The binding of N-trifluoroacetyl chito-oligosaccharides to wheat-germ agglutinin: a fluorescence investigation

We describe the synthesis of N-trifluoroacetyl chito-oligosaccharides and their use as ligands to probe the binding sites of wheat-germ agglutinin, a lectin specific for N-acetylglucosamine. The binding is monitored using intrinsic protein fluorescence, which is due to tryptophan side-chains. We present arguments purporting to show the presence of a fluorophore close to each of the four sites. The binding of chito-oligosaccharides to wheat-germ agglutinin is complex and can only be approximately described by an independent and equivalent sites model. This model applies when the ligand concentration range is restricted to higher values. The possible role of ligand-mediated protein aggregation and of site inequivalence is discussed. We find that the affinity of trifluoroacetylated chito-oligosaccharides for wheat-germ agglutinin is higher than that of the N-acetylated parent compounds, the difference increasing with chain length. Our results are in agreement with a model of the binding site previously proposed by Clegg et al. (Biochemistry 22 (1983) 4797-4804).     

Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.     

A chitotetrose specific lectin fromLuffa acutangula: Physico-chemical properties and the assignment of orientation of sugars in the lectin binding site

A chitooligosaccharide specific lectin (Luffa acutangula agglutinin) has been purified from the exudate of ridge gourd fruits by affinity chromatography on soybean agglutinin-glycopeptides coupled to Sepharose-6B. The affinity purified lectin was found homogeneous by polyacrylamide gel electrophoresis, in sodium dodecyl sulphate-polyacrylamide gels, by gel filtration on Sephadex G-100 and by sedimentation velocity experiments. The relative molecular weight of this lectin is determined to be 48,000 ±1,000 by gel chromatography and sedimentation equilibrium experiments. The sedimentation coefficient (S₂₀, w) was obtained to be 4.06 S. The Stokes’ radius of the protein was found to be 2.9 nm by gel filtration. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis the lectin gave a molecular weight of 24,000 in the presence as well as absence of 2-mercaptoethanol. The subunits in this dimeric lectin are therefore held by non-covalent interactions alone. The lectin is not a glycoprotein and circular dichroism spectral studies indicate that this lectin has 31% α-helix and no β-sheet. The lectin is found to bind specifically to chitooligosaccharides and the affinity of the lectin increases with increasing oligosaccharide chain length as monitored by near ultra-violet-circular dichroism and intrinsic fluorescence titration. The values of ΔG, ΔH and ΔS for the binding process showed a pronounced dependence on the size of the oligosaccharide. The values for both ΔH and ΔS show a significant increase with increase in the oligosaccharide chain length showing that the binding of higher oligomers is progressively more favoured thermodynamically than chitobiose itself. The thermodynamic data is consistent with an extended binding site in the lectin which accommodates a tetrasaccharide. Based on the thermodynamic data, blue shifts and fluorescence enhancement, spatial orientation of chitooligosaccharides in the combining site of the lectin is assigned.     

Effect of a serine-to-aspartate replacement on the recognition of chitin oligosaccharides by truncated hevein. A 3D view by using NMR

The interaction of a synthetically prepared mutant peptide of hevein (a well known chitin-binding lectin) Hev32S19D with chitin oligosaccharides (and chitosan analogues) has allowed us to estimate their affinity constants and associated thermodynamic data. The mutant peptide is able to bind chitin oligomers, but with significant decreases in the association constants with chito-oligosaccharides. The determination of the three-dimensional structure of the peptide mutant, by using NMR, has permitted us to deduce that the topology of the backbone is very similar to that of the parent Hev32 peptide. The same is true regarding the orientations of the key aromatic residues Trp21, Trp23, and Tyr30. The decrease in the association constants can be attributed to the different topological orientation of key side chains and to the importance of protein-sugar intermolecular essential hydrogen bonds and CH-π stacking interactions. The analysis has permitted us to infer the free energy of binding associated with these interactions as well as to estimate the corresponding binding enthalpy.     

Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.     

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum

Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their alpha- but not beta-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic alpha-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.     

