Further characterization of the combining sites of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4).

Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4)(GS I-A(4)), which is cytotoxic to the human colon cancer cell lines, is one of two lectin families derived from its seed extract. It contains only a homo-oligomer of subunit A, and is most specific for GalNAcalpha1-->. In order to elucidate the GS I-A(4)-glycoconjugate interactions in greater detail, the combining site of this lectin was further characterized by enzyme linked lectino-sorbent assay (ELLSA) and by inhibition of lectin-glycoprotein interactions. This study has demonstrated that the Tn-containing glycoproteins tested, consisting of mammalian salivary glycoproteins (armadillo, asialo-hamster sublingual, asialo-ovine, -bovine, and -porcine submandibular), are bound strongly by GS I-A(4.)Among monovalent inhibitors so far tested, p-NO2-phenylalphaGalNAc is the most potent, suggesting that hydrophobic forces are important in the interaction of this lectin. GS I-A(4)is able to accommodate the monosaccharide GalNAc at the nonreducing end of oligosaccharides. This suggests that the combining site of the lectin is a shallow cavity. Among oligosaccharides and monosaccharides tested as inhibitors of the binding of GS I-A(4), the hierarchy of potencies are: GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Forssman pentasaccharide) > GalNAcalpha1-->3(LFucalpha1-->2)Gal (blood group A)()> GalNAc > Galalpha1-->4Gal > Galalpha1-->3Gal (blood group B-like)> Gal.

Multi-antennary Gal beta1-->4GlcNAc and Gal beta1-->3GalNAc clusters as important ligands for a lectin isolated from the sponge Geodia cydonium.

The affinity of a lectin from the sponge Geodia cydonium (GCL-I) for multi-antennary Gal beta1-->4GlcNAc and Gal beta1-->3GalNAc ligands was studied by both the biotin/avidin-based microtiter plate lectin binding assay and the inhibition of lectin-glycoform interaction. Among the glycoforms tested for binding, GCL-I reacted strongly with three multi-antennary Gal beta1-->4GlcNAc clusters containing glycoproteins (asialo human and bovine alpha1-acid gps and asialo fetuin), T (Gal beta1-->3GalNAc) rich glycoprotein from porcine salivary gland, asialo bird nest gp, and human blood group A active cyst gp, while human and bovine alpha1-acid gps, fetuin, and Tn containing gps were inactive. Among the haptens tested for inhibition, tri-antennary Gal beta1-->4GlcNAc (Tri-II) was about 1500, 72, and 72 times more active than GalNAc, Gal beta1-->4GlcNAc (II), and Gal beta1-->3GalNAc (T), respectively. Based on the present and previous results, it is proposed that tri-antennary Gal beta1-->4GlcNAc and Gal beta1-->3GalNAc clusters, in addition to GalNAc alpha1-->3GalNAc and GalNAc alpha1-->3Gal, are also important ligands for binding; and sialic acid of glycoprotein does interfere with binding.

Frequent occurrence of identical heavy and light chain Ig rearrangements.

Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated at least 10 different types of VH region rearrangements, involving three distinct germline genes, among FACS-sorted PtC-binding B-1 cells from three strains of mice (C57BL/6J, BALB/c and C.B-17). The predominant rearrangement, VH11-DSP-JH1 (VH11 type 1), has been previously found in anti-PtC hybridomas in several studies. We show that within each of six mice from two strains (C57BL/6J and BALB/c), unique instances of IgH/IgL pairing arose either from different B cell progenitors prior to IgH rearrangement or from pre-B cells which expanded after IgH rearrangement but prior to IgL rearrangement. Together with other recurrent rearrangements described here, our findings demonstrate that clonal expansion of mature B cells cannot account for all repeated rearrangements. As suggested by initial studies of dominant idiotype expression, these findings confirm that clonal expansion is only one of the mechanisms contributing to the establishment of recurrent rearrangements.

Further characterization of the binding properties of a GalNAc specific lectin from Codium fragile subspecies tomentosoides.

