Alexander M. Lawson

Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate β-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in β1,3- or both β1,3- and β1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate “designer” microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular β-glucans and compete with zymosan binding and triggering of tumor necrosis factor-α secretion by a Dectin-1-expressing macrophage cell line.

Neoglycolipids: probes of oligosaccharide structure, antigenicity, and function.

Publisher Summary Neoglycoproteins have found considerable use in studies of carbohydrate-binding proteins and monoclonal antibodies and are available as commercial products. The special feature of the neoglycolipid technology is that it is applicable not only to mixtures of oligosaccharides released from proteins by established procedures [hydrazinolysis ( N -linked chains) or alkaline borohydride degradation ( O -linked chains) or by various partial degradation procedures, including endoglycosidase digestions] but is also applicable to any desired oligosaccharide sequence, be it natural or synthetic. By a microconjugation procedure each oligosaccharide is joined to a lipid molecule. The resulting neoglycolipids serve as immobilized probes on silica gel chromatograms, on plastic plates, or as microparticulate probes after incorporation into liposomes, and they are readily amenable to assay procedures established for glycosphingolipids. In the form of neoglycolipids, the oligosaccharides are presented at the matrix surface in a clustered state. This is an important requirement for interactions with carbohydrate-binding proteins, including antibodies, which frequently have low affinities and give no detectable binding to monovalent carbohydrate ligands under conventional binding assay conditions. The lipid most extensively used for generating neoglycolipids has been the aminophospholipid L -1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine.

Oligosaccharide-mediated interactions of the envelope glycoprotein gp120 of HIV-1 that are independent of CD4 recognition.

In this study carbohydrate-mediated interactions of the envelope glycoprotein, gp120, of HIV-1 were investigated. Oligosaccharide probes (neoglycolipids), prepared from the N-glycosidically-linked chains of the natural and recombinant forms of gp120, were used in conjunction with the intact glycoprotein to investigate reactivities with a soluble carbohydrate-binding protein (lectin) known as mannose-binding protein in human serum. Evidence is presented that the high-mannose-type oligosaccharides with seven, eight and nine mannose residues from both forms of gp120 are recognized by the serum lectin, and that these reactivities are unrelated to CD4 recognition. Reactivities of the two forms of envelope glycoprotein with macrophages derived from human blood monocytes and with the mannose-specific macrophage endocytosis receptor isolated from human placental membranes were also investigated. Evidence is presented that both forms of gp120 bind to the macrophage surface by multiple interactions in addition to CD4 binding, and that among these interactions is a carbohydrate-mediated binding to the endocytosis receptor. We propose that such carbohydrate-mediated interactions could form the basis of viral attachment to a variety of healthy and diseased tissues.

High-sensitivity structural analyses of oligosaccharide probes (neoglycolipids) by liquid-secondary-ion mass spectrometry

The sensitivity of detection and extent of information on structure obtainable by liquid-secondary-ion mass spectrometry (l.s.i.-m.s.) of neoglycolipids on the conventional target probe and directly from the surface of silica plates following t.l.c. has been assessed. Neoglycolipids were derived from malto-oligosaccharides, chitin oligosaccharides, and a range of deoxyhexose-, hexose-, 2-acetamido-2-deoxyhexose-, and sialic acid-containing mammalian oligosaccharides by reductive amination using phosphatidylethanolamine dipalmitoate (PPEADP). Sub-pmol amounts of the maltopentaose-PPEADP derivative applied directly to the target probe provided information on molecular weight, whereas approximately 1 pmol was required when analysed on the silica gel t.l.c. plate. With a biantennary octasaccharide derivative, the sensitivity of detection was 20-50 times lower and the other oligosaccharides had intermediate sensitivities. Information on composition and sequence was obtained readily from fragment ions, using 5 pmol of the maltopentaose derivative and 50 pmol of the octasaccharide derivative on the target probe, and 50 and 200 pmol, respectively, on the silica gel chromatogram. The optimised conditions formed the basis for characterising the structures of the components of mixtures of oligosaccharides generated from glycoproteins.

Neoglycolipid Technology: Deciphering Information Content of Glycome

Publisher Summary Neoglycolipid technology was introduced in 1985 to enable direct binding studies to be conveniently performed with oligosaccharides of glycoproteins. At that time, there was a lack of microscale methods for examining the carbohydrate–protein interactions, after release of the oligosaccharides from the carrier proteins. Most carbohydrate–protein interactions are of such low affinities that di- or multivalence of both oligosaccharide and recognition protein is required for detection by precipitation, radioimmunoassay, or enzyme-linked immunosorbent assay experiments. It was clear that a method was required to examine the released oligosaccharides in a multivalent state. The existing methods for examining the recognition of specific oligosaccharides by antibodies and other carbohydrate-recognizing proteins involved purification of the oligosaccharides, often from highly heterogeneous mixtures, and testing them individually as inhibitors of the binding of the recognition proteins to macromolecules or cells. The amounts of oligosaccharides required for inhibition of binding were frequently prohibitive. The approach chosen to address the problem was chemical conjugation of oligosaccharides to lipid. The artificial glycolipids, neoglycolipids, formed would enable the presentation of the oligosaccharides in the clustered state. The conjugation principle selected was reductive amination to a phosphatidylethanolaminetype aminolipid. For O-glycans released by reductive alkali treatment, a mild periodate oxidation procedure was included to generate reactive aldehydes for the conjugation. A major advantage of conjugating each oligosaccharide in a mixture to a lipid molecule rather than to a protein, such as bovine serum albumin, is that each component remains discrete and can be isolated.

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.

