass spectrometric and nuclear magnetic resonance spectroscopic analyses supplemented by TLC-MS analyses of derived neoglycolipids. A family of Manol-terminating oligosaccharides has been revealed which includes novel sequences with a 2, 6-disubstituted Manol: In contrast to the Manol-terminating HNK-1 antigen-positive chains described previously that occur as a minor population [Yuen, C.-T., Chai, W., Loveless, R.W., Lawson, A.M., Margolis, R.U. & Feizi, T. (1997) J. Biol. Chem. 272, 8924-8931], the above oligosaccharides are abundant. The ratio of these compounds to the classical N-acetylgalactosaminitol-terminating oligosaccharides is about 1 : 3. Thus, there appears to be in higher eukaryotes a major alternative pathway related to the yeast-type protein O-mannosylation, the enzymatic basis and functional importance of which now require investigation.

Animals↗
PubMed1998

Characterization of heparin oligosaccharide mixtures as ammonium salts using electrospray mass spectrometry.

Among glycosaminoglycans, polysulfated heparin chains provide the greatest challenge to characterization due to high polarity, structural diversity, and sulfate lability. The present report demonstrates how electrospray mass spectrometry (ESMS) can be used to derive compositional information from pure and mixed fractions of heparin tetra- to decasaccharide fragments prepared by controlled digestion of heparin with heparinase I. It also describes an improved procedure for fractionation of heparin oligosaccharides up to octadecasaccharides. Ammonium salts prove to be superior to sodium salts, particularly for analysis of mixed components. In the mass spectrum of a hexasaccharide fraction, the identification of six major mass peaks that represent the known hexasaccharide structures confirms that ESMS analysis of heparin oligosaccharide fragments gives a close representation of their constituent composition. In addition to the previously identified components, several unreported oligosaccharides were detected in the spectra of octa- and decasaccharide fractions. The ESMS identification of the three major species in a decasaccharide fraction was confirmed after HPLC subfractionation and reanalysis. ESMS detection sensitivity of low picomole amounts of oligosaccharides can be readily achieved.

Chemical Fractionation↗
PubMed1998

Structural characterisation of two hexasaccharides and an octasaccharide from chondroitin sulphate C containing the unusual sequence (4-sulpho)-N-acetylgalactosamine-beta1-4-(2-sulpho)-glucuronic acid-beta1-3-(6-sulpho)-N-acetylgalactosamine.

The structures of two hexasaccharides and an octasaccharide, isolated from shark cartilage chondroitin sulphate C after partial chondroitin ABC lyase digestion, have been elucidated by disaccharide analysis, liquid secondary-ion mass spectrometry, and 1H/13C NMR spectroscopy. In the case of the octasaccharide, the order of the internal disaccharide units was deduced from a novel one-dimensional TOCSY/ROESY excitation sculpting experiment [Gradwell M. J., Kogelberg, H. & Frenkiel, T. A. (1997) J. Magn. Reson. 124, 267-270] which incorporates two double-pulsed field-gradient spin-echo sequences. The oligosaccharides each contain the unusual motif GalNAc(4SO3-)beta1-4GlcA(2SO3-)beta1-3GalNAc(6SO3++ +-). From the adjoining sequences in the oligosaccharides, it is apparent that this motif occurs interspersed with typical chondroitin sulphate-C disaccharides, GlcAbeta1-3GalNAc(6SO3-). Such differentially sulphated domains along the glycosaminoglycan chains are potential sites for specific recognition processes.

Carbohydrate Sequence↗
PubMed1998

Inhibition of binding of malaria-infected erythrocytes by a tetradecasaccharide fraction from chondroitin sulfate A.

Adherence of parasite-infected erythrocytes (IEs) to the microvascular endothelium of various organs, a process known as sequestration, is a feature of Plasmodium falciparum malaria. This event is mediated by specific adhesive interactions between parasite proteins, expressed on the surface of IEs, and host molecules. P. falciparum IEs can bind to purified chondroitin sulfate A (CS-A), to the proteoglycan thrombomodulin through CS-A side chains, and to CS-A present on the surface of brain and lung endothelial cells and placental syncytiotrophoblasts. In order to identify structural characteristics of CS-A important for binding, oligosaccharide fragments ranging in size from 2 to 20 monosaccharide units were isolated from CS-A and CS-C, following controlled chondroitin lyase digestion, and used as competitive inhibitors of IE binding to immobilized ligands. Inhibition of binding to CS-A was highly dependent on molecular size: a CS-A tetradecasaccharide fraction was the minimum length able to almost completely inhibit binding. The effect was dose dependent and similar to that of the parent polysaccharide, and the same degree of inhibition was not found with the CS-C oligosaccharides. There was no effect on binding of IEs to other ligands, e.g., CD36 and intercellular adhesion molecule 1. Hexadeca- and octadecasaccharide fractions of CS-A were required for maximum inhibition of binding to thrombomodulin. Analyses of oligosaccharide fractions and polysaccharides by electrospray mass spectrometry and high-performance liquid chromatography suggest that the differences between the activities of CS-A and CS-C oligosaccharides can be attributed to differences in sulfate content and sulfation pattern and that iduronic acid is not involved in IE binding.

