Jerry Liao

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.

Immunochemical studies on blood groups—LXIX. The conformation of the trisaccharide determinant in the combining site of anti-I Ma (group 1)

Abstract Further studies of the inhibition of the monoclonal anti-I Ma (group 1) antibody, in quantitative precipitin assays, by synthetic oligosaccharides derived from N-acetyllactosamine (LacNAc) are reported. The results confirm that the antibody binds about the βLacNAc-OCH2CHO-portions of such structures as βLacNAc(1→6)β d Gal or βLacNAc(1→6)α d GalNAc. The trisaccharides βLacNAc(1→6)-7-deoxy- d -glycero- d -galacto-heptose (7) and βLacNAc(1→6)-7-deoxy- l -glycero- d -galacto-heptose (8), in contrast to βLacNAc(1→6) d Gal (1), were found (1HNMR) to possess well-defined conformations. Since 7 proved to be a 2.2 times better inhibitor than 1, whereas 8 was a poor inhibitor, it is concluded that the antibody binds 1 in the conformation which is preferred by 7. On this basis, it is noted that 1 would reside in a conformation that possesses a region amenable to hydrophobic bonding that extends between the C-5 groupings of the two d Gal units and over the acetamido group of the central β d GlcNAc residue with the 3-hydroxyl of this unit hydrogen bonded to O-5 of the terminal β d Gal unit. It is suggested that it is this region which becomes bound to a hydrophobic site of the antibody. Thus, nine of the hydroxyl groups in 1 would remain available for interaction with the solvent water and it would follow that the combining site is an hydrophobic cleft.

Immunochemical characterization of the specificities of two human monoclonal IgM's reacting with chondroitin sulfates.

We have studied the specificities of two human monoclonal, IgM containing sera, s/IgMMAC and s/IgMFIS, from patients with polyneuropathy. s/IgMMAC precipitates only with chondroitin sulfate C and not with A and B whereas s/IgMFIS is precipitated by chondroitins A, B (dermatan sulfate), and C. Inhibition assays using 2-acetamido-2-deoxy-3-O-(4-deoxy-beta-L-threo-hex-4-enopyranosyluroni c acid)-D-galactose and its 6- and 4-sulfate derivatives showed that the disaccharide 6-sulfate was the best inhibitor of precipitation of s/IgMMAC by chondroitin sulfate C, and the disaccharide 4-sulfate the best inhibitor of precipitation of s/IgMFIS by either chondroitin sulfates C or B. The nonsulfated disaccharide was a good inhibitor in each instance. D-Glucose 6-sulfate, Na2SO4, several sugar phosphates, and phosphate buffer also inhibited but to different extents with the s/IgMMAC and s/IgMFIS. All studies were carried out in 0.15M NaCl. The data indicate that both monoclonal proteins are antibodies comparable to the phosphorylcholine-binding myeloma proteins, and that the reactions show specificities above and beyond charge effects. The relation of various cross-reacting macromolecules to the monoclonal antibody was studied by diffusion in gels.

Variable region cDNA sequences of three mouse monoclonal anti-idiotypic antibodies specific for anti-α(1→6)dextrans with groove- or cavity-type combining sites☆

The variables regions of three syngeneic anti-idiotypic antibodies (Ab2s) were cloned and sequenced. They are encoded by different VL genes, two are from different members of V kappa-Ox1 superfamily. The H chains are encoded by VH genes belonging to three different VH families, J558, Q52 and 7183. Together with a previous report from this laboratory, the nucleotide sequences of four Ab2s to anti-alpha(1----6)dextrans have been presented. They are derived from a number of unrelated germline genes, and differ from similar studies in anti-NP, anti-GAT and anti-Ars systems. Three of four Ab2s in the anti-alpha(1----6)dextran system appear to have D-D fusions, which has also been reported in several other Ab2s.

A monoclonal IgMκ from a blood group B individual with specificity for α-galactosyl epitopes on partially hydrolyzed blood group B substance

Abstract We have characterized a human monoclonal IgMκ, designated IgM DON , from a blood group B individual. IgM DON is specific for α-galactosyl residues on blood group B substance; its fine specificity as defined by hemagglutination, quantitative precipitin, and inhibition ELISA assays was for the defucosylated terminal Gal(α1–3)Gal epitope. Gal(α1–3)Gal epitopes are also found on a variety of normal and pathogenic intestinal bacteria, and polyclonal IgG antibodies with the same specificity are found in the serum of nearly all normal individuals. The specificity of IgM DON was also quite similar to that of a human antiserum, serum 262, obtained by immunizing an individual with blood group B substance that had been subjected to mild acid hydrolysis (BP1). The possible ways whereby IgM DON might have arisen are discussed.