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PubMed1983

Changes in glycosaminoglycan biosynthesis during differentiation in vitro of human monocytes.

Monocytes isolated from human blood were maintained in vitro on plastic culture dishes. After 3-4 days, adherent cells displayed morphological changes previously attributed to differentiation of the cells into histiocytes. 35S-labelled glycosaminoglycans were isolated after incubation of the cells with inorganic [35S]sulphate. Polysaccharide recovered from the culture medium after labelling from day 0 to day 2 or from day 5 to day 7 in vitro was approximately 90% galactosaminoglycan (resistant to deamination by HNO2), irrespective of labelling period. Whereas day-0-2 material was extensively degraded to disaccharide on incubation with the bacterial eliminase chondroitinase AC, a significant portion, about 30%, of the day-5-7 material resisted degradation under the same conditions. The resistant portion was readily depolymerized by treatment with chondroitinase ABC and may be dermatan sulphate. Paper electrophoresis and paper chromatography of the disaccharides obtained by eliminase digestion identified the day-0-2 labelled galactosaminoglycan as chondroitin 4-sulphate. In contrast, the corresponding day-5-7 material yielded approximately 20% disulphated disaccharide, both on digestion with chondroitinase AC and on subsequent enzymic degradation of the chondroitinase AC-resistant fraction. Further treatment of the disulphated disaccharide with chondro-4-sulphatase and chondro-6-sulphatase indicated that both sulphate groups were located on the N-acetylgalactosamine residue. In accordance with these findings, the day-5-7 polysaccharide showed a higher negative charge density than the day-0-2 material on ion-exchange chromatography. It is concluded that the novel properties acquired by the monocyte during prolonged culturing on plastic include the ability to synthesize glycosaminoglycan(s) containing 4,6-disulphated N-acetylgalactosamine units.

Carbohydrates↗
PubMed1985

Structure of heparin-derived tetrasaccharides.

The structure of heparin was examined by characterizing a disaccharide and five of the more than a dozen tetrasaccharide components obtained by its depolymerization with flavobacterial heparinase. Enzymic depolymerization of porcine mucosal heparin results in a mixture of di-, tetra-, hexa- and higher oligo-saccharides. The di- and tetra-saccharide components represent 75mol/100mol of these heparin fragments. Ion-exchange chromatography indicates the presence of only one disaccharide, deltaIdu2S(1----4)-alpha-D-GlcNS6S (where Idu is iduronic acid, deltaIdu is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is glucosamine, GlcA is glucuronic acid and S is sulphate), but results in the isolation of five major and at least seven minor tetrasaccharide components. The structures of the disaccharide and five major tetrasaccharides were determined by chemical, enzymic, electrophoretic and spectroscopic methods, including 13C, 1H n.m.r. and fast atom bombardment-m.s. The structure of these five tetrasaccharides are: delta Idu2S(1----4)-alpha-D-GlcNS6S(1---4)-alpha-L-Idu2S(1-- --4)-alpha -D-GlcNS6S; delta Idu2S(1----4)-alpha-D-GlcNS6S(1----4)-beta-D-GlcA(1--- -4)- alpha-D-GlcNS6S; delta Idu2S(1----4)-alpha-D-GlcNS(1----4)-beta-D-GlcA delta Idu2S(1----4)-alpha-D-GlcNAc(1----4)-beta-D-GlcA(1----4)- alpha-D-GlcNS6S; and delta Idu2S(1----4)-alpha- D-GlcNAc(1----4)-alpha-L-Idu(1----4)-alpha-D-GlcNS6S. Biological activity for the disaccharide and the five major tetrasaccharides was examined, and none of them were found to possess significant anticoagulant activity.

Animals↗
PubMed1995

Structure of heparan sulphate from human brain, with special regard to Alzheimer's disease.

