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We conducted 16 parallel in vitro selection experiments to isolate catalytic DNAs from a common DNA library for the cleavage of all 16 possible dinucleotide junctions of RNA incorporated into a common DNA/RNA chimeric substrate sequence. We discovered hundreds of sequence variations of the 8-17 deoxyribozyme--an RNA-cleaving catalytic DNA motif previously reported--from nearly all 16 final pools. Sequence analyses identified four absolutely conserved nucleotides in 8-17. Five representative 8-17 variants were tested for substrate cleavage in trans, and together they were able to cleave 14 dinucleotide junctions. New 8-17 variants required Mn2+ to support their broad dinucleotide cleavage capabilities. We hypothesize that 8-17 has a tertiary structure composed of an enzymatic core executing catalysis and a structural facilitator providing structural fine tuning when different dinucleotide junctions are given as cleavage sites.

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1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)>13,14-dihydro-15-keto PGD(2)>15-deoxy-Delta(12,14)-PGJ(2)>PGJ(2)>Delta(12)-PGJ(2)>15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)>PGJ(2)>Delta(12)-PGJ(2)>15-deoxy-Delta(12,14)-PGJ(2) >>>13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.

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The carbohydrate binding specificity of the seed lectin from Artocarpus integrifolia, artocarpin, has been elucidated by the enzyme-linked lectin absorbent assay [Misquith, S., et al (1994) J. Biol. Chem. 269, 30393-30401], wherein it was demonstrated to be a Man/Glc specific lectin with high affinity for the trisaccharide present in the core of all N-linked oligosaccharide chains of glycoproteins. As a consequence of this characterization, the binding epitopes of this trisaccharide, 3, 6-di(alpha-D-mannopyranosyl)-D-mannose, for artocarpin were investigated by isothermal titration calorimetry using its monodeoxy as well as Glc and Gal analogues. The thermodynamic data presented here implicate 2-, 3-, 4-, and 6-hydroxyl groups of the alpha(1-3) Man and alpha(1-6) Man residues, and the 2- and 4-OH groups of the central Man residue, in binding to artocarpin. Nevertheless, alpha(1-3) Man is the primary contributor to the binding affinity, unlike other Man/Glc binding lectins which exhibit a preference for alpha(1-6) Man. In addition, unlike the binding reactions of most lectins reported so far, the interaction of mannotriose involves all of its hydroxyl groups with the combining site of the lectin. Moreover, the free energy and enthalpy contributions to binding of individual hydroxyl groups of the trimannoside estimated from the corresponding monodeoxy analogues show nonlinearity, suggesting differential contributions of the solvent and protein to the thermodynamics of binding of the analogues. Thus, this study not only provides evidence for the extended site recognition of artocarpin for the trimannoside epitope but also suggests that its combining site is best described as a deep cleft as opposed to shallow indentations implicated in other lectins.

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The thermodynamics of binding of various saccharides to artocarpin, from Artocarpus integrifolia seeds, a homotetrameric lectin (M(r) 65, 000) with one binding site per subunit, was determined by isothermal titration calorimetry measurements at 280 and 293 K. The binding enthalpies, DeltaH(b), are the same at both temperatures, and the values range from -10.94 to -47.11 kJ mol(-1). The affinities of artocarpin as obtained from isothermal titration calorimetry are in reasonable agreement with the results obtained by enzyme-linked lectin absorbent essay, which is based on the minimum amount of ligand required to inhibit horseradish peroxidase binding to artocarpin in enzyme-linked lectin absorbent essay (Misquith, S., Rani, P. G., and Surolia, A. (1994) J. Biol. Chem. 269, 30393-30401). The interactions are mainly enthalpically driven and exhibit enthalpy-entropy compensation. The order of binding affinity of artocarpin is as follows: mannotriose>Manalpha3Man>GlcNAc(2)Man(3)>MealphaMan>Man>M analpha6Man> Manalpha2Man>MealphaGlc>Glc, i.e. 7>4>2>1.4>1>0.4>0.3>0.24>0.11. The DeltaH for the interaction of Manalpha3Man, Manalpha6Man, and MealphaMan are similar and 20 kJ mol(-1) lower than that of mannotriose. This indicates that, while Manalpha3Man and Manalpha6Man interact with the lectin exclusively through their nonreducing end monosaccharide with the subsites specific for the alpha1,3 and alpha1,6 arms, the mannotriose interacts with the lectin simultaneously through all three of its mannopyranosyl residues. This study thus underscores the distinction in the recognition of this common oligosaccharide motif in comparison with that displayed by other lectins with related specificity.

