Effect of partial deglycosylation on catalytic characteristics and stability of an avocado peroxidase

An isoperoxidase (EC 1.11.1.7) purified from avocado ( Persea americana Mill. cv. Topa Topa) leaves, lost 35% of its glycosidic moiety after incubation with glycopeptidase F (EC 3.2.2.18). The partial removal of carbohydrate chains caused a decrease in the K_m for reductor substrates tguaiacol, syringaldazine and indole-3-acetic acid) and favoured the enzyme activation by Ca²⁺. A decrease in stability against temperature was also observed. Our results are consistent with the hypothesis that the carbohydrate moiety located on the surface of the peroxidase molecule acts as a molecular shield.     

Deglycosylation of ovalbumin prohibits formation of a heat-stable conformer

To study the influence of the carbohydrate-moiety of ovalbumin on the formation of the heat-stable conformer S-ovalbumin, ovalbumin is deglycosylated with PNGase-F under native conditions. Although the enzymatic deglycosylation procedure resulted in a complete loss of the ability to bind to Concavalin A column-material, only in about 50% the proteins lost their complete carbohydrate moiety, as demonstrated by mass spectrometry and size exclusion chromatography. Thermal stability and conformational changes were determined using circular dichroism and differential scanning calorimetry and demonstrated at ambient temperature no conformational changes due to the deglycosylation. Also the denaturation temperature of the processed proteins remained the same (77.4 +/- 0.4 degrees C). After heat treatment of the processed protein at 55 degrees C and pH 9.9 for 72 h, the condition that converts native ovalbumin into the heat-stable conformer (S-ovalbumin), only the material with the intact carbohydrate moiety forms this heat-stable conformer. The material that effectively lost its carbohydrate moiety appeared fully denatured and aggregated due to these processing conditions. These results indicate that the PNGase-F treatment of ovalbumin prohibits the formation and stabilization of the heat-stable conformer S-ovalbumin. Since S-ovalbumin in egg protein samples is known to affect functional properties, this work illustrates a potential route to control the quality of egg protein ingredients.     

Partial deglycosylation of an anionic isoperoxidase from peach seeds – effect on enzyme activity, stability and antigenicity

An anionic isoperoxidase (EC 1.11.1.7) purified from peach seeds ( Prunus persica L. Batsch cv. Merry) was partially deglycosylated by glycopeptidase F (EC 3.2.2.18) treatment. A 40% deglycosylation resulted in an activity loss of 50% when assayed with o -dianisidine. 60% with guaiacol and 78% with 2,2'-azino-bis(3-ethyl)benzethiozoline-6-sulfonic acid (ABTS) as substrate. The indole-3-acetic acid oxidase activity loss was close to 55%. The partially deglycosylated isoperoxidase also showed a higher K_m value for H₂O₂ and higher values for Arrhenius activation energy and enthalpy of activation. There was a decrease in enzyme stability at 4°C after deglycosylation. Native and partially deglycosylated isoperoxidase reacted equally well in an enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal antibodies raised against the native enzyme. The carbohydrate moiety of this peach seed isoperoxidase appears to be important for enzyme activity and stability.     

Effect of pH,temperature and alcohols on the stability of glycosylated and deglycosylated stem bromelain

The biological significance of the carbohydrate moiety of a glycoprotein has been a matter of much speculation. In the present work, we have chosen stem bromelain fromAnanas comosus as a model to investigate the role of glycosylation of proteins. Stem bromelain is a thiol protease which contains a single hetero-oligosaccharide unit per molecule. Here, the deglycosylated form of the enzyme was obtained by periodate oxidation. The differences in the glycosylated and deglycosylated forms of the glycoprotein have been studied at various temperatures and pH values, using probes such as loss of enzyme activity and by the changes in fluorescence and circular dichroism spectra. Deglycosylated bromelain showed decreased enzyme activity and perturbed fluorescence and circular dichroism spectra. In addition to this, a comparative study of their activities in different organic solvents showed a marked decrease in case of deglycosylated form of the enzyme. It is thus concluded that glycosylation contributes towards the functional stability of glycoenzymes.     