Heparan sulphate (HS) was isolated after proteolytic digestion of cerebral cortex, obtained at autopsy, of patients with Alzheimer's disease (AD) and of control subjects. Deaminative cleavage in combination with selective radiolabelling procedures showed that the N-acetylated regions in the intact polysaccharides ranged from isolated residues to approximately 10 consecutive N-acetylated disaccharide units, without any apparent difference between AD and control HS. The yield of disaccharide deamination products was slightly higher with AD than with control HS, suggesting a differential distribution of N-sulphate groups. Separation of the disaccharides by anion-exchange h.p.l.c. yielded four mono-O-sulphated and one di-O-sulphated disaccharide; these components occurred in strikingly similar proportions in all cerebral HS preparations (except polysaccharide from neonatal brain) irrespective of the age of the individual and the histopathology of the cortex specimen. No significant difference was noted between HS obtained from control and from AD tissue. By contrast, the composition of HS isolated from brain differed significantly from that of HS preparations derived from other human organs.

Acetylation↗
PubMed1998

Characteristic hexasaccharide sequences in octasaccharides derived from shark cartilage chondroitin sulfate D with a neurite outgrowth promoting activity.

A mouse brain chondroitin sulfate (CS) proteoglycan, DSD-1-PG, bears the DSD-1 epitope and has neurite outgrowth promoting properties. Shark cartilage CS-C inhibits the interactions between the DSD-1-specific monoclonal antibody 473HD and the CS chains of the DSD-1-PG, which is expressed on the mouse glial cells (Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C., and Schachner, M. (1994) J. Cell Biol. 126, 783-799). On the other hand, several hexasaccharides isolated from commercial shark cartilage CS-D, which contains a higher proportion of characteristic D units (GlcUA(2-sulfate)beta1-3GalNAc(6-sulfate)) as compared with CS-C, has the A-D tetrasaccharide sequence composed of an A disaccharide unit (GlcUAbeta1-3GalNAc(4-sulfate)) and a D disaccharide unit (Nadanaka, S. and Sugahara, K. (1997) Glycobiology 7, 253-263). In this study, the biological activities and the structure of shark cartilage CS-D were investigated. CS-D inhibited the interactions between monoclonal antibody 473HD and DSD-1-PG and also promoted neurite outgrowth of embryonic day 18 hippocampal neurons. Eight octasaccharide fractions were isolated from CS-D after partial digestion with bacterial chondroitinase ABC by means of gel filtration chromatography and anion-exchange high performance liquid chromotography to investigate the frequency and the arrangement of the A-D tetrasaccharide unit in the polymer sequence. Structural analysis performed by a combination of enzymatic digestions with 500-MHz 1H NMR spectroscopy demonstrated that the isolated octasaccharides shared the common core structure DeltaHexAalpha1-3GalNAcbeta1-4(GlcUAbeta1-3GalNAc)3 with four, five, and six sulfate esters at various hydroxyl groups in different combinations. In the structure, DeltaHexA and GlcUA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and glucuronic acid, respectively. No D-D tetrasaccharide sequence was found, and discrete D disaccharide units were demonstrated exclusively as A-D tetrasaccharide units in either an A-D-A or an A-D-C hexasaccharide sequence in the five octasaccharides that represented about 5.0% (w/w) of the starting polysaccharides (C denotes the disaccharide GlcUAbeta1-3GalNAc(6-sulfate)). It remains to be determined whether such characteristic hexasaccharide sequences present in shark cartilage CS-D serve as functional domain structures recognized by some protein ligands.

Animals↗
PubMed2000

Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate.

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.

Animals↗
PubMed2002

Characterization of a novel lipid-A from Rhizobium species Sin-1. A unique lipid-A structure that is devoid of phosphate and has a glycosyl backbone consisting of glucosamine and 2-aminogluconic acid.