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Two mannose-binding lectins, Allium sativum agglutinin (ASA) I (25 kDa) and ASAIII (48 kDa), from garlic bulbs have been purified by affinity chromatography followed by gel filtration. The subunit structures of these lectins are different, but they display similar sugar specificities. Both ASAI and ASAIII are made up of 12.5- and 11.5-kDa subunits. In addition, a complex (136 kDa) comprising a polypeptide chain of 54 +/- 4 kDa and the subunits of ASAI and ASAIII elutes earlier than these lectins on gel filtration. The 54-kDa subunit is proven to be alliinase, which is known to form a complex with garlic lectins. Constituent subunits of ASAI and ASAIII exhibit the same sequence at their amino termini. ASAI and ASAIII recognize monosaccharides in mannosyl configuration. The potencies of the ligands for ASAs increase in the following order: mannobiose (Manalpha1-3Man) < mannotriose (Manalpha1-6Manalpha1-3Man) approximately mannopentaose << Man9-oligosaccharide. The addition of two GlcNAc residues at the reducing end of mannotriose or mannopentaose enhances their potencies significantly, whereas substitution of both alpha1-3- and alpha1-6-mannosyl residues of mannotriose with GlcNAc at the nonreducing end increases their activity only marginally. The best manno-oligosaccharide ligand is Man9GlcNAc2Asn, which bears several alpha1-2-linked mannose residues. Interaction with glycoproteins suggests that these lectins recognize internal mannose as well as bind to the core pentasaccharide of N-linked glycans even when it is sialylated. The strongest inhibitors are the high mannose-containing glycoproteins, which carry larger glycan chains. Indeed, invertase, which contains 85% of its mannose residues in species larger than Man20GlcNAc, exhibited the highest binding affinity. No other mannose- or mannose/glucose-binding lectin has been shown to display such a specificity.

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Jacalin and artocarpin, the two lectins from jackfruit (Artocarpus integrifolia) seeds, have different physicochemical properties and carbohydrate-binding specificities. However, comparison of the partial amino-acid sequence of artocarpin with the known sequence of jacalin indicates close to 50% sequence identity. Artocarpin crystallizes in two forms, both monoclinic P2(1), with one and two tetramic molecules, respectively, in the asymmetric units of form I (a = 69.9, b = 73.7, c = 60.6 A and beta = 95.1 degrees ) and form II (a = 87.6, b = 72.2, c = 92.6 A and beta = 101.1 degrees ). Both the crystal structures have been solved by the molecular replacement method using the known structure of jacalin as the search model and one of them partially refined, confirming that the two lectins are indeed homologous.

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Artocarpin, a mannose-specific lectin, is a homotetrameric protein (M(r) 65,000) devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range 5-6.5. Investigations of its carbohydrate binding specificity reveal that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man alpha 1-3[Man alpha 1-6]Man) and mannopentaose are the strongest ligands followed by Man alpha 1-3Man. Extension of these ligands by GlcNAc at the reducing ends of mannooligosaccharides tested remarkably improves their inhibitory potencies, while substitution of both the alpha 1-3 and alpha 1-6 mannosyl residues of mannotriose and the core pentasaccharide of N-linked glycans (Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc) by GlcNAc or N-acetyllactosamine in beta 1-2 linkage diminishes their inhibitory potencies. Sialylated oligosaccharides are non-inhibitory. Moreover, the substitution of either alpha 1-3 or alpha 1-6 linked mannosyl residues of M5Gn or both by mannose in alpha 1-2 linkage leads to a considerable reduction of their inhibitory power. Addition of a xylose residue in beta 1-2 linkage to the core pentasaccharide improves the inhibitory activity. Considering the fact that artocarpin has the strongest affinity for the xylose containing hepasaccharide from horseradish peroxidase, which differs significantly from all the mannose/glucose-specific lectins, it should prove a useful tool for the isolation and characterization of glycoproteins displaying such structure.

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