N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase

To study the influence of N-linked carbohydrate moiety on the catalytic and biochemical properties of glycosylated enzyme, a recombinant β-d-glucuronidase (PGUS-P) from Penicillium purpurogenum as a model glycoprotein, was deglycosylated with peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic deglycosylation procedure resulted in the complete removal of carbohydrate moiety. Compared with the glycosylated PGUS-P, the deglycosylated PGUS-P exhibited 20-70% higher activity (p<0.05) within pH 6-9, but 15-45% lower activity (p<0.05) at 45-70°C. The apparent decrease in the thermal stability of the deglycosylated enzyme was reflected by a decrease in the denaturation temperature (T(d)) values determined by differential scanning calorimetry (DSC). The removal of N-linked glycans also reduced enzyme's sensitivity to certain metal ions. The deglycosylated PGUS-P displayed lower K(m) vaules, but higher k(cat)/K(m) ratios than the glycosylated isoform towards glycyrrhizin. The consequent conformational changes were also determined by circular dichroism (CD) and fluorescence spectroscopy which revealed no significant difference in the secondary but a slight dissimilarity between the tertiary structures of both isoforms of PGUS-P.     

Properties of the deglycosylatedβ-fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524

Summary The carbohydrate moiety of -fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524 was largely removed by exposure of the enzyme to endo--N-acetylglucosaminidase F; the total carbohydrate content of the enzyme was decreased from 53% (w/w) to 15% (w/w). The stability of the deglycosylated enzyme at pH 4 to 7 and 40 to 50°C was decreased and theK _m value for sucrose was increased from 0.65 to 1.43 M. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is concluded that the carbohydrate moiety of -fructofuranosidaseP-2 contributes to the stability of the enzyme as well as its affinity for sucrose.     

Chemical deglycosylation of hen ovomucoid: protective effect of carbohydrate moiety on tryptic hydrolysis and heat denaturation

Hen ovomucoid was chemically deglycosylated by treatment with trifluoromethanesulfonic acid at 0 degrees C for 60 min. About 75 mol% of the carbohydrate moiety was removed from the glycoprotein without changing its amino acid composition, and its trypsin inhibitory activity and immunoreactivity with specific antibodies remained unchanged. The deglycosylated ovomucoid was inactivated and degraded easily by an excess amount of trypsin, whereas the native glycoprotein was not. Furthermore, the biological and immunological activities of the deglycosylated ovomucoid were lowered by heat treatment more easily than those of the native ovomucoid. These results suggest that the carbohydrate moiety of ovomucoid contributes to the stability of the ovomucoid molecule against tryptic hydrolysis and heat denaturation.     

The function of the human factor V carbohydrate moiety in blood coagulation

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5-2-fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin-induced activation, whereas deglycosylated factor V activation was impaired. Lectin-blot experiments with sialic-acid-specific Limax flavus agglutinin (LFA), galactose-specific Ricinus communis agglutinin (RCA-II) and mannose-specific concanavalin A on thrombin-induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1F2 contained terminal galactose residues. LFA and RCA-II inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation.     

The common alpha subunit of bovine glycoprotein hormones: limited formation of native structure by the totally nonglycosylated polypeptide chain

The folding of the bovine glycoprotein hormone alpha subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process. The bacterially derived alpha polypeptide (bac-alpha), extracted from E. coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay. Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18-fold. Approximately 2% of the refolded bac-alpha preparation combines with the beta subunit of human chorionic gonadotropin (hCG beta) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac-alpha (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1%), it appears that correct folding of alpha subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin alpha subunit (LH alpha) and chemically deglycosylated LH alpha (which retains two asparagine-linked N-acetyl glucosamine residues per alpha oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced alpha subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LH alpha fully regained its native structure. The partially deglycosylated LH alpha also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HCG beta to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional alpha subunit.     

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.     

Enzymatic generation of triplet acetone by deglycosylated horseradish peroxidase

A study of the effects of deglycosylation of horseradish peroxidase on protein conformation, as well as on its catalytic activity of oxidation of isobutyraldehyde or its enol form to triplet acetone and formic acid, was performed. The loss of carbohydrates leads to structural modifications of this enzyme. This is confirmed by a change in the circular dichroism spectrum, an increase in tryptophan's environment polarity, and a loss of the chiral specificity toward D- and L-tryptophan. Deglycosylation does not destroy either the peptide backbone or the amino acid residues and does not affect the heme group content of the protein. The rates of oxygen uptake and light emission observed when horseradish peroxidase oxidizes isobutyraldehyde or the trimethylsilyl enol ether form of the latter are reduced when the enzyme is 70% deglycosylated. Concomitantly, the acting deglycosylated enzyme becomes inactivated during the course of the reaction. It appears that the carbohydrate moiety plays an important role in the protection of the peroxidase from damaging effects induced by triplet acetone and in the stabilization of the three-dimensional structure of this enzyme.     

