Crystal state and solution conformation of the B blood group trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)]-beta-D-Galp-OCH3.

The crystal structure of the human B blood group related trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)-beta-D-Galp-OCH3 (1) has been determined. The solution structure of 1 was studied by two-dimensional NMR techniques at 600 MHz in D2O solution and the conformational properties were analyzed in terms of the torsional angles phiH and psiH, derived from 3JCH coupling constants, and 10 inter-residue proton-proton distances. 3JCH could be accurately measured by a recently introduced two-dimensional heteronuclear correlation experiment (EXSIDE). The nuclear Overhauser enhancement-derived distances and the calculated torsion angles were compared with the same information available from the crystal structure. The agreement is excellent, indicating that the trisaccharide adopts a restricted conformation in solution, which was also predicted by the Hard Sphere Exo-Anomeric forcefield. The data of 1 are complemented by NMR studies of the closely related alpha-L-Fucp-(1-->2)-[6-deoxy-alpha-D-Galp]-(1-->3)-beta-D-Galp O-(CH2 )7CH3 trisaccharide (2).

The recognition of three different epitopes for the H-type 2 human blood group determinant by lectins of Ulex europaeus, Galactia tenuiflora and Psophocarpus tetragonolobus (winged bean).

The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc alpha (1-2)Gal beta (1-4)GlcNAc beta Me) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc alpha (1-2)Gal beta (1-4)Xyl beta Me was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction.

Substrate recognition by amyloglucosidase: evaluation of conformationally biased isomaltosides.

Amyloglucosidase catalyzes the hydrolysis of methyl beta-maltoside (1) 30-50 times more rapidly than methyl alpha-isomaltoside (2). It is established that OH-6', OH-4', and OH-4 which are involved in key polar interactions with the enzyme in the case of isomaltoside. Conformational analyses based on HSEA calculations indicate that the dispositions in space of OH-3 of maltose relative to OH-4' and OH-6' in the preferred conformation for the maltoside (1) is energetically more readily achieved by methyl 6R-C-methyl-alpha-isomaltoside (3), than for its 6-S-isomer (4). A kinetic evaluation of the hydrolysis in fact has shown that the R-compound is more strongly bound by the enzyme (Km = 0.9 mM) than the parent isomaltoside (Km = 24.5 mM), whereas the S-compound has the weakest enzyme binding (Km = 90 mM). Since the kcat values were all within the range 0.85 +/- 0.20 s-1, it is evident that the relative rates of hydrolysis are related to the relative ease for the compounds to achieve an interaction of a hydroxyl group in the aglycon of an alpha-D-glucopyranoside with the enzyme for the formation of the enzyme-substrate complex. The relative rates of hydrolysis of the alpha-glucosides of the 1,3-dihydroxy-trans-decalins, 5 and 6, provide further support for this highly desirable but not necessary recognition for the orientation of the reducing glucose unit in the active site.

Involvement of water in host-guest interactions.

As predicted by inhibition studies the X-ray crystal structure of the complex formed between the tetrasaccharide alpha-L-Fuc(1----2)-beta-D-Gal(1----3) [alpha-L-Fuc-(1----4)]-beta-D-GlcNAc- OMe (Leb-OMe) and the lectin IV of Griffonia simplicifolia (GS-IV) shows three hydroxyl groups (referred to as the polar key) hydrogen bonded within the combining site and flanked by hydrophobic surfaces. Apart from OH-6 of the beta-D-GlcNAc unit, the six other hydroxyl groups reside at or near the periphery of the combining site. Linear enthalpy-entropy compensation is observed for complex formation with monodeoxy and other derivatives of Leb-OMe involving one of these six hydroxyl groups. Decreases in both the thermodynamic parameters (- delta H 0 and - delta S 0) are largest when a hydroxyl group is in contact with water at the periphery of the combining site. The experimental evidence indicates that the binding reactions involve very similar if not identical changes in the conformations of both the lectin and the ligands; it is therefore proposed that the enthalpy-entropy compensations arise because water molecules hydrogen bonded to the amphiphilic surfaces of the unbound oligosaccharide and the protein are more mobile (higher entropy content) and less strongly hydrogen bonded than are water molecules in bulk solution. Monte Carlo simulations of the hydration of Leb-OMe appear to support this idea. In accordance with this proposal the association of complementary amphiphilic molecular surfaces from aqueous solution is driven by the release of the water molecules from both non-polar and polar regions of the amphiphiles to form stronger hydrogen bonds in bulk water. In the case of highly amphiphilic molecules such as the oligosaccharide Leb-OMe the negative contributions to entropy change dominate positive contributions that may arise from hydrophobic effects. The GS-IV(Leb-OMe)2 complex is stabilized by the hydrogen-bonding networks involving an asparate, an asparagine and a serine residue within the combining site and the above-mentioned key hydroxyl groups. Improved packing of the molecules may also be involved.