The structure of the lipid-A from Rhizobium species Sin-1, a nitrogen-fixing Gram-negative bacterial symbiont of Sesbania, was determined by composition, nuclear magnetic resonance spectroscopic, and mass spectrometric analyses. The lipid-A preparation consisted of a mixture of structures due to differences in fatty acylation and in the glycosyl backbone. There were two different disaccharide backbones. One disaccharide consisted of a distal glucosaminosyl residue beta-linked to position 6 of a proximal 2-aminoglucono-1,5-lactonosyl residue, and in the second disaccharide, the proximal residue was 2-amino-2,3-dideoxy-d-erythro-hex-2-enono-1,5-lactone. For both disaccharides, the distal glucosamine was acylated at C-2' primarily with beta-hydroxypalmitate (beta-OHC16:0) which, in turn, was O-acylated with 27-hydroxyoctacosanoic acid. For some of the lipid-A molecules, the distal glucosaminosyl residue was also acylated at C-3' with beta-hydroxymyristate (beta-OHC14:0), whereas other molecules were devoid of this acyl substituent. Both the 2-aminoglucono-1,5-lactonosyl and 2-amino-2,3-dideoxy-d-erythro-hex-2-enono-1,5-lactonosyl residues were acylated at C-2, primarily with beta-OHC16:0. Minor amounts of lipid-A molecules contained beta-OHC14:0 at C-3 and/or beta-hydroxystearate (beta-OHC18:0) or beta-hydroxyoctadecenoate (beta-OHC18:1) as the C-2 and C-2' N-acyl substituents.

Lipid A↗
PubMed2002

Evidence for a lectin activity for human interleukin 3 and modeling of its carbohydrate recognition domain.

We demonstrate that human interleukin 3 (IL-3) is a lectin recognizing specifically the glycosaminoglycan part of a chondroitin sulfate proteoglycan (PGS3; Normand, G., Kuchler, S., Meyer, A., Vincendon, G., and Zanetta, J. P. (1988) J. Neurochem. 51, 665-676) isolated from the adult rat brain. The specificity of the interaction of this particular proteoglycan with IL-3 is due to the abundance of GlcA(2S)beta 1,3GalNAc(4S)beta 1 disaccharide units as suggested by (1)H NMR. Computational docking experiments of the lower energy conformers of the different disaccharides from chondroitin sulfates reveal a privileged binding site for GlcA(2S)beta 1,3GalNAc(4S)beta 1 (involving His-26, Arg-29, Asn-70, and Trp-104) localized in an area of IL-3 different from the receptor-binding domain previously identified by others (Bagley, C. J., Phillips, J., Cambareri, B., Vadas, M. A., and Lopez, A. F. (1996) J. Biol. Chem. 271, 31922-31928). Molecular modeling of the mutation P33G, described as increasing the biological activity of IL-3 without affecting its receptor binding (Lokker, N. A., Movva, N. R., Strittmatter, U., Fagg, B., and Zenke, G. (1991) J. Biol. Chem. 266, 10624-10631) provokes a change of the three-dimensional structure of IL-3, especially in the area of the putative carbohydrate recognition domain defined above. Computational docking experiments of the different disaccharides of chondroitin sulfates indicate a loss of affinity for the previous ligand but a higher affinity for the classic disaccharide of chondroitin-4-sulfate. This change from a rare and specific ligand to a more abundant constituent of proteoglycans could induce an increased quantitative association between the IL-3 receptors and its ligands and, consequently, an increased signaling.

Animals↗
PubMed2005

Distinct substrate specificities of bacterial heparinases against N-unsubstituted glucosamine residues in heparan sulfate.

The rare N-unsubstituted glucosamine (GlcNH(3)(+)) residues in heparan sulfate have important biological and pathophysiological roles. In this study, four GlcNH(3)(+)-containing disaccharides were obtained from partially de-N-sulfated forms of heparin and the N-sulfated K5 polysaccharide by digestion with combined heparinases I, II, and III. These were identified as DeltaHexA-GlcNH(3)(+),DeltaHexA-GlcNH(3)(+)(6S),DeltaHexA(2S)-GlcNH(3)(+), and DeltaHexA(2S)-GlcNH(3)(+)(6S). Digestions with individual enzymes revealed that heparinase I did not cleave at GlcNH(3)(+) residues; however, heparinases II and III showed selective and distinct activities. Heparinase II generated DeltaHexA-GlcNH(3)(+)(6S),DeltaHexA(2S)-GlcNH(3)(+), and DeltaHexA(2S)-GlcNH(3)(+)(6S) disaccharides, whereas heparinase III yielded only the DeltaHexA-GlcNH(3)(+) unit. Thus, the action of heparinase II requires O-sulfation, whereas heparinase III acts only on the corresponding non-sulfated unit. These striking distinctions in substrate specificities of heparinases could be used to isolate oligosaccharides with novel sequences of GlcNH(3)(+) residues. Finally, heparinases were used to identify and quantify GlcNH(3)(+)-containing disaccharides in native bovine kidney and porcine intestinal mucosal heparan sulfates. The relatively high content of O-sulfated GlcNH(3)(+)-disaccharides in kidney HS raises questions about how these sequences are generated.

