A study of human-type ABH antigens on erythrocytes and in saliva of sheep

The results of serological investigations of human type ABH antigens and antibodies of 116 sheep are presented. Traces of ABH antigens on sheep erythrocytes are recognized by elution. Agglutinins with anti-AA1, anti-B and sometimes anti-H specificity besides of heteroagglutinins were differentiated by cross-absorption experiments. It was established that the sheep saliva also contains H antigens and sometimes A and/or A1 antigens. On the basis of their serological characteristics the sheep divided into 11 serological groups. In order to explain some serological peculiarities in sheep the existence of genes for regulation, the production of ABH antigens in glucolipid form and genes for regulation of the same antigens in glucoprotein form were postulated.     

Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-blood group family

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.     

Semiquantitative immunohistochemical studies of blood group antigen A,B,H, Lea, Leb structures and Ii backbone chains in the normal human cervix and in cervical adenocarcinoma

Summary Epithelia frequently express blood group antigens and these are often perturbed in neoplasia. This study has characterized the range of expression of ABH and Lewis terminal structures and the Ii backbone chains in the normal human cervix by semiquantitative immunohistochemistry. Effects of the secretor gene were defined by determination of salivary secretor status. Modifications of blood group antigen expression in cervical adenocarcinoma were also addressed.Normal cervical squamous and glandular epithelia showed a range of expression of the antigens studied. Lewis-gene-negative cases showed no expression of Lewis antigens. Secretor status had no effect on ABH expression in squamous epithelium, but it did have a marked effect on ABH expression in glands and on Le^b expression in both squamous and glandular epithelia. Patterns of expression of i chains in squamous epithelium suggest that these may be the carriers of ABH and Lewis antigens in a proportion of cases. Distinct patterns of expression were seen in glandular tubal metaplasia and in endothelium.Adenocarcinomas showed topographical rather than quantitative changes in blood group antigen expression with more extensive luminal expression of ABH, Lewis and Ii structures than that seen in normal glands. This change is distinct from those usually associated with malignancy.     

Serological relationships among antigens of the BoLA and the bovine M blood group systems

Alloimmunizations with either lymphocytes or red cells from donor cows positive for BoLA w16 and blood group M' antigens into recipients negative for these antigens produced antisera reactive in the cytotoxic test with w16-positive lymphocytes and in the haemolytic test with M'-positive erythrocytes. Similarly, alloimmunizations of blood group M1-negative recipients with either lymphocytes or red cells from donor cows possessing the M1 blood group factor produced antisera specifically reactive with lymphocytes and erythrocytes from M1-positive cattle. Absorptions with either lymphocytes or erythrocytes from individual animals of the same M antigenic type as the donor removed all haemolytic and cytotoxic reactivity. The results indicate that blood group M' and BoLA w16 share a similar antigenic structure. Likewise, blood group M1 has an antigenically similar counterpart which is also part of the BoLA system.     

Norwalk virus-like particles bind specifically to A,H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids

Noroviruses and norovirus virus-like particles (VLPs) exhibit strain specific patterns in their binding to ABH and Lewis histo-blood group antigens. In this study we demonstrate for the first time specific binding of Norwalk virus VLPs to type 1 and type 2 chain glycosphingolipids (GSLs) carrying ABH and Lewis antigens. N-succinimidyl-3-tributylstannyl benzoate (ATE) was precursor labeled with ¹²⁵I and then conjugated to VLPs. The ¹²⁵I-VLPs were used in GSL thin-layer chromatogram binding assays and displayed binding to H type 1, Lewis b, A type 1, A Lewis b GSLs but no binding to B type 1 or B Lewis b GSLs. For the type 2 chain GSLs the Norwalk VLPs bound to H type 2, Lewis y, A type 2 and A Lewis y. In addition, the VLPs bound to several complex GSLs from blood group O and A, but not from blood group B red blood cells.     

Enzymatic synthesis of blood-group Lewis-specific glycolipids.

A blood-group Lewis precursor glycolipid was isolated from the plasma of a Lewis-negative individual [Le(a--b--)] and treated with fucosyltransferases from human gastric mucosa and GDP-fucose. Subsequently the glycolipid was adsorbed onto Le(a--b--) erythrocytes and the presence of blood-group Lewis antigens was assessed by passive hemagglutination with anti-Lewis sera. It was shown that the precursor glycolipid was enzymatically transformed to blood-group Lewis a (Lea) and Lewis b (Leb) specific glycolipids. Leb-glycolipid was also synthesized by fucosylation of an isolated Lea-glycolipid. Moreover Le(a--b--) erythrocytes were shown to develop Lea and Leb activities when subjected to enzymatic fucosylation, thus showing that Lewis-negative cells carry blood-group Lewis precursor glycolipid on the surface of their membrane. Le(a + b--) erthrocytes, upon enzymatic fucosylation, acquired Leb activity.     

