Interaction of the alpha-mannosidase from Canavalia ensiformis with the lectin from the same plant, concanavalin A

The specific interaction between the lectin concanavalin A and the alpha-mannosidase from the Leguminosa Canavalia ensiformis was studied by means of laser nephelometry and affinity chromatography. Both proteins react optimally within a certain stoichiometrical range. Interaction is restricted to a narrow pH interval (around pH 5) and to low ionic strengths (less than 10mM NaCl). Neither the sugar-binding site of the lectin nor the catalytic and the hydrophobic sites of the enzyme participate in the interaction. The conformation of the enzyme at pH 5 which favours the interaction can be arrested by immobilization. After this, the enzyme is able to bind the lectin even at pH 8 where no interaction takes place between the dissolved proteins.     

In vivo binding partners of the Lens culinaris lectin

The immobilized lectin from the lentil (Lens culinaris) specifically binds two fractions out of the L. culinaris seed globulins. Both fractions are displaced from the lectin at low pH values. In addition, fraction I fails to interact at high ionic strengths, and fraction II in the presence of glucose or other lectin-specific sugars. The behaviour in zonal isoelectric precipitation and electrophoretical patterns indicate that both fractions represent subpopulations of the storage proteins. The interaction as demonstrated by affinity chromatography is corroborated by nephelometry: If the dissolved proteins (lectin plus fraction I or fraction II) are mixed under proper conditions the solutions become turbid. An even more pronounced interaction is observed if the lectin is reacted with both fractions at the same time. Seed albumins able to interact with the immobilized lectin include the dissolved lectin and two glycosidases (alpha-mannosidase, alpha-galactosidase) all of which are located in the protein bodies. A third glycosidase (beta-galactosidase) from outside of the protein bodies does not bind to the lectin. The results are discussed in view of the possibility that lectins may serve as packaging aids for other proteins in the protein bodies.     

Evidence for the occurrence of calmodulin in the yeasts Candida albicans and Saccharomyces cerevisiae

The lectin extracted from Vicia graminea seeds has been purified by conventional techniques but such procedures did not give a satisfactory yield. We describe a new purification which involves 3 steps after obtention of the crude extract. The first step is based on affinity chromatography on con A—Sepharose. Further purification steps were performed on DEAE-Sephacel chromatography and ultrogel AcA₄₄ gel filtration. The homogeneity of the lectin was demonstrated by polyacrylamide gel electrophoresis. Purification of the lectin by this new method was less time consuming, the yield was higher and the specific activity increased.     

Relationship between lectin, alpha-, beta-glucosidase, and beta-galactosidase activities of Azospirillum]

The activities of alpha-glucosidase, beta-glucosidase, and beta-galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol-acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied among the azospirilla studied. The cofunction of the A. brasilense Sp7 lectin and beta-galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum 59b lectin and glucosidases was revealed. The lectin from A. lipoferum 59b may possess saccharolytic activity.     

Nuclear protein kinase activity in glucagon-stimulated perfused rat livers (Short Communication)

A simple method of purification of alpha-mannosidase from jack-bean meal is described which yields a product free of beta-N-acetylglucosaminidase activity.     

The purification and properties of extracellular glycosidases of the cellular slime mould Dictyostelium discoideum

The purification of beta-N-acetylhexosaminidase, alpha-glucosidase, alpha-mannosidase and beta-glucosidase from the spent growth medium of Dictyostelium discoideum strain Ax-2 myxamoebae is described. beta-N-Acetylhexosaminidase and alpha-glucosidase were obtained in high yield and as homogeneous preparations whereas the alpha-mannosidase preparation consisted of two electrophoretically distinct isoenzymes. The physical, chemical and kinetic properties of these enzymes are described.     

Utility of pentose colorimetric assay for the purification of potato lectin, an arabinose-rich glycoprotein

Potato lectin (Solanum tuberosum agglutinin, STA) is an unusual glycoprotein containing approximately 50% carbohydrates by weight. Of the total carbohydrates, 92% is contributed by L-arabinose, which are O-linked to hydroxyproline residues. The ferric chloride-orcinol assay (Bial’s test), which is specific for pentoses has so far been used only for the determination of free pentoses in biological samples. However, this colorimetric assay has not been used for the detection of pentoses in bound form as it occurs in Solanaceae lectins (potato, tomato, and Datura lectins). Utilizing the pentose colorimetric assay for monitoring the presence of potato lectin, a simpler and shorter procedure for the purification of this lectin from potato tubers has been developed. The yield of potato lectin (1.73 mg per 100 g potato tuber) is twice compared to the yields reported in earlier procedures. Although potato lectin is well known for its specificity to free trimers and tetramers of N-acetyl-D-glucosamine (GlcNAc), it possesses a similar specificity to the core (GlcNAc)₂ of N-linked glycoproteins. The utilization of the pentose assay in the purification of arabinose-rich lectins/agglutinins obviates the necessity for the use of agglutination assay in the various purification steps. The pentose assay appears to be a simple and convenient colorimetric assay for detecting any pentose-rich glycoprotein in plant extracts. The utility of the pentose assay appears to have a significant potential in the detection of hydroxyproline-rich glycoproteins (HRGPs), which are generally O-arabinosylated.     

