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Biomedical subjects

A M Lawson

Publications and source records attributed to A M Lawson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Nonreductive release of O-linked oligosaccharides from mucin glycoproteins for structure/function assignments as neoglycolipids: application in the detection of novel ligands for E-selectin.

The neoglycolipid technology comprises several microprocedures involving the generation of lipid-linked oligosaccharide probes for carbohydrate recognition studies in conjunction with oligosaccharide sequence determination by mass spectrometry. Although applicable to any desired oligosaccharides, procedures are greatly facilitated if the oligosaccharides are nonreduced, as conjugation is by reductive amination of a reducing end aldehyde to a phosphatidylethanolamine. Using bovine submaxillary mucin as a model for release of O-glycans in the reducing state, and based on yields of neoglycolipids and side-products from "peeling" reactions and degradation, aqueous ethylamine 70% w/v at 22 degrees C for 48 h has been selected in preference to other conditions, triethylamine, sodium hydroxide, and hydrazine. The integrity of the main acidic and neutral oligosaccharides released under these conditions, di- to octasaccharides, was established by analyses of free oligosaccharides by liquid secondary ion mass spectrometry (LSIMS) and of the derived neoglycolipids by TLC-LSIMS; the repertoire compared favorably with that of the oligosaccharide alditols generated by conventional reductive alkaline borohydride treatment. More forcing conditions of ethylamine 70% w/v at 65 degrees C for 6 h were required to release oligosaccharides from porcine gastric mucin; di- to nonasaccharides were obtained of which about one-third had an intact core GalNAc. Relative to yields after reductive alkaline hydrolysis, the overall yields for these two glycoproteins were 20% and 40-50% for acidic and neutral oligosaccharides, respectively. Among O-glycans released from an ovarian cystadenoma glycoprotein using ethylamine, three variants of the sulfated Le(a/x) sequences were identified as ligands for the endothelial adhesion molecule E-selectin, one of which is based on the unusual backbone Gal-3/4GlcNAc-3Gal-3Gal.

Animals↗
PubMed1996

Generation and structural characterization of a range of unmodified chondroitin sulfate oligosaccharide fragments.

Chondroitin sulfate C has been used to demonstrate an approach of generating a range of unmodified glycosaminoglycan oligosaccharide fragments. This involves cleavage by oxymercuration treatment of the nonreducing terminal 4,5-unsaturated uronic acid (DeltaUA) residues from the fragments produced by enzymatic digestion of chondroitin sulfate with chondroitinase ABC. Carrying out the reaction on the unfractionated digestion mixture produces a range of mono- to tridecasaccharides, the compositions of which were established by liquid secondary ion mass spectrometry (LSIMS) and their chromatographic patterns compared with oligosaccharides in the untreated digest. Ten of the main sequences, tri- to octasaccharides, isolated by HPLC from the treated and untreated digests were fully characterized by a combination of LSIMS and 1H NMR. Of these, 6 are homologs of the series with structures DeltaUA1-[3GalNAc(6S)beta1- 4GlcAbeta1]n-3Gal-NAc(6S) and [GalNAc(6S)beta1-4GlcAbeta1]n- 3GalNAc(6S), where n = 1-3. The other 4 sequences, DeltaUA1-[3Gal-NAc(6S)beta1-4GlcAbeta1]n-3GalNAc(4S) and [GalNAc(6S)beta1-4GlcAbeta1]n-3GalNAc(4S), where n = 1 and 2, contain the alternative 4-sulfated GalNAc at the reducing terminal. These results establish that oligosaccharides generated by oxymercuration treatment retain their integrity and only lack the terminal DeltaUA residue.

Chondroitin Sulfates↗
PubMed1996

Two families of murine carbohydrate ligands for E-selectin.