The biochemical aspects of blood group antigens]

The biochemical aspects of the immunodominant structures of blood groups antigens are mainly restricted to the following: ABH and Lewis in secretory fluids or on the red blood cells; P system (P1, P, Pk antigens); MN antigens and related; Tn and Tn antigens; Some hypothesis may be put forward for the I, i antigens. Many other antigens seem to be on the dependence of interactions between proteins and lipids of the red cell membrane; such immunodominant structures are not yet known. Except for the ABH and Lewis groups, the biosynthesis pathways are at present unclear.     

H-deficient blood groups ( Bombay) of Reunion Island.

Forty-two H-deficient individuals (lacking H antigen on erythrocytes) with anti-H in their sera were found on Reunion Island. A, B, and AB Bombay subjects had small but detectable amounts of A and/or B antigens on erythrocytes. All the H-deficient phenotypes tested were nonsecretors of ABH in their saliva, and one-third were Lewis negative. Fifty-three of the 108 (49%) unaffected members in the 14 Bombay pedigrees analyzed were se/se, showing that the families were selected for the nonsecretor trait, and suggesting that the Bombay probands used to select the families have se/se genotype. In accordance with this concept, all the children from Bombay nonsecretor x unaffected nonsecretor matings were se/se. Segregation of H and Se is compatible with the genetic model proposing that Se and H are closely linked structural genes, and the analysis of the present and previously published Bombay pedigrees strongly supports this model.     

A new anti-H lectin from the seeds ofGalactia tenuiflora

A new anti-blood group H lectin was isolated from the seeds ofGalactia tenuiflora. This lectin is mostly specific for the H type 2 trisaccharide but it shows some cross-reactivity with the H type 4 and H type 3 trisaccharides. Differences between this lectin and lectin 1 fromUlex europaeus are described. These differences concern the respective abilities of the lectins to recognize erythrocytes from some H deficient phenotypes, the inhibitions by salivas and the tissue distribution of the antigens recognized by the two lectins. The most important differences were noted in the surface epithelium of the stomach. This area is known to express ABH antigens under the control of theSe gene as defined by theUlex europaeus lectin 1, yet it is always strongly labelled by theGalactia tenuiflora lectin irrespective of the secretor status of the tissue donor.     

Subcellular localization of blood group substances ABH in human salivary glands

We investigated the subcellular localization of ABH antigens in human submandibular, sublingual, and buccal glands by applying a post-embedding immunogold method using monoclonal antibodies specific for A, B, and H antigens. In most glands the immunoreactivity was usually restricted to mucous cells, in which only secretory granules and sometimes Golgi cisternae were specifically labeled. A and B antigens were demonstrated only in the glands of type A, B, and AB subjects, while H antigen was visualized in glands from individuals of all blood types. Moreover, differences were observed in the relative distribution of ABH antigens, depending on the type of gland.     

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.     

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.     

Immunodetection of Helicobacter sp. and the associated expression of ABO blood group antigens in the gastric mucosa of captive and free-living New World primates in the Amazon region

The histo-blood group ABH antigens were first described in humans. These antigens are only present on erythrocytes from great apes and humans, while in more primitive animals they are found in tissues and body fluids. The ABH antigens are mainly distributed in tissues exposed to the external environment and potentially serve as ligands for pathogens or inhibitors of tissue connections. The objective of this paper was two-fold: (i) to determine the presence of Helicobacter sp. in the gastric mucosa of 16 captive and 24 free-living New World monkeys and (ii) to evaluate the presence of histopathological alterations related to bacterial infection and the associated expression of ABH antigens in the tissue. Stomach tissues from 13 species of monkey were assessed using haematoxylin-eosin and modified Gram staining (Hucker) methods. An immunohistochemical analysis of the tissue revealed the presence of infectious bacteria that were characteristic of the genus Helicobacter sp. The results demonstrate that various species of monkey might be naturally infected with the Helicobacter sp. and that there is an increased susceptibility to infection. This study serves as a comparative analysis of infection between human and non-human primates and indicates the presence of a new species of Helicobacter.     