Previous study on the binding properties of a lectin isolated from Codium fragile subspecies tomentosoides (CFT) indicates that this lectin recognizes the GalNAc alpha1--> sequence at both reducing and nonreducing ends. In this study, the carbohydrate specificity of CFT was further characterized by quantitative precipitin (QPA) and inhibition of lectin-enzyme binding assays. Of the glycoforms tested for QPA, all asialo-GalNAc alpha1--> containing glycoproteins reacted well with the lectin. Asialo hamster and ovine submandibular glycoproteins, which contain almost exclusively Tn (GalNAc alpha1-->Ser/Thr) residues as carbohydrate side chains, and Streptococcus type C polysaccharide completely precipitated the lectin added, while the GalNAc beta1-->containing Tamm-Horsfall Sd(a+) glycoprotein and its asialo product were inactive. Among the oligosaccharides tested for inhibiting lectin-glycoprotein interaction, GalNAc alpha1-->3GalNAc beta1-->3Gal alpha1-->4Gal beta1--> 4Glc(Fp) and Gal beta1-->3GalNAc alpha1-->benzyl (T alpha) were the best, and about 125-fold more active than GalNAc. They were about 3.3, 6.6, and 43 times more active than Tn containing glycopeptides, GalNAc alpha1-->3(LFuc alpha1--> 2)Gal(Ah) and Gal beta1-->3GalNAc(T), respectively. From the present and previous results, it is concluded that the combining site of CFT is probably of a groove type that recognizes from GalNAc alpha1--> to pentasaccharide(Fp). The carbohydrate specificity of this lectin can be constructed and summarized in decreasing order by lectin determinants as follows: Fp and T alpha > Tn cluster > Ah >> I/II.

Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A4, reacting with Tn (Ga1NAc alpha1 --> Ser/Thr) or galabiose (Ga1 alpha1 --> 4Ga1) containing ligands.

Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A4(GS I-A4) reacting with the Tn(GalNAc alpha1 --> Ser/Thr) sequence or human blood group Pk active disaccharide (E, Gal alpha1 --> 4Gal, galabiose) was studied by quantitative precipitin (QPA) and precipitin-inhibition assays. When human blood group P1 or Tn active glycoproteins were tested by QPA, GS I-A4 reacted strongly with both the Tn active glycoproteins purified from asialo porcine, ovine and armadillo submandibular glands and a P1 active glycoprotein isolated from sheep hydatid fluid. They precipitated over 80% of the lectin nitrogen added. The asialo porcine salivary glycoprotein-GS I-A4 interaction was inhibited by both Tn containing glycopeptides and Gal alpha1 --> 4Gal indicating that GS I-A4 not only reacts with human blood group A(GalNAc alpha1 --> 3Gal) and B(Gal alpha1 --> 3Gal) active disaccharides, but also recognizes the Tn sequence and the E(Gal alpha1 --> 4-Gal) ligand. From these results, the carbohydrate specificity of GS I-A4 can be defined as A, Tn > or = B and E.

Characterization of a human monoclonal immunoglobulin M (IgM) antibody (IgMBEN) specific for Vi capsular polysaccharide of Salmonella typhi.

A search for human monoclonal antibodies to protective antigens of bacteria revealed an immunoglobulin M lambda chain [IgM(lambda); designated IgMBEN] reactive with the Vi capsular polysaccharide of Salmonella typhi. Vi, a linear homopolymer of alpha(1-->4)GalApNAc that is O acetylated at C-3, is a licensed vaccine for typhoid fever. Immunologic properties of IgMBEN were compared to those of burro globulin prepared by intravenous injections of S. typhi (B339-340). IgMBEN and B339-340 yielded identical precipitin lines with Vi by double immunodiffusion. IgMBEN and B339-340 produced similar precipitation results with Vi and its derivatives prepared by de-O-acetylation, carboxyl reduction, and removal or replacement of the N-acetyl at C-2 with O-acetyl. B339-340 yielded maximal precipitation with Vi (0.41 mg of antibody per ml with 1.4 micrograms of Vi); next was carboxyl-reduced, O-acetylated Vi, which precipitated 0.325 mg of antibody per ml with 2.5 micrograms of Vi. IgMBEN yielded maximal precipitation with de-O-acetylated, carboxyl-reduced Vi (approximately 11.0 mg of antibody per ml with approximately 1.3 micrograms of antigen); next were de-O-acetylated Vi (9.89 mg/ml) and Vi (9.19 mg/ml). The precipitin curves and equivalence points of these three antigens were similar. Pneumococcus type 1, which contains GalApNAc, did not precipitate with Vi or its derivatives. These slight differences in specificity between IgMBEN and B339-340 were related to our proposed structure of Vi. We plan to use IgMBEN as a reference for measurement of vaccine-induced Vi antibodies.