Computational analysis of multivalency in lectins: structures of garlic lectin-oligosaccharide complexes and their aggregates

Multivalency in lectins is a phenomenon that has been discussed at considerable length. The structural basis for the role of multivalency in garlic lectin has been investigated here through computational studies. Biochemical studies have shown that the binding affinity of garlic lectin for high mannose oligosaccharides is orders of magnitude greater than that for mannose. Modeling and energy calculations clearly indicate that such increase in affinity cannot be accounted for by binding of these oligosaccharides at any of the six sites of a garlic lectin dimer. These studies also indicate that a given oligosaccharide cannot bind simultaneously to more than one binding site on a lectin dimer. The possibility of a given oligosaccharide simultaneously binding to and hence linking two or more lectin molecules was therefore explored. This study showed that trimannosides and higher oligomers can cross-link lectin dimers, amplifying the protein-oligosaccharide interactions severalfold, thus explaining the role of multivalency in enhancing affinity. A comprehensive exploration of all possible cross-links posed a formidable computational problem. Even a partial exploration involving a carefully chosen region of the conformational space clearly showed that a given dimer pair can be cross-linked not only by a single oligosaccharide molecule but also simultaneously by two oligosaccharides. The number of such possible double cross-links, including those forming interesting tetrameric structures, generally increases with the size of the oligosaccharide, correlating with the biochemical data. In addition to their immediate relevance to garlic lectin, these studies are of general interest in relation to lectin-oligosaccharide interactions.     

Allozymic, morphological, and phenological diversity in cultivatedLuffa acutangula (Cucurbitaceae) from China, Laos, and Nepal, and Allozyme Divergence betweenL. acutangula andL. Aegyptiaca

The immature fruits of domesticated Luffa acutangula (L.) Roxb. are a common vegetable in Asia and India. To learn more about traditional cultivars, accessions were collected from southern Yunnan Province of China, northern Laos, and southeastern Nepal, and assessed for various parameters of genetic diversity. The size and shape of the fruits and seeds varied considerably. A form that we found cultivated only in Nepal bore clusters of small fruits that are produced by hermaphrodite flowers. Plants produced male flowers first, and the first node to bear flowers varied from the second to the twenty-seventh. Twenty-nine allozyme loci were assayed. Within L. acutangula one allozyme locus was polymorphic. Luffa acutangula andL. aegyptiaca are fixed for different alleles at nine loci, indicating that they are completely reproductively isolated from each other.     

Quantitative determination of binding affinity of delta-subunit in Escherichia coli F1-ATPase: effects of mutation, Mg2+, and pH on Kd

To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of delta-subunit to Escherichia coli F(1)-ATPase. The signal used is that of the natural tryptophan at residue delta28, which is enhanced by 50% upon binding of delta-subunit to alpha(3)beta(3)gammaepsilon complex. K(d) for delta binding is 1.4 nm, which is energetically equivalent (50.2 kJ/mol) to that required to resist the rotor strain. Only one site for delta binding was detected. The deltaW28L mutation increased K(d) to 4.6 nm, equivalent to a loss of 2.9 kJ/mol binding energy. While this was insufficient to cause detectable functional impairment, it did facilitate preparation of delta-depleted F(1). The alphaG29D mutation reduced K(d) to 26 nm, equivalent to a loss of 7.2 kJ/mol binding energy. This mutation did cause serious functional impairment, referable to interruption of binding of delta to F(1). Results with the two mutants illuminate how finely balanced is the stator resistance function. delta' fragment, consisting of residues delta1-134, bound with the same K(d) as intact delta, showing that, at least in absence of F(o) subunits, the C-terminal domain of delta contributes zero binding energy. Mg(2+) ions had a strong effect on increasing delta binding affinity, supporting the possibility of bridging metal ion involvement in stator function. High pH environment greatly reduced delta binding affinity, suggesting the involvement of protonatable side-chains in the binding site.     

Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis. Comparison with other sialic acid-specific lectins

The sialic acid-specific leukoagglutinating lectin from the seeds of Maackia amurensis (MAL) has been studied by the techniques of quantitative precipitin formation, hapten inhibition of precipitation, hapten inhibition using an enzyme-linked immunosorbent assay, and lectin affinity chromatography. The ability of the immobilized lectin to fractionate oligosaccharides based on their content of sialic acid has also been investigated. Our results indicate that MAL reacts with greatest affinity with the trisaccharide sequence Neu5Ac/Gc alpha 2,3Gal beta 1,4GlcNAc/Glc. The lectin requires three intact sugar units for binding and does not interact when the beta 1,4-linkage is replaced by a beta 1,3-linkage nor when the "reducing sugar" of the trisaccharide is reduced. Results from enzyme-linked immunosorbent assays show that an N-acetyllactosamine repeating sequence is not required; however, the N-acetyllactosamine repeating sequence does appear to enhance the binding of MAL to a series of glycolipids. In addition, the sialic acid may be substituted with either N-acetyl or N-glycolyl groups without reduction in binding. The C-8 and C-9 hydroxyl groups of sialic acid do not play a role in binding as shown by the strong reaction of periodate-treated glycoproteins. Comparison of the specificity of the three sialic acid-binding lectins indicates that Limax flavus agglutinin binds to Neu5Ac in any linkage and in any position in a glycoconjugate, Sambucus nigra lectin requires a disaccharide of the structure Neu5Ac alpha 2,6Gal/GalNAc, and MAL has a binding site complimentary to the trisaccharide Neu5Ac alpha 2,3Gal beta 1,4GlcNAc/Glc, to which sialic acid contributes less to the total binding affinity than for either S. nigra lectin or L. flavus agglutinin.     