Molecular-mass heterogeneity of Griffonia simplicifolia lectin IV subunits. Differences in the oligosaccharide moieties in the N-terminal region.

Lectin IV of Griffonia simplicifolia (Mr approximately 56,000), which has a strong affinity for both the Lewis b and Y blood-group determinants, is a dimeric protein of two subunits, alpha (29 kDa) and beta (27 kDa), separable by SDS/PAGE and containing covalently linked oligosaccharide. After digestion with N-glycanase, the protein migrates as a single band with a mobility identical with that of the beta-subunit. After cleavage with hydroxylamine of 3H-labelled, but otherwise intact, lectin, the radioactively labelled oligosaccharide was found to be associated with two blocked N-terminal peptides separable by h.p.l.c. and having identical amino acid compositions. One of these had three or four glucosamine residues per molecule, whereas the other had only one or two. Sequence analyses of these, as well as of a 21 kDa hydroxylamine-cleaved fragment and of the intact lectin pretreated with pyroglutamate aminopeptidase, have provided a unique sequence for residues 1-62 of the two subunits. Evidence is presented for two sites of N-linked oligosaccharide attachment at Asn-5 and Asn-18. Whereas the alpha-subunit has oligosaccharide linked to both sites, the beta-subunit has carbohydrate associated with only one (Asn-18). Sugar analyses of the whole lectin reveal a monosaccharide composition of (Xyl)3(Fuc)2(Man)10(GlcNAc)6, representing 6.4% of the mass of the molecule. Taken together with the susceptibility of the Asn-5 linkage (but not of Asn-18) to N-glycanase digestion, the observations indicate that the structures of the oligosaccharides at residues 5 and 18 are different.

The binding of the Lewis-a human blood group determinant by two hybridoma monoclonal anti-Lea antibodies.

The combining sites of two hybridoma monoclonal antibodies with specificities for the Lewis-a human blood group determinant, beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1---- , were probed by use of a wide range of inhibitors of the reaction between an 125I-labelled artificial Lewis-a antigen and the antibodies under the conditions of a solid-phase radioimmunoassay. Amongst the inhibitors examined were the eight possible monodeoxy derivatives of the trisaccharide methyl glycoside. As was previously found for other antibodies and lectins, the results indicated that, in each case, a cluster of polar groups provide a key polar interaction with the protein. The further involvement of large lipophilic regions of the trisaccharide was also indicated. These regions are expected to provide an essentially nonpolar interaction for complex formation, and appear to include intramolecularly hydrogen-bonded hydroxyl groups. Although the features of the trisaccharide that are recognized are similar in kind, the patterns are very different. For example, in the case of antibody AH8-34, the key polar group is provided by OH-2 and OH-3 of the beta-D-Galp group, whereas for the other antibody (CF4-C4) OH-3 and OH-4 of the beta-D-Galp group and OH-4 of the alpha-L-Fucp group provide the key polar interaction. It appears that, for AH8-34, the trisaccharide is accepted with OH-2 of the beta-D-Galp group and OH-3 of the alpha-L-Fucp group intramolecularly hydrogen-bonded to neighboring proton acceptors. For CF4-C4, such intramolecular hydrogen-bonding appears to involve OH-4 and OH-6 of the beta-D-Galp group.

