Purification and characterization of a Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc-specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus

A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on beta-D-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human alpha(2)-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches, i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds beta-D-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acalpha2, 6Galbeta1,4Glc/GlcNAc (6'-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for alpha2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.     

Properties of the lectin from the hog peanut (Amphicarpaea bracteata)

An N-acetyl-D-galactosamine-specific lectin has been isolated from the two seed forms of the hog peanut (Amphicarpaea bracteata) using an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1,3)-[alpha-L-Fuc-(1,2)]-beta-D-Gal (Synsorb A). The affinity-purified lectin appears to be identical in both seed types. Gel filtration on Sephadex G-200 gives a single symmetrical peak corresponding to Mr 135,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows four subunit forms, each of which contains carbohydrate. Limited amino terminal sequencing indicates heterogeneity in two of the first 10 residues. The lectin contains no cysteine. There are four equivalent, noninteracting GalNAc binding sites per 135,000-Da molecule, having an association constant for methyl N-acetyl-alpha-D-galactosaminide of 4.0 X 10(4) M-1. Precipitin and hapten inhibition studies show the lectin to be specific for terminal, nonreducing D-GalNAc units, with a preference for the alpha-anomer and enhanced specificity for the disaccharide, GalNAc alpha 1,3GalNAc. There is also a single adenine binding site per Mr 135,000 lectin molecule with an association constant of 1.3 X 10(6) M-1.     

Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.     

The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.     

The binding characteristics and utilization of Aleuria aurantia,Lens culinaris and few other lectins in the elucidation of fucosyltransferase activities resembling cloned FT VI and apparently unique to colon cancer cells

Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.     

Bowringia milbraedii agglutinin. Specificity of binding to early processing intermediates of asparagine-linked oligosaccharide and use as a marker of endoplasmic reticulum glycoproteins

We have purified a protein with hemagglutinating activity from the seeds of a West African legume, Bowringia milbraedii. The purified protein, designated BMA, has a native Mr = 38,000 on gel filtration and a subunit size of Mr = 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions. Hemagglutination was inhibited most effectively by Man alpha 1----2 linked sugars. Affinity chromatography of oligosaccharides on BMA-Sepharose showed that Man alpha 1----2Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol (where GlcNAcol is N-acetylglucosaminitol) and Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol were retarded on the column, whereas Man alpha 1----3Man beta 1----4GlcNAcol did not bind. Oligomannosidic-type glycans obtained by treatment of [3H] mannose-labeled baby hamster kidney cells with endo-beta-N-acetylglucosaminidase H bound more strongly to BMA-Sepharose and required 10 or 200 mM methyl-alpha-mannoside for elution. Oligosaccharides bearing the sequence Man alpha 1----2Man alpha 1----6Man alpha 1----6Man, i.e. Man9GlcNAc and certain isomers of Man8GlcNAc and Man7GlcNAc, bound more tightly than other Man8 GlcNAc and Man7GlcNAc isomers lacking this sequence. Man6GlcNAc and Man5GlcNAc were weakly bound. These results suggest that BMA binds preferentially to glycoproteins that are subjected to early steps of oligosaccharide processing in the endoplasmic reticulum but not to glycoproteins that are exposed to more extensive processing by Golgi mannosidases. Staining of permeabilized cells with BMA-chromophore conjugates revealed a reticular cytoplasmic pattern consistent with a preferential visualization of the endoplasmic reticulum. BMA staining was less evident in the juxtanuclear regions that were stained brightly with wheat germ agglutinin, a lectin that binds preferentially to sialylated glycoproteins located in Golgi compartments.     

Properties of Lectins in the Root and Seed of Lotononis bainesii

A lectin was purified from the root of Lotononis bainesii Baker by affinity chromatography on Sepharose-blood group substance A + H. The molecular weight of the lectin was estimated by gel filtration to be 118,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lectin was a tetramer composed of two slightly different subunits with respective molecular weights of 32,000 and 35,000. The lectin had a hexose content of 12% (w/w) and contained the sugars fucose, glucosamine, mannose, and xylose. Root lectin hemagglutination was preferentially inhibited by disaccharides with terminal nonreducing galactose residues. Antigens capable of cross-reaction with root lectin antibody were not detected in the seed of L. bainesii.

A lectin from the seed of L. bainesii was partially purified by adsorption to pronase-treated rabbit erythrocytes. The lectin preparation had a molecular weight of approximately 200,000. Galactose and galactono-1,4-lactone inhibited seed lectin hemagglutination but lactose was ineffective. There was no evidence that the root of L. bainesii contained material antigenically related to the seed lectin.

     

Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein. Its carbohydrate-binding specificity was investigated both by sugar-hapten inhibition of hemagglutination and by enzyme-linked immunosorbent assay. The inhibition tests showed the affinity of the lectin to be weakly directed toward sialic acid and lactose, and the enhanced affinity toward trisaccharides containing the NeuAcα2,3Galβ-structure. Importantly, the lectin strongly interacted with glycoconjugates containing NeuAcα2,3Galβ1,3GlcNAc-/GalNAc sequences. This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides.

alpha1,3galactosyltransferase (alpha1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Galalpha1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Galalpha1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8(Galbeta1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing alpha1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Galbeta1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 microM vs. Km = 190 microM for Galbeta1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-beta-D-galactopyranosyl-(1, 4)-2-acetamindo-2-deoxy-beta-D-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of alpha1,3GalT. alpha1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 microM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that alpha1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for alpha1,3GalT activity.     

Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin- Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.      

Sialoforms of dipeptidylpeptidase IV from rat kidney and liver

Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and endopeptidase found in the cell surface membrane of many tissues, was employed as a model membrane glycoprotein to study the expression of sialoforms on cell surface glycoproteins. Native, enzymatically active DPP IV was purified from plasma membranes of kidney and liver by lectin affinity chromatography in conjunction with crown ether anion exchange chromatography. The enzyme was gradient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing (IEF) showed that it consists of several polypeptides of different isoelectric points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and subsequent isoelectric focusing revealed that the differences in isoelectric points were due to differences in the degree of sialylation. Differences in the degree of sialylation between the fractions were also demonstrated by SDS-PAGE under nonreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro -desialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzyme was predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each other by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.     

Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T)-antigen

A lectin (Amaranthin) present in the seeds of Amaranthus caudatus has been isolated by fractionation on DEAE-cellulose followed by affinity chromatography on Synsorb-T beads (Gal beta 1,3GalNAc alpha-O-R-Synsorb). The lectin appeared homogeneous by gel electrophoresis at pH 4.3 and gave a single protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr = 33,000-36,000. A native Mr = 54,000 was determined by gel filtration suggesting that amaranthin exists as a homodimer. Compositional analysis revealed high amounts of acidic and hydroxyamino acids and relatively large amounts of lysine, methionine, and tryptophan for a plant protein. Amaranthin formed a precipitate with asialo-bovine submaxillary mucin, asialo-ovine submaxillary, porcine submaxillary mucin, asialo-fetuin and asialoglycophorin. Hapten inhibition of precipitate formation between amaranthin and asialo-ovine submaxillary indicated that the T-disaccharide and its alpha-linked glycosides (Gal beta 1,3GalNAc alpha-O-R; R = OH, methyl, -(CH2)8-COOCH3, allyl, o-nitrophenyl, or benzyl) were the best inhibitors. N-Acetylgalactosamine, the only monosaccharide which inhibited precipitation, was 350-fold less effective than Gal beta 1,3GalNAc alpha-O-R. Hapten inhibition with derivatives of the T-disaccharide suggested that the C'-4 axial hydroxyl group of the galactosyl moiety, and the C-4 axial hydroxyl group, and the C-2 acetamido group of the GalNAc unit are the most important loci for lectin interaction. NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-(CH2)8CO2CH3 was as potent an inhibitor as Gal beta 1,3GalNAc alpha-O-(CH2)8CO2-CH3, and amaranthin was precipitated by NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-BSA (where BSA is bovine serum albumin), indicating that the amaranthin-combining site tolerates substitutions at the C'-3 hydroxyl group. Amaranthin was precipitated by a Gal beta 1,3GalNAc alpha-O-BSA glycoconjugate but not by the anomeric Gal beta 1,3GalNAc beta-O-BSA glycoconjugate illustrating that the disaccharide must be linked alpha in order to interact with the lectin. Metal ions do not appear to be required for lectin activity. A study of pH dependence showed significant precipitate formation between pH 4 to 9 with a maximum at pH 5. Hapten inhibition and glycoconjugate precipitation assays were also conducted for peanut (Arachis hypogaea) agglutinin. A comparison between the carbohydrate-binding specificities of amaranthin and peanut (Arachis hypogaea) agglutinin is discussed.     

Separation and characterization of two alpha 1,2-mannosyltransferase activities from Saccharomyces cerevisiae

Two GDP-mannose-dependent mannosyltransferase activities (designated M1MT-I and M2MT-I) from Triton X-100 extracts of Saccharomyces cerevisiae mnn1 microsomes were separated by concanavalin A lectin chromatography and partially purified. The two transferases were distinguished by differences in concanavalin A affinity and in carbohydrate acceptor specificity. Analyses of the reaction products indicate that both enzymes are alpha 1,2-mannosyltransferases. M1MT-I utilizes mannose or methyl-alpha-mannoside as acceptor while M2MT-I catalyzes the transfer of mannose from GDP-mannose to unsubstituted nonreducing alpha 1,6-linked mannose residues in the acceptor molecule. M2MT-I activity correlates with the presence of a single alpha 1,2-linked mannose residue at the nonreducing terminus of mnn2mnn9 and mnn2mnn10 outer chain oligosaccharides, and the enzyme may be involved in regulating outer chain elongation.     

