Characterization of sugar chain structures of human alpha-fetoprotein by lectin affinity electrophoresis. ibarahp@oka.urban.ne.jp

Alpha-Fetoprotein (AFP) glycoforms, defined as AFP with different chemical structures of carbohydrate, were analyzed by affinity electrophoresis with several lectins of known specificities against complex-type oligosaccharides. Serum AFP samples from cord blood on full-term delivery and from patients with hepatocellular carcinoma and extrahepatic malignancies including gastrointestinal tumors and yolk sac tumors were used. Two-dimensional lectin affinity electrophoresis and also lectin affinity chromatography coupled with lectin affinity electrophoresis were employed. More than ten AFP glycoforms were identified or characterized using the above-mentioned AFP samples. Known specificities of the lectins against complex-type oligosaccharides were refined or their additional specificities were found in this study. Lectin appeared to have specificity against carbohydrates by recognizing not only specific residues but also the whole carbohydrate molecule containing the residues, resulting in differential affinities for the lectin.     

Purification and characterization of the agglutinins from the sponge Aaptos papillata and a study of their combining sites

The lectins from the sponge Aaptos papillata were isolated by affinity chromatography using polyleucyl blood group A + H substances from hog stomach linings as an absorbent and eluting with 3 M MgCl2. Further separation on diethylaminoethylcellulose and preparative disc electrophoresis on polyacrylamide gave the three fractions, Aaptos lectins I, II, and III. They were essentially homogenous in polyacrylamide electrophoresis and sedimentation analysis: a small second component was seen in lectins I and II in immunoelectrophoresis at high concentration. The S20,W0 values for Aaptos lectins I, II, and III were 3.5, 6.0, and 5.5. By electrophoresis in sodium dodecyl sulfate with an without beta-mercaptoethanol Aaptos lectin I showed two bands corresponding to molecular weights of 12 000 and 21 000; Aaptos lectins II and III gave only one band of molecular weight of 16 000. In isoelectric focusing, Aaptos lectin I showed bands at pH 4.7 and 5.4 and in the range between 6.8 and 7.6, while Aaptos lectins II and III were almost identical with bands at pH 3.8, 4.7 to 4.9, and 5.3. Aaptos lectin I differed from II and III in amino acid composition but the latter two were very similar. They contained no significant carbohydrate. Aaptos lectin I reacted best with blood group substances with terminal nonreducing N-acetyl-D-glucosamine residues precipitating about two-thirds of the lectin N added while blood group substances with terminal nonreducing DGalNAc were almost inactive. However, Aaptos lectin II was completely precipitated by blood group substances and glycoproteins containing terminal DGalNAc, DGlcNAc, or sialic acid residues. Aaptos lectin III had a precipitation pattern similar to Aaptos lectin II. DGlcNAc but not DGalNAc inhibited precipitation of Aaptos lectin I by blood group substances and N, N', N', N'-tetraacetylchitotetraose was the best inhibitor and was 2000 times more active than DGlcNAc. Precipitin reactions with Aaptos lectin II were inhibited by equal amounts of DGlcNAc and by sialic acid which were four times more potent than DGalNAc. N,N',N'-triacetylchiotriose was the best inhibitor and was 13 times better than DGlcNAc. At 37 degrees C three to four times higher amounts of inhibitor were necessary to inhibit precipitation of Aaptos lectin II than were needed at 4 degrees C, indicating higher affinity of blood group substances for Aaptos lectin II with increasing temperature. Aaptos lectin I was precipitated by the monofunctional hapten p-nitrophenyl-alphaDGalNAc, while p-nitrophenyl-betaDGalNAc did not precipitate and was a good inhibitor. Both phenomena indicate involvement of hydrophobic bonds.     

Multimodal application of lectin affinity electrophoresis of alpha-fetoprotein

Multimodal application of lectin affinity electrophoresis of alpha-fetoprotein (AFP) glycoforms is reviewed. Crossed affinity immunoelectrophoresis developed by B?g-Hansen and others was extended to tandem-lectin affinity electrophoresis by tandem lining of two different lectin gels and to mixed-lectin affinity electrophoresis. By introducing an antibody-affinity blotting technique for detection of separated glycoforms of AFP, several two-dimensional combinations of lectin affinity electrophoresis became possible: two different lectins for the first and second dimension electrophoresis, lectin-gradient affinity electrophoresis, and electrophoretic separation of lectin isoforms in the first-dimension electrophoresis, followed by affinity electrophoresis against the separated lectin isoforms. Usefulness of the different modalities of lectin affinity electrophoresis for several analytical purposes has been described.     