Chromatography, High Pressure Liquid↗
PubMed1991

Dietary manipulation for treating infants with prolonged dehydrating diarrhoea: a comparison of four different formulae.

Dietary manipulation is often the first step in the treatment of infants with persistent acute dehydrating diarrhoea. This usually entails elimination of lactose, but other disaccharides or whole protein may be causing the disease. Serial elimination of these takes time and it may be preferable to use a whole protein and disaccharide-free formula as the first feed change. This study assessed the effect on stool weight of a change from cow's milk formula feeds to one of four different formulae in infants with severe diarrhoea persisting after 3 days in hospital. Two feeds were lactose-free soy formulae containing sucrose, one was disaccharide-free soy formula and one a disaccharide-free protein hydrolysate. Regardless of which feed the infants received, diarrhoea resolved in approximately 50% following the change in diet. Comparing those who got better with those who did not, the former were generally better nourished and had an initial lower stool output, but it was impossible to predict on clinical grounds which individual would respond to the removal of cow's milk. The results suggest that elimination of lactose in infants with persistent severe diarrhoea will benefit a significant number in the early stage of the disease. As there is no additional benefit from eliminating sucrose or whole protein at this stage, the cheapest available lactose-free formula should be used initially.

Diarrhea, Infantile↗
PubMed1992

Synthesis of four novel trisaccharides by induction of loose acceptor specificity in Gal beta 1----4 transferase (EC 2.4.1.22): Galp(beta 1----4)Glcp(X)Glc where X = beta 1----3: beta 1----4: beta 1----6: alpha 1----4.

Development of tandem mass spectral methods for direct linkage determination in oligosaccharides requires sets of trisaccharides differing only in one structural parameter. In this case, we chose the position of linkage to the reducing-end hexose. These sets of compounds would also be useful for the development of high-resolution separation techniques geared to resolve linkage types. Conventional organic synthesis of such a set could take as long as 2-5 months for each member of the set. Each trisaccharide would require 10-20 steps of synthesis. Instead, we utilized low pH to induce a loose acceptor specificity for bovine milk galactosyltransferase (lactose synthase: EC 2.4.1.22) and by this method, within 2 weeks, generated four novel oligosaccharides for NMR and mass spectral studies. The disaccharides cellobiose (beta 1----4), laminaribiose (beta 1----3), gentiobiose (beta 1----6) and maltose (alpha 1----4) acted as acceptors for EC 2.4.1.22 under these conditions. The beta 1----2-linked disaccharide, sophorose, was not commercially available and is not included in this study. The alpha-linked disaccharides were also examined, but except for the alpha 1----4 disaccharide maltose, were very poor acceptors under a variety of conditions. From these four acceptors, the following four novel trisaccharides were synthesized in micromole amounts, suitable for studies of linkage position using low-energy collision-induced-dissociation tandem mass spectrometry (FAB-MS-CID-MS), and for NMR: Galp(beta 1----4)Glcp(beta 1----3)-Glc, Galp(beta 1----4)Glcp(beta 1----4)Glc, Galp(beta 1----4)Glcp(beta 1----6)-Glc and Galp(beta 1----4)Glcp(alpha 1----4)Glc.

Carbohydrate Conformation↗
PubMed1983

Blood insulin, glucose, fructose and gastric inhibitory polypeptide levels in carbohydrate-sensitive and normal men given a sucrose or invert sugar tolerance test.

