Blood group A glycolipid antigen expression in kidney, ureter, kidney artery, and kidney vein from a blood group A1Le(a-b+) human individual. Evidence for a novel blood group A heptaglycosylceramide based on a type 3 carbohydrate chain

Kidney, ureter, kidney artery, and kidney vein tissue were obtained from a single human transplant specimen. The donors erythrocyte blood group phenotype was A1Le(a-b+). Total non-acid glycolipid fractions were isolated and individual glycolipid components were identified by immunostaining thin layer plates with a panel of monoclonal antibodies and by mass spectrometry of the permethylated and permethylated-reduced total glycolipid fractions. The dominating glycolipids in all tissues were mono- to tetraglycosylceramides. In the kidney, ureter, and artery tissue less than 1% of the glycolipids were of blood group type, having more than 4 sugar residues. In contrast, 14% of the vein glycolipids were of blood group type, and the dominating components were type 1 chain blood group H pentaglycosylceramides and A hexaglycosylceramides. Trace amounts of structurally different blood group A glycolipids (type 1 to 4 core saccharide chains) with up to 10 sugar residues were found in the kidney, ureter, and vein tissues, including evidence for a novel blood group A heptaglycosylceramide based on the type 3 chain in the vein. The only detected A glycolipid antigen in the artery tissue was the blood group A difucosyl type 1 chain heptaglycosylceramide (ALeb) structure. Blood group Lewis and related antigens (Lea, Leb, and ALeb) were expressed in the kidney, ureter, and artery, but were completely lacking in the vein, indicating that the Le gene-coded alpha 1-4-fucosyltransferase was not expressed in this tissue. The X and Y antigens (type 2 chain isomers of the Lea and Leb antigens) were detected only in the kidney tissue.     

Histochemical localization and analysis of blood group-related antigens in human pancreas using immunostaining with monoclonal antibodies and exoglycosidase digestion

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.     

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.     

Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.     

Extended type 1 chain glycosphingolipids: dimeric Lea (III4V4Fuc2Lc6) as human tumor-associated antigen

Glycolipid extracts from various human cancer tissues and cell lines showed the presence of a slow-migrating glycolipid component which was strongly reactive with monoclonal antibody (mAb) NCC-ST-421 (raised against human gastric adenocarcinoma) and weakly cross-reactive with anti-Lea mAbs. The slow-migrating glycolipid was isolated from human colonic adenocarcinoma cell line Colo205 grown in nude mice, and was purified by high-performance liquid chromatography followed by preparative thin-layer chromatography. Its structure was elucidated by sequential enzymatic degradation and thin-layer chromatography immunostaining of the degradation products with various mAbs, 1H NMR spectroscopy, positive-ion fast atom bombardment mass spectrometry, and methylation analysis. The major slow-migrating component reacting with mAb ST-421 was identified as dimeric Lea, with the structure as follows. [formula: see text] Antigens containing this structure and various analogous structures (including enzymatically synthesized Lea/Lex hybrid antigen) were tested with ST-421. While the mAb was equally reactive with dimeric Lea and Lea/Lex, only the former was chemically detectable as the slow-migrating glycolipid from the tumor extract. ST-421 showed less reactivity with simple Lea (III4FucLc4) or extended Lea (V4FucLc6, and/or IV3Gal beta 1----3[Fuc alpha 1----4]GlcNAcnLc4), and was not reactive with Lex/Lex (dimeric Lex). It was concluded, therefore, that the major tumor-associated slow-migrating glycolipid reacting with ST-421 has the dimeric Lea structure shown above. Since extension of lacto-series structure has been shown to be limited to type 2 chain in normal cells and tissues, extended elongation of type 1 chain as shown in this structure represents a novel tumor-associated epitope.     

Molecular mechanisms of expression of Lewis b antigen and other type I Lewis antigens in human colorectal cancer.