In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin in mouse, glycolipids from tissues and the neutrophilic cell line 32D c13 were tested for E-selectin binding. Kidneys of BALB/c and NMRI mice (but not CBA) and the 32D c13 cells were found to contain minor glycolipid populations that support strongly the binding of murine E-selectin. By chromatogram binding experiments and in situ liquid secondary ion mass spectrometry (LSIMS) with neoglycolipids derived from their endoglycoceramidase-released oligosaccharides, in conjunction with compositional and linkage analyses, one of the glycolipid ligands in kidney was identified as the Le(x)-active extended globo-glycolipid: [formula: see text] Neoglycolipids enriched for the ligand structures were obtained from oligosaccharides released by endo-beta-galactosidase from the 32D c13 cells. By TLC-LSIMS and antibody binding, the main E-selectin binding determinant on these was identified as sialyl-Le(a).

Animals↗
PubMed1996

The Le(x) carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development.

The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex-type N-glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N-linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5-positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N-glycans containing a 3-fucosyl N-acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid-linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3-fucosyl N-acetyllactosamine sequence [Lewisx (Le(x)) or X-hapten] also recognized by anti-SSEA-1 and anti-CD15. Histochemical studies with different antibodies recognizing the Lex sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti-SSEA-1 and anti-CD15.

Animals↗
PubMed1995

Characterisation by LSI-MS and 1H NMR spectroscopy of tetra-, hexa-, and octa-saccharides of porcine intestinal heparin.

The characterisation of oligosaccharide fragments isolated from enzymatically depolymerised porcine intestinal heparin is required in order to probe structure/function relationships of heparin in anticoagulation, antiangiogenesis and antiviral activity. We have used both LSI-MS and 600-MHz 1H NMR with chemical shift assignment by comprehensive 1H-1H TOCSY experiments to fully characterise the major oligosaccharide components including 4 tetrasaccharides, 3 hexasaccharides, and 2 octasaccharides. One of the octasaccharides has not been identified previously and has the structure: delta UA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S)-IdoA(2S)- GlcNS(6S)-GlcA-GlcNS(6S), where delta UA is 4,5-unsaturated uronic acid (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid), GlcN is --> 4)-alpha-D-glucosamine, IdoA is --> 4)-alpha-L-iduronic acid, GlcA is --> 4)-beta-D-glucuronic acid, and 2-O-, 6-O-, and 2-N-sulfate are abbreviated to 2S, 6S, and NS, respectively. Nearly complete NMR proton chemical shifts are reported for this data set. In addition a novel approach involving oxymercuration-lipid conjugation was used to independently assign sulfate substitution on the delta UA residues.

Animals↗
PubMed1995

TLC-LSIMS of neoglycolipids of glycosaminoglycan disaccharides and of oxymercuration cleavage products of heparin fragments that contain unsaturated uronic acid.

Heparin and chondroitin sulfate disaccharides have been investigated by high-performance (HP) TLC and liquid secondary-ion mass spectrometry (LSIMS) after conversion to neoglycolipid derivatives by reductive-amination with an aminolipid (dihexadecyl phosphatidylethanolamine, DHPE). Mobility on HPTLC was largely determined by the number of sulfate groups present, but was also influenced by the position of sulfate, monosaccharide composition and linkage. The mass spectra acquired directly from the TLC plate provided quasimolecular and fragment ions from which composition, including sulfate content, and sequence information was obtained at high sensitivity. Lipid DHPE conjugation and TLC-LSIMS were performed to analyse products of the oxymercuration reaction used to cleave unsaturated uronic acid (delta UA) residues from glycosaminoglycan (GAG) fragments produced by enzymatic degradation with glycan lyases. Previously the identification of the product from delta UA and the integrity of the remaining structures from oligosaccharides larger than disaccharide have not been made. Multiple and characteristic products of the cleaved delta UA were detected and these can be used for identification of terminal delta UA and its sulfate content. It was established with several disaccharides and a tetrasaccharide that glycosidic linkages and O- and N-sulfate groups are preserved in the remaining structures after removal of delta UA. These results indicate that the oxymercuration reaction will be applicable to generating series of GAG fragments containing unmodified sequences for biological activity studies.

Carbohydrate Sequence↗
PubMed1995

Studies on the order and site specificity of GalNAc transfer to MUC1 tandem repeats by UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase from milk or mammary carcinoma cells.

