The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Fractionation of oligosaccharides liberated by hydrazinolysis and structural studies of the neutral oligosaccharides

The asparagine-linked sugar chains of the plasma membrane glycoproteins of rat erythrocytes were released as oligosaccharides by hydrazinolysis and labeled by NaB³H₄ reduction. The radioactive oligosaccharides were separated into a neutral and at least four acidic fractions by paper electrophoresis. The neutral oligosaccharide fraction was separated into at least 11 peaks upon Bio-Gel P-4 column chromatography. Structural studies of them by sequential exoglycosidase digestion in combination with methylation analysis revealed that they were a mixture of three high mannose-type oligosaccharides and at least 11 complex type oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their cores and Galβ1 → 4GlcNAc, Galβ1 → 3Galβ1 → 4GlcNAc, and various lengths of Galβ1 → 4GlcNAc repeating chains in their outer chain moieties. Most of the complex-type Oligosaccharides were biantennary, and the tri- and tetraantennary Oligosaccharides contain only the Galβ1 → 3Galβ1 → 4GlcNAc group in their outer chain moieties.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Structural studies of the acidic oligosaccharides

Among the four acidic oligosaccharide fractions obtained by paper electrophoresis of the hydrazinolysate of the plasma membrane glycoproteins of rat erythrocytes, one was further separated into two by prolonged paper electrophoresis using 120-cm paper. Three fractions were mixtures of monosialyl oligosaccharides and two of disialyl oligosaccharides. After desialylation, their neutral portions were fractionated by Bio-Gel P-4 column chromatography and by affinity chromatography using a Con A-Sepharose column. Structural studies of the neutral oligosaccharides, thus obtained, indicated that at least 26 different complex-type oligosaccharides are present as a neutral portion of the acid oligosaccharides. Structurally they can be classified into bi-, tri-, and tetraantennary oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc_OT as their common cores. Galβ1 → 3Galβ1 → 4GlcNAc, Siaα2 → 3Galβ1 → 4GlcNAc, Siaα2 → 6Galβ1 → 4GlcNAc, and a series of Siaα2 → (Galβ1 → 4GlcNAcβ1 → 3)_n · Galβ1 → 4GlcNAc were found as their outer chains. Their structures together with the structures of neutral oligosaccharides reported in the preceding paper indicated that the outer chain moieties of the asparagine-linked sugar chains of rat erythrocyte membrane glycoproteins are formed not by random concerted action of glycosyl transferases in Golgi membrane but by the mechanism in which the formation of one outer chain will regulate the elongation of others.     

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. Lewis-active and Lewis-like substances.

High resolution nuclear magnetic resonance spectra were recorded in a chloroform solution of six Lewis-active or Lewis-like glycosphingolipids in permethylated and permethylated-reduced (LiAlH₄) form. The samples were selected to cover the presently known structural variants of α-fucose linked to galactose and N-acetylglucosamine. Fucα1 → 2Gal, Fucα1 → 3GlcNAc, and Fucα1 → 4GlcNAc gave characteristic and well-separated anomeric resonances. Furthermore, upon reduction there was a strong deshielding effect on Fucα1 → 3GlcNAc and Galβ1 → 3GlcNAc (linkage vicinal to reduced amide), which makes it possible to differentiate type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains. This improved method of nuclear magnetic resonance spectroscopy is discussed in relation to sequence analysis by mass spectrometry, two microscale structural methods using the same type of derivatives and needing no degradations before analysis.     

Structural studies of the carbohydrate moiety of human antithrombin III

Human antithrombin III contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively released as radioactive oligosaccharides from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB³H₄ reduction. All of the oligosaccharides, thus obtained, contain N-acetylneuraminic acid. A same neutral nonaitol was released from all acidic oligosaccharides by sialidase treatment. By combination of the sequential exoglycosidase digestion and methylation analysis, their structures were elucidated as NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6-(NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc, Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6(NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manαl → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc, and NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6(Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc.     

Redefinition of the Carbohydrate Binding Specificity of Helicobacter pylori BabA Adhesin

Certain Helicobacter pylori strains adhere to the human gastric epithelium using the blood group antigen-binding adhesin (BabA). All BabA-expressing H. pylori strains bind to the blood group O determinants on type 1 core chains, i.e. to the Lewis b antigen (Fucα2Galβ3(Fucα4)GlcNAc; Le(b)) and the H type 1 determinant (Fucα2Galβ3GlcNAc). Recently, BabA strains have been categorized into those recognizing only Le(b) and H type 1 determinants (designated specialist strains) and those that also bind to A and B type 1 determinants (designated generalist strains). Here, the structural requirements for carbohydrate recognition by generalist and specialist BabA were further explored by binding of these types of strains to a panel of different glycosphingolipids. Three glycosphingolipids recognized by both specialist and generalist BabA were isolated from the small intestine of a blood group O pig and characterized by mass spectrometry and proton NMR as H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer), Globo H hexaglycosylceramide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), and a mixture of three complex glycosphingolipids (Fucα2Galβ4GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, Fucα2Galβ3GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, and Fucα2Galβ4(Fucα3)GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). In addition to the binding of both strains to the Globo H hexaglycosylceramide, i.e. a blood group O determinant on a type 4 core chain, the generalist strain bound to the Globo A heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), i.e. a blood group A determinant on a type 4 core chain. The binding of BabA to the two sets of isoreceptors is due to conformational similarities of the terminal disaccharides of H type 1 and Globo H and of the terminal trisaccharides of A type 1 and Globo A.     