A new chitooligosaccharide specific lectin from snake gourd (Trichosanthes anguina) phloem exudate. Purification, physico-chemical characterization and thermodynamics of saccharide binding

A new lectin has been purified to homogeneity from the phloem exudate of snake gourd (Trichosanthes anguina) by affinity chromatography on chitin. The snake gourd phloem lectin (SGPL) specifically binds chitooligosaccharides and their inhibitory potency increased with increase in size. PAGE and SDS-PAGE studies indicate that SGPL is a heterodimer, in which the two subunits (48 and 53 kDa) are joined by disulfide bonds. Consistent with this, electrospray-ionization mass spectrum yielded the exact mass of the protein as 104,621.8 Daltons. CD studies showed that SGPL contains about 9% α-helix, 39% β-sheet, 20% β-turns and 32% unordered structures and that saccharide binding does not significantly affect its secondary and tertiary structures. Titration calorimetric studies indicate that the dimeric lectin binds two ligand molecules [(GlcNAc)_3–6] with association constants determined at 25 °C being 1.7 × 10⁵ and 3.6 × 10⁵ M⁻¹, for chitotriose and chitohexaose, respectively. Binding of all the chitooligosaccharides is governed by enthalpic forces, whereas the contribution from binding entropies was unfavorable. These results suggest that the SGPL-saccharide interaction is stabilized by hydrogen bonding and van der Waals’ interactions. Enthalpy–entropy compensation was observed for the SGPL-chitooligosaccharide interaction, suggesting that water molecules play a key role in the binding process.     

Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation

Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.     

GalNAc/Gal-binding Rhizoctonia solani agglutinin has antiproliferative activity in Drosophila melanogaster S2 cells via MAPK and JAK/STAT signaling

Rhizoctonia solani agglutinin, further referred to as RSA, is a lectin isolated from the plant pathogenic fungus Rhizoctonia solani. Previously, we reported a high entomotoxic activity of RSA towards the cotton leafworm Spodoptera littoralis. To better understand the mechanism of action of RSA, Drosophila melanogaster Schneider S2 cells were treated with different concentrations of the lectin and FITC-labeled RSA binding was examined using confocal fluorescence microscopy. RSA has antiproliferative activity with a median effect concentration (EC(50)) of 0.35 μM. In addition, the lectin was typically bound to the cell surface but not internalized. In contrast, the N-acetylglucosamine-binding lectin WGA and the galactose-binding lectin PNA, which were both also inhibitory for S2 cell proliferation, were internalized whereas the mannose-binding lectin GNA did not show any activity on these cells, although it was internalized. Extracted DNA and nuclei from S2 cells treated with RSA were not different from untreated cells, confirming inhibition of proliferation without apoptosis. Pre-incubation of RSA with N-acetylgalactosamine clearly inhibited the antiproliferative activity by RSA in S2 cells, demonstrating the importance of carbohydrate binding. Similarly, the use of MEK and JAK inhibitors reduced the activity of RSA. Finally, RSA affinity chromatography of membrane proteins from S2 cells allowed the identification of several cell surface receptors involved in both signaling transduction pathways.     

The rat ovarian lutropin receptor. Purification, hormone binding properties, and subunit composition

The luteinizing hormone/human choriogonadotropin (hCG) receptor from superovulated rat ovary was purified to homogeneity. A novel scheme based on reverse immunoaffinity chromatography using immobilized antibodies to membrane proteins from receptor down-regulated ovary and subsequent two-step affinity purification on hCG-Sepharose was used to isolate homogeneous receptor. The purification method was also compared to an alternate scheme involving lectin affinity chromatography followed by hCG affinity chromatography. The purified receptor obtained by the latter method was heterogeneous and highly aggregated. The hormone binding properties, molecular size, and subunit composition of the purified receptor obtained by either method were identical. The stability of the receptor during and following solubilization was markedly improved by using 20% glycerol. The pure receptor consists of four nonidentical subunits of molecular weight 79,300 (alpha), 66,400 (beta), 55,300 (gamma), and 46,700 (delta) as indicated by polyacrylamide gel electrophoresis under reducing conditions. All receptor subunits generally, but occasionally excepting the alpha-subunit, were specifically labeled with iodinated hCG in membrane and soluble receptor preparations using bifunctional cross-linking agents. Analysis of the cross-linked hormone-receptor complexes under nonreducing conditions showed the molecular mass of the undissociated receptor to be 268,000 daltons. Hormone binding studies demonstrated that the isolated receptor retained all of the specific binding characteristics expected for the luteinizing hormone/hCG receptor. In combination, these results indicate that the functional and structural properties of the receptor were not altered during purification.     