Twelve carbohydrate-sensitive and 12 normal men were selected for the study. Carbohydrate-sensitivity was based on an abnormal insulin response to a sucrose load. The subjects were fed a diet consisting of 45% of the calories as carbohydrate, 40% fat and 15% protein for 5 days prior to a sucrose or invert sugar tolerance test. In a crossover design, subjects were given 2 g/kg body weight of sucrose or invert sugar, and responses of insulin, glucose, fructose and gastric inhibitory polypeptide (GIP) were determined. Blood samples were taken at 0, 0.5, 1, 2 and 3 hours after being given the test loads. Insulin and glucose levels were significantly higher in carbohydrate-sensitive as compared to normal men. Glucose and GIP did not show any significant differences between the two carbohydrate loads. At 1 hour, the carbohydrate-sensitive men given sucrose had significantly higher insulin levels than carbohydrate-sensitive men given invert sugar (disaccharide effect). At 1, 2 and 3 hours, the disaccharide effect was shown in the fructose levels of the carbohydrate-sensitive men. In normal men, the disaccharide effect with levels of fructose was seen at 0.5 and 3 hours. This study indicates that the disaccharide effect on blood insulin cannot be explained by differences in gastric inhibitory polypeptide in unadapted human subjects.

Adult↗
PubMed1984

Thrombin-inhibitory activity of whale heparin oligosaccharides.

Whale heparin was partially digested with a purified heparinase and the oligosaccharide fractions with 8-20 monosaccharide units were isolated from the digest by gel filtration on Sephadex G-50, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. A marked difference in the inhibitory activity for thrombin in the presence of antithrombin III was observed between the high-affinity fractions for antithrombin III of octasaccharide approximately hexadecasaccharide and those of octadecasaccharide approximately eicosasaccharide. The disaccharide compositions of these hexadeca-, octadeca-, and eicosasaccharides were analyzed by high-performance liquid chromatography after digestion with a mixture of purified heparitinases 1 and 2 and heparinase. The analytical data indicated that the proportions of trisulfated disaccharide (IdUA(2S)alpha 1----4GlcNS(6S)) and disulfated disaccharide (UA1----4GlcNS(6S)) increased with the manifestation of high thrombin-inhibitory activity, while that of monosulfated disaccharide (UA1----4GlcNS) decreased. The present observations, together with those so far reported, suggest that the presence of the former structural elements, specifically IdUA(2S)alpha 1----4GlcNS(6S), as well as the antithrombin III-binding pentasaccharide at the proper positions in the molecules of whale heparin oligosaccharides is essential for the manifestation of high inhibitory activity for thrombin in the presence of antithrombin III. The structural bases for the manifestation of the anticoagulant activity of whale and porcine heparins and their oligosaccharides are also discussed.

Animals↗
PubMed2007

Concentration dependence of vibrational properties of bioprotectant/water mixtures by inelastic neutron scattering.

Neutron scattering has been demonstrated to be a powerful tool for characterizing the structure and dynamics of biological molecules and for investigating the physical and chemical mechanisms of biophysical processes. The aim of the present work is to investigate by inelastic neutron scattering (INS) the vibrational behaviour of a class of bioprotectant systems, such as homologous disaccharides, trehalose, maltose and sucrose, in water mixtures. INS measurements have been performed on trehalose/H2O, maltose/H2O and sucrose/H2O mixtures at very low temperature as a function of concentration by using the thermal original spectrometer with cylindrical analyzers (TOSCA) spectrometer at the ISIS Facility (DRAL, UK). The findings allow the analyses of the vibrational features of the INS spectra in order to study the effect of disaccharides on the H2O hydrogen-bonded tetrahedral network. The obtained neutron scattering findings point out that disaccharides, and in particular trehalose, have a destructuring effect on the water tetrahedral network, as emphasized by the analysis of the librational modes region from 50 to 130 meV energy transfer. On the other hand, the analysis of the bending modes region (130-225 meV) shows a locally ordered structure in the disaccharide/H2O mixtures.Finally, the observed experimental evidences are linked to the different bioprotective effectiveness of disaccharides as a function of concentration.

Cryoprotective Agents↗
PubMed2004

Characterization of splenic glycosaminoglycans accumulated in vivo in experimentally induced amyloid-susceptible and amyloid-resistant mice.

