Leb-active glycolipid in human plasma: Measurement by radioimmunoassay

A radioimmunoassay that measures Le^b-active glycolipids in human plasma has been developed using antiserum from a goat immunized with a Le^b blood group hapten, lacto-N-difucohexaose I, conjugated to polylysine. Binding by the antiserum of lacto-N-difucohexaose I conjugated to ¹²⁵I-labeled bovine serum albumin is specifically inhibited by Le^b-active ceramide hexasaccharide. Plasma levels of the glycolipid are quantitated by comparing the inhibitory activity of plasma with that of the purified Le^b-active glycolipid. Plasma samples from 35 blood group O Le(a ? b +) individuals contain Le^b-active ceramide hexasaccharide at an average concentration of 0.9 μg/ml (range: 0.2 to 2.5 μg/ml); no Le^b-active glycolipid (less than 0.02 μg/ml) could be detected in plasma from blood group O Le(a + b?) or O Le(a? b?) individuals. Plasma from A₁ Le(a ? b+) individuals contains less Le^b-active glycolipid than plasma from A₂ Le(a? b+) individuals: its level in 19 samples of A, Le(a? b+) plasma averages 0.2 μg/ml (range: 0.1 to 0.45 μg/ml), and its level in 9 samples of A₂ Le(a? b+) plasma averages 1.1 μg/ml (range 0.8 to 1.3 μg/ml). About one-third of the total Le^b-active glycolipid in whole blood is associated with erythrocytes and the rest is found in plasma.     

Preparation of precipitating antigens by coupling oligosaccharides to polylysine

Oligosaccharides isolated from human milk when coupled to polylysine by a mixed anhydride procedure are effective precipitating antigens. The lacto-N-fucopentaose II conjugate specifically precipitates antibody directed against the human Le^a blood group antigen while the lacto-N-difucohexaose I conjugate specifically precipitates antibody directed against the human Le^b blood group antigen. The derivatives were used to define the specificity of a human anti-I cold agglutinin.     

N-Deacetylation of 2-acetamido-2-deoxy-hexosecontaining oligosaccharides and polysaccharides by calcium in liquid ammonia

N-Deacetylation of 2-acetamido-2-deoxy-hexose residues is accomplished in liquid ammonia containing calcium. Oligosaccharides, lacto-N-fucopentaose II and lacto-N-difucohexaose I, containing 3,4-disubstitutedN-acetylhexosamine residues are quantitativelyN-deacetylated. When applied to polysaccharides, however, only partialN-deacetylation was achieved.Author for correspondence. AXRD     

New derivatization and separation procedures for reducing oligosaccharides

4-Trifluoroacetamidoaniline was reacted with reducing oligosaccharides in the presence of sodium cyanoborohydride to give aminoalditol derivatives, useful for linkage to proteins or solid matrices. A mixture of reducing oligosaccharides, difficult to separate by HPLC, was treated in the same way. The resulting derivatives were easily separated by HPLC.Abbreviations TFAN 4-trifluoroacetamidoaniline - LcOse₄ lacto-N-tetraose - IV²Fuc-LcOse₄ lacto-N-fucopentaose l - III⁴Fuc-LcOse₄ lacto-N-fucopentaose II - III³Fuc-nLcOse₄ lacto-N-fucopentaose III - IV²Fuc, III⁴Fuc-LcOse₄ lacto-N-difucohexaose I - II⁶Galß1-4GlcNAc-LcOse₄ lacto-N-hexaose - II³NeuAc-Lac 3-sialyllactose - GlcNAcß1-4GlcNAcß1-4GlcNAc chitotriose - GalNac1-3|Fuc1-2|Galß1-4Glc A-tetrasaccharide     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Trade-off relationships between some reproductive characteristics in plants with special reference to life history stragegy