Affinity of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin B4 for Gal alpha 1-->4 Gal ligand.

The affinity of Bandeiraea (Griffonia) simplicifolia lectin-I isolectin B4 (BSI-B4) for the isomer of human blood group B active disaccharide (B, Gal alpha 1-->3Gal), the Gal alpha 1-->4Gal galabiose ligand, was studied by quantitative precipitin (QPA) and precipitin-inhibition assays. When human blood group B, P1 and H active glycoproteins were tested by OPA. BSI-B4 reacted strongly with both the B active glycoprotein purified from human ovarian cyst fluid and a P1 active glycoprotein isolated from sheep hydatid fluid and precipitated over 86% of the lectin nitrogen added. The P1 active glycoprotein-BSI-B4 interaction was inhibited by both Gal alpha 1-->3Gal alpha 1-->methyl and Gal alpha 1-->4Gal disaccharide indicating that BSI-B4 is not only reacting with Gal alpha 1-->3Gal disaccharide, but also recognizing Gal alpha 1-->4Gal. The galabiose sequence is frequently found in the carbohydrate chains of many glycosphingolipids located at the mammalian cell membranes such as intestinal and red blood cell membranes, for E. coli ligand binding and toxin attachment.

Human and mouse monoclonal antibodies to blood group A substance, which are nearly identical immunochemically, use radically different primary sequences.

A human monoclonal antibody (HuA) specific for blood group A substance with two fucose groups was found to be immunochemically almost identical with that of a previously characterized mouse monoclonal anti-A, AC-1001. The VH and VL chain cDNAs of HuA were sequenced and compared with those of AC-1001. The human and mouse antibodies used VH and Vk genes that came from different families and shared minimal nucleotide and amino acid sequence identity. Thus, two antibodies from two different species can use evolutionarily unrelated sequences to bind the same carbohydrate epitope. The cloned HuA VH and VL genes were then transfected into a mouse myeloma cell line and re-expressed, together, and each separately with an irrelevant VH or VL. Only the original HuA VH and Vk had anti-A activity, demonstrating that both the heavy and light chains contributed to specificity.

Microheterogeneity of mouse antidextran monoclonal antibodies.

Mouse antidextran monoclonal antibodies showed microheterogeneity which was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Not only the heavy (H) chains but also the light (L) chains were heterogeneous in terms of isoelectric point (pI). The higher the pI, the more prominent the H chain spots. To demonstrate the cause of the microheterogeneity an IgG1 monoclonal antibody (mAb 35.8.2H) was examined especially for involvement of the sugar moiety in the microheterogeneity. The glycosylated region was determined in the Fc portion from serine 239 to methionine 309 by a glycan detection method using mild periodate oxidation, which confirms that the sugar chain is attached to the conserved glycosylation site of asparagine 297. However, charge heterogeneity of the H chain was not entirely attributed to the Fc because the papain digest of the antibody was separated into two Fc spots, a few Fd spots and two L chain spots by 2-D PAGE. This indicates that factors other than the sugar moiety are responsible for charge heterogeneity of IgG monoclonal antibody. On the other hand, the H chain isoforms of lower pI were shown to be more susceptible to V8 protease by peptide mapping. This result strongly suggests the occurrence of deamidation at glutamine or asparagine residues.

Agglutination-induced erythrocyte deformation by two blood group A-specific lectins: studies by light and electron microscopy.

The adhesive energy in lectin-induced agglutination can be assessed by the deformation of erythrocytes in aggregates. Helix pomatia (HPA) and Dolichos biflorus (DBA) specifically agglutinated blood group A erythrocytes and induced a change in curvatures of cells at the end of the aggregates. The curvatures changed from concavity to convexity with increasing lectin concentrations. HPA-induced aggregates achieved the theoretical maximal end cell curvature of 0.27 microns-1 at 2-3 micrograms/ml; DBA-induced aggregates approached 0.23 microns-1, requiring 400 micrograms/ml. At any given lectin concentration, HPA showed greater surface binding, caused higher cell curvatures, induced larger aggregate size, and had greater adhesive energies, as compared to DBA. HPA and DBA are globular proteins with a diameter of approximately 5.5 nm and approximately 6.1 nm, respectively, as revealed by the negative staining electron microscopy. The former induced uniform intercellular spacing (approximately 15 nm), whereas the latter induced both smooth (approximately 20 nm) and ruffled spacings. The uniform intercellular spacing was not a function of lectin concentrations. Microscopic studies of erythrocyte deformation provided morphological correlates of biophysical findings of differential adhesive energies induced by these two blood group A-specific lectins.