Blood group i and I activities of "lacto-N-norhexaosylceramide" and its analogues: the structural requirements for i-specificities.

A pure straight chain ceramide hexasaccharide (“lacto-N-norhexaosylceramide” Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glc→Ceramide) showed strong i-activity determined by hemagglutination inhibition and by radioimmunoassay with five out of six anti-i antisera. Two repeating Galβ1→4GlcNAc residues and GlcNAcβ1→3Gal residues could be essential for the full expression of this activity; eleven closely related analogues including those derived by chemical modification had lower or no detectable activity. The same structure reacted also with some anti-I antisera. The strong i-activity and the moderate I-activity were both abolished by elimination of the terminal Gal or by removal of the N-acetyl groups of the two GlcNAc residues.     

Different asparagine-linked sugar chains on the two polypeptide chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) purified from placenta, like urinary hCG, is shown to have the sialylated forms of three neutral oligosaccharides: Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4(Fucα1→6)GlcNAc (N-1), Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-2) and Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-3). Gel permeation chromatographic analysis of oligosaccharides released from α- and β-subunits of placental hCG has revealed that the α-subunit has one each of sialylated N-2 and N-3, while the β-subunit has one each of sialylated N-1 and N-2.     

The hapten structure of a developmentally regulated glycolipid antigen (SSEA-1) isolated from human erythrocytes and adenocarcinoma: a preliminary note

Glycolipid antigen reacting to the monoclonal antibody directed to the developmentally regulated antigen SSEA-1 was isolated from human erythrocytes and colonic adenocarcinoma. The antigens have the Le^x (Galβl→4[Fucα]→3]GlcNAcβl→R) or Le^y (Fucαl→2Galβl→4[Fucαl→3]GlcNAcβl→R) structure at the termini of the branched polylactosaminolipid. In addition, a novel polyfucosyl structure locating exclusively at the internal GlcNAc was detected in the tumor antigen. The antibody reacts with a simple monovalent Le^x glycolipid (Galβl→4[Fucαl→3]GlcNAcβl→3Galβl→4Glcβl→Cer) previously isolated from colonic carcinoma when presented at a high density on liposomes. The antibody therefore may react to the bivalent or multivalent Le^x or Le^y structure.     

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. Blood group ABH-active substances.

High resolution nuclear magnetic resonance spectra of permethylated and permethylated-reduced (LiAlH₄) derivatives were recorded in chloroform solution for the following glycosphingolipids with known structure: lactotriaosylceramide, neolactotetraosylceramide (paragloboside), two blood group H-active pentaglycosylceramides (type 1 and type 2 saccharide chains, respectively), a B-active hexaglycosylceramide, an A-active hexaglycosylceramide, and an A-active octaglycosylceramide. Good quality and resolution allow a clear-cut diagnosis of α-anomeric protons of Fuc, Gal, and GalNAc, and in most cases of all β protons. Upon reduction there is a strong deshielding effect on H-1 of Gal of Galβ1 → 3GlcNAc but not on Gal of Galβ1 → 4GlcNAc. It is therefore possible to differentiate type 1 and type 2 chains by this method, a structural difference of importance for serological specificity. Nuclear magnetic resonance spectroscopy may therefore provide conclusive information on the anomeric structure of the immunodeterminant of blood group-active glycolipids using the same derivatives as for sequence analysis by mass spectrometry.     

Structural characterization of glycolipids of rat small intestine having one to eight hexoses in a linear sequence

