Biochemical and functional characterization of the Tn-specific lectin fromSalvia sclarea seeds

Andrea Medeiros1, Sergio Bianchi1, Juan J. Calvete2, Henia Balter3, Sylvie Bay4, Ana Robles3,DanieÁ le CantacuzeÁ ne4, Manfred Nimtz5, Pedro M. Alzari6 and Eduardo Osinaga1

1Depto. de BioquõÂmica, Facultad de Medicina, Montevideo, CP, Uruguay; 2Instituto de Biomedicina, CSIC, Valencia, Spain;3Depto de Radiofarmacia, Facultad de Ciencias, Montevideo, Uruguay; 4Unite de Chimie Organique, Institut Pasteur, Paris, France;5Gesellschaft fuÈr Biotechnologische Forschung MbH, Braunschweig, Germany; 6Unite de Biochimie Structurale, Institut Pasteur, Paris, France

SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antigen (GalNAca-O-Ser/Thr), a specific

marker of many human carcinomas. Two-dimensional electrophoresis, amino-acid and amino-sugar

analysis, and MALDI-TOF MS showed that SSL is an acidic (pI 5.5), 60±61-kDa dimeric glycoprotein

composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass

of SSL in solution determined by equilibrium sedimentation analytical ultracentrifugation was

59 ^ 9 kDa. This value did not change in the pH range 2.5±8.5, indicating that SSL does not associate

into higher order structures. Tandem mass spectrometry and methylation analysis of N-glycans released

from SSL by hydrazinolysis indicated that SSL possesses 2±3 glycosylation sites occupied with the

typical plant glycans Mana1±6[(Mana1±3)(Xylb1±2)]Manb1±4-GlcNAcb1±4(Fuca1±3)GlcNAc and

[(Mana1±3/6)(Xylb1±2)]Manb1±4-GlcNAcb1±4(Fuca1±3)GlcNAc. The influence of adjacent Tn structures

on the binding of two Tn-specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti-Tn

monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to

the synthetic Tn glycopeptides was independent of the density of Tn structures. On the other hand, mAb 83D4

only reacted with glycopeptides displaying two or three consecutive Tn structures.

Keywords: Salvia scalrea lectin; Tn antigen-binding lectins; synthetic Tn glycopeptides; anti-Tn monoclonal

antibody; glycan structure.

In animal cells, mucin-type O-glycosylation of proteinsis initiated in the Golgi apparatus by the transfer of aN-acetylgalactosamine (GalNAc) residue to the hydroxyl side-chain of a serine or threonine residue. This reaction is catalyzedby GalNAc transferase (EC 2.4.1.41). Subsequent stepwiseenzymatic elongation by specific glycosyltransferases yieldsthe eight O-linked core structures identified to date [1]. Inparticular transformed cells, this elongation does not occurgenerating the Tn antigenic determinant (GalNAca-O-Ser/Thr).First discovered by Dausset et al. [2] at the red blood cellsurface of a patient with hemolytic anemia, this antigen isresponsible for the polyagglutination of erythrocytes caused byantibodies reactive with Tn commonly present in all human sera(Tn syndrome). It is now recognized that this antigen, expressedin an unmasked form in about 90% of human carcinomas [3], isone of the most specific human cancer-associated structures [4].A direct link has been demonstrated between carcinomaagressiveness (e.g. extent of tissue spread and vessel invasion)

and the cell-surface density of the Tn antigen [5]. Moreover,mice immunized against Tn-rich asialo ovine submaxillarymucin (aOSM) were able to develop an effective immunereponse, which protected them against the Tn-expressing tumorTA3-HA [6]. Although the Tn determinant is a glycosylatedamino-acid structure, it has been recently reported that someTn-glycopeptides are capable of binding to proteins of themajor histocompatibility complex, inducing T-cell lymphocyteactivation [7], and providing thus a potentially importanttherapeutic target [8]. The Tn determinant has also beenimplicated in tumor cell organotropic metastasis [9] and in HIVinfection [10], stressing the relevance of the characterization ofthe interactions between the Tn antigen and Tn-bindingproteins.

Several proteins have been described that specificallyrecognize the Tn determinant, including monoclonal antibodies[11,12], plant lectins [13,14], human macrophage lectins [15],and glycosyltransferases [16]. As part of our structural studiesof Tn-binding proteins, we have constructed a single-chain Fvfragment of the anti-Tn mAb 83D4, and have demonstrated thatthis recombinant polypeptide retains Tn-binding properties[17]. Molecular modelling of the 83D4 molecule indicated thatthe antibody combining site may be primarily defined by thecomplementarity-determining region H1 and H2 loops. Wehave also reported the amino-acid sequence and the crystalstructure of the isolectin B4 from Vicia villosa (VVLB4) seeds[18], a homotetrameric glycoprotein that specifically aggluti-nates Tn-exposing red blood cells [13]. The tertiarystructure of the VVLB4 subunit displays the characteristiclegume lectin fold consisting of a b-sandwich formed by a

Eur. J. Biochem. 267, 1434±1440 (2000) q FEBS 2000

Correspondence to E. Osinaga, Depto. de BioquõÂmica, Facultad de

Medicina, Av. Gral. Flores 2125, Montevideo CP 11800, Uruguay.

Fax: + 598 2924 95 63, Tel.: + 598 2924 95 62,

E-mail: eosinaga@lobbm.fmed.edu.uy

Abbreviations: aOSM, asialo ovine submaxillary mucin; VVLB4,

isolectin B4 from Vicia villosa; SSL, Salvia sclarea lectin;

NEPHGE, nonequilibrium pH gradient electrophoresis.; DIEA,

N,N-diisopropylethylamine; HOBT, N-hydroxybenzotriazole; TBTU,

2-1H(benzotriazole)-1,1,3,3-tetramethyluronium tetrafluoroborate.