Reaction of germinal centers in the T-cell-independent response to the bacterial polysaccharide alpha(1-->6)dextran.

Primary immunization of BALB/c mice with alpha(1-->6)dextran (DEX), a native bacterial polysaccharide, induces an unexpected pattern of splenic B-cell responses. After a peak of antibody-secreting B-cell response at day 4, deposition of dextran-anti-dextran immune complexes, as revealed by staining with both dextran and antibodies to dextran, occurs and persists in splenic follicles until at least the fourth week after immunization. Antigen-specific B cells appear and proliferate in such follicles, leading by day 11 to development of DEX-specific germinal centers as characterized by the presence of distinct regions of DEX+ peanut agglutinin-positive (PNA+) cells. At this time, fluorescence-activated cell sorter analysis also reveals the appearance of a distinct population of DEX+ PNA+ splenic B cells. In contrast, DEX+ PNA- cells, characterized by intense cytoplasmic staining, are present outside of splenic follicles, peak at day 4 to day 5, and persist until at least day 28. The frequency of these cells correlates with DEX-specific antibody-secreting cells, as detected by the ELISA-spot assay. Thus, in addition to the expected plasma cellular response, the typical T-cell-independent type II antigen, DEX, surprisingly also elicits the formation of antigen-specific germinal centers. These observations raise fundamental questions about the roles of germinal centers in T-cell-independent immune responses.

Modeling study of antibody combining sites to (alpha 1-6)dextrans. Predictions of the conformational contribution of VL-CDR3 and J kappa segments to groove-type combining sites.

The shuffling of the V kappa-Ox1 light chain joined to J kappa 4 of J kappa 5 instead of J kappa 2 reduced or abolished antigen binding of three groove-type anti-(alpha 1-6)dextran monoclonal antibodies, raising questions as to the structural roles of J kappa in antibody combining sites. The J kappa 4 light chain used contains Pro95A at the V kappa-Ox1-J kappa 4 junction, as well as a Phe to Ile substitution at the beginning of this J kappa 4 segment. To predict whether the defect in antigen binding is a consequence of the J kappa replacement, the Pro insertion or the Phe to Ile substitution, model-building studies were performed. As shown by the surface representation of antibody combining sites, the models with length variation in the VL-CDR3 loop by only 1 residue altered the shape of the combining site dramatically; whereas those with replacement of J kappa or having amino acid substitutions in VL-CDR3 affect the combining site less extensively. A distinct loop configuration of VL-CDR3 appears in models having either a Pro, Gly, or Ala insertion at position 95A. These results indicate that the length of VL-CDR3 is crucial for its loop conformation and may, therefore, have played a major role in abolishing dextran binding activity of the J kappa 4 variants. The potential of V kappa-Ox1 genes in generating conformational diversity in the loop of VL-CDR3 and its influence in forming different combining sites are discussed.

Length distribution of CDRH3 in antibodies.

Sequences of the third complementarity determining region of antibody heavy chains (CDRH3s) are listed according to their length. Human sequences vary from 2 to 26 amino acids residues, but less extensively in other species. When combined with the other five complementarity determining regions, this enormous length variation of CDRH3, together with amino acid substitutions in their sequences, can provide a very large number of antibody specificities and can influence the shape of antibody combining sites.

A monoclonal IgM kappa from a blood group B individual with specificity for alpha-galactosyl epitopes on partially hydrolyzed blood group B substance.

We have characterized a human monoclonal IgM kappa, designated IgMDON, from a blood group B individual. IgMDON is specific for alpha-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(alpha 1-3)Gal epitope. Gal(alpha 1-3)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 IgMDON 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 IgMDON might have arisen are discussed.