Precipitation of concanavalin A by a high mannose type glycopeptide

The interactions of a high mannose type glycopeptide with Concanavalin A has been investigated by quantitative precipitation analysis. The equivalence points of the precipitin curves indicate that the glycopeptide is bivalent for lectin binding. These results and others demonstrate that there are two lectin binding sites per molecule of the glycopeptide: one site on the alpha (1-6) arm of the core beta-mannose residue involving a trimannosyl moiety, and another site on the alpha (1-3) arm of the core beta-mannose residue involving an alpha (1-2) mannobiosyl group. The two sites are unequal in their affinities, and bind by different mechanisms. These results are related to the possible structure-function properties of high mannose type of glycopeptides on the surface of cells.     

Studies on hemagglutinins (lectins) from plasma of murrel fish, family Channidae

Hemagglutinating activity can be identified in the plasma of different species of murrel fish. This activity may be divided into four types according to their agglutinability towards erythrocytes from different sources. Type I plasma agglutinates human blood group A erythrocytes, type II can agglutinate neuraminidase treated human A B O erythrocytes, type III shows no agglutinating activity towards human erythrocytes, while type IV agglutinates human erythrocytes non-specifically. All of them bind to DEAE-cellulose but elute out by different salt concentrations. Type IV plasma is found to be a combination of three separate hemagglutinins, which are separable by sequential binding to human A B O erythrocytes. Blood group A specific lectin activity is purified from this plasma using formalinised A group erythrocytes. The apparent homogeneity of this purified lectin is established by polyacrylamide gel electrophoresis, isoelectric focusing and immunodiffusion. This agglutinin is antigenically identical with that isolated from type I plasma by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose column. Their molecular weights are also found to be identical (Mr 140,000) in polyacrylamide gel electrophoresis, having two identical subunits. Forssman glycolipid (0.03 mM) was found to be the most potent inhibitor of agglutination, although Gal beta 1-3 GalNAc (0.09 mM) is also a good inhibitor. Exhaustive dialysis of the purified lectin (hemagglutinin) against EDTA denatures it irreversibly by dissociating it to its subunit structure. Thus human A group agglutinating activity isolated from type I and type IV plasma are identical.     

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.     

Purification to homogeneity and properties of mannosidase II from mung bean seedlings

Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.     

Purification and Characterization of the Acidic Lectin from Winged Bean Seeds

Winged bean acidic lectin was purified by DEAE-Sephadex A-50 and affinity chromatography on N-acetylgalactosamine-agarose gel. The purified lectin was a glycoprotein homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and gel filtration. The molecular weight of the lectin was 52,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single component of molecular weight of 27,000. Its isoelectric point was 5.5. The acidic lectin was rich in acidic amino acids, and contained 2mol of methionine but no cystine. It also agglutinated both trypsinized and untreated human erythrocytes (types A, B, AB and O), but not rabbit erythrocytes. The hemagglutination was inhibited by d-galactose and related sugars. Modification of the acidic lectin with N-bromosuccinimide caused a concomitant loss of the hemagglutinating activity with oxidation of tryptophan residue. The acidic lectin was immunologically different from the purified winged bean basic lectin by double immunodiffusion using antiserum raised against the basic lectin.     

One-step purification of murine IgM and human alpha 2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 M methyl alpha-D-mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl alpha-D-mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. alpha 2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man(alpha 1,3)Man units are bound to the immobilized lectin.     

Subunit structure of the purified human placental insulin receptor. Intramolecular subunit dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Insulin receptors purified from human placental membranes by gel-filtration and insulin-agarose affinity chromatography were found to be composed of eight different high molecular weight complexes as identified by nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The subunit stoichiometry of these different high molecular weight forms of the insulin receptor were determined by comparisons of silver-stained gel profiles with the autoradiograms of 125I-insulin specifically cross-linked to the alpha subunit and [gamma-32P]ATP specifically autophosphorylated beta subunit gel profiles. Two-dimensional SDS-polyacrylamide gel electrophoresis in the absence and presence of reductant confirmed the subunit stoichiometries as alpha 2 beta 2, alpha 2 beta beta 1, alpha 2 (beta 1)2, alpha 2 beta, alpha 2 beta 1, alpha 2, alpha beta, and beta, where alpha is the Mr = 130,000 subunit, beta is the Mr = 95,000 subunit, and beta 1 is the Mr = 45,000 subunit. Treatment of the insulin receptor preparations with oxidized glutathione or N-ethylmaleimide prior to SDS-polyacrylamide gel electrophoresis increased the relative amount of the alpha 2 beta 2 complex concomitant with a total disappearance of the alpha 2 beta, alpha 2 beta 1, alpha 2, and free beta forms. The effects of oxidized glutathione were found to be completely reversible upon extensive washing of the treated insulin receptors. In contrast, the effects of N-ethylmaleimide were totally irreversible by washing, consistent with known sulfhydryl alkylating properties of this reagent. The formation of these lower molecular weight insulin receptor subunit complexes was further demonstrated to be due to SDS/heat-dependent intramolecular sulfhydryl-disulfide exchange occurring within the alpha 2 beta 2 complex. These studies demonstrate that the largest disulfide-linked complex (alpha 2 beta 2) is the predominant insulin receptor form purified from the human placenta with the other complexes being generated by proteolysis and by internal subunit dissociation.