Guanidine Hydrochloride Denaturation of Glycosylated and Deglycosylated Stem Bromelain

Glycosylation is one of the major naturally occurring covalent modifications of proteins. We have used stem bromelain, a thiol protease with a single, N-glycosylated polypeptide chain as a model to investigate the role of glycosylation of proteins. Periodate oxidation was used to obtain the deglycosylated form of the enzyme. Denaturation studies in the presence of guanidine hydrochloride (Gn·HCl) were performed using fluorescence and circular dichroism spectroscopy. The glycosylated stem bromelain was found to be stabilized by 1.9 kcal/mol as compared to the deglycosylated one. At a given concentration of denaturant, the fraction of denatured protein was higher in the case of deglycosylated stem bromelain. In short, deglycosylated bromelain showed more susceptibility towards guanidine hydrochloride denaturation, indicating the contribution of the carbohydrate part of the glycoprotein to the stability of the enzyme.     

Role of the carbohydrate part of yeast acid phosphatase

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.     

Characterization of deglycosylated human urinary colony-stimulating factor (CSF-1)

1. Isolation of CSF-1 from human urine was performed through five purification steps. These include concentration by dialysis, silica gel absorption, hydrophobic chromatography and phenyl-Sepharose CL-6B, Fast Protein Liquid Chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. These methods have been reported in a previous paper (Tao et al., 1987). 2. The isolated CSF-1 which exhibits a one band pattern on SDS-PAGE under non-reducing conditions after Coomassie Blue and silver stainings. CSF-1 was purified 100,000-fold and has a specific activity of 2.16 x 10(7) units/mg protein. Its apparent Mr is 57,000 with an isoelectric point pI = 5.8-6.0 CSF-1 is a glycoprotein with 40% of carbohydrate (w/w). 3. An almost complete removal of the carbohydrate moiety from CSF-1 was obtained after treatment with trifluoromethanesulfonic (TFMS) acid followed by gel filtration on Sephadex G-25 (Fine). The deglycosylated (DG) CSF-1 possesses an apparent Mr of 38,000 and an isoelectric point, pI: 6.2 as compared to native-CSF-1 (N-CSF-1), Mr = 57,000 and pI = 5.8 respectively. 4. The TFMS treatment did not alter the activities of CSF-1 as shown by biological assay and receptor binding assay. The thermostability experiment revealed that DG-CSF-1 was less stable than N-CSF-1. The circular dichroism spectra (CD) of N-CSF-1 and DG-CSF-1 were different. 5. The features of interaction of iodinated-N-CSF-1 and iodinated-DG-CSF-1 with single cell suspensions from human peritoneal macrophage were studied. The binding activity of peritoneal macrophage was the highest among all cells examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immobilization of glycoenzymes through carbohydrate side chains.

Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N-hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.     

N-Deglycosylation of fructosyltransferase and invertase from Fusarium oxysporum decreases stability but has little effect on kinetics and synthetic specificity

Most of the carbohydrate moiety of invertase and fructosyltransferase (FTF) from F. oxysporum IMI 172464 was removed by peptide-N-glycosidase F. The molecular weights of native invertase and FTF were 260 kDa and 210 kDa respectively. Deglycosylation lowered the molecular sizes by 42% and 23%, respectively. The K values for sucrose remained unchanged by deglycosylation. However the stability of both enzymes at their optimum pH (4.0 for invertase, 5.0 for FTF) and optimum temperature (45°C for invertase, 35°C for FTF) was decreased: their sensitivity to protease digestion was increased by 36% and 41%, respectively. The synthetic specificity of deglycosylated FTF remained unchanged. The carbohydrate moiety of invertase and FTF contributes to the stability of the enzymes but is not essential in their catalytic function and plays no part in determining their specificity.     

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.     

Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.     

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH₄)₂SO₄, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the V _max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V _max and a 2-fold decrease in K _m were observed. Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997     

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.