Immunochemical studies on the combining site of the A + N blood type specific Moluccella laevis lectin.

The specificity of the anti A+N lectin of Moluccella laevis (MLL) was examined by hemagglutination experiments with enzyme-modified human erythrocytes and by inhibition of hemagglutination. In addition, binding to various glycoproteins and inhibition by different sugars and glycoproteins were examined by enzyme immunoassay with antibodies to the lectin. Treatment of AMM erythrocytes with proteolytic enzymes increased their agglutinability by MLL 4-16-fold; similar treatment of ONN cells decreased their agglutinability 8-16-fold. This is in line with the known location and enzyme sensitivity of A and N specificity determinants. Treatment of the erythrocytes with sialidase increased their agglutinability and abolished the distinction between N and M cells. Hapten inhibition of hemagglutination of AMM and ONN erythrocytes by the lectin, and its binding to glycoproteins measured by enzyme immunoassay, confirmed the high specificity of MLL for N-acetyl-D-galactosamine (200-500 times more than for D-galactose) and suggested the presence of hydrophobic interactions around HO-2 of the D-galactose unit. The methyl alpha-glycosides of D-galactose and of N-acetyl-D-galactosamine were better inhibitors than the corresponding beta-glycosides; this preference was abolished, and sometimes reversed, when the p-nitrophenyl glycosides of the same monosaccharides were tested, stressing again the importance of hydrophobic interactions in the binding of carbohydrates to MLL. The lectin reacted well with ONN substance and with glycophorin A of the N phenotype (GPAN), but did not react with OMM substance or GPAM. The strongest inhibitor was asialo ovine submaxillary mucin, which contains many unsubstituted alpha-D-GalpNAc-(1-->3)-Ser/Thr residues; calculated per N-acetyl-D-galactosamine residue, it was 1500 stronger than free N-acetyl-D-galactosamine. In accordance with this result, it was found that the lectin strongly agglutinates Tn cells. The specificity of MLL can, thus, be defined as anti-Tn, crossreactive with blood types A and N, and with sialosyl-Tn. The N-specificity can best be explained by assuming that GPAN contains a small number of unsubstituted or partially sialylated alpha-D-GalpNAc-(1-->3)-Ser/Thr residues, which are present in smaller proportions, if at all, in GPAM.

Possible use of similar framework region amino acid sequences between human and mouse immunoglobulins for humanizing mouse antibodies.

We have previously noted that a specific amino acid sequence could form the second framework region of human, mouse and rabbit immunoglobulin light chains, suggesting that this sequence has been preserved for 80 million years. Through divergent evolution, each species has acquired a different set of framework region sequences; however, these sets still share a few similar or identical amino acid sequences. In the present study, we have identified such sequences for all four framework regions between human and mouse immunoglobulin light and heavy chains. They may be useful in humanizing or reshaping mouse or rat antibodies for therapeutic applications in human patients.

Variable region cDNA sequences of three mouse monoclonal anti-idiotypic antibodies specific for anti-alpha(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.

Subgroups of Tcr alpha chains and correlation with T-cell function.

T-cell receptor (Tcr) alpha chains are classified into four subgroups (I, II, III, and miscellaneous) based on the amino acid residues at positions 61 and 62. Subgroup I has Gly Phe at these positions, subgroup II has Arg Phe, subgroup III has Arg Leu, and subgroup miscellaneous has several other combinations. Variability plots for subgroups I, II, and III sequences show higher values around positions 93-103, 105, 108, 111, 113, and 115, suggesting that these positions may interact with the processed antigen molecules. Smaller peaks are present at various other regions which may bind the major histocompatibility complex class I or II molecules. The patterns of variability within one subgroup are similar for all species, for human alone, and for mouse alone. These subgroup patterns appear much less complicated than patterns for sequences in all subgroups taken together, implying that subgroups may be related to Tcr functions. Among 83 mouse chains, 15 are from cytotoxic cells and 40 from helper cells. Of the 15 from cytotoxic cells, 11, 2, 0, and 2 are in subgroups I, II, III, and miscellaneous; and of the 40 from helper cells, 9, 16, 12, and 3 are in subgroups I, II, III, and miscellaneous, respectively. Thus, a correlation between sequence and function of Tcr alpha chains seems possible.