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha- mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.      

Purification of an N-acetyl-D-glucosamine specific lectin (P.B.A.) from epidermal cell membranes of Pieris brassicae L

We report the isolation and the purification of an N-acetyl-D-glucosamine specific lectin capable of agglutinating either fixed trypsinized rabbit erythrocytes or chitin particles. An agglutinin assay based on the affinity of this lectin for the chitin was devised with fluorescent particles of scorpion cuticle to measure lectin activity during purification steps. Lectin was isolated from epidermal cell membranes; its molecular weight was determined by gel filtration and polyacrylamide electrophoresis in sodium dodecyl sulfate. Mr was estimated to be 43,000. Lectin could be constituted by two subunits. Mr of which was estimated to be 23,000. The specificity of this lectin against N-acetyl-D-glucosamine and its oligomers suggests a possible role in the dynamics of these saccharides during the cuticle cycle.     

Isolation and properties of beta-D-galactoside-specific lectin from chick embryo thigh muscle

A beta-galactoside-specific lectin, capable of agglutinating trypsinized rabbit erythrocytes, was isolated from 13-day-old embryonic chick thigh muscle and purified 1000-fold by affinity chromatography on asialofetuin/Sepharose and Sephadex G-100. A quantitative hemagglutinin assay based on the disappearance of single erythrocytes in a Coulter electronic particle counter was devised to measure lectin activity at different steps of purification. The molecular weight of the lectin was determined by gel filtration to be approximately 31,000, whereas polyacrylamide gel electrophoresis in sodium dodecyl sulfate gave a value of approximately 15,000, suggesting that the lectin is a dimer. The lectin is unstable below pH 5, and it requires the presence of dithiothreitol for the retention of maximal activity. The major portion of this lectin is membrane-bound; only 50% of the activity present in the muscle homogenate could be isolated in soluble form by extraction of muscle acetone powder with a buffer of high ionic strength. In view of the lack of a calcium requirement for its activity, the role of this lectin in myoblast fusion, a calcium-dependent phenomenon, is not clear.     

The soluble form of rat liver alpha-mannosidase is immunologically related to the endoplasmic reticulum membrane alpha-mannosidase

The soluble alpha-mannosidase of rat liver, originally described as a cytoplasmic alpha-mannosidase, has been purified to homogeneity by conventional techniques. The purified enzyme has an apparent molecular weight of 350,000 and is composed of 107-kDa subunits. The soluble alpha-mannosidase has the same enzymatic properties as the endoplasmic reticulum (ER) membrane alpha-mannosidase of rat liver (Bischoff, J., and Kornfeld, R. (1983) J. Biol. Chem. 258, 7909-7910) which is believed to play a role in oligosaccharide processing in the rough ER. Like the membrane-bound ER alpha-mannosidase, the soluble alpha-mannosidase can hydrolyze alpha-linked mannose from both p-nitrophenyl alpha-mannoside (Km = 0.14 mM) and high mannose oligosaccharides, is not inhibited by the mannose analogues swainsonine and 1-deoxymannojirimycin, is stabilized by MnCl2 or CoCl2, and does not bind to concanavalin A-Sepharose. A goat polyclonal antibody raised against the purified soluble alpha-mannosidase specifically recognizes the rat liver membrane-bound ER alpha-mannosidase, leading us to propose that they are two forms of the same enzyme and that the soluble form is derived from the ER membrane alpha-mannosidase by proteolysis. The antibody also cross-reacts with both the soluble and membrane-bound forms of ER alpha-mannosidase activity in cultured Chinese hamster ovary cells and rat H35 hepatoma cells. Since the ER alpha-mannosidase is presumed to be involved in the early steps of oligosaccharide processing, the action of the purified soluble form of the enzyme on high mannose oligosaccharides was examined. Surprisingly, the enzyme released free mannose from oligosaccharides ranging in size from Glc1Man9GlcNAc to Man5GlcNAc with almost equal efficiency. However, a long term incubation of the enzyme with Man9GlcNAc led to the accumulation of Man7GlcNAc and produced only small amounts of Man6GlcNAc and Man5GlcNAc. Structural analysis of these reaction products indicated that the purified soluble form of ER alpha-mannosidase shows little specificity for which mannose residues it removes from Man9GlcNAc. In contrast, as shown in the accompanying paper, the intracellular action of ER alpha-mannosidase on glycoprotein-bound Man9GlcNAc2 is highly specific.     