Several reproductive triats in plants were studied in more than 200 populations of 61 wild species from diverse ecological conditions. As a result, it was found that there occur three distinct types of plants in the energy allocation patterns to reproductive structures (RA) and the propagule output per plant (P_N), i.e. (1) the number of propagules per plant increases in response to the increase in RA (Type I), (2) the number of propagules decreases in response to the increase in RA (Type II), and (3) the RA remains constant despite the great differences in the propagule number per plant. A conspicuous trade-off relationship was also discovered to occur between the RA to a single propagule (R_A) and the propagule output per plant (P_N), such that log R_A=logC−blot P_N, or R_A=C/P_N ^b =CP_N ^−b , where C is a constant. The three different ranges ofb-values were recognized, i.e.b<1.0,b>1.0, andb=1.0, which correspond to Type I, Type II, and Type III, respectively. Related problems to the concept ofr- andK-strategy are also discussed.     

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous G_M1- and G_D1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (G_M3, G_D1a, G_T1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (G_M1, G_M2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH mouse ascites hepatoma 22a - LDL low density lipoprotein - ASM anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine] - RITC rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: G_M3, II³NeuAc-LacCer, II³-N-acetylneuraminosyllactosylceramide - G_M2 II³-NeuAc-GgOse₃Cer, II³-N-acetylneuraminosylgangliotriaosylceramide - G_M1 II³-NeuAc-GgOse₄Cer, II³-N-acetylneuraminosylgangliotetraosylceramide - G_D1a, II³ IV³(NeuAc)₂-GgOse₄Cer, II³, IV³-di(N-acetylneuraminosyl)gangliotetraosylceramide - G_T1b II³(NeuAc)₂, IV³ NeuAc-GgOse₄Cer, II³-di-N-acetylneuraminosyl, IV³-N-acetylneuraminosylgangliotetraosylceramide     

Complex <Emphasis Type="Italic">N</Emphasis>-glycans or core 1-derived <Emphasis Type="Italic">O</Emphasis>-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or Le<Superscript>Y</Superscript> in the preimplantation mouse embryo

The glycan epitopes termed stage-specific embryonic antigens (SSEA) occur on glycoproteins and glycolipids in mammals. However, it is not known whether these epitopes are attached to N- or O-glycans on glycoproteins and/or on glycolipids in the developing mouse embryo. In this paper the expression of the antigens SSEA-1, SSEA-3, SSEA-4 and Le^Y was examined on ovulated eggs, early embryos and blastocysts lacking either complex and hybrid N-glycans or core-1 derived O-glycans. In all cases, antigen expression determined by fluorescence microscopy of bound monoclonal antibodies to embryos at the stage of development of maximal expression was similar in mutant and control embryos. Thus, none of these developmental antigens are expressed solely on either complex N- or core 1-derived O-glycans attached to glycoproteins in the preimplantation mouse embryo. Furthermore, neither of these classes of glycan is essential for the expression of SSEA-1, SSEA-3, SSEA-4 or Le^Y on mouse embryos.     

Characterization of the binding specificity ofAnguilla anguilla agglutinin (AAA) in comparison toUlex europaeus agglutinin I (UEA-I)

Using immunochemical and immunohistochemical methods, the binding site ofAnguilla anguilla agglutinin (AAA) was characterized and compared with the related fucose-specific lectin fromUlex europaeus (UEA-I). In solid-phase enzyme-linked immunoassays, the two lectins recognized Fuc1-2Gal-HSA. AAA additionally cross-reacted with neoglycolipids bearing lacto-N-fucopentaose (LNFP) I [H type 1] and II [Le^a] and lactodifucotetraose (LDFT) as glycan moieties. UEA-I, on the other hand, bound to a LDFT-derived neoglycolipid but not to the other neoglycolipids tested. Binding of AAA to gastric mucin was competitively neutralized by Le^a-specific monoclonal antibodies. UEA-I binding, on the other hand, was reduced after co-incubation with H type 2- and Le^y-specific monoclonal antibodies. According to our results, AAA reacts with fucosylated type 1 chain antigens, whereas UEA-I binds only to the 1-2-fucosylated LDFT-derived neoglycolipid. In immunohistochemical studies, the reactivity of AAA and UEA-I in normal pyloric mucosa from individuals with known Lewis and secretor status was analysed. AAA showed a broad reaction in the superficial pyloric mucosa from secretors and non-secretors, but AAA reactivity was more pronounced in Le^(a+b-) individuals. On the other hand, UEA-I stained the superficial pyloric mucosa only from secretor individuals. A staining of deep mucous glands by the lectins was found in all specimens. Both reacted with most human carcinomas of different origin. Slight differences in their binding pattern were observed and may be explained by the different fine-specificities of the lectins.     