Synthetic, conformational, and immunochemical studies of modified Lewis b and Y human blood-group determinants to serve as probes for the combining site of the lectin IV of Griffonia simplicifolia.

Syntheses of the methyl glycosides of the Lewis b [alpha-L-Fuc-(1----2)-beta-D-Gal-(1----3) [alpha-L-Fuc-(1----4)]-beta-D-GlcNAc-] and Y [alpha-L-Fuc-(1----2)-beta-D-Gal-(1----4) [alpha-L-Fuc-(1----3)]-beta-D- GlcNAc-] human blood-group determinants and both their 6a-deoxy and N-deacetylated derivatives are reported. In the case of the Lewis b structure (Leb-OMe), the 6a-O-mesyl and 6a-deoxy-6a-iodo derivatives were also prepared. The conformational preferences predicted by HSEA calculation are shown to be in good agreement with expectations based on 1H- and 13C-n.m.r. spectroscopy. The immunochemical data based on inhibition and thermodynamic studies require that the binding of Leb-OMe and Y-OMe by the lectin IV of Griffonia simplicifolia does not involve recognition of the OMe, NHAc, or 6a-OH group and, consequently, occurs at a cleft at the surface of the protein. The complex formed between the lectin and 6a-deoxy-6a-iodo-Leb-OMe provided the heavy nuclei required for the solution of the X-ray crystal structure.

Crystallization of the lectin IV of Griffonia simplicifolia and its complexes with the Lewis b and Y human blood group determinants.

Single crystals of the lectin IV from Griffonia simplicifolia have been grown in the tetragonal crystal system. The space group is P4(2)2(1)2 with a = 78.95(5) A and c = 89.01(2) A, and there is one subunit of the dimeric glycoprotein in the crystallographic asymmetric unit. The crystals diffract to at least 2.5 A d spacings and are stable in the x-ray beam for 3 weeks. Crystals of the complex with the Lewis b (Leb) and Y human blood group determinants as the methyl glycosides, alpha-L-Fuc(1----2)beta-D-Gal(1----3)[alpha-L-Fuc(1----4)]beta-D-GlcNAc-OMe (where Fuc is fucose and -OMe is methoxy) and alpha-L-Fuc(1----2)beta-D-Gal(1----4)[alpha-L-Fuc(1----3)]-beta-D-GlcNAc- OMe, respectively, have also been grown and found to be isomorphous with the native lectin. Crystals have also been obtained with several derivatives of the Lewis b-OMe tetrasaccharide including that which has the 6-hydroxyl of the beta-D-GlcNAc unit replaced by iodine. In the latter case, the presence of the iodine atoms was established.

The presence of at least two different H-blood-group-related beta-D-gal alpha-2-L-fucosyltransferases in human serum and the genetics of blood group H substances.