Eight different fractions containing glycolipids with 1 to 8 hexoses in a linear sequence were isolated from rat small intestine. The structure of the major components was established by mass spectrometry and proton nuclear magnetic resonance spectroscopy of the permethylated and permethylated-reduced (LiAlH₄) derivatives and by gas-liquid chromatography of degradation products of the native and permethylated or permethylated-reduced glycolipids. The major compounds were glucosylceramide, lactosylceramide, globotriaosylceramide, and a novel tetrahexosylceramide with the structure Gal α 1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer. In addition four minor compounds having five to eight hexoses were identified with the probable structures Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Galα1 → 3Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer, and Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer. In the pentahexosylceramide fraction a novel fucolipid was also present having the probable structure Fucα1 → 2Galα1 → 3Galα1 → 4Galβ1 → 4Galβ1 → 1Cer. The lipophilic part of the glycolipids was composed of trihydroxy 18:0 and dihydroxy 18:1 long-chain bases in combination with nonhydroxy and hydroxy 16:0–24:0 fatty acids. Glycolipid studies of isolated mucosal epithelial cells and the nonepithelial intestinal residue revealed a specific cell distribution of these hexosyl compounds. The two major components, glucosylceramide and globotriaosylceramide, were mainly located in the epithelial cells together with small amounts of lactosylceramide and tetrahexosylceramide. The epithelial cells practically lacked however the penta- to octahexosylceramides. The nonepithelial residue contained all hexosyl compounds. The fucolipid was exclusively present in the epithelial cells.     

N-linked glycopeptides with blood group determinants lacking neuraminic acid from the epithelial cells of rat small and large intestine.

The N-linked type of glycans were prepared as their glycopeptides after pronase digestion of the epithelial cells from the small and large intestine of two inbred strains of rat. These glycopeptides were analysed for sugar composition, for blood-group activity, by 1H-NMR spectroscopy, and after permethylation by electron-impact mass spectrometry. The glycopeptides were of the triantennary and tetraantennary types with intersected GlcNAc. The terminal parts were, in contrast to most N-linked glycans, devoid of neuraminic acid residues. Instead they contained blood-group determinants. Blood-group-H types 1 (Fuc alpha 1-2Gal beta 1-3GlcNAc) and 2(Fuc alpha 1-2Gal beta 1-4GlcNAc) were found in the small and large intestines of both strains, although type-1 predominated. One rat strain (GOT-W) did not express blood-group-A glycopeptides in the small intestine, but the large intestine from the same strain did. The other strain (GOT-BW) expressed blood-group-A determinants in the small intestine. The lack of neuraminic acid residues in the small and large intestine and of blood-group-B activity in the large intestine differed from that found in glycosphingolipids obtained from the same organs.     

Immunochemistry of the Lewis-blood-group system: Proton nuclear magnetic resonance study of plasmatic Lewis-blood-group-active glycosphingolipids and related substances

The Le^a-, Le^b-, and H-type 1 (Le^dH)-blood-group-active glycosphingolipids, as well as H-I-type 2 glycolipid, lactotetraosyl ceramide, and neo-lactotetraosyl ceramide were examined by ¹H nuclear magnetic resonance at 360 MHz in dimethyl-d₆ sulfoxide as solvent. The resonances of almost all protons of the sugar rings were assigned with the aid of spin decoupling and nuclear Overhauser difference spectroscopy. The latter technique was also applied to establish the sequences and sites of glycosidic linkage. This information, combined with the chemical shift-structure correlations established in our previous work, led to an independent identification of those six glycolipids. Type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains can be distinguished by this approach. Some deviations from additivity in chemical shifts, calculated for oligosaccharides from the data on their constituent sugar residues, furnished information on the conformational changes in crowded glycolipid molecules.     

Tissue specificity of glycosphingolipids as expressed in pancreas and small intestine of blood group A and B human individuals

A chemical investigation has been done on blood group active glycosphingolipids of both small intestine and pancreas from two individuals, one blood group A and one blood group B. Total non-acid glycolipid fractions were prepared and the major blood group fucolipids present were purified and structurally characterized by mass spectrometry, proton NMR spectroscopy, and degradation methods. The glycolipid structures identified were a blood group Leb hexaglycosylceramide, a B-hexaglycosylceramide with a type 1 (Gal beta 1 leads to 3GlcNAc) carbohydrate chain, A-hexaglycosylceramides with types 1 and 2 (Gal beta 1 leads to 4GlcNAc) carbohydrate chains, a B-heptaglycosylceramide with a type 1 carbohydrate chain, and A-heptaglycosylceramides with type 1 and 2 carbohydrate chains. In addition several minor glycolipids having more than seven sugar residues were detected by thin-layer chromatography. The small intestine and pancreas had some distinct differences in their expression of the major fucolipids. The small intestine contained only glycolipids based upon type 1 carbohydrate chain while the pancreas had both type 1 and type 2 structures. The intestines contained mainly difucosyl compounds while the pancreas tissues contained both mono- and difucosyl glycolipids. Monofucosylglycolipids based on both types 1 and 2 saccharides were present in one pancreas while the other one contained only monofucosylcomponents based on type 1 chain. The ceramides of the intestinal glycolipids were found to be more hydroxylated (trihydroxy long-chain base, hydroxy fatty acids) compared to the pancreas glycolipids (dihydroxy long-chain base, non-hydroxy fatty acids).     