Lewis b (Leb) antigens are gradiently expressed from the proximal to the distal colon, i.e., they are abundantly expressed in the proximal colon, but only faintly in the distal colon. In the distal colon, they begin to increase at the adenoma stage of cancer development and then increase with cancer progression. We aimed to clarify the molecular basis of Leb antigen expression in correlation with the expression of other type I Lewis antigens, such as Lewis a (Lea) and sialylated Lewis a (sLea), in colon cancer cells. Considering the Se genotype and the relative activities of the H and Se enzymes, the amounts of Leb antigens were proved to be determined by both the H and Se enzymes in noncancerous and cancerous colon tissues. But the Se enzyme made a much greater contribution to determining the Lebamounts than the H enzyme. In noncancerous colons, the Se enzyme were gradiently expressed in good correlation with the Leb expression, while the H enzyme was constantly expressed throughout the whole colon. In distal colon cancers, the H and Se enzymes were both significantly upregulated in comparison with in adjacent noncancerous tissues. In proximal colon cancers, expression of the H enzyme alone was highly augmented. The augmented expression of Leb antigens in distal colon cancers is caused mainly by upregulation of the Se enzyme and partly by the H enzymes, while it is caused by upregulation of the H enzyme alone in proximal colon cancers. The Se gene dosage profoundly influences the amounts of the Leb, Lea, and sLea antigens in whole colon tissues, regardless of whether they are noncancerous or cancerous tissues. It suggests that the Se enzyme competes with alpha2,3 sialyltransferase(s) and the Le enzyme for the type I acceptor substrates.     

Glycosylation-dependent interactions of C-type lectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells

Dendritic cells (DCs) are APCs that play an essential role by bridging innate and adaptive immunity. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is one of the major C-type lectins expressed on DCs and exhibits high affinity for nonsialylated Lewis (Le) glycans. Recently, we reported the characterization of oligosaccharide ligands expressed on SW1116, a typical human colorectal carcinoma recognized by mannan-binding protein, which is a serum C-type lectin and has similar carbohydrate-recognition specificities as DC-SIGN. These tumor-specific oligosaccharide ligands were shown to comprise clusters of tandem repeats of Lea/Leb epitopes. In this study, we show that DC-SIGN is involved in the interaction of DCs with SW1116 cells through the recognition of aberrantly glycosylated forms of Lea/Leb glycans on carcinoembryonic Ag (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1). DC-SIGN ligands containing Lea/Leb glycans are also highly expressed on primary cancer colon epithelia but not on normal colon epithelia, and DC-SIGN is suggested to be involved in the association between DCs and colorectal cancer cells in situ by DC-SIGN recognizing these cancer-related Le glycan ligands. Furthermore, when monocyte-derived DCs (MoDCs) were cocultured with SW1116 cells, LPS-induced immunosuppressive cytokines such as IL-6 and IL-10 were increased. The effects were significantly suppressed by blocking Abs against DC-SIGN. Strikingly, LPS-induced MoDC maturation was inhibited by supernatants of cocultures with SW1116 cells. Our findings imply that colorectal carcinomas affecting DC function and differentiation through interactions between DC-SIGN and colorectal tumor-associated Le glycans may induce generalized failure of a host to mount an effective antitumor response.     

Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities

Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP-[14C]fucose as catalyzed by cell-free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum-free medium. Purified lactotetraosyl ceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or IV2 Fuc alpha LcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4 Fuc alpha LcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP-fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H-1 fucosylation migrated as discrete and distinct bands on high-performance thin-layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fuc alpha 1----2Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4IV2 (Fuc alpha) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co-migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H-1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme-limiting conditions, we obtained a 12-19-fold purification of the LcOse4Cer and H-1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH-cytochrome-c reductase and UDP-GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different (p less than 0.01) by statistical tests, 2.4 microM and 0.5 microM, respectively. These apparent Km values were much lower (10(3) X) and the pH optima were lower (4.8-5.3), than the corresponding properties reported for the alpha 1----3/alpha 1----4 fucosyl transferase purified from human milk. Our results suggest a role for the non-glycosidic moieties of the acceptors and/or the tissue-specific or primitive expression of these fucosyl transferase activities.     

Isolation and purification of Lea blood-group active and related glycolipids from human plasma of blood-group A Lea individuals

From 8 1 of human plasma of blood-group A Le^a nonsecretors three different Le^a blood-group active ceramide pentasaccharides (a total of 4.65 mg) have been isolated, all revealing glucose, galactose, N-acetylglucosamine and fucose in molar ratios of 1 : 2 : 1 : 1 as determined by gas liquid chromatography. A fourth blood-group active fraction (0.72 mg) represents a mixture of a Le^a active ceramide pentasaccharide and an A active ceramide hexasaccharide (molar ratio 7.7 : 2.3 as calculated from the content of different aminosugars). Additionally, two different globosides, two different hematosides and a new N-acetylglucosamine containing ceramide tetrasaccharide were obtained. All 9 glycolipid fractions demonstrated homogeneity in analytical high performance thin layer chromatography (HPTLC) using 4 different solvent systems. 0.2 μg of each Le^a active glycolipid completely inhibited the agglutination of O Le(a + b ?) erythrocytes by 50 μl of 4 hemagglutinating units of caprine anti Le^a serum. At least 0.04 μg of each Le^a antigen are sufficient for incubation to convert 9 × 10⁷ O Le(a?b?) erythrocytes into Le^a-positive cells. Mainly due to the relatively low content of the blood-group A glycolipid in plasma (0.17 mg/8 1), previously negative erythrocytes readily become agglutinable by anti Le^a sera and not by anti A sera after incubation with appropriate plasma.     