Interactions between lonidamine and beta- or hydroxypropyl-beta-cyclodextrin

Equilibrium dialysis and Scatchard plots were used to establish that human and rabbit paraoxonases both have two calcium binding sites. Independent-site and stepwise constant analyses were used to calculate a higher affinity site (Kd1) of 3.6 +/- 0.9 x 10(-7) M for human A paraoxonase, and 1.4 +/- 0.5 x 10(-8) M for rabbit paraoxonase, and a lower affinity site (Kd2) of 6.6 +/- 1.2 x 10(-6) M for human A paraoxonase, and 5.3 +/- 0.94 x 10(-6) M for rabbit paraoxonase. In both species, the higher affinity sites were found to be essential to maintain hydrolytic activity; complete removal of calcium led to irreversible inactivation. The lower affinity sites were required for catalytic activity, and their binding of calcium was reversible. Experimentally estimated values of Kd2 based on the concentration of calcium required to obtain half the maximum enzymatic activity were 3 microM for human A and B paraoxonases, and also in the order of 3 microM for rabbit paraoxonase, using three different substrates. Calcium was the only metal found that protects against denaturation and also confers hydrolytic activity with these two mammalian paraoxonases.     

Mag-Fura-2 (Furaptra) exhibits both low (microM) and high (nM) affinity for Ca2+

Based on studies using high-affinity Ca2+ probes (dissociation constant (Kd) = 0.15-0.3 microM), steady-state [Ca2+]in is believed to be in the nanomolar range in most cells. However, probes with lower affinity indicate that [Ca2+]in may increase to micromolar levels during activation of specific cell functions, e.g., contraction. These conclusions rely on accurate knowledge of the Kd of the dyes for Ca2+. Mag-Fura-2 (also known as Furaptra) is a low-affinity Ca2+ indicator (Kd ca. 50 microM) which has been used for such studies. In the present work, Mag-Fura-2 is shown to respond to changes in cytosolic Ca2+ in the submicromolar range. In vitro, and in situ titration of Mag-Fura-2 in A7r5 cells, demonstrate that Mag-Fura-2 exhibits both high- and low-affinity for Ca2+. Moreover, pH affects both high and low affinity Ca2+ binding site. Since Mag-Fura-2 has been used to study Ca2+ within specific subcellular compartments, the present observations indicate that knowledge of factors such as ambient pH of these compartments is required to accurately interpret Ca2+ responses. Furthermore, the sensitivity of Mag-Fura-2 at submicromolar levels must be considered for accurate determination of Ca2+ in specific compartments believed to exhibit high micromolar levels of Ca2+.     

Comparison of carbohydrate structures of serum alpha-fetoprotein by sequential glycosidase digestion and lectin affinity electrophoresis

Serum alpha-fetoprotein (AFP) is a glycoprotein of which the sugar chain is considered to show structural changes with malignancies. Microheterogeneity of the serum AFP carbohydrate structure was studied in samples from 35 patients with benign and malignant diseases. Sera were digested directly, extensively, and sequentially with sialidase. beta-galactosidase and beta-N-acetylhexosaminidase. Before and after digestion, sera were examined by means of lectin affinity electrophoresis using eight lectins. Relationships between AFP carbohydrate structures and liver diseases were elucidated by the lectin-reactive profiles and the effect of glycosidase digestion. More than 94% of the AFP carbohydrate structures found in patients with benign and malignant liver diseases were biantennary complex-type oligosaccharides. Changes in the AFP carbohydrate structures at the early stage of hepatocellular carcinoma revealed the addition of alpha 1-->6 fucose to the reducing terminal N-acetylglucosamine and monosialylated AFPs. In both advanced hepatocellular carcinoma and AFP producing extrahepatic malignancies, AFP carbohydrate structures were characterized as the further addition of beta 1-->4 N-acetylglucosamine and heterogeneity in the galactose and N-acetylglucosamine residues. Sequential glycosidase digestion and lectin affinity electrophoresis is useful for analysing the carbohydrate structures of serum glycoprotein.     

Microalbuminuria, arterial hypertension and the cardiovascular risk

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc-derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.     