The common structure in fucosyllactosaminolipids accumulating in human adenocarcinomas,and its possible absence in normal tissue

Two major glycolipids accumulating in a human primary liver adenocarcinoma, but absent in normal liver, were characterized as lacto-N-fucopentaosyl(III)ceramide and difucosyllacto-N-$\text{nor}$-hexaosylceramide, (Galβ1→4[Fucα1→3]GlcNAcβ1→3Galβ1→4[Fucα1→3]GlcNAcβ1→3Galβ1→4Glcβ1→1Cer), a new type of glycolipid with Le^x-determinant. Comparison of glycolipids bearing Le^x-determinant in various cases of human colonic adenocarcinoma, in adjacent normal mucosa tissue, and in erythrocytes reveals a possibility that glycolipids accumulating in human adenocarcinoma, but not in normal tissue, have a common structural unit as identified below:

     

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Partial $\text{in}\text{vitro}$ sialylation of biantennary and triantennary glycopeptides of α₁-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution ¹H-NMR spectroscopic analysis of the isolated products enabled the assignment of the $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→3)}\text{Man}$ branch as the most preferred substrate site for sialic acid attachment. The $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→6)}\text{Man}$ branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal $\text{N}$-acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.     

Chemical structures of oligosaccharides in milk of the raccoon (<Emphasis Type="Italic">Procyon lotor</Emphasis>)

In this study on milk saccharides of the raccoon (Procyonidae: Carnivora), free lactose was found to be a minor constituent among a variety of neutral and acidic oligosaccharides, which predominated over lactose. The milk oligosaccharides were isolated from the carbohydrate fractions of each of four samples of raccoon milk and their chemical structures determined by ¹H-NMR and MALDI-TOF mass spectroscopies. The structures of the four neutral milk oligosaccharides were Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (lacto-N-fucopentaose IV), Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (fucosyl para lacto-N-neohexaose) and Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (difucosyl lacto-N-neohexaose). No type I oligosaccharides, which contain Gal(β1–3)GlcNAc units, were detected, but type 2 saccharides, which contain Gal(β1–4)GlcNAc units were present. The monosaccharide compositions of two of the acidic oligosaccharides were [Neu5Ac]₁[Hex]₆[HexNAc]₄[deoxy Hex]₂, while those of another two were [Neu5Ac]₁[Hex]₈[HexNAc]₆[deoxy Hex]_3. These acidic oligosaccharides contained α(2–3) or α(2–6) linked Neu5Ac, non reducing α(1–2) linked Fuc, poly N-acetyllactosamine (Gal(β1–4)GlcNAc) and reducing lactose.     

Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides

Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galβ1 ? 3GlcNAc (type 1) or Galβ1 ? 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)₃-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galβ1 ? 4GlcNAc(6S)β1 ? 3Galβ1 ? 4GlcNAc(6S)β1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galβ1 ? 3GlcNAcβ1 ? 3Galβ1 ? 4GlcNAcβ1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125–1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galβ1 ? 3GlcNAc(6S)β1 ? 3Galβ1 ? 4GlcNAc(6S)β1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galβ1 ? 3GlcNAcβ1 ? 3Galβ1 ? 4GlcNAcβ1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only “type 2-type 2 keratan sulfate” but also “type 1-type 2 keratan sulfate”, significantly.     

The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant)

One of the monoclonal (AH-6) antibodies prepared by hybridoma technique against human gastric cancer cell line MKN74 was found to react with a series of glycolipids having the Y determinant (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc). The structure of one such glycolipid isolated from human colonic cancer and from dog intestine was identified as lactodifucohexaosyl-ceramide (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide; IV3,III3Fuc2nLc4Cer). The hapten glycolipid did not react with monoclonal antibodies directed to Lea, Leb, and X-hapten structures, and the AH-6 antibody did not react with the X-hapten ceramide pentasaccharide (Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), H1 glycolipid (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), nor with glycolipids having the Leb (Fuc alpha 1 leads to 2Gal beta 1 leads to 3[Fuc alpha 1 leads 4]GlcNAc beta 1 leads to R) determinant. The antibody reacted with blood group O erythrocytes, but not with A erythrocytes. Immunostaining of thin layer chromatography with the monoclonal antibody AH-6 indicated that a series of glycolipids with the Y determinant is present in tumors and in O erythrocytes.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-acetyllactosamine-containing oligosaccharides,galectin ligands

The following spacered oligosaccharides were synthesized: GlcNAcβ1-3Galβ1-4GlcNAcβ-sp, GlcNAcβ1-6Galβ1-4GlcNAcβ-sp, GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, and Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp (sp = O(CH₂)₂NH₂). They represent N-acetyllactosamines substituted with N-acetylglycosamine or N-acetyllalctosamine residue at O3, O6, or at both positions of galactose. Glycosylation was achieved by coupling with N-trichloroethoxycarbonyl-protected glucosamine bromide in the presence of silver triflate.