Twin Siblings with a Reye's-Like Syndrome Associated with an Abnormal Organic Aciduria, Hypoglycemia, Diarrhea, and Vomiting with Close Similarities to Jamaican Vomiting Sickness

Summary: Seventeen-month-old identical Caucasian male twins developed vomiting and diarrhea, and both were admitted to hospital 30 hr after the onset of symptoms. One was dead on arrival. Hepatic histology showed fatty degeneration affecting all parts of the lobule. Death was due to aspiration of gastric contents. Electron microscopic examination of the intestine showed the presence of replicating adenovirus. The other infant was comatose with undetectable blood glucose. He was maintained on IV glucose for 48 hr and made a complete recovery with no further hypoglycemia even after an overnight fast. Eighteen months later, he was a normally developed child for his age. The clinical diagnosis made in both infants was Reyes syndrome.Urinary organic acids were determined by gas chromatography and identified by gas chromatography-mass spectrometry. Greatly increased concentrations of adipic acid, 4-hydroxyphenylacetic acid, suberic acid, and of a previously unrecorded urinary organic acid which has been identified as 5-hydroxyhexanoic acid were observed. There were also increased concentrations of octanoic, sebacic, decenedioic, octenedioic, and 3-hydroxyisovaleric acids, and greatly reduced concentrations of the aldonic and deoxyaldonic acids and acids of the tricarboxylic acid cycle. Lactic acid excretion was not increased, and there was only a moderate increase in the concentrations of 3-hydroxybutyric and acetoacetic acids. No gross increase in concentrations or abnormal acids were observed in the urinary volatile short chain fatty acids.The organic acids observed in the urine of the present patients have been compared to those observed in patients with Jamaican vomiting sickness and those with Reyes syndrome. The similar dicarboxylic C6-C10 aciduria and profound hypoglycemia observed in the present patients and in those with Jamaican vomiting sickness is of interest and probably occurs due to a similar mechanism, inhibition of β-oxidation of medium-chain fatty acids by a chemical toxin. Some differences between the organic aciduria observed in these cases and in patients with Jamaican vomiting sickness suggest that the nature of the possible toxin, although closely similar, is not identical. The occurrence in both of the present patients of high concentrations of 5-hydroxyhexanoic acid may indicate the nature of the causative xenobiotic agent, and it has been suggested that this could be hex-4-enoic acid.Speculation: The occurrence of profound hypoglycemia and abnormal organic aciduria in twin English siblings with a Reyes-like syndrome and close clinical and biochemical similarities to Jamaican vomiting sickness suggests that some cases of Reyes syndrome in infants are due to causes similar to that involved in Jamaican vomiting sickness, that is, organic acid chemical toxins related to metabolites of hypoglycin.

Characterisation of oligosaccharides released from human-blood-group O erythrocyte glycopeptides by the endo-β-galactosidase of Bacteroides fragilis

Desialylated human blood group O erythrocyte glycopeptides were digested with the endo-beta-galactosidase of Bacteroides fragilis and the enzyme-released products reduced with NaBH4 and purified by Bio-Gel P-4 chromatography. Three linear and six branched oligosaccharides of poly(N-acetylllactosamine) type, which together accounted for 90% of the oligosaccharide alditols, were characterised by fast-atom-bombardment mass spectrometry and gas-liquid chromatography/mass spectrometry. Linkage and composition data were obtained for the remaining material. The salient findings were (a) the branched oligosaccharide alditols each contained the sequence: (Formula: see text) and (b) there was no evidence for the terminal branch-point sequence: (Formula: see text). Together these observations indicate that, as with erythrocyte glycolipids described previously [Scudder, P., Hanfland, P., Uemura, K. & Feizi, T. (1984) J. Biol. Chem. 259, 6586-6592], the endo-beta-galactosidase of Bacteroides fragilis cannot hydrolyse branch-point beta-galactosidic linkages on erythrocyte membrane glycopeptides.

Specificity of mild periodate oxidation of oligosaccharidealditols: 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.

Characterisation of minor tetra- to hepta-saccharides O-linked to human meconium glycoproteins by t.l.c.—m.s. microsequencing of neoglycolipid derivatives in conjunction with conventional m.s. and 1H-n.m.r. spectroscopy

As part of the establishment of a data base for core and backbone sequences of O-linked oligosaccharides of human meconium glycoproteins, the minor tetra- to hepta-saccharides released from mild-acid treated blood group [corrected] H-active glycoproteins have been studied. These oligosaccharides are heterogeneous and difficult to isolate, and a t.l.c.-m.s. microsequencing procedure has been applied to the neoglycolipid derivatives, in conjunction with 1H-n.m.r. spectroscopy, methylation analysis, and mass spectrometry (m.s.) of native and methylated oligosaccharides. Among an array of oligosaccharides characterised are those having the branched beta-GlcNAc-(1----6)[beta-Gal- (1----3)]-GalNAcol core, and others with the following linear sequences not characterised previously from this source: beta-Gal-(1----3)-beta-GlcNAc-(1----3)-beta-Gal-(1----3)-GalNAcol, beta-Gal-(1----4)-beta-GlcNAc-(1----3)-beta-Gal-(1----3)-GalNAcol, alpha-GalNAc- (1----3)-beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)-GalNAcol, beta-GlcNAc- (1----3)-beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)- GalNAcol, beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)-beta-Gal-(1----3/4)-beta- GlcNAc-(1----3)-beta-Gal-(1----3)-GalNAcol, and beta-GlcNAc-(1----3)-beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)-beta-Gal- (1----3/4)-beta-GlcNAc-(1----3)-GalNAcol.