Binding of human liver hydrolases by immobilized lectins.

The binding of 22 human liver hydrolase activities by immobilized lectins of six different carbohydrate specificities, namely alpha-D-mannose (glucose), D-N-acetylglucosamine, D-N-acetylgalactosamine, L-fucose, alpha-D-galactose and beta-D-galactose, were examined. Differences in binding among these enzymes and within specific enzymes were observed. For example, the neutral forms of alpha-mannosidase and beta-xylosidase were bound by the Ulex europaeus lectin I (specific for L-fucose), whereas the acidic forms were not. Bandierea simplicifolia lectin (specific for alpha-galactose) bound 65% of beta-glucuronidase activity; recycling experiments demonstrated complete binding of the enzyme that had been eluted with the competitor D-galactose and no binding of the fraction that was not initially bound. These results suggested the presence of two forms of this enzyme. Similar data were obtained for acidic beta-galactosidase activity. These experiments may provide the basis for the expanded use of immobilized lectins for purification and characterization of hydrolases and other glycoproteins.     

Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.

Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.     

Characterization of alpha-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

alpha-mannosidase from Erythrina indica seeds is a Zn(2+) dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 degrees C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol(-1). N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3-8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of alpha-helical structure in the enzyme. alpha-Mannosidase from E indica exhibits immunological identity with alpha-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with alpha-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of alpha-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

Purification of an N-acetyl-d-glucosamine specific lectin (P. B. A.) from epidermal cell membranes of Pieris brassicae L

We report the isolation and the purification of an N-acetyl-d-glucosamine specific lectin capable of agglutinating either fixed trypsinized rabbit erythrocytes or chitin particles. An agglutinin assay based on the affinity of this lectin for the chitin was devised with fluorescent particles of scorpion cuticle to measure lectin activity during purification steps.Lectin was isolated from epidermal cell membranes; its molecular weight was determined by gel filtration and polyacrylamide electrophoresis in sodium dodecyl sulfate. Mr was estimated to be 43,000. Lectin could be constituted by two subunits, Mr of which was estimated to be 23,000. The specificity of this lectin against N-acetyl-d-glucosamine and its oligomers suggests a possible role in the dynamics of these saccharides during the cuticle cycle.     

Carbohydrate-binding properties of an immobilized alpha-D-galactopyranosyl-binding protein (lectin) from the seeds of Bandeiraea simplicifolia.

The alpha-D-galactopyranosyl-binding lectin from Bandeiraea simplicifolia has been coupled to cyanogen bromide-activated Sepharose 4B. Using this immobilized system, we have been able to study the interaction of the lectin with model carbohydrate-protein conjugates and polysaccharides, and to reaffirm this protein's carbohydrate-binding specificity. The opportunity for the isolation of biopolymers containing alpha-D-galactopyranosyl end-groups is demonstrated by the single-step purification of a new galactomannan from the seeds of Cassia alata.     

Alpha-mannosidases involved in N-glycan processing show cell specificity and distinct subcompartmentalization within the Golgi apparatus of cells in the testis and epididymis.

The Golgi apparatus is enriched in specific enzymes involved in the maturation of carbohydrates of glycoproteins. Among them, alpha-mannosidases IA, IB and II are type II transmembrane Golgi-resident enzymes that remove mannose residues at different stages of N-glycan maturation. alpha-Mannosidases IA and IB trim Man9GlcNAc2 to Man5GlcNAc2, while alpha-mannosidase II acts after GlcNAc transferase I to remove two mannose residues from GlcNAcMan5GlcNAc2 to form GlcNAcMan3GlcNAc2 prior to extension into complex N-glycans by Golgi glycosyltransferases. The objective of this study is to examine the expression as well as the subcellular localization of these Golgi enzymes in the various cells of the male rat reproductive system. Our results show distinct cell-and region-specific expression of the three mannosidases examined. In the testis, only alpha-mannosidase IA and II were detectable in the Golgi apparatus of Sertoli and Leydig cells, and while alpha-mannosidase IB was present in the Golgi apparatus of all germ cells, only the Golgi apparatus of steps 1-7 spermatids was reactive for alpha-mannosidase IA. In the epididymis, principal cells were unreactive for alpha-mannosidase II, but they expressed alpha-mannosidase IB in the initial segment and caput regions, and alpha-mannosidase IA in the corpus and cauda regions. Clear cells expressed alpha-mannosidase II in all epididymal regions, and alpha-mannosidase IB only in the caput and corpus regions. Ultrastructurally, alpha-mannosidase IB was localized mainly over cis saccules, alpha-mannosidase IA was distributed mainly over trans saccules, and alpha-mannosidase II was localized mainly over medial saccules of the Golgi stack. Thus, the cell-specific expression and distinct Golgi subcompartmental localization suggest that these three alpha-mannosidases play different roles during N-glycan maturation.     