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.     

Lewis blood group antigens defined by monoclonal anti-colon carcinoma antibodies

Monoclonal antibodies directed against human cancer cells were prepared by the murine hybridoma technique. These antibodies detect Lewis blood group antigens as determined by indirect solid-phase radioimmunoassay, hapten inhibition studies, and chromatogram binding assay. One monoclonal antibody is specific for the Lea terminal carbohydrate of Gal beta 1----3Glc NAc(4----1 alpha Fuc) beta 1----3LacCer. Five monoclonal antibodies react with the Leb terminal carbohydrate sequence of Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer, and four of these antibodies are highly specific for this glycolipid and do not react with other similar di- and monofucosylated glycolipids. One of the anti-Leb antibodies cross-reacts with blood group H glycolipid and has binding properties similar to those of the previously described antibody NS-10-17 [M. Brockhaus, J. L. Magnani, M. Blaszczyk, Z. Steplewski, H. Koprowski, K.-A. Karlsson, G. Larson, and V. Ginsburg (1981) J. Biol. Chem. 256, 13223-13225]. Two antibodies react with both the Lea and Leb antigens, though both bind preferentially to Leb.     

Expression of Lewis histo-blood group glycolipids in the plasma of individuals of Le(a+b+) and partial secretor phenotypes

Red cell Lewis antigens are carried by glycosphingolipids passively absorbed from plasma. Plasma was collected from a spectrum of individuals with normal and unusual Lewis/secretor phenotypes in order to investigate the glycolipid basis for the unusual phenotypes. Samples were obtained from: a Le(a+b–) ABH nonsecretor who secreted Lewis substances; a Le(a+b–) partial secretor; Le(a+b+) partial secretors; Le(a+b+) secretors; and a full range of normal Lewis/secretor phenotypes as controls. The Le(a+b+) samples represented Polynesian, Asian and Réunion Island ethnic backgrounds. Nonacid glycolipids were prepared, separated by thin-layer chromatography, and then immunostained with potent monoclonal antibodies of known specificity. Despite different serological profiles of the Le(a+b–) and Le(a+b+) Polynesian samples, their plasma glycolipid expressions were very similar, with both Le^a and Le^b co-expressed. The copresence of Le^a and Le^b in Le(a+b+) samples is in marked contrast to Caucasians with normal Lewis phenotypes, who have predominantly either Le^a or Le^b. These results suggest that there is a range of the secretor transferases in different individuals, possibly due to different penetrance or to several weak variants. We also show that Lewis epitopes on longer and/or more complex core chains appear to be predominant in the Polynesian Le(a+b+) samples. The formation of these extended glycolipids is compatible with the concept that in the presence of reduced secretor fucosyltransferase activity, increased elongation of the precursor chain occurs, which supports the postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.Abbreviations CBA chromatogram binding assay - TLC thin-layer chromatography     

Blood group A determinants with mono- and difucosyl type 1 chain in human erythrocyte membranes

Application of a monoclonal antibody defining monofucosyl type 1 chain A (AH21) revealed the presence of a glycolipid having the same thin-layer chromatography mobility as Aa but showing a clear reactivity with AH21. This glycolipid was detectable in Lea-b- erythrocytes but not in Lea+b- or Lea-b+ erythrocytes. Another monoclonal antibody defining difucosyl type 1 chain A (HH3) detected the presence of a glycolipid component reacting with this antibody in Lea-b+ erythrocytes but not in Lea+b- or Lea-b- erythrocytes. The component defined by monoclonal antibody AH21 and that defined by HH3 were isolated and characterized by 1H NMR spectrometry and methylation analysis as having the structures (Formula: see text) The 1H NMR spectra of these glycolipid antigens were characterized by resonances for anomeric protons that are identical with those of glycolipids with type 1 chain previously isolated but distinctively different from those of type 2 chain analogues. Resonances reflecting ceramide composition are characteristic for these antigens from human erythrocytes and are distinguishable from those of the same antigen from other sources.     

