Flavio Gruezo

Immunochemical studies on the combining site of the blood group H-Specific lectin 1 from Ulex europeus seeds

Abstract The specificity of purified Ulex lectin I has been studied by quantitative precipitin, quantitative precipitin inhibition, and competitive binding assays using tritium-labeled hog mucin H substance. The lectin is precipitated by human and hog H substances, by human A 2 substances, by a cow substance with A activity, and by B substances of human and horse origin. The lectin did not precipitate with A 1 substances, with Le a substances, with a precursor substance with I activity, or with PI fractions obtained by mild acid hydrolysis of blood group B substance. Ulex lectin I was most specific for the blood group H oligosaccharide HR L 0.75, having the structure l Fucαl ↓ 2 d Galβ1 → 4 d GlcNAcβ1 → 6R; this was determined by inhibition of precipitation of the lectin with a human H substance and by inhibition of the binding of tritiated hog H substance to the Ulex lectin I coupled to Sepharose beads using blood group and milk oligosaccharides as inhibitors. The lectin was also reactive with l Fucα1 → 2 d Galβ1 → 4[ l Fucαl → 3] d GlcNAcβ1 → 6R, 2′-fucosyllactose, lactodifucotetraose, and methyl α l Fuc, all of which had essentially parallel slopes by each assay method. l Fuc and fucosyl oligosaccharides with type 1 chains gave lines of different slopes but had the same range of activity. The Ulex lectin I site differs from the Lotus tetragonolobus site, which does not react at all with H oligosaccharides with the type 1 chain. Inhibition assays using tritiated hog H substance and various oligosaccharides were about 60 to 80 times more sensitive than assays by inhibition of precipitation.

Immunochemical studies on the combining site of the D-galactopyranose and 2-acetamido-2-deoxy-D-galactopyranose specific lectin isolated from Bauhinia purpurea alba seeds.

Abstract The combining site of the Bauhinia purpurea alba lectin was studied by quantitative precipitin and precipitin inhibition assays. Of 45 blood group substances, glycoproteins, and polysaccharides tested, 35 precipitated over 75% of the lectin. Precursor blood group substances with I activity (Cyst OG 10% from 20% and Cyst OG 20% from 10%), desialized fetuin, and desialized ovine salivary glycoprotein, in which more than 75% of the carbohydrate side chains have d GalN Ac linked through α1 → to the OH group of Ser or Thr of a protein core, completely precipitated the lectin. The poorly reactive blood group substances after mild acid hydrolysis or Smith degradation, as well as sialic acid-containing glycoproteins after removal of sialic acid, had substantially increased activity so that more than 80% of the lectin was precipitated. Precipitability with various blood group substances and glycoproteins is ascribable to the terminal nonreducing d GalNAc, d Galβ1 → 3 d GalNAc, d Galβ1 → 3 or 4 d GlcNAc, and d Galβ1 → 3 or 4 d GlcNAcβ1 → 3 d Gal determinants on the carbohydrate moiety. Of the monosaccharides tested for inhibition of precipitation, d GalNAc and its p -nitrophenyl and methyl α-glycosides were best. These compounds were four to five times better than the corresponding d Gal compounds but methyl β DGalNAc p was only about 40% more active than methyl β d Galp. The α-anomers of p -nitrophenyl DGalNAc p and d Galp, were twice as active as the corresponding β-anomers. Methyl αDGalNAc p was four times as active as the β-anomer but the inhibitory power of the methyl α- and β-anomers of d Gal were about equal. Among the oligosaccharides tested, d Galβ1 → 3 d GalNAc and its tosyl derivatives were most active, the tosyl glycosides being about twice as active as d Galβ1 → 3 d GalNAc, which was somewhat more active than d GalNAcα1 → 6 d Gal and d GalNAc, and 2.5 and 5 times as active as d GalNAcα1 → 3 d Galβ1 → 3 d GlcNAc and d GalNAcαl → 3 d Ga1, respectively (blood group A specific). These findings suggest that a subterminal d GalNAc β-linked and substituted on carbon 3 plays an important role in binding. Consistent with this inference are the findings that d Galβ1 → 3 d GlcNAc and d Galβ1 → 6 d Gal were poorer inhibitors although d Galβ1 → 3 d GlcNAc was two to three times as active as glycosides of d Gal. Oligosaccharides with terminal nonreducing d Gal and subterminal α-linked d Gal were as active or less active than d Gal. d Galβ1 → 3 d GlcNAcβ1 → 3 d Galβ1 → 4 d Glc (lacto- N -tetraose) and d Galβ1 → 3 d GlcNAcβ1 → 3 d Gal-β1-O-(CH 2 ) 8 COOCH 3 were equally active and 1.5 times as potent as d Galβ1 → 3 d GlcNAc whereas d Galβ1 → 3 d GlcNAcβ1 → 6 d Gal was only 40% as potent as d Galβ1 → 3 d GlcNAc suggesting that a third sugar may be part of the determinant. Substitution of d Galβ1 → 3 d GlcNAcβ1 → 3 d Galβ1 → 4 d Glc on the subterminal d GlcNAc by l Fucα1 → 4 in lacto- N -fucopentaose II reduced activity fourfold; if the nonreducing d Gal is substituted by l Fucα1 → 3 as in lacto- N -fucopentaose I its activity is almost completely abolished. This suggests that a terminal nonreducing d Gal as well as subterminal d GlcNAc are contributing to binding. The β → 3 linkage of the terminal d Gal to the subterminal amino sugar is significant since d Galβ1 → 4 d GlcNAc is a poorer inhibitor. Although the available data suggest that the combining site of the lectin Bauhinia purpurea alba may be most complementary to the structure d Galβ1 → 3 d GalNAcβ1 → 3 d Gal, several other possibilities remain to be tested when suitable oligosaccharides become available.

Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation, liberation, and fractionation of oligosaccharides and reaction with lectins

Abstract Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain d GalNAc, d Galβ1 → 3 d GalNAc, d Galβ1 → 4 d GlcNAc, d Galβ1 → 3 d GlcNAcβ1 → 3 d Gal and/or d Galβ1 → 4 d GlcNAcβ1 → 6 d Gal and/or d Galβ1 → 4 d GlcNAcβ1 → 6 d GalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that d GalNAc, d Galβ1 → 3 d GalNAc, d Galβ1 → 4 d GlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, d Galβ1 → 4 d GlcNAcβ1 → d 6 d Gal and/or d Galβ1 → 4 d GlcNAcβ1 → 6 d GalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with 3H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB3H4 at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.

Purification and characterization of lectin II from Ulex europaeus seeds and an immunochemical study of its combining site

Abstract The lectin II from Ulex europaeus seeds was purified by adsorption on insoluble polyleucyl hog A + H blood group substance and elution with 35% ethylene glycol, and by chromatography on ϵ-aminocaproyl-fucosyl-amine-agarose. In immunodiffusion against rabbit antiserum to the crude extract, the isolated lectin formed one line which fused with one of the five formed by crude extract. The purified lectin showed two bands on acrylamide electrophoresis under alkaline or acid conditions but only one band of molecular weight 23,000 if the electrophoresis was in the presence of 0.1% sodium dodecyl sulfate at pH 8.8. The agglutinating and precipitating abilities are abolished by EDTA and can be restored by bivalent cations. The purified lectin precipitated to different extents with blood group A1, A2, B, HLeb, Lea, and I precursor substances and with acid- or Smith-degraded substances. Inhibition of precipitation indicated that the lectin site was unusual in that it interacted most strongly with the h -specific oligosaccharide and with 2′-fucosyllactose, followed by β1 → 4 linked oligomers of d GlcNAc. Molecular models showed that all these inhibitors have a similarity in three-dimensional structures that could account for their activities.

Immunochemical studies on the N-acetyllactosamine β-(1→6)-linked trisaccharide specificity of Ricinus communis agglutinin

The combining site of Ricinus communis agglutinin (RCA1) was studied by quantitative precipitin and precipitin inhibition assays. Of 31 complex carbohydrates tested, all except active and inactive antifreeze glycoproteins, Streptococcus group C polysaccharide, and native rat salivary glycoprotein, reacted strongly, and 22 completely precipitated the lectin, indicating that RCA1 has both a broad range of affinity and a low solubility of its carbohydrate-bound complex. Of the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl beta-D-galactopyranoside was the best. It was about 6.4 times better than methyl beta-D-galactopyranoside. The beta anomer of glycosides of D-galactose was much more potent than the corresponding alpha anomer. Among the oligosaccharides tested, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal was the best inhibitor, which was approximately 2/3 as active as p-nitrophenyl beta-D-galactopyranoside. It was approximately 1.4 times as active as beta-D-Gal-(1----4)-D-GlcNAc (N-acetyllactosamine), twice as active as beta-D-Gal-(1----3)-D-GlcNAc, and 4.5 times more active than lacto-N-tetraose. From the results, it can be concluded that; (a) hydrophobic interaction is important for binding; (b) the combining site of this lectin is at least as large as a trisaccharide; and (c) of the compounds studied, the trisaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal was the most complementary to the human blood group I Ma determinant beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal.

Immunochemical studies on blood groups. L. Structures of some oligosaccharides obtained by mild acid hydrolysis of human ovarian cyst blood group A substance.

Mild acid hydrolysis of a human ovarian cyst blood group A substance folowed by preparative chromatography resulted in the isolation of two disaccharides, β D Gal(1→3) D GNAc, β D Gal(1→4) D GNAc, a trisaccharide, β D GNAc(1→3)β D GNAc(1→3) D Gal, and two tetrasaccharides, β D Gal(1→3)β D GNAc(1→3)β D Gal(1→3) D GNAc, and β D Gal(1→3)β D GNAc(1→3)β D GNAc(1→3)β D Gal(1→4) D GNAc. Their structure were established by analyses, periodate oxidation, methylation, alkaline degradation, and ORD spectra. The oligosaccharides appear to be derived from the interior of the megalosaccharide chains of the blood group active glycoprotein. A procedure for establishing linkages of internal D GNAc residues in oligosaccharides by gas chromatography following BaO methylation, methanolysis, and O -acetylation has been developed. Methylation analyses are also presented for four oligosaccharides obtained from alkaline borodeuteride degradation of a precursor blood group substance, OG, which, unlike the other oligosaccharides, contained 4-substituted internal D GNAc residues. Although the two tetrasaccharide fractions from A substance were only obtained as mixtures, the methods developed for methylation analysis and base-borohydride degradation were capable of elucidating each structure in the mixtures.