CD36 Antigens↗
PubMed1997

Sialyl-Lewis(x) sequence 6-O-sulfated at N-acetylglucosamine rather than at galactose is the preferred ligand for L-selectin and de-N-acetylation of the sialic acid enhances the binding strength.

Oligosaccharide sequences based on sialyl-Lewis(x) with 6-O-sulfation at galactose (6'-sulfo) or at N-acetylglucosamine (6-sulfo) and expressed on high endothelial venules are considered likely endogenous ligands for the leukocyte adhesion molecule, L-selectin. In the course of high performance TLC of three hexaglycosylceramides 6'-sulfo sialyl Lewis(x), 6-sulfo sialyl Lewis(x), and 6',6-bis-sulfo sialyl Lewis(x), synthesized chemically for selectin recognition studies, two minor byproducts were detected and isolated from each parent compound. By liquid secondary ion mass spectrometry these were identified as isomers containing a de-N-acetylated sialic acid or having a modified carboxyl group. Binding experiments with the parent compounds and the non-sulfated sialyl Lewis(x) glycolipid show that 6-sulfation potentiates, whereas 6'-sulfation virtually abolishes L-selectin binding. Thus the hierarchy of binding strengths were 6-sulfo sialyl > sialyl = 6',6-bis-sulfo sialyl >> 6'-sulfo sialyl Lewis(x). Whereas modification of the sialic acid carboxyl group markedly impaired L-selectin binding, de-N-acetylation resulted in enhanced binding. The natural occurrence on high endothelial venules of this 'super-active' de-N-acetylated form of 6-sulfo sialyl Lewis(x), and related structures, now deserves investigation.

Acetylation↗
PubMed1997

Localization of O-glycosylation sites on glycopeptide fragments from lactation-associated MUC1. All putative sites within the tandem repeat are glycosylation targets in vivo.

Since there is no consensus sequence directing the initial GalNAc incorporation into mucin peptides, O-glycosylation sites are not reliably predictable. We have developed a mass spectrometric sequencing strategy that allows the identification of in vivo O-glycosylation sites on mucin-derived glycopeptides. Lactation-associated MUC1 was isolated from human milk and partially deglycosylated by trifluoromethanesulfonic acid to the level of core GalNAc residues. The product was fragmented by the Arg-C-specific endopeptidase clostripain to yield tandem repeat icosapeptides starting with the PAP motif. PAP20 glycopeptides were subjected to sequencing by post-source decay matrix-assisted laser desorption ionization mass spectrometry or by solid phase Edman degradation to localize the glycosylation sites. The masses of C- or N-terminal fragments registered for the mono- to pentasubstituted PAP20 indicated that GalNAc was linked to the peptide at Ser5,Thr6 (GSTA) and Thr14 (VTSA) but contrary to previous in vitro glycosylation studies also at Thr19 and Ser15 located within the PDTR or VTSA motifs, respectively. Quantitative data from solid phase Edman sequencing revealed no preferential glycosylation of the threonines. These discrepancies between in vivo and in vitro glycosylation patterns may be explained by assuming that O-glycosylation of adjacent peptide positions is a dynamically regulated process that depends on changes of the substrate qualities induced by glycosylation at vicinal sites.

Acetylgalactosamine↗
PubMed1997

Brain contains HNK-1 immunoreactive O-glycans of the sulfoglucuronyl lactosamine series that terminate in 2-linked or 2,6-linked hexose (mannose).

The monoclonal antibody HNK-1 originally raised to an antigenic marker of natural killer cells also binds to selected regions in nervous tissue. The antigen is a carbohydrate that has attracted much interest as its expression is developmentally regulated in nervous tissue, and it is found, and proposed to be a ligand, on several of the adhesive glycoproteins of the nervous system. It is also expressed on glycolipids and proteoglycans, and is the target of monoclonal auto-antibodies that give rise to a demyelinating disease. The epitope, as characterized on glycolipids isolated from the nervous system, is expressed on 3-sulfated glucuronic acid joined by beta1-3-linkage to a neolacto backbone. Here we exploit the neoglycolipid technology, in conjunction with immunodetection and in situ liquid secondary ion mass spectrometry, to characterize HNK-1-positive oligosaccharide chains derived by reductive alkaline release from total brain glycopeptides. The immunoreactive oligosaccharides detected are tetra- to octasaccharides that are very minor components among a heterogeneous population, each representing less than 0.1% of the starting material. Their peripheral and backbone sequences resemble those of the HNK-1-positive glycolipids. An unexpected finding is that they terminate not with N-acetylgalactosaminitol but with hexitol (2-substituted and 2,6-disubstituted). In a tetrasaccharide investigated in the greatest detail, the hexitol is identified as 2-substituted mannitol.

Animals↗