Sera from H normal, secretors and nonsecretors (H/-, Se/- and H/-, se/se), as well as from H-deficient secretors (h/h, Se/- or Bombay secretors) contain enzyme(s) for the transfer of L-fucose in the alpha-configuration to the 2-position of suitable beta-D-galactopyranosyl units. Sera from H-deficient nonsecretors (h/h, se/se; i.e., Bombay nonsecretors) are devoid of such beta-D-Gal alpha-2-L-fucosyltransferase(s). In order to study these enzymes, a comparison was made of the kinetic properties of the enzymes present in the sera of H-normal nonsecretors (H/-, se/se) with those of H-deficient secretors (h/h, Se/se) with those of H-deficient secretors (h/h, Se/-). These studies revealed a clear difference between the two sources of enzyme: (1) the apparent Km for GDP-fucose was four times lower with the H-normal nonsecretor serum (0.008 mM) than with the H-deficient secretor serum (0.028 mM); (2) acceptors with a type 1 or type 3 chain proved to be better than acceptors with a type 2 chain or than phenyl-beta-D-galactopyranoside for the enzyme present in the serum of H-deficient secretor individuals. Indeed, the synthetic type 2 compound, betaDGal (1-->4)-3-deoxy-beta-DGlcNAc-1-OCH3, which cannot act as an acceptor of beta DGlcNAc alpha-3/4-L-fucosyltransferases, remained unchanged in the serum of an H-deficient secretor but was a good acceptor in the serum of an H-normal nonsecretor, and (3) the alpha-2-L fucosyltransferease activity of the H-deficient secretor serum was more sensitive to heat inactivation than that of the H-normal nonsecretor serum (t1/2 at 46 degrees C were 10 min and 75 min, respectively). These results show that at least two distinct alpha-2-L-fucosyltransferases are present in human serum. It is concluded that the enzymatic activity found in the H-deficient secretor serum (h/h, Se/-) could be the product of the Se gene and the enzymatic activity found in the H-normal nonsecretor serum (H/-, se/se) could be the product of the H gene. This conclusion correlates well with the finding that H and Se genes are closely linked and might have derived by gene duplication in the course of evolution.

The combining site of anti-I Ma (group 1).

Evidence is provided that the trisaccharide beta DGal(1----4)beta DGlcNAc(1----6)beta DGal is bound by the monoclonal anti-I Ma antibody beginning with a basically nonpolar cleft at the surface of the protein which comes into contact with a weakly polar region of the trisaccharide that extends from the C-5 methylene group of the reducing unit about the surfaces involving the acetamido grouping and the OH-3' of the beta DGlcNAc unit intramolecularly hydrogen bonded to the O-5'' of the nonreducing beta DGal unit and up to the C-5'' methylene group. The combining site then terminates with a polar grouping at its periphery which is disposed to react with OH-6'' and likely OH-4'' in a highly specific manner. The hydroxyl groups at positions 1, 2, 3, 4, 6', 3'' and 4'' remain in contact with the aq. phase. This conclusion was deduced from the relative potencies as inhibitors of a wide number of synthetic compounds that bear varying structural relationships to the trisaccharide. It appears that the stability of the complex is mainly related to attractive interactions between two large complementary and essentially hydrophobic surfaces relative to those when these surfaces are exposed to water.

Synthesis and conformational properties of methyl 6,6-di-C-methyl-beta-D-galactopyranoside. Probes for the combining sites of D-galactosyl-binding proteins.

The binding of D-galactopyranosyl groups by lectins and antibodies can involve the 5-hydroxymethyl group. In order to examine the nature of these binding reactions, it was of interest to synthesize 6,6-di-C-methyl-D-galactose which was found to exist, like D-galactose, extensively in the pyranose forms. 2,3,4,6-Tetra-O-acetyl-7-deoxy-6-C-methyl-alpha-D-galacto-heptopyranosyl bromide was prepared under standard conditions and converted into methyl 6,6-di-C methyl-beta-D-galactopyranoside (6). Evidence based on 13C-n.m.r. studies indicates that the favored conformer of 6 has O-4 and O-6 in syn-axial-like relationship. General comments are presented on the nature of the binding of oligosaccharides by proteins.

Immunochemical studies on the combining sites of Forssman hapten reactive hemagglutinins from Dolichos biflorus, Helix pomatia, and Wistaria floribunda.