Expression of ABH and X (Lex) antigens in various cells

Using a panel of reagents specific to the various subtypes of ABH antigens, it could be demonstrated that platelets carry ABH type 2 monofucosylated determinants on intrinsic glycoproteins. The presence of these antigens is controlled by the H gene and correlates with the presence of alpha-2-L-fucosyltransferase and the absence of alpha-3-L-fucosyltransferase. In contrast, intrinsic ABH antigens were not found on mononuclear cells, correlating with the absence of alpha-2-L-fucosyltransferase on these cells. However, after transformation with the Epstein-Barr virus and stimulation with 12-O-tetradecanoylphorbol-13-O-acetate (TPA), B lymphocytes were found to express the H antigen under control of the H gene and not the Se gene. The lymphoblastoid cell lines also expressed the X and sialylated X antigens which are normally markers of the myeloid lineage. These antigens are also normally found in epithelial cells of the digestive tract, kidney proximal convoluted tubules and hepatocytes. The alpha-3-L-fucosyltransferase responsible for the synthesis of this antigen is present in the serum but we report the existence of two individuals, a mother and her daughter, who lack more than 90% of this serum enzyme. The young girl suffers from a congenital kidney anomaly: oligomeganephronic hypoplasia. Her kidney tubules are devoid of X antigen. However, she and her mother have the X antigen on their granulocytes and its sialylated form on their monocytes. It therefore appears that there are distinct genetic controls for the expression of antigen X in different body compartments. This would be quite similar to the H and Se gene controls in tissues of distinct embryological origins.     

Aberrant expression of histo-blood group A type 3 antigens in vascular endothelial cells in inflammatory sites.

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1-4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.     

Blood group antigen expression is involved in <Emphasis Type="Italic">C. albicans</Emphasis> interaction with buccal epithelial cells

Human blood group polymorphisms are known to be determined by the expression of A, B or H antigens and the Lewis antigens. Protection against microbial infections has been associated with inheritance of polymorphisms in genes encoding and regulating the expression of ABH and Lewis antigens in bodily secretions and epithelial tissue surfaces, subsequently resulting in the presentation of different glycosylated terminal antigens on the cell surface. We investigated the role of blood group antigens in diversifying the glycosylation of buccal epithelial cells (BEC) that line the oral cavity. Specifically, we characterized and statistically evaluated the expression of histo-blood group (A, B, O) antigens on N-and O-linked glycans from BEC membrane proteins of various individuals that represented different blood group type and secretor status using a porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry (PGC-LC-ESI-MS) based glycomics approach. From these BEC membrane proteins a total of 77 N-glycan and 96 O-glycan structures were structurally characterized from 19 individuals and relatively quantitated. The N-glycans from the secretor individuals did not express any A/B blood group determinants, but contained several terminal H-antigens. Apart from the non-secretors, the N-glycan profiles of BEC from all blood groups displayed similar glycan types, while varying in their relative intensities between individuals. However, multivariate analysis of the O-glycans from individuals displayed segregation patterns clearly associated with their blood group type and secretor status. In adhesion assays the oral pathogen Candida albicans showed a significantly higher interaction to blood group O type BECs relative to other blood groups.     

Role of ABO secretor status in mucosal innate immunity and H. pylori infection

The fucosylated ABH antigens, which constitute the molecular basis for the ABO blood group system, are also expressed in salivary secretions and gastrointestinal epithelia in individuals of positive secretor status; however, the biological function of the ABO blood group system is unknown. Gastric mucosa biopsies of 41 Rhesus monkeys originating from Southern Asia were analyzed by immunohistochemistry. A majority of these animals were found to be of blood group B and weak-secretor phenotype (i.e., expressing both Lewis a and Lewis b antigens), which are also common in South Asian human populations. A selected group of ten monkeys was inoculated with Helicobacter pylori and studied for changes in gastric mucosal glycosylation during a 10-month period. We observed a loss in mucosal fucosylation and concurrent induction and time-dependent dynamics in gastric mucosal sialylation (carbohydrate marker of inflammation), which affect H. pylori adhesion targets and thus modulate host-bacterial interactions. Of particular relevance, gastric mucosal density of H. pylori, gastritis, and sialylation were all higher in secretor individuals compared to weak-secretors, the latter being apparently "protected." These results demonstrate that the secretor status plays an intrinsic role in resistance to H. pylori infection and suggest that the fucosylated secretor ABH antigens constitute interactive members of the human and primate mucosal innate immune system.     