The pathogenesis of the amyloid deposition diseases is poorly understood. The CE/J mouse, which is naturally protected from amyloid A (AA) protein amyloidosis, has provided a tool to study mechanisms that may be implicated in amyloid deposition diseases by means of comparison of findings with those in an AA-susceptible mouse strain. We have compared proteoglycan/glycosaminoglycan accumulation in vivo in amyloid-protected CE/J mouse strain and in AA-susceptible CBA/J mouse strain in homeostasis and when injected with amyloid-inducing agents. Results indicate that there is an overall increase in [(35)S]proteoglycan/glycosaminoglycan accumulation in the spleens of both strains of mice, but with a specific increase in heparan sulfate in only CBA/J mouse spleens that are rich in amyloid. Further, we report the absence of heparan sulfate in the splenic perifollicular areas of amyloid-free CE/J mouse, whereas in the amyloid-laden CBA/J mouse there is co-localization of heparan sulfate with the AA deposits. We have also examined the glycosaminoglycan disaccharide products in both these strains of mice for their sulfation positions and found no differences in the disaccharide composition of chondroitin/dermatan sulfate and heparan sulfate isolated from the control CBA/J and control CE/J mice. There were no differences in chondroitin/dermatan sulfate in both strains after experimental induction. However, analysis of the heparan sulfate disaccharides by means of capillary high-performance liquid chromatography linked to microelectrospray ionization time-of-flight mass spectrometry indicated that the disaccharide composition of the splenic heparan sulfate obtained from the treated CBA/J mice that had developed amyloid was markedly different from that obtained from the control CBA/J mice and the treated amyloid-resistant CE/J mice. These findings suggest that unique heparan sulfates play a fundamental role in the pathogenesis of amyloid.

Amyloidosis↗
PubMed1985

Use of synthetic antigens with the carbohydrate structure of asialoglycophorin A for the specification of Thomsen-Friedenreich antibodies.

The panagglutination phenomenon described by Thomsen and Friedenreich (TF) is due to the reaction of naturally occurring TF-antibodies with the carbohydrate group beta-D-Gal-(1-3)-D-GalNAc of desialylated glycophorin A, the major glycoprotein component of the erythrocyte membrane. The specificity of human TF-antibodies reacting with this disaccharide was investigated by hemagglutination inhibition assay and radioimmunoassay using various synthetic oligosaccharides and neoglycoproteins as well as asialoglycophorin A. The results indicate that TF-antibodies represent a heterogeneous mixture of carbohydrate-specific antibodies. The disaccharide beta-D-Gal-(1-3)-D-GalNAc is the common structure recognized by all TF-antibodies. However, the conjugation mode of the carbohydrate to the carrier protein is important for defining the specificity of different subpopulations of TF-antibodies. The immunological reaction depends on the configuration of the glycosidical linkage as well as on the chemical nature of the aglycon, which is coupled to the disaccharide. These findings suggest that the heterogeneity of natural TF-antigens is due to the wide distribution of the carbohydrate structure beta-D-Gal-(1-3)-D-GalNAc. The characterization of TF- or TF-like antibodies directed to particular natural TF-antigens (e.g. asialoglycophorin A, tumor TF-antigens, glycolipids, bacterial antigens) requires TF-analogues, which contain the additional molecular regions together with the TF-disaccharide. These structures, apart from the TF-hapten, are obviously important for defining the immunodeterminant group of TF-antigens of different origin.

Antibodies↗
PubMed1976

Immunochemical detection of the Thomsen-Friedenreich antigen (T-antigen) on the pig lymphocyte plasma membrane.

A method for the isolation of lymphocytes from pig peripheral blood and preparation of plasma membranes was developed. The method resulted in a ten-fold increase in 5'-nucleotidase activity and neuraminic acid, relative to protein. A cholesterol/phospholipid ratio of 0.93 was obtained. Alkaline borohydride treatment of isolated plasma membrane, after desialylation, released the disaccharide beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol (the immunodominant group of the T-antigen), which was identified by gas chromatography using a crystalline standard. The disaccharide was not found without prior desialylation, indicating that all the disaccharide units were substituted by neuraminic acid. Serological evidence using the agglutinins from Arachis hypogoea and Helix pomatia confirmed the presence of this disaccharide (T-antigen) in the lymphocyte membrane and indicated that it was the major alkali-labile oligosaccharide of the pig lymphocyte membrane.