A synthetic peptide [TAP25, (T1aAPPAHGVT9S10APDT14RPAPGS20)T1bAPPA5b] corresponding to one repeat (T1a-S20) and five overlapping amino acids (T1b-A5b) of the MUC1 core protein served as an acceptor substrate for in vitro glycosylation. TAP25 was glycosylated using the detergent-solubilized UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases from the breast carcinoma cell line T47D, the colon carcinoma cell line HT29 and from human premature skim milk. The glycosylated peptides were isolated by ultrafiltration, purified by reverse-phase HPLC and further analysed by liquid secondary ion mass spectrometry (LSIMS). Three different glycosylation species, mono-, di- and triglycosylated peptides were identified. Automated Edman sequencing and LSIMS of proteolytic fragments independently revealed the sites of GalNAc incorporation and confirmed that the threonine residues Thr9 and Thr1b are the preferred sites of glycosylation independent of the enzyme source, while Thr14 remained non-glycosylated even with the enzyme preparation from milk. In addition, evidence was obtained that at least 20% of the glycosylated peptides exhibited GalNAc incorporation at Ser20. On the basis of kinetic studies a preferred sequence of GalNAc addition to the three acceptor sites has been concluded (Thr9-->Thr1b-->Ser20). Although Thr14 within the PDTRP motif of the tandem repeats remained non-glycosylated, the introduction of GalNAc into adjacent positions significantly decreased the immunoreactivity of antibodies SM-3, HMFG-1 and HMFG-2 defining overlapping epitopes of this motif. It is assumed that glycosylation at Thr9, Thr1b and Ser20 distorts the peptide conformation of the binding epitope.

Amino Acid Sequence↗
PubMed1995

Familial giant cell hepatitis with low bile acid concentrations and increased urinary excretion of specific bile alcohols: a new inborn error of bile acid synthesis?

A 9-wk-old infant with familial giant cell hepatitis and severe intrahepatic cholestasis had low plasma concentrations of chenodeoxycholic acid and cholic acid and elevated plasma concentrations of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 zeta-tetrol, and 5 beta-cholest-24-ene-3 alpha,7 alpha,12 alpha-triol. Analysis of the urine by fast atom bombardment mass spectrometry and by gas chromatography-mass spectrometry after treatment with Helix pomatia glucuronidase/sulfatase showed that the major cholanoids in urine were the glucuronides of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 zeta-tetrol. These results are consistent with an inborn error of the 25-hydroxylase pathway for bile acid synthesis, specifically one of the enzymes responsible for conversion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25-pentol to cholic acid and acetone. Treatment with chenodeoxycholic acid was tried on two occasions. On the first it appeared to precipitate a rise in bilirubin, on the second the liver function tests improved and the improvement was maintained when the treatment was modified to a combination of chenodeoxycholic acid and cholic acid and finally, cholic acid alone. Despite the normalization of liver function tests, a liver biopsy at 1.25 y showed an active cirrhosis. Nonetheless, the child is thriving at the age of 3.5 y, whereas an affected sibling died at 13 mo.

Bile Acids and Salts↗
PubMed1994

Oligosaccharide ligands for NKR-P1 protein activate NK cells and cytotoxicity.

A diversity of high-affinity oligosaccharide ligands are identified for NKR-P1, a membrane protein on natural killer (NK) cells which contains an extracellular Ca(2+)-dependent lectin domain. Interactions of such oligosaccharides on the target cell surface with NKR-P1 on the killer cell surface are crucial both for target cell recognition and for delivery of stimulatory or inhibitory signals linked to the NK cytolytic machinery. NK-resistant tumour cells are rendered susceptible by preincubation with liposomes expressing NKR-P1 ligands, suggesting that purging of tumour or virally infected cells in vivo may be a therapeutic possibility.

Animals↗
PubMed1994

Differential recognition by conglutinin and mannan-binding protein of N-glycans presented on neoglycolipids and glycoproteins with special reference to complement glycoprotein C3 and ribonuclease B.