Human-type blood groups and Gm factors in gibbons (Hylobates)

Blood specimens from 69 gibbons (63Hylobates lar, 4Hylobates concolor, and 2Hylobates pileatus) were tested for human-type ABO, MN, and Rh blood groups. AmongH. lar, three phenotypes were noted in the ABO and MN blood groups respectively, but all fourH. concolor were grouped as AM. All group A gibbons were of subgroup A₁; subgroups A₂B and A₁₂B were observed at a low frequency in group AB gibbons. Le^b antigen was detected in about 30% of the red cell samples fromH. lar, but all the samples were negative for Le^a. All the gibbons tested had c(hr) antigen but no other Rh antigens (D, C, E, and e) in their red cells. Some selected blood samples fromH. lar were also tested for some other blood group antigens and for the Gm and Inv factors. The Jk^a antigen was detected in all the red cell samples tested, but the S, s, U, K, k, and Fy^a antigens were not. In the tests of plasma with anti-Gm (1),H. lar could be divided into two groups, i.e., Gm(1)^Gi and Gm(–1)^Gi; Gm(2), Gm(4), and Inv(1) were absent in the species.     

Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

Helicobacter pylori infection can change the intensity of gastric Lewis antigen expressions differently between adults and children

This study tested whether there were different expressions of gastric Lewis antigens between children and adults with Helicobacter pylori infection, and whether the difference was related to the infection outcome. About 68 dyspeptic children and 110 dyspeptic adults were enrolled to check H. pylori infection, its colonization density, and the related histology. Gastric Lewis antigens b (Le^b), x (Le^x), and sialyl-Lewis x (sialyl-Le^x) were immunohistochemically stained and scored for the intensity. The H. pylori-infected adults, but not the children, had a lower Le^b intensity over the antrum (p = 0.019) but higher Le^b intensity over the corpus (p = 0.001) than the non-infected ones. Over the antrum, both the H. pylori-infected children and adults had a lower Le^x and higher sialyl-Le^x intensity than those non-infected ones (p < 0.05). The H. pylori-infected adults had a higher bacterial density (p = 0.004) and Le^b intensity (p = 0.016) over the corpus than the H. pylori-infected children. For the H. pylori-infected adults, but not children, the corpus had a higher Le^b (p = 0.038) and lower Le^x (p = 0.005) intensity than the antrum. Furthermore, the H. pylori-infected adults expressed a higher Le^b and had a higher bacterial density than those with weak Le^b (antrum, p < 0.001; corpus, p = 0.001). In conclusion, H. pylori infection is associated with the intensity change of Lewis antigen expressions in the stomach. The changes of gastric Lewis antigen expressions are different between adults and children with H. pylori infection, which may exert different H. pylori colonization over the corpus between adults and children.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

The Lectin from the Crustacean Liocarcinus depurator Recognizes O-acetylsialic Acids