Characterization and representative structures of N-oligosaccharides bound to apolipoprotein H

We studied the structure of N-linked carbohydrates bound to apolipoprotein H by a combination of two methods which make use of lectins. Digoxigenin-labelled lectins are used for the structural characterization of carbohydrate chains of glycoproteins. Concanavalin A lectin affinity chromatography was used to analyse apolipoprotein H according to the characteristics of its carbohydrate chain inner to sialic acid residues. Our results from digoxigenin-labelled lectins analysis showed that apolipoprotein H gave positive bands to SNA, DSA, GNA, PNA and AAA lectins. Apolipoprotein H gave a negative band when reacted with MAA lectin. When we applied apolipoprotein H onto the Concanavalin A lectin column no detectable amounts of protein were eluted with Concanavalin A buffer. After adding a buffer with low sugar concentration (10 mM glucoside) a large amount of apolipoprotein H was recovered. These molecules of apolipoprotein H weakly bound to the lectin. When a higher sugar concentration (500 mM mannoside) was added most of the sample applied was eluted. These molecules of apolipoprotein H firmly bound to the column having high affinity for the lectin. These results combined with those coming from the digoxigen-labeled lectins method enable us to understand the inner structure of carbohydrate chains with their outer branches. Molecules of apolipoprotein H which weakly bind to Concanavalin A could bear complex N-glycans organized in biantennary or truncated hybrid structures. Firmly bound apolipoprotein H referred to molecules rich in N-glycan hybrid structures. They have an outer branch belonging to the high mannose carbohydrate chains which explain the ability to bind to the column and an other main branch bearing the sequence galactose beta-(1-4)-N-acetylglucosamine beta-(1-2) mannose. Galactose could be the terminal sugar or, alternatively, be masked with sialic acid alpha-(2-6) terminally linked.     

Pig heart fumarase contains two distinct substrate-binding sites differing in affinity

A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (Kd being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which Kd is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (Kd = 0.6 to 2.2 microM, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).     

Macromolecular recognition: effect of multivalency in the inhibition of binding of yeast mannan to concanavalin A and pea lectins by mannosylated dendrimers

The synthesis and binding properties of a new family of high affinity alpha-D-mannopyranoside ligands are described. The synthesis of the new multivalent ligands is based on the scaffolding of multiantennary branches of L-lysine residues having electrophilic N-chloroacetylated end groups as core structures. An alpha-D-mannopyranoside with p-substituted aryl aglycon ending with a thiol group was prepared and covalently attached to each of the branches of the dendritic structures. The resulting glycodendrimers with 2 (12), 4 (14), 8 (16), and 16 (18) mannoside residues were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl and p-nitrophenyl alpha-D-mannopyranosides as standards. Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohemagglutinin (Canavalia ensiformis, concanavalin A) and to pea lectin (Pisum sativum) were determined. Analogous mannosylated copolyacrylamides were also prepared for comparison. The IC50 values were also plotted as a function of dendrimer valencies. The inhibitions showed 16-mer 18 to be approximately 600- and 2000-fold more potent than methyl alpha-D-mannopyranoside, and 66- and 1383-fold more potent than p-nitrophenyl alpha-D-mannopyranosides with Con A and pea lectins, respectively. Even when these numbers are expressed relative to single mannopyranoside residues per dendrimers, the relative potencies against the aromatic mannoside are still 4- and 86-fold better against Con A and pea lectins. These results unequivocally indicate that the optimum inhibitory binding properties of the new mannosylated dendrimers vary with both dendrimers and lectin valencies.     

Rheologic characterization of vegetal lectins by dissociation of induced erythrocyte agglutinates

Energy evolved from hemagglutination reaction or spent in dissociating erythrocyte agglutinates has been proved to be an excellent parameter for analyzing cell-cell interactions mediated by bridging molecules such as antibodies or lectins. We developed a new rheo-optical method to estimate the energy of dissociation of red blood cell agglutinates. In a Couette shear field agglutinates can be dissociated until a suspension of monodispersed cells is obtained. Intensity of light backscattered by suspended agglutinates increases during their mechanical dissociation. Variation of backscattered light intensity correlates with the energy spent in the process. The adhesive energy of erythrocyte agglutination induced by lectins has been estimated by applying this method. Two specific lectins (Dolichus Biflorus agglutinin and Ulex Europaeus agglutinin) and a new lectin obtained from Amarantus Cruentus seeds which specificity is unknown were studied. Results obtained in this work for Dolichus Biflorus lectin are comparable with values published by other authors. An asymptotic decrease of adhesive energy was observed when the mechanical dissociation was applied several times on the same sample. Our results suggest that the cell detachment is accompanied by the extraction of membrane receptors. This finding is consistent with results obtained by other authors.     