A sialic acid-binding lectin from ovine placenta: purification,specificity and interaction with actin

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since in binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of action to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.     

Serum inhibitors of desialylated glycoprotein binding to hepatocyte membranes.

Identification of the material present in human serum which is responsible for inhibition of binding of desialylated glycoproteins to rat hepatocyte membranes was accomplished by means of affinity chromatography using Sephadex to which the galactose-specific lectin, Ricinus Communis Agglutinin (RCAI) was covalently bound. RCAI-Sephadex was capable of extraction of virtually all of the inhibitory activity from cirrhotic serum. The RCA I-bound inhibitory activity could be eluted with 0.05 M D-galactose. The D-galactose eluate when subjected to radioimmunoelectrophoresis against a number of specific antibodies to human serum glycoproteins produced arcs corresponding to alpha 1-acid glycoprotein, alpha2-macroglobulin, IgG, IgA, and IgM. In another experiment putative terminal galactosyl groups of desialylated glycoproteins in the D-galactose eluate from cirrhotic serum exposed to RCAI-Sephadex were labelled with tritiated borohydride after treatment with galactose oxidase. Subsequent gel electrophoresis showed peaks of radioactivity throughout the area of the gel corresponding to protein molecular weights of the 19 S, 7 S, and 4 S classes. It thus appears that a heterogeneous population of desialylated serum glycoproteins accounts for the inhibition of binding of desialylated glycoprotein to the hepatocyte membrane and that these desialylated glycoproteins are present in small amounts in normal human serum and in greatly increased quantities in serum from patients with cirrhosis.     

Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.     

免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素

 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果．哺乳动物红细胞能够特异地吸附凝集素．用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体．再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果显示,该凝集素的分子量约为７３ ｋ Ｄ．这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的．为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法．     

The distribution and localization of the fucose-binding lectin in rat tissues and the identification of a high affinity form of the mannose/N-acetylglucosamine-binding lectin in rat liver

A small-scale affinity chromatographic procedure was developed to screen for the presence of fucose and mannose/N-acetylglucosamine-binding lectins in small amounts of rat tissues. Of all tissues examined, only the liver contained the fucose-binding lectin, whereas both liver and blood serum contained the mannose/N-acetylglucosamine lectin. By means of immunocytological methods using antibodies to hepatic lectins, the fucose lectin was shown to be uniquely present in Kupffer cells and absent in all other types of rat macrophages examined. The binding and uptake of different neoglycoproteins by nonparenchymal cell fractions of liver indicated that the fucose-binding lectin was either not responsible for the uptake or that more than one lectin was acting. With the identification of another lectin (Mr = 180,000) by the above screening procedure for hepatic lectins and the results of studies in the following paper (Haltiwanger, R.S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7440-7444) two lectins appear to be involved. A small amount of the hepatic mannose/N-acetylglucosamine lectin was found by the above screening procedure to have a higher affinity for L-fucosyl-bovine serum albumin-Sepharose than the majority of the lectin in hepatocytes. This lectin, called the high affinity form, was purified and its properties examined. On a weight basis the high affinity form bound 7-12 times more ligand than the normal form. Its Ka for L-fucosyl-bovine serum albumin was 2.3 X 10(9) M-1 compared to 3.5 X 10(8) M-1 for the normal form. Moreover, the concentrations of monosaccharides required to inhibit the high affinity form were about 3 times less than those required to inhibit binding of the normal form. The two forms, however, have identical molecular weights (32,000) under reducing and nonreducing conditions, bind anti-lectin antibodies in the same way, and give identical peptide maps after V-8 protease digestion. The structural basis for the different binding affinities of the two forms remains unknown.     

Synthesis of cluster mannosides via a Ugi four-component reaction and their inhibition against the binding of yeast mannan to concanavalin A

The Ugi four-component reaction (U-4CR) was utilized to prepare divalent and trivalent cluster mannosides with different scaffolds. The glycoclusters obtained were tested for their relative inhibitory potency against the binding of yeast mannan to concanavalin A by solid-phase enzyme-linked lectin assays (ELLA) using methyl alpha-D-mannopyranoside as a standard. Among them, a divalent mannoside containing aromatic groups showed the strongest binding affinity to concanavalin A.