Purification of acetyllactosamine-specific tomato lectin by erythroglycan-sepharose affinity chromatography

Tomato lectin is specific for oligomers of poly-N-acetyllactosamine containing 3 repeating Gal(beta 1-4)GlcNAc (beta 1-3)-disaccharides. As such it is highly useful for purifying oligosaccharides or glycopeptides with poly-N-acetyllactosamine character. We have found the lectin very useful as an affinity reagent for isolating glycoproteins or glycoprotein domains having poly-N-acetyllactosamine glycosylation. Conventional preparation of tomato lectin by ovomucoid-Sepharose affinity chromatography was found to be unsatisfactory due to instability of column and bleeding of ovomucoid into eluents requiring the necessity for additional purification steps following affinity chromatography. We prepared a column of human erythrocyte band 3 carbohydrate glycopeptide (erythroglycan) attached to Sepharose as an affinity matrix. The purification of tomato lectin to homogeneity in one step on this column matrix is described in this report.     

Maturation of asparagine-linked oligosaccharides in Dictyostelium discoideum analyzed with modification-specific probes

Lysosomal enzymes in Dictyostelium discoideum contain high mannose oligosaccharides that contain mannose 6-phosphate and several unusual structures. The synthesis and distribution of these post-translational modifications were studied using probes for different carbohydrate groups. These probes include lectin-like antibodies directed to two distinct sulfated and one nonsulfated N-linked determinants, the lectin Con A, and the mammalian 215-kDa phosphomannosyl receptor. Only Con A binds to newly synthesized alpha-mannosidase present in the rough endoplasmic reticulum. The other modifications are acquired at different rates and are first detected on protein in light density Golgi-like membranes. Mutations which prevent protein transport to Golgi membranes block synthesis of these moieties, but inhibitors which prevent later transport steps have no effect. The majority of modified proteins are in lysosomes but significant amounts are delivered to nonlysosomal destinations. Different lysosomal proteins contain unequal amounts of each modification.     

The lectin from the mushroom Pleurotus ostreatus: a phosphatase-activating protein that is closely associated with an α-galactosidase activity: A part of this paper has been presented as a preliminary report at the 17th Interlec. Meeting 1997 in Würzburg,Germany

The lectin from the mushroom Pleurotus ostreatus described earlier [F. Conrad, H. Rüdiger, The lectin from Pleurotus ostreatus: purification, characterization and interaction with a phosphatase, Phytochemistry 36 (1994) 277–283] was further characterized. Determination of the isoelectric point by capillary electrophoresis gave a value of 7.6. The dissociation constant of the lectin-α-lactose-1-phosphate complex determined by capillary electrophoresis is 3 mM. The activation of an endogenous phosphatase by the lectin as found earlier for the pseudosubstrate p-nitrophenylphosphate was confirmed also for naturally occurring substrates as ADP and ATP. We observed that at all purification steps the lectin is accompanied by an α-galactosidase activity. Both activities could neither be resolved by electrophoresis under non-denaturing conditions nor by affinity chromatography. However, carbohydrate binding by the lectin and carbohydrate processing by the enzyme are not due to the same site since: (i) the lectin accepts both α- and β-glycosides whereas the enzyme activity is restricted to the α-anomer; (ii) the interaction with erythrocytes leads to a stable agglutinate, i.e. no ‘clot-dissolving activity’ [C.N. Hankins, J.I. Kindinger, L.M. Shannon, Legume α-galactosidases which have hemagglutinin properties, Plant Physiol. 65 (1980) 618–622] is observed; (iii) the α-galactosidase activity is inhibited by galactose but not by a β-galactoside. Therefore, lectin and enzymatic activities are either properties of two tightly associated proteins, or of just one molecule. The kinetic parameters of the lectin-associated α-galactosidase activity for p-nitrophenyl-α-galactopyranoside are: K_M=2.5 mM, k_cat=66 s⁻¹, and K_I=20 mM for the inhibitor d-galactose.