Study of human erythrocyte membrane protein interactions by selective solubilization of Triton-skeletons.

The membrane skeleton, responsible for shape and mechanical properties of the red cell, was purified by the Triton extraction procedure in presence of 5 mM, 150 mM or 600 mM NaCl. The proportion of spectrin, protein 4.1 and actin present in erythrocyte skeletons does not depend on the molarity of NaCl used. In contrast ankyrin, protein band 3 and protein 4.2 are removed from skeletons as the ionic strength increased. Solubilization assays of membrane skeletons were used to study protein interactions inside the skeleton. Solubilization was performed by Tris, a non-selective disruptive reagent, or by p-mercuribenzene sulfonic acid (PMBS), which principally release spectrin and actin. Tris action was assessed by calculation of the percentage of solubilized proteins, which increased proportionally with Tris molarity. PMBS action was kinetically determined as the decrease in skeleton turbidity. With these two reagents, we observed a lower dissociation of skeletons prepared with high ionic strength buffer. Erythrocyte pretreatment with okadaic acid, an inhibitor of serine-threonine phosphatases, revealed a phosphorylation-induced skeleton gelation and a better resistance to Tris-solubilization.

Specific and nonspecific inhibition of adhesion of oral actinomyces and streptococci to erythrocytes and polystyrene by caseinoglycopeptide derivatives.

Various caseinoglycopeptide derivatives prepared from mammalian milk were evaluated as inhibitors of hemagglutinations mediated by Actinomyces viscosus Ny1, Streptococcus sanguis OMZ9, and, for comparative purposes, plant lectins from Arachis hypogaea and Bauhinia purpurea. It was found that recognition of the beta-D-galactose-(1----3)-2-acetamido-2-deoxy-D-galactose carbohydrate chain by Actinomyces viscosus Ny1 organisms and Arachis hypogaea and B. purpurea agglutinins had similar structural requirements; in all cases, the desialylated bovine caseinoglycomacropeptide, on which several units of the above mentioned disaccharide are clustered, behaved as the most potent hemagglutination inhibitor. By contrast, none of the preparations tested inhibited erythrocyte agglutination by S. sanguis OMZ9. Thus, the desialylated bovine caseinoglycomacropeptide acts as a potent and specific inhibitor of oral Actinomyces adhesion to cell membranes (a soft surface) and could be used as a probe for the study of recognition mechanisms mediated by Actinomyces galactose-binding lectins. During the present study, both native and desialylated variants of the same bovine glycomacropeptide also totally prevented the adhesion of Actinomyces viscosus Ny1, S. sanguis OMZ9, and S. mutans OMZ176 to polystyrene surfaces. Comparative evaluations of various structurally different compounds gave the following results. Neither mono- nor disaccharides related to caseinoglycopeptide carbohydrates prevented adhesion; highly positively or negatively charged polypeptides and polysaccharides were either not or only moderately active. Besides these glycomacropeptides, an inhibitory activity was also exhibited by other mucin-type glycoproteins carrying short O-linked carbohydrate chains (including bovine submaxillary mucin), polyethylene glycol, and bovine serum albumin. Consequently, caseinoglycopeptide prevention of oral bacterial adhesion to polystyrene tubes (a hard surface) takes place with no species specificity and can be compared to nonspecific inhibition exhibited by various polymers with very different structural characteristics.

Preliminary investigation of the structure of the carbohydrate component of Vicia graminea lectin, a plant glycoprotein.

Purified Vicia graminea lectin, isolated from seeds, was found to contain D-mannose, 2-acetamido-2-deoxy-D-glucose, L-fucose, D-galactose, and D-xylose in the molar ratios approximately 3.9:1.5:1.2:1.1:1.0. The oligosaccharides, obtained after hydrazinolysis of Vg-lectin, were N-reacetylated, reduced with sodium borohydride, and fractionated into two peaks. The first peak contained D-galactose, D-mannose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-glucitol in the molar ratios 6:3:2:0.7. After h.p.l.c. fractionation into five oligosaccharides, the second peak contained D-mannose, D-xylose, L-fucose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-glucitol. Methylation analysis suggested the following general structure for these oligosaccharides: (formula; see text).