Conglutinin and mannan-binding protein are serum proteins that have similar carbohydrate binding specificities toward high mannose-type oligosaccharides, and yet only conglutinin binds the complement glycoprotein iC3b, which contains oligosaccharides of this type. In the present study, the interactions of conglutinin and mannan-binding protein were evaluated with the complement glycoprotein C3, including various physiologically derived fragments of this glycoprotein, and neoglycolipids prepared from oligosaccharides released from C3 and its isolated alpha and beta chains. Several conclusions can be drawn. First, the interaction of conglutinin is profoundly influenced by the state of the protein moiety of the alpha chain in the vicinity of the glycosylation site Asn-917. Second, the binding to the C3-derived glycoprotein iC3b appears to be exclusively mediated through the Man8 or Man9 oligosaccharide on the alpha chain; there is no evidence for other N-linked oligosaccharides on C3 that are uniquely bound by conglutinin. Third, although conglutinin shows a more restricted binding relative to mannan-binding protein toward the oligosaccharides free of protein, it has a broader binding pattern toward the oligosaccharides as presented on C3-derived glycoproteins. From these and additional observations with RNase B, which contains high mannose-type oligosaccharides at Asn-34, it is clear that the protein moieties of these glycoproteins markedly influence the presentation of the oligosaccharides such that biological specificity is mediated by the commonly occurring high mannose-type oligosaccharides in the context of specific carrier proteins.

Blood Proteins↗
PubMed1994

Biliary bile acid profiles in patients with familial adenomatous polyposis before and after colectomy.

The development of colorectal polyps and cancer in patients with familial adenomatous polyposis (FAP) is directly linked to inactivation of the APC gene. Other, epigenetic, mechanisms may be involved in tumorigenesis and a previous study suggested that an intrinsic difference in the biliary bile acid profile of untreated patients with FAP persisted after colectomy. Gas chromatography and gas chromatography-mass spectrometry were used to examine the biliary bile acid profiles of four groups of patients with normal gallbladders: 20 patients with an intact colon comprising 12 with FAP and eight controls; and 26 patients after colectomy comprising 12 with FAP and 14 controls. Comparison of ten different bile acids from both amidate fractions (glycine and taurine) revealed a small increase in the molar percentage of a minor bile acid (12-oxolithocholic acid) in patients with FAP and an intact colon compared with the matching control group. Colectomy was associated with a dramatic reduction in levels of secondary bile acids but with little difference between patients with FAP and controls.

Adenomatous Polyposis Coli↗
PubMed1993

Core-typing of O-linked glycans from human gastric mucins. Lack of evidence for the occurrence of the core sequence Gal1-6GalNAc.

Mucins from the pooled gastric juice of Lewis-positive secretors were investigated to establish their glycosylation patterns with particular reference to the type and abundance of the glycan-core structures. Following reductive beta-elimination, the neutral glycan alditols from these mucins were fractionated by ion exchange and size-exclusion chromatographies and subjected to structural analyses. It was possible to gain insights into the core sequences of the neutral O-linked glycan alditols by matching (a) composition data from liquid secondary-ion mass spectrometry of the native alditol fractions, (b) specific structural information on the core sequences by thin-layer-chromatography mass spectrometry of alditol-derived neoglycolipids and (c) data from electron-impact mass spectrometry of permethylated glycan alditols or their partially methylated alditol acetates. The predominant core structures detected among the neutral glycans representing about 77% (by mass) of the total carbohydrates released from gastric mucins were core 1, Gal beta 1-3GalNAc (Ac, acetyl) and core 2, Gal beta 1-3(GlcNAc beta 1-6)GalNAc in the approximate ratio 1:2. Core 3, GlcNAc beta 1-3GalNAc, and core 4, GlcNAc beta 1-3(GlcNAc beta 1-6)GalNAc, were much less abundant (< 10%), while core 5, GalNAc alpha 1-3GalNAc, core 6, GlcNAc beta 1-6GalNAc, and a recently described sequence GalNAc alpha 1-6GalNAc (core 7) were not detected. This investigation also addressed the question of the presence of the sequence Gal beta 1-6GalNAc which has been reported previously to occur as a core-structure element in gastric mucins. This was greatly assisted by the availability of the authentic chemically synthetized disaccharide alditol which, when converted into a neoglycolipid after mild periodate oxidation, gives diagnostic ions in mass spectrometry and can be detected with high sensitivity. No evidence was found for the presence of this unusual sequence among the oligosaccharides in gastric mucins.

