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.     

New affinity procedure for the isolation and further characterization of the blood group B specific lectin from the red marine alga Ptilota plumosa

The red marine alga Ptilota plumosa has been shownto contain an anti-human blood group B lectin. We report here a new isolationprocedure by affinity chromatography on Sephadex G-200 and characterisation ofthe isolated lectin. The M _r , determined by gelfiltration, was 52,500. SDS-PAGE revealed a single protein band withM _r 17,440, indicating the native lectin was atrimer of subunits with the same M_r, as reported for the lectinsfromtwo other Ptilota species, P.filicinaand P. serrata. Analysis of amino acid composition showedslightly more basic than acidic amino acids. This was in contrast to theP. filicina and P. serrata lectinspreviously found to contain a higher proportion of acidic than basic aminoacids. Haemagglutination inhibition tests showed the P.plumosa lectin was inhibited by galactose, glucose and theirderivatives with p-nitrophenyl--D-galactoside moststrongly inhibitory. All glycoproteins tested failed to inhibit the lectin. Theamino acid composition, human blood group-B specificity and lack of inhibitionby glycoproteins indicate the lectin from P. plumosapossesses unique characteristics among marine algal lectins.     

PA-I and PA-II lectin interactions with the ABO(H) and P blood group glycosphingolipid antigens may contribute to the broad spectrum adherence ofPseudomonas aeruginosa to human tissues in secondary infections

Pseudomonas aeruginosa may cause serious infections in most human tissues/organs. Its adherence to them is mediated by a battery of adhesins including the PA-I and PA-II lectins, which are produced in this bacterium in high quantities. PA-I binds to thed-galactose of the erythrocyte glycosphingolipids exhibiting highest affinities for B and P^k (followed by P₁) antigens, while PA-II preferentially binds to thel-fucose of H, A and B antigens. IntactP. aeruginosa cells also exhibit a clear P^k and P₁ over p preference. Such affinities for the most common human ABH and P system antigens may underlie the widespread tissue infectivity and pathogenicity of this bacterium.     

Monoclonal antibodies specific for type 3 and type 4 chain-based blood group determinants: Relationship to the A1 and A2 subgroups

Two monoclonal antibodies, specific for A type 3 and A type 4 blood group determinants, are described. These antibodies recognized A1 but not A2 erythrocytes. A third monoclonal antibody showing specificity for A type 3 and A type 4, and also for H type 3 and H type 4, did not discriminate between A1 and A2 erythrocytes. On red cells these three antibodies recognized glycosphingolipids and binding to glycoproteins could not be demonstrated. On paraffin-embedded tissue sections the three antibodies labelled a supranuclear area, characteristic of the Golgi apparatus, of all cells producing A antigens. This labelling occurred irrespective of the A1, A2 status.The results suggest that glycolipids of erythrocytes and possibly of other cell types bear the A type 3/4 determinant specific for the A1 subgroup and that A type 3/4 determinants of glycoproteins might be present in both A1 and A2 subgroups on short oligosaccharide chains which are only detectable at the level of the Golgi apparatus.     

Identification of a new adhesin‐like protein from Lactobacillus mucosae ME‐340 with specific affinity to the human blood group A and B antigens

Aims: To identify and characterize a new adhesin‐like protein of probiotics that show specific adhesion to human blood group A and B antigens. Methods and Results: Using the BIACORE assay, the adhesion of cell surface components obtained from four lactobacilli strains that adhered to blood group A and B antigens was tested. Their components showed a significant adhesion to A and B antigens when compared to the bovine serum albumin (BSA) control. The 1 mol l^?1 GHCl fraction extracted from Lactobacillus mucosae ME‐340 contained a 29‐kDa band (Lam29) using SDS–PAGE. The N‐terminal amino acid sequence and homology analysis showed that Lam29 was 90% similar to the substrate‐binding protein of the ATP‐binding cassette (ABC) transporter from Lactobacillus fermentum IFO 3956. The complete nucleotide sequence (858 bp) of Lam29 was determined and encoded a protein of 285 amino acid residues. Phylogenetic analysis and multiple sequence alignments indicated this protein may be related to the cysteine‐binding transporter. Conclusions: The adhesion of ME‐340 strain to blood group A and B antigens was mediated by Lam29 that is a putative component of ABC transporter as an adhesin‐like protein. Significance and Impact of the Study: Lactobacillus mucosae ME‐340 expressing Lam29 may be useful for competitive exclusion of pathogens via blood group antigen receptors in the human gastrointestinal mucosa and in the development of new probiotic foods.     

Glycosylation with thioglycosides activated by dimethyl(methylthio)sulfonium tetrafluoroborate: synthesis of two trisaccharide glycosides corresponding to the blood group A and B determinants

Two trisaccharide glycosides,p-trifluoroacetamidophenylethyl 3-O-(2-acetamido-2-deoxy--d-galactopyranosyl)-2-O-(-l-fucopyranosyl)--d-galactopyranoside andp-trifluoroa-cetamidophenylethyl 2-O-(-l-fucopyranosyl)-3-O-(-d-galactopyranosyl)--d-galactopyranoside, corresponding to the human blood group A and B determinants, were synthesized. A key fucosylgalactosyl disaccharide derivative was glycosylated with galactosaminyl or galactosyl donors, respectively. Dimethyl (thiomethyl)sulfonium tetrafluoroborate was used for thioglycoside activation in coupling reactions.     

Enzyme-linked lectin assay (ELLA): Use of alkaline phosphatase-conjugated Griffonia simplicifolia B4 isolectin for the detection of α- -galactopyranosyl end groups

Alkaline phosphatase has been coupled to Griffonia simplicifolia I B₄ isolectin using a one-step glutaraldehyde conjugation procedure. This enzyme-lectin conjugate (AP-GS I-B₄) has been used to specifically detect plastic-bound natural and synthetic glycoproteins bearing α- -galactopyranosyl end groups. The extent of reactivity of the AP-GS I-B₄ with the glycoproteins appears to be proportional to the number of terminal galactosyl residues present. Furthermore, this assay, termed ELLA (enzyme-linked lectin assay), is specifically inhibitable by low-molecular-weight sugars containing terminal α- -galactosyl groups. The ELLA reactions may be assayed rapidly and objectively by the use of commercially available ELISA-plate readers using standard filters.     

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.     

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.     

Importance of molecular studies on major blood groups—Intercellular adhesion molecule-4, a blood group antigen involved in multiple cellular interactions

Several blood groups, including the LW-blood group were discovered in the first part of last century, but their biochemical characteristics and cellular functions have only more recently been elucidated. The LW-blood group, renamed ICAM-4 (CD242), is red cell specific and belongs to the intercellular adhesion molecule family. ICAM-4 binds to several integrin receptors on blood and endothelial cells and is thus able to form large cellular complexes containing red cells. Its physiological function(s) has remained incompletely understood, but recent work shows that macrophage integrins can bind red cells through this ligand. In this article we discuss molecular properties of major blood group antigens, describe ICAM-4 in more detail, and show that phagocytosis of senescent red cells is in part ICAM-4/β₂-integrin dependent.