Proton efflux in human skeletal muscle during recovery from exercise

In recovery from exercise, phosphocreatine resynthesis results in the net generation of protons, while the net efflux of protons restores pH to resting values. Because proton efflux rate declines as pH increases, it appears to have an approximately linear pH-dependence. We set out to examine this in detail using recovery data from human calf muscle. Proton efflux rates were calculated from changes in pH and phosphocreatine concentration, measured by 31P magnetic resonance spectroscopy, after incremental dynamic exercise to exhaustion. Results were collected post hoc into five groups on the basis of end-exercise pH. Proton efflux rates declined approximately exponentially with time. These were rather similar in all groups, even when pH changes were small, so that the apparent rate constant (the ratio of efflux rate to pH change) varied widely. However, all groups showed a consistent pattern of decrease with time; the halftimes of both proton efflux rate and the apparent rate constant were longer at lower pH. At each time-point, proton efflux rates showed a significant pH-dependence [slope 17 (3) mmol x l(-1) x min(-1) x pH unit(-1) at the start of recovery, mean (SEM)], but also a significant intercept at resting pH [16 (3) mmol x l(-1) x min(-1) at the start of recovery]. The intercept and the slope both decreased with time, with halftimes of 0.37 (0.06) and 1.4 (0.4) min, respectively. We conclude that over a wide range of end-exercise pH, net proton efflux during recovery comprises pH-dependent and pH-independent components, both of which decline with time. Comparison with other data in the literature suggests that lactate/proton cotransport can be only a small component of this initial recovery proton efflux.     

Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C

BACKGROUND: Isoelectric focusing (IEF) of tear proteins has not yet been carried out in a satisfactory way. Two-dimensional (2D) electrophoresis, especially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tear proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. METHODS: Ampholines, covering a broad range from pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubation with a group II lectin and two group V lectins was performed. RESULTS: Tear proteins could be separated into 31 single bands. Tear-specific pre-albumin (TSPA), lactoferrin, sIgA, IgG and lysozyme were found to be main components. Isoelectric points (IEPs, pls) of all proteins separated were determined by comparison with IEF standards. 2D patterns of IEF and SDS electrophoresis were obtained for the main subunit components of lactoferrin, sIgA, TSPA, and lysozyme. An additional new component of considerable concentration was focused at pI 8.6 with a subunit MW of 14 kDa. With s-WGA a component at an IEP of 5.2 was visualized, representing transferrin. With SNA, lactoferrin stained as a sharp main band at pI 5.1 with three additional weaker bands at IEPs from 4.8 to 4.9. At IEPs between 4.4 and 6.1, multiple components of sIgA were stained with MAA. The sugar specificity of transferrin at pI 5.2 was beta-GlcNAc. Lactoferrin showed glycation with NANA-alpha-2-6-Gal or NANA-alpha-2-6-GalNAc, whereas the sugar specificity of sIgA was NANA-alpha-2-3-Gal. CONCLUSIONS: The investigative strategy applied here, including IEF alone, in combination with SDS-electrophoresis, and SDS-electrophoresis followed by lectin staining proved to be a reproducible method for tear protein analysis of hitherto unexperienced capacity. Lectin-stained bands of native tear proteins are not uniformly glycated by one sugar residue, but show various sugar specificities. IgA as a whole molecule is specifically glycated with NANA-alpha-2-3-Gal.     