A lectin that recognized sialic acids and agglutinated mouse erythrocytes was purified from hemolymph of the crab Liocarcinus depurator. It consisted of 38-kDa subunits and had a pI about 6.0. The specificity of the lectin was assayed by hemagglutination inhibition. N-acetylneuraminic acid (Neu5Ac) was a good inhibitor and its N-acetyl group at C-5 was critical for lectin-ligand interaction. Substitution of the C-9 hydroxyl on Neu5Ac with an O-acetyl group (9-O-Ac-Neu5Ac) increased the inhibitory potency of this molecule. Furthermore, O-acetyl substitution of all the hydroxyl groups yielded even better inhibitors (2,4,7,8,9-O-Ac-Neu5Ac and its 1-O-methyl ester). Removal of the hydroxyl or O-acetyl group connected to C-2 reduced the potency of these inhibitors. The lectin agglutinated and stimulated human but not mouse lymphocytes. It was also inhibited by Escherichia coli (O111:B4) lipopolysaccharide and agglutinated specific gram-negative bacteria. In vitro labeling with [³⁵S]methionine indicated that the lectin was synthesized in hepatopangreas of L. depurator. Immunofluorescence showed that among hemocytes it localized mainly in the large-granule population.     

Effect of heterologous bacteria and combined nitrogen on the adsorption ofRhizobium meliloti to lucerne seedling roots

Summary The binding ofRhizobium meliloti strains A₂ (effective) and V₆ (ineffective),Agrobacterium tumefaciens strain B₆S₃ andR. trifolii strain TL₅ to lucerne seedling roots was studied by using¹⁴C or³H-labelled bacteria. When added singly or in combination with the heterologous bacteria, the number of A₂ cells attached to the roots was significantly less than the number of B₆S₃ or TL₅ cells. However, the presence of the heterologous bacteria did not decrease the proportion of A₂ cells added in the inoculum that bind to the roots, suggesting thatR. meliloti is attached to specific sites. In fact, the same number of A₂ or V₆ cells bind to the roots and in mixed inoculation the 2 strains share equally the binding sites. When added to the seedlings growth medium NO ₃ ^– at 5 or 16 mM significantly decreased the number of A₂ cells adhering to lucerne seedling roots. The results suggest that the lectin-recognition hypothesis is probably involved in the attachment ofR. meliloti to lucerne seedling roots.Contribution No. 252 Station de recherches, Agriculture Canada     

Identification of Leb antigens in meconium of a phenotypically a Le(a+b−) non-secretor individual

Blood group active fucolipids of human meconium have been shown to correlate to the ABH and Lewis blood groups and to the secretor status of the corresponding children. Using a monoclonal anti-Le^b antibody and an antibody to chromatogram binding assayy the presence of Le^b antigens in meconium of a phenotypically A Le(a⁺b^?) non-secretor indivual is here demonstrated. Phenotype was determined on cord blood and saliva obtained 2 years after birth.     

In vivo activity of recombinant human lewis fucosyltransferase III in leaves of Nicotiana tabacum L.

Fucosylation in plants occurs in glycoproteins and polysaccharides but the function of fucosylation is largely unknown. We aimed to analyze the effects of over-expression of human fucosyltransferase III (hFucT III) on in vivo N-glycan accumulation in tobacco plant leaves and focused on comparing the amount of Lewis a (Le^a)-epitope accumulation in transgenic and in wild-type plants. Fucosyltransferase assays, Western blot and mass spectrometry were used to identify, quantify and analyse Le^a N-glycans. We found that constitutive overexpression of hFucT III activity had no effect on Le^a complex type N-glycans accumulation. Our results suggest that tobacco recombinant hFucT III acts more as a hydrolase than as a transferase.     

Identification of Le antigens in meconium of a phenotypically a Le(ab) non-secretor individual

Blood group active fucolipids of human meconium have been shown to correlate to the ABH and Lewis blood groups and to the secretor status of the corresponding children. Using a monoclonal anti-Le^b antibody and an antibody to chromatogram binding assayy the presence of Le^b antigens in meconium of a phenotypically A Le(a⁺b⁻) non-secretor indivual is here demonstrated. Phenotype was determined on cord blood and saliva obtained 2 years after birth.     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.