Animals↗
PubMed1977

The action of lysozyme on peptidoglycan with N-unsubstituted glucosamine residues. Isolation of glycan fragments and their susceptibility to lysozyme.

1. A peptidoglycan preparation N-acetylated at about 30% of glucosamine residues was obtained by the treatment of the lysozyme-resistant cell wall paptidoglycan of Bacillus cereus with acetic anhydride at pH 7. Fractionation of dialyzable material resulting from lysozyme digestion of the glycan component of this peptidoglycan preparation yielded five oligosaccharides designated as S1 to S5 besides the disaccharide GlcNAc-MurAc. 2. Oligosaccharide S3, which accounted for about 30% of the disaccharide units recovered as disaccharides and oligosaccharides, was identified as GlcN-MurAc-GlcNAc-MurAc. Oligosaccharide S1, accounting for about 20% of the disaccharide units recovered, was characterized as GlcN-MurAc-GlcN-MurAc-GlcNAc-MurAc, while oligosaccharide S2, present in a smaller amount, as GlcNAc-MurAc-GlcN-MurAc-glcNAc-MurAc. Oligosaccharides S4, and S5, present in small amounts, were identified as GlcNAc-MurAc-GlcNAc-MurAc and MurAc-GlcNAc-MurAc, respectively. 3. Oligosaccharides S1, S3 and S5 proved to be completely insusceptible to lysozyme, whereas S2 was digsted by lysozyme to produce GlcNAc-MurAc and S3. S1 was found to act as a more potent inhibitor than S3 in lysozyme-catalyzed digestion of polysaccharides. 4. The results obtained show that the lysozyme-catalyzed hydrolysis of peptidoglycan oligosaccharides had an obligatory requirement for the N-acetyl group on the glucosamine residue located in subsite C in the enzyme-substrate complex.

Bacillus cereus↗
PubMed1983

Beta-agarases I and II from Pseudomonas atlantica. Substrate specificities.

Beta-Agarase I and II were characterised by their action on agar-type polysaccharides and oligosaccharides. Beta-Agarase I, an endo-enzyme, was specific for regions containing a minimum of one unsubstituted neoagarobiose unit [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose], hydrolysing at the reducing side of this moiety. Yaphe demonstrated that agar was degraded by this enzyme to neoagaro-oligosaccharides limited by the disaccharide but with a predominance of the tetramer [Yaphe, W. (1957) Can. J. Microbiol. 3, 987-993]. Beta-Agarase I slowly degraded neoagarohexaose but not the homologous tetrasaccharide. [1-3H]Neoagarohexaitol was cleaved to neoagarotetraose and [1-3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to methylated, sulphated and unsubstituted neoagaro-oligosaccharides which were invariably terminated at the reducing end by unsubstituted neoagarobiose. The novel enzyme, beta-agarase II, was shown to be an endo-enzyme. Preliminary evidence indicated this enzyme was specific for sequences containing neoagarobiose and/or 6(1)-O-methyl-neoagarobiose. It degraded agar to neoagaro-oligosaccharides of which the disaccharide was limiting and predominant. Beta-Agarase II rapidly degraded isolated neogarotetraose and neoagarohexaose to the disaccharide. With [1-3H]neoagarohexaitol, exo-action was observed, the alditol being cleaved to neoagarobiose and [1-3H]neoagarotetraitol. Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol. Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24% of the starting carbohydrate. This fraction was almost exclusively disaccharides (22.4%) containing neoagarobiose (7.4%) and 6(1)-O-methyl-neoagarobiose (15%). Beta-Agarase II is probably the 'beta-neoagarotetraose hydrolase' reported by Groleau and Yaphe as an exoenzyme against neoagaro-oligosaccharides [Groleau, D. and Yaphe, W. (1977) Can. J. Microbiol. 23, 672-679].

Agar↗