Interaction of Vicia graminea anti-N lectin with cell surface glycoproteins from erythrocytes with rare blood group antigens.

The erythrocyte receptors for Vicia graminea (Vg) anti-N lectin have been investigated after 125I-labelling of the purified lectin and binding to membrane components separated by dodecyl sulphate polyacrylamide gel electrophoresis. GP alpha (synonym glycophorin A or MN glycoprotein) and GP delta (synonym glycophorin B or Ss glycoprotein) are the main Vg receptors of native human blood group NN and MN erythrocytes whereas Vg lectin only binds to GP delta from MM red cells. The glycoprotein of 28 kDa present in Mi III erythrocytes (a presumed variant of GP delta) carries Vg receptors. Both binding studies and agglutination experiments with this lectin suggest that the delta Mi III gene might produce more glycoprotein molecules than the normal delta gene. Binding of Vg lectin to hybrid glycoproteins [from Mi V, St(a+) and Dantu (+) donors] produced by unequal crossing-over between alpha and delta genes, may occur if the molecules exhibit N activity. The lectin does not bind to sialic acid- and galactose-deficient glycoproteins from Tn erythrocytes and no binding could be detected in the region of GP delta of erythrocytes from S-s-U-individuals. Addition of N-acetylgalactosamine residues to the alkali-labile oligosaccharides attached to GP alpha and GP delta, as found in Cad erythrocytes, decrease the binding capacity for Vg lectin. Finally the absence of Vg lectin binding sites on native GP alpha molecule from MgMg and McM erythrocytes, which carry well defined variants of this glycoprotein, supports the view that the binding site of the lectin on native glycoproteins is located at the N-terminal end of glycoprotein (GP alpha and GP delta) with N specificity (N-terminus = Leu).

Miltenberger Class I and II erythrocytes carry a variant of glycophorin A.

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.

Subunit structure of Vicia graminea anti-(blood-group N) lectin.

The subunit of the Vicia graminea lectin with blood-group-N specificity was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in 6M-guanidinium chloride, and its molecular weights was found to be 25 000. The unique N-terminal sequence fof the first nine residues of the lectin confirmed that Vicia lectin consists of four identical chains non-covalently linked. Finally the microheterogeneity of the lectin shown by analytical isoelectric focusing is discussed.

[Structural characteristics of the blood group N determinant, tested with anti-N lectin from Vicia graminea]. / Caractéristiques structurales du déterminant de groupe sanguin N, explorées par la lectine anti N de Vicia graminea.

Crude extracts of Vicia graminea seeds agglutinate human N erythrocytes as anti-N immunsera. The anti-N lectin is purified after precipitations with ammonium sulphate of crude extracts, DE52 Whatman chromatography and sephadex G150 gel filtration. Its homogeneity is demonstrated by physical and immunological methods. The structure determinant for the Vicia graminea anti-N activity was investigated: --with the major glycoprotein of N erythrocytes. --with glycoconjugates isolated from urine of normal human N-blood group as urinary glycoconjugates are probably related to the membrane glycoprotein catabolism. Purification and characterization of glycoconjugates are undertaken by gel filtration and non-exchange chromatography. This purification is checked by hemagglutination-inhibition test with V. graminea lectin. Biochemical characterization of active glycoconjugates gives way to the carbohydrate determinant recognized by anti-N antisera and Vicia graminea lectin.

Purification and characterization of a lectin (plant hemagglutinin) with N blood group specificity from Vicia graminea seeds.

A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes.

[Properties of a hemagglutinin isolated from the seeds of Vicia graminea]. / Propriétés d'une hémagglutinine isolée des graines de Vicia graminae

A lectin is isolated from Vicia graminea seeds; it is purified from a crude extract after a precipitation with ammonium sulfate "DEAE"-cellulose chromatography and "sephadex G 150" gel filtration. Its homogeneity is demonstrated by different methods. It is a glycoprotein with 7.3% of carbohydrates. The native lectin is found to have a molecular weight about 100 000 and the subunit molecular weight is estimated to be 25 000 daltons. The lectin agglutinates only N erythrocytes and it is not mitogenic. The Vicia graminea lectin is the first anti N lectin which has been purified and characterized.