Glycolipids of human large intestine: difference in glycolipid expression related to anatomical localization, epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes of the donors

Human large intestine specimens were obtained during elective surgery from donors of known blood group ABO, Lewis and secretor phenotypes. The intestinal epithelial cells were isolated from the non-epithelial tissue in one case and in another case mucosa tissue was obtained by scraping. Total non-acid glycolipid and ganglioside fractions were isolated from the tissue specimens, analyzed by thin-layer chromatography and detected by chemical reagents and autoradiography after staining the plate with various blood group monoclonal antibodies and bacterial toxins. The amount of non-acid glycolipids present in the large intestine epithelial cells was 3.9 micrograms/mg of cell protein and in the non-epithelial tissue 0.39 mg/g dry tissue weight. The epithelial cells contained monoglycosylceramides and blood group Lea pentaglycosylceramides as major compounds together with small amounts of diglycosylceramides. In addition, trace amounts of tri- and tetra-glycosylceramides together with more complex glycolipids were present. The non-epithelial tissue contained mono-, di-, tri- and tetra-glycosylceramides as major non-acid components. Blood group ABH glycolipids were present in trace amounts in the non-epithelial part of the large intestine. Lea pentaglycosylceramide was the major blood group glycolipid present in all Le-positive individuals independent of the secretor status. Leb glycolipids were present in trace amounts in secretor individuals but completely lacking in non-secretors. Trace amounts of X antigens were found in all individuals, while Y antigens were only present in secretor individuals. The Lea, Leb, X and Y glycolipids were located in the epithelial cells. The gangliosides were present mainly in the non-epithelial tissue (65-350 nmol of sialic acid/g dry weight) and only trace amounts (less than 0.014 nmol/mg of cell protein) were found in the epithelial cells. The major gangliosides of the non-epithelial tissue were identified as GM3, GM1, GD3, GD1b, GT1b and GQ1b. In addition, several minor gangliosides were also present. Binding of cholera toxin to the thin-layer plate revealed trace amounts of the GM1 ganglioside in the epithelial cell ganglioside fraction.     

Expression of Lewis antigenic determinants in colorectal adenocarcinomas

Expression of type 1 and type 2 chain Lewis antigens was studied in 32 rectal adenocarcinoma specimens; the results were correlated with the patients' Lewis phenotype and secretor status. In addition, the pattern of expression of these antigens was analyzed in adjacent and distant normal mucosa. We used an indirect immunofluorescence technique with p-phenylenediamine counterstaining (Oriol technique) and a panel of monoclonal antibodies directed against the different antigenic specificities. Normal distal colonic mucosa only expresses monofucosylated structures (Lea and X) arising from activity of the alpha 1-3,4-fucosyltransferase coded by the Le gene. Rectal adenocarcinomas also show Lea and X, but also reexpress blood group antigens ABH and exhibit difucosylated determinants (Leb and Y). The accumulation of mono- and difucosylated type 2 chain in neoplastic processes, independently of the Le and Se genes, could be due to the enzymes coded by reactivation of the H and X genes. Blood group antigens form a complex signal code, genetically regulated, which intervenes in differentiation, growth and cellular recognition processes, and which may undergo important modifications during malignant transformation. These alterations could be useful in the diagnosis and prognosis of some types of carcinoma.     

Action of glycosyl transferases upon "Bombay" (Oh) erythrocytes. Conversion to cells showing blood-group H and A specificities.

Individuals of the rare "Bombay" (Oh) blood-group phenotype lacking, due to a genetic defect, the alpha(1-2)fucosyl transferase, which is responsible for converting blood-group H precursor substances to H-specific structures. Treatment with GDP-fucose and alpha(1-2)fucosyl transferase prepared from gastric mucosa of O individuals to transform native or ficin-treated "Bombay" erythrocytes into cells phenotypically resembling O cells. The transformation was achieved, however, after prior incubation of the "Bombay" erythrocytes with neuraminidase, indicating that blood-group H precursor molecules on the surface of these cells are masked by sialyl residues. Blood-group A specificity was conferred upon neuraminidase-treated "Bombay" cells by enzymatic transfer of alpha-N-acetylgalactosamine residues, in addition to alpha-fucose residues.     

Scope and limitations of rotating-frame nuclear Overhauser enhancement spectroscopy applied to oligosaccharides

Two-dimensional rotating-frame nuclear Overhauser enhancement spectroscopy has been applied to milk oligosaccharides containing the blood-group determinants Lea, Led, or Leb. The results indicate a large number of dipolar interactions that are consistent with the preferred conformations described for these blood-group determinants. Despite careful experimental conditions, coherent transfer processes are not suppressed completely, allowing only a qualitative evaluation of the nuclear Overhauser data. The 1H- and 13C-n.m.r. spectra are assigned by comparison with literature data and by application of two-dimensional 13C-1H shift-correlation spectroscopy.     