The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea)

Peanut agglutinin was purified by affinity chromatography on Sepharose-epsilon-aminocaproyl-beta-D-galactopyranosylamine. The purified lectin obtained in a yield of 150 mg/100 g of defatted peanut was homogeneous on polyacrylamide gel electrophoresis, ultracentrifugation, and gel filtration. This intrinsic sedimentation coefficient (So20,w) and the intrinsic diffusion coefficient (Do20,w) were estimated at pH 7.4 as 5.7 +/- 0.1 S and 5.0 X 10(-7) cm2s(-1), respectively. The molecular weight of the agglutinin, determined by sedimentation and diffusion and by gel filtration, was found to be 110,000. Disc gel electrophoresis and gel filtration, both in the presence of sodium dodecyl sulfate, gave a single component of Mr = 27,500 suggesting that the lectin is a tetramer composed of four subunits. Four alanine residues per 110,000 g were found by NH2-terminal analysis and the sequence of the five NH2-terminal amino acids was: ALa-Glu-Ser-Val-Thr. Each cycle in a sequenator gave a single amino acid, suggesting that the four subunits are identical. Peanut agglutinin does not contain covalently bound sugar; it is devoid of cysteine and cystine, low in methionine, histidine, and tryptophan, but rich in acidic and hydroxyamino acids. The lectin agglutinated erthrocytes of human ABO blood types equally well, but only after they have been treated with neuraminidase. Of the monosaccharides tested for inhibition of hemagglutination only D-galactose and alpha- and beta-D-galactosides were active. High inhibitory activity was found with the Discaccharide DGalbeta(1 in equilibrium 3)DGalNAc and with the disialylated glycoproteins: alpha1-acid glycoprotein, fetuin, glycophorin, and human blood group NN or MM antigen. These desialylated glycoproteins also reacted with the lectin to form precipitin bands in Ouchterlony double diffusion in agar.     

Garlic (Allium sativum) lectins bind to high mannose oligosaccharide chains

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.     

Clinical pharmacokinetics of vasodilators. Part II

The legume lectins are a large family of homologous carbohydrate binding proteins that are found mainly in the seeds of most legume plants. Despite their strong similarity on the level of their amino acid sequences and tertiary structures, their carbohydrate specificities and quaternary structures vary widely. In this review we will focus on the structural features of legume lectins and their complexes with carbohydrates. These will be discussed in the light of recent mutagenesis results when appropriate. Monosaccharide specificity seems to be achieved by the use of a conserved core of residues that hydrogen bond to the sugar, and a variable loop that determines the exact shape of the monosaccharide binding site. The higher affinity for particular oligosaccharides and monosaccharides containing a hydrophobic aglycon results mainly from a few distinct subsites next to the monosaccharide binding site. These subsites consist of a small number of variable residues and are found in both the mannose and galactose specificity groups. The quaternary structures of these proteins form the basis of a higher level of specificity, where the spacing between individual epitopes of multivalent carbohydrates becomes important. This results in homogeneous cross-linked lattices even in mixed precipitation systems, and is of relevance for their effects on the biological activities of cells such as mitogenic responses. Quaternary structure is also thought to play an important role in the high affinity interaction between some legume lectins and adenine and a series of adenine-derived plant hormones. The molecular basis of the variation in quaternary structure in this group of proteins is poorly understood.     

Diocleinae lectins are a group of proteins with conserved binding sites for the core trimannoside of asparagine-linked oligosaccharides and differential specificities for complex carbohydrates

The seed lectin from Dioclea grandiflora and jack bean lectin concanavalin A (ConA) are both members of the Diocleinae subtribe of Leguminosae lectins. Both lectins have recently been shown to possess enhanced affinities and extended binding sites for the trisaccharide, 3,6-di-O-(alpha-D-mannopyranosyl)-D-mannose, which is present in the core region of all asparagine-linked carbohydrates (Gupta, D., Oscarson, S., Raju, S., Stanley, P. Toone, E. J. and Brewer, C. F. (1996) Eur. J. Biochem. 242, 320-326). In the present study, the binding specificities of seven other lectins from the Diocleinae subtribe have been investigated by hemagglutination inhibition and isothermal titration microcalorimetry (ITC). The lectins are from Canavalia brasiliensis, Canavalia bonariensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. Hemagglutination inhibition and ITC experiments show that all seven lectins are Man/Glc-specific and have high affinities for the core trimannoside, like ConA and D. grandiflora lectin. All seven lectins also exhibit the same pattern of binding to a series of monodeoxy analogs and a tetradeoxy analog of the trimannoside, similar to that of ConA and D. grandiflora lectin. However, C. bonariensis, C. floribunda, D. rostrata, and D. violacea, like D. grandiflora, show substantially reduced affinities for a biantennary complex carbohydrate with terminal GlcNAc residues, while C. brasiliensis, D. guianensis, and D. virgata, like ConA, exhibit affinities for the oligosaccharide comparable with that of the trimannoside. Thermodynamic data obtained by ITC indicate different energetic mechanisms of binding of the above two groups of lectins to the complex carbohydrate. The ability of the lectins to induce histamine release from rat peritoneal mast cells is shown to correlate with the relative affinities of the proteins for the biantennary carbohydrate.     