Carbohydrate Sequence↗
PubMed1993

Use of a porous graphitised carbon column for the high-performance liquid chromatography of oligosaccharides, alditols and glycopeptides with subsequent mass spectrometry analysis.

HPLC using a porous graphitised carbon (PGC) column eluted in acetonitrile-aqueous trifluoroacetic acid has been shown to give complementary chromatography to reversed-phase (ODS) HPLC for separation of peptides and glycopeptides. The PGC column can also be used for separation of oligosaccharides and oligosaccharide alditols released from protein by enzymes (N-linked chains) or base-borohydride degradation (O-linked chains). The advantages are that peptides, glycopeptides, reducing oligosaccharides, sialylated oligosaccharides and oligosaccharide alditols can be chromatographed under the same conditions. The samples can be readily recovered by evaporation for sensitive liquid secondary ion mass spectrometric (LSI-MS) analysis and there is no contamination or deterioration of chromatography from column leakage. LSI-MS analysis revealed that complete peak separation of all of the possible oligosaccharide components of the standard glycoproteins fetuin and bovine submaxillary mucin was not achieved. However, PGC remains as a useful adjunct to other HPLC profiling and separation techniques in particular where subsequent MS analysis is desired.

Amino Acid Sequence↗
PubMed1993

Possible intermediates in the biosynthesis of mycoside B by Mycobacterium microti.

Two lipids were isolated from Mycobacterium microti which became labelled when the cells were grown in the presence of [2-14C]propionate. They were purified by thin-layer chromatography and studied by chemical degradation and mass spectrometry. The lipids were identified as phenolphthiocerol dimycocerosate and phenolphthiodiolone dimycocerosate, the aglycosyl derivatives of mycoside B, the phenolic glycolipid produced by M. microti. Cell-free extracts of the organism were able to glycosylate the lipids to form mycoside B in vitro. It is probable that the lipids are intermediates in the biosynthesis of phenolic glycolipids by mycobacteria.

Carbon Radioisotopes↗
PubMed1993

Specificity of mild periodate oxidation of oligosaccharide-alditols: relevance to the analysis of the core-branching pattern of O-linked glycoprotein oligosaccharides.

The specificity of mild periodate oxidation of 3- and 6-substituted 2-acetamido-2-deoxy-D-galactitols and 4- and 6-substituted D-glucitols has been investigated. The products were reacted with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine and the derivatives were analysed by application of liquid secondary-ion mass spectrometry directly to the TLC plates. There was > 95% specificity of cleavage of the C-4-C-5 bond (threo-diol) for the GalNAcol derivatives. The major sites of oxidation for the Glcol derivatives also involved threo-diols. For alpha-Neu5Ac-(2-->6)-GalNAcol, approximately 30% of the products of oxidation involved the sialic acid side chain, and approximately 60% were cleaved at the C-4-C-5 bond of the GalNAcol moiety. The mild periodate oxidation reaction forms part of a strategy for determining the patterns of branching of the cores of O-linked glycoprotein oligosaccharides and other oligosaccharide-alditols.

Carbohydrate Sequence↗
PubMed1993

An integrated procedure for the direct detection of characteristic lipids in tuberculosis patients.

An integrated method is described for the sensitive detection of tuberculostearic, mycocerosic and mycolic acids in infected materials from tuberculosis patients. Tuberculostearic acid is analysed by two-dimensional gas chromatography of pentafluorobenzyl esters, the key component being switched from a short non-polar column to a high resolution polar column with final electron capture detection. Mycocerosic acids are identified by simple one-dimensional electron capture gas chromatography of pentafluorobenzyl esters, with the use of negative-ion chemical ionisation gas chromatography in indecisive cases. Conversion of mycolic acids to anthrylmethyl esters produces compounds which are suitable for sensitive detection by fluorescence high-performance liquid chromatography. Application of all three of these methods to sputum samples from tuberculosis patients gave profiles characteristic of Mycobacterium tuberculosis.

Chromatography, Gas↗