Structural and immunochemical identification of Lea, Leb, H type 1, and related glycolipids in small intestinal mucosa of a group O Le(a-b-) nonsecretor

Total nonacid glycosphingolipids were isolated from small intestine mucosal scrapings of a red cell blood group O Le(a-b-) nonsecretor cadaver. Glycolipids were extracted and fractionated into five fractions based on chromatographic and immunostaining properties. These glycolipid fractions were then analysed by thin-layer chromatography for Lewis activity with antibodies reactive to the type 1 precursor (Lec), H type 1 (Led), Lea and Leb epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and EI-MS/MS-TOF) and proton NMR spectroscopy. EI-MS/MS-TOF allowed for the identification of trace substances in fractions containing several other glycolipid species. Consistent with the red cell phenotype, large amounts of lactotetraosylceramide (Lec-4) were detected. Inconsistent with the red cell phenotype, small quantities of Lea-5, H-5-1 and Leb-6 glycolipids were immunochemically and structurally identified in the small intestine of this individual. By EI-MS/MS-TOF several large glycolipids with 9 and 10 sugar residues were also identified. The extensive carbohydrate chain elongation seen in this individual with a Lewis negative nonsecretor phenotype supports the concept that Lewis and Secretor blood group fucosylation may be a mechanism to control type 1 glycoconjugate chain extension. Abbreviations: FUT1, H gene; FUT2, Secretor gene, (gene bank accession no. U17894); FUT3, Lewis gene or Fuc-TIII gene, (gene bank accession no. X53578); FUT5, Fuc-TV gene; [Imm]+, immonium ion; Lea-5, Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Leb-6, Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Lec-4, Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Led or H-5-1, Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Lex-5, Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; MAb, monoclonal antibody; MS, mass spectrometry; CID, collision-induced dissociation; EI, electron impact ionisation; MS/MS-TOF, tandem mass spectrometry using a time-of-flight mass spectrometer as the second mass spectrometer: m/Cz, mass-to-charge ratio; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; TLC, (high performance) thin layer chromatography. Saccharide types are abbreviated to Hex for hexose, HexNAc for N-acetylhexosamine and dHex for deoxyhexose (fucose). Ceramide is abbreviated to Cer, and ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids This revised version was published online in November 2006 with corrections to the Cover Date.     

Lea-active heptaglycosylceramide, a hybrid of type 1 and type 2 chain, and the pattern of glycolipids with Lea, Leb, X (Lex), and Y (Ley) determinants in human blood cell membranes (ghosts). Evidence that type 2 chain can elongate repetitively but type 1 chain cannot

Two major glycolipids reactive with the monoclonal anti-Lea antibody have been isolated from human blood cell membranes. One component was identified as lactofucopentaosyl(II)ceramide and the other as a ceramide heptassaccharide with the structure described below: (formula; see text) The structure includes the Lea determinant (type 1 chain) linked to lactoneotetraosylceramide (type 2 chain); thus, it is regarded to be a hybrid between type 1 and 2 chain. In addition, a minor component having the thin-layer chromatographic mobility of a ceramide nonasaccharide, which was reactive to anti-Lea antibody, was detected. No other component with a thin-layer chromatographic mobility slower than the above components and reactive to the anti-Lea antibody was detected. In contrast, a series of slowly migrating glycolipids having X (Lex) determinant (Gal beta 1----4(Fuc alpha 1----3)GlcNAc) was detected. A similar series of long chain glycolipids having Y (Ley) determinant (Fuc alpha 1----2Gal beta 1----4(Fuc1----3)GlcNAc) was detected in human blood cells; in contrast, only one major Leb glycolipid was found with the mobility of a ceramide hexasaccharide. No glycolipid with a long carbohydrate chain composed exclusively of type 1 chain was detected. Thus, chain elongation may proceed through type 2 chain, but not through type 1 chain. Lea and X (Lex) haptens are distributed equally among blood group A, B, and O red blood cells, whereas the quantity of Leb and Y (Ley) haptens is much lower in A and B blood cells than in O blood cells.     

The Lewis blood group system among Chinese in Taiwan.

The nonsecretor gene se is absent (or very rare) among Chinese in Taiwan and the previously reported Le(a+b-) phenotype in this population is in fact Le(a+b+) as proven by the presence of small amounts of Leb antigen on red blood cells. Salivary ABH substances in this phenotype are usually (although not always) markedly reduced. The Chinese Le(a+b+) phenotype is postulated to be the result of a weak secretor gene Se omega. Although the Le(a+b+) phenotype is very rare in Caucasians, it has a frequency of 25% in Chinese. All Le(a-b-) Chinese are ABH secretors and have varying amounts of Lea and/or Leb substances in saliva.