A probarley lectin processing enzyme purified from Arabidopsis thaliana seeds

An aspartic proteinase was purified from the seeds of Arabidopsis thaliana (ecotype RLD) using affinity chromatography on pepstatin-agarose and ion exchange chromatography. The purified enzyme is optimally active at pH 3.5 and completely inhibited by pepstatin A. The purified Arabidopsis aspartic proteinase contains four subunits (apparent molecular weights 31, 28.5, 15 and 6 kDa), two of which are probably linked by disulfide bridges. These properties are similar to the aspartic proteinase previously isolated from barley seeds. The amino acid sequence of the peptide subunits corresponds exactly with the sequence of the previously isolated cDNA for the Arabidopsis aspartic proteinase. The Arabidopsis enzyme processed probarley lectin in vitro at the carboxy-terminus between phenylalanine and alanine, the same place where the barley enzyme cleaves the lectin in vitro. The aspartic proteinase appears to be the major enzyme processing the lectin in seeds as pepstatin A inhibited this activity in a crude seed extract.     

Distribution and localization of galectin purified from Rana catesbeiana oocytes

Galectins are a family of lectins that recognize beta-D-galactosides independently of calcium ions, and are widely distributed in animals. To characterize a galectin previously purified from oocytes of Rana catesbeiana (American bullfrog), we studied its distribution and localization in several tissues from this frog. Hemagglutination assay and western blotting showed that this lectin is present in many tissues including the liver, skin, kidney, skeletal muscle, and sciatic nerve, but is particularly concentrated in the ovary. Light microscopic immunohistochemistry showed that this lectin is localized in such places as cell-cell junctions, basement membranes, extracellular matrix, or secretory substances in several organs, indicating that this galectin is mainly distributed extracellularly. However, in the ovary, light microscopy showed that this lectin is present in or associated with the yolk platelet. Electron microscopy further revealed that it is localized in the periphery of the yolk platelet (the yolk plasm), but not in the cortical granule. These results indicate that Rana oocytes contain abundant galectin in their yolk platelets in contrast to Xenopus laevis oocytes, which have been found not to contain galectins but other classes of lectins in their yolk platelets and cortical granules.     

Lectin-induced apoptosis of tumour cells

The mechanisms of cytotoxic activity of Griffonia simplicifolia 1-B4 (GS1B4) and wheat germ agglutinin (WGA) lectins against various murine tumour cell lines were studied. Tumour cells that lack lectin-binding carbohydrates were resistant to lysis by these lectins. However, YAC-1 cells that expressed GS1B4 lectin-binding sites showed low sensitivity to lysis. To further analyse the relative importance of cell surface carbohydrates in lectin cytotoxicity, BL6-8 melanoma cells, which do not express the alpha 1,3 galactosyltransferase (alpha 1,3GT) gene and cell surface alpha-galactosyl epitopes reacting with GS1B4 lectin, were transfected with cDNA encoding alpha 1,3GT. After transfection, BL6-8 cells expressed high levels of GS1B4-binding alpha-galactosyl epitopes, but remained resistant to lysis by GS1B4 lectin, suggesting that the presence of lectin-binding epitopes, while essential, is not sufficient for tumour cell lysis and probably some intracellular mechanisms are involved in the regulation of lectin-mediated cytotoxicity. We found that the GS1B4 and WGA lectins induced apoptosis with DNA fragmentation of sensitive, but not resistant, tumour cell lines. DNA fragmentation, as well as tumour cell lysis, was blocked in the presence of the specific inhibitory sugar. To determine whether binding of the lectin to cell surface carbohydrates is sufficient to trigger tumour cell lysis, lectin-sensitive CL8-1 melanoma cells were incubated with GS1B4 lectin immobilized on agarose beads. Although these tumour cells bind to the immobilized lectin, it failed to trigger tumour cell death, suggesting that only soluble lectin is capable of tumour cell lysis and lectin internalization is probably required for their lysis.(ABSTRACT TRUNCATED AT 250 WORDS)