The lectin of Dolichos biflorus, a hemagglutinin previously considered to be blood group A specific, is now found to react much more strongly with the terminal disaccharide unit [alpha DGalNAc(1 leads to 3) beta DGalNAc] of the Forssman antigenic determinant. In contrast, the relative reactions of the lectins of Helix pomatia (which also agglutinates A erythrocytes) and Wistaria floribunda (which agglutinates A, B, and O erythrocytes) with the Forssman pentasaccharide were substantially weaker than that of Dolichos biflorus. The combining site of the lectin of Helix pomatia has a broader affinity for terminal 2-acetamido-2-deoxy-alpha-D-galactopyranose (alpha DGalNAc) residues than does that of Dolichos biflorus. The reactions of the lectin with terminal alpha DGalNAc units are strongly dependent on the nature of the aglycon and remain ill defined. The lectin may also react with appropriately presented terminal 2-acetamido-2-deoxy-beta-D-glucopyranose units. The broad affinity of the lectin of Wistaria floribunda which reacts both with a range of blood group specific glycoproteins (A, B, H, Lea, and Leb) and with non blood group glycoproteins [Sugii, S., & Kabat, E.A. (1980) Biochemistry 19, 1192-1199] appears best assigned to a combining site that favors pauci- or multivalent cooperative effects of clustered terminal beta-D-galactopyranose units. An attempt is made to rationalize certain of the inhibition data in terms of topographical features at the surfaces of the carbohydrate structures which are considered compatible for binding within essentially hydrophobic combining sites.

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant.

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.

The conformational analysis of oligosaccharides by H-NMR and HSEA calculation.

The application of 1H-nuclear Overhauser enhancement, 1H-spin-lattice-relaxation-time and 1H-chemical shift measurements for the assessment of the conformational preferences of oligosaccharides are briefly reviewed. It is demonstrated that additivity rules, for the correlation of the chemical shifts of similar hydrogen atoms in different oligosaccharides, can be useful in the conformational analysis of oligosaccharides when the differential chemical shifts are greater than 0.1 ppm. These often can be attributed to specific interunit deshielding of a hydrogen atom by an oxygen atom with which it is in strong nonbonded interaction. HSEA calculations are used to demonstrate that differential chemical shifts of less than 0.1 ppm can have origins that are not significant to the overall conformational preferences of the oligosaccharides which are being compared. Both shielding and deshielding effects can arise from a change in the orientation of a substituent group as the result of the introduction of a sugar on a neighboring unit. It is demonstrated that substituent groups, such as hydroxymethyl and acetamido groups, on occasions, should be treated in HSEA calculations as freely rotating about their linkage to a pyranose ring.

Competition between ABO and Le gene specified enzymes. II. Quantitative analysis of A and B antigens in saliva of ABH nonsecretors.

One B- and two A-specific radioimmunoassays detected A and B antigenic determinants in saliva of ABH nonsecretors. The A or B antigens found in saliva of nonsecretors had lower efficiency and higher heterogeneity, in the inhibition of the A or B assays, as compared to A or B antigens in saliva of ABH secretors. In the 3 assays, samples of nonsecretor Lewis positive donors (Lea) had less A or B antigens than samples of nonsecretor Lewis negative donors (Lec), suggesting that there is a competition between A or B and Le gene-specified products.

Competition between ABO and Le gene specified enzymes. I. A Lewis related difference in the amount of A antigen in saliva of A1 and A2 secretors.

Radioimmunoassays were prepared using two anti-A and one anti-B reagents. The specificity of the procedures was assessed with 13 artificial antigens. The amounts of A and B natural antigens in saliva of ABH secretors of known Lewis phenotype were measured with these assays. The results confirmed that the average amount of A antigen is lower in Lewis-positive (Leb) than in Lewis-negative (Led) donors and in A2 than in A1 donors. However, the differences among the four combined A and Lewis phenotypes were only supported by significantly lower amounts of A antigenic determinants in A2Leb as compared to the other three phenotypes (A1Leb, A1Led and A2Led) that had similar amounts of A antigenic determinants. No Lewis-related difference could be detected in the amounts of B antigens between BLeb and BLed donors. The results are discussed in terms of competition between A, B and Lewis-gene-specified enzymes for their common acceptors. The difference in the efficiency of the A2 enzyme as compared to that of the A1 enzyme is proposed as a possible explanation for the A1-A2 phenotypic difference.