Molecular behavior of mutant Lewis enzymes in vivo

The expression of type-1 Lewis antigens on erythrocytes and in digestive organs is determined by a Lewis type alpha(1,3/1, 4)-fucosyltransferase (Lewis enzyme) encoded by the Fuc-TIII gene ( FUT3 gene; Lewis gene). We have classified the Lewis alleles in the Japanese population into four types, the wild-type allele ( Le ) and three mutated alleles, i.e., le1, which has missense mutations T59G and G508A, le2, which has T59G and T1067A, and le3, which has only T59G. Here we carried out an extensive study on the biological properties of the three mutant Lewis enzymes, the le1, le2, and le3 enzymes, using native tissues and obtained the following results. (1) In in vivo and in vitro experiments, the le1 and le2 enzymes were found to be susceptible to protease digestion probably because the one missense mutation in the catalytic domains, i.e., Gly170 to Ser in the le1 enzyme and Ile356 to Lys in the le2 enzyme, makes the three-dimensional structures of the enzymesunstable, while the le3 and wild-type Lewis enzymes wereresistant to protease digestion. (2) The le1 and le2 enzymes cannot synthesize type 1 Lewis antigens on either glycolipids or mucins. The le3 enzyme cannot synthesize Lewis-active glycolipids, which result in the Lewis antigen-negative phenotype of erythrocytes, while it can synthesize Lewis antigens on mucins in normal and cancerous colon tissues. The missense mutation, Leu20 to Arg, in the transmembrane domain reduces retention of the le3 enzyme in the Golgi membrane resulting in an apparent reduction of enzyme activity as revealed by the lack of Lewis antigen synthesis. (3) The Lewis gene dosage actually has effects in vivo on the amount of the Lewis enzyme, its activity, and finally the amounts of Lewis carbohydrate antigens. This is the first article that clearly demonstrates the gene dosage effects on the amount of the glycosyltransferase protein, its activity, and the amounts of carbohydrate products in vivo.     

Role of ABH blood group antigens in the stimulation of a DIDS-sensitive Ca2+ influx pathway in human erythrocytes by Ulex europaeus agglutinin I and a monoclonal anti A1 antibody

Of eleven agglutinating lectins tested, only one, Ulex europaeus agglutinin I (UEA1), stimulated Ca2+ uptake in quin2-loaded erythrocytes by about 2-fold. UEA1 is known to be an alpha-L-fucose and ABH blood group specific lectin. The 45Ca2+ influx induced by UEA1 was absent in the presence of extracellular fucose (5 and 15 mM) and depended on the ABH blood group of the donor, the stimulatory potency of the lectin decreasing in the order H greater than A2 greater than A1. Ca2+ entry blockers, such as cobalt and verapamil, did not affect the 45Ca2+ influx induced by UEA1. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited dose-dependently with a Ki of 1-2 microM. 10 microM DIDS, 10 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 20 microM dipyridamole fully blocked the 45Ca2+ influx induced by UEA1. The effect of UEA1 on 45Ca2+ influx was absent in K+ and Mg2+ media and was less pronounced in choline than in Na+ media. The 45Ca2+ influx induced by the lectin was abolished by preincubation with 12-O-tetradecanoylphorbol 13-acetate (TPA, 60 ng/ml). A monoclonal antibody raised against A1 erythrocytes (Bric 54) accelerated 45Ca2+ influx in quin2 loaded A1 erythrocytes by about 2-fold. No effect was seen in A2 and H erythrocytes. The 45Ca2+ influx elicited by Bric 54 exhibited a sensitivity towards inhibition by DIDS and TPA, as well as a dependence on the cation composition of the incubation medium similar to that observed with UEA1. The effects of UEA1 and Bric 54 were not additive. These observations suggest that the Ca2+ influx induced by UEA1 and Bric 54 is mediated by the same transport pathway. Since both the lectin and the antibody exhibit ABH blood group specificity, it appears reasonable to conclude that ABH antigens can serve as recognition sites for activation of a Ca2+ influx pathway in human erythrocytes, which is sensitive to inhibitors of the band 3 anion-exchanger.     

ABH and Lewis histo-blood group antigens, a model for the meaning of oligosaccharide diversity in the face of a changing world

Antigens of the ABH and Lewis histo-blood group family have been known for a long time. Yet their biological meaning is still largely obscure. Based on the available knowledge about the genes involved in their biosynthesis and about their tissue distribution in humans and other mammals, we discuss here the selective forces that may maintain or propagate these oligosaccharide antigens. The ABO, alpha 1,2fucosyltransferase and alpha 1,3fucosyltransferase enzyme families have been generated by gene duplications. Members of these families contribute to biosynthesis of the antigens through epistatic interactions. We suggest that the highly polymorphic genes of each family provide intraspecies diversity that allows coping with diverse and rapidly evolving pathogens. In contrast, the genes of low frequency polymorphism are expected to play roles at the cellular level, although they may be dispensable at the individual level. In addition, some members of these three gene families are expected to be functionally redundant and may either provide a reservoir for additional diversity in the future or become inactivated. We also discuss the role of the ABH and Lewis histo-blood group antigens in pathologies such as cancer and cardiovascular diseases, but argue that it is merely incidental and devoid of evolutionary impact.