(Received 28 October 1999, revised 7 January 2000, accepted

10 January 2000)

q FEBS 2000 Characterization of Salvia sclarea lectin. (Eur. J. Biochem. 267) 1435

flat six-stranded b-sheet and a curved seven-stranded b-sheetinterconnected by loops of variable length [19]. The tetramercan be described as a dimer of dimers, as has been frequentlyobserved in the crystal structures of Phaseolus vulgarisleukoagglutinin, soybean agglutinin, and the seed lectin ofDolichos biflorus [19,20]. The amino-acid residues defining themetal- and sugar-binding sites of leguminous lectins are highlyconserved in VVLB4 structure, indicating that residues outsidethe carbohydrate-binding pocket and/or quaternary associationdeterminants may modulate the affinity for the Tn glycopep-tide. Among GalNAc-specific lectins, the lectin from Salviasclarea seeds (SSL) [14] is one of the most specific lectin forthe GalNAca-O-Ser/Thr structure [21]. However, the biochem-ical properties of SSL are poorly understood. The aim of thepresent work was to characterize molecular features of SSL andto compare its binding characteristics with those of the VVLB4,and the monoclonal antibody 83D4 to synthetic Tn glyco-peptides of defined Tn epitope density.

M A T E R I A L S A N D M E T H O D S

Tn-binding proteins

Salvia sclarea seeds, obtained from the Conservatoire NationalDes Plantes Medicinales et Aromatiques (Milly La-Foret,France), were extracted as described previously [14]. Afterprecipitation with cold ethanol (80% v/v), the pellet waslyophilized, redissolved in water and applied to a GalNAc-agarose (Sigma Chemical Co.) column equilibrated in 10 mmphosphate, 135 mm NaCl, pH 7.3 (NaCl/Pi) containing 0.5 mNaCl. The lectin was eluted with the same buffer containing0.1 m GalNAc. Free GalNAc was removed by dialysis and theSSL was stored at 220 8C in NaCl/Pi. Purified VVLB4 wasfrom Sigma. Anti-Tn monoclonal antibody 83D4 (IgM, k) [22]was precipitated from ascitic fluids with ammonium sulfate,dissolved in 1.0 m NaCl in NaCl/Pi, and purified by gel-filtration chromatography on a Sephacryl S-200 2.5 � 85 cmcolumn.

Electrophoretic analysis

SDS/PAGE (12.5%) was carried out as described previously[23]. Two-dimensional electrophoretic analysis [first dimen-sion, nonequilibrium pH gradient electrophoresis (NEPHGE);second dimension, SDS/PAGE] was performed by the methodof O'Farrell et al. [24]. NEPHGE was carried out on 6.5-cmgels containing 2% ampholines (pH range 3.5±9.5, Pharmacia).The gels were equilibrated for 30 min at room temperature inSDS/PAGE electrode buffer (25 mm Tris/HCl, 192 mm glycine,0.1% SDS, pH 8.3) prior to separation in the second dimension.Proteins were identified by silver staining.

Compositional analyses.

Amino-acid and amino-sugar analyses of purified SSL werecarried out with an AlphaPlus (Pharmacia) amino-acid analyzerafter sample hydrolysis in sealed, evacuated ampoules at110 8C with 6 m HCl for 24 h and with 4 m HCl for 4 h,respectively.

Analytical ultracentrifugation.

Determination of molecular masses by analytical ultracentri-fugation was performed at 20 8C using a Beckman XL-Acentrifuge with absorption optics using six-channel cells.

Samples contained initially around 1 mg´mL21 SSL in thefollowing buffers supplemented with 1 mm of CaCl2 andMgCl2: 20 mm Tris/HCl, 0.1 m NaCl, pH 8.5 (a) and pH 7.5(b); 20 mm sodium citrate, 0.1 m NaCl at pH values 6.5, 5.5,4.5, and 3.5 .

Mass spectrometry

The molecular masses of: (a) native SSL; (b) SSL denatured in100 mm Tris/HCl, pH 8.6, 6 m guanidine hydrochloride for5 min at 90 8C, and incubated with a fivefold molar excess ofiodoacetamide over cysteine residues for 1 h at roomtemperature; and (c) SSL reduced in 100 mm Tris/HCl,pH 8.6, 6 m guanidine hydrochloride, 2% (v/v) 2-mercapto-ethanol at 100 8C for 2 min, and alkylated with a fivefold molarexcess of iodoacetamide over reducing reagent for 1 h at roomtemperature, were determined by MALDI MS using a BrukerREFLEXTM TOF instrument equipped with a reflectronsystem and a N2 laser (337 nm). The sample (1 mL;containing 5±25 pmol´mL21) was mixed with an equal volumeof sinnapinic acid (10 mg´mL21 in 10% ethanol), spotted ontothe stainless steel tip and dried at room temperature. Spectrawere recorded under an acceleration voltage of 30 kV.

Carbohydrate composition and methylation analysis.

N-glycans were released from SSL (approximately 250 mg) byautomated hydrazinolysis using a Glycoprep 1000 instrument(N mode, Oxford Glycosystems). After methanolysis, re-N-acetylation and trimethylsilylation, monosaccharides wereanalyzed as the corresponding methyl glycosides by gaschromatography on a Carlo Erba Mega Series instrumentusing a 30-m DB1 capillary column [25]. For methylationanalysis, oligosaccharides were permethylated, purified, hydro-lyzed, reduced, and peracetylated [26]. Separation andidentification of partially methylated alditol acetates wereperformed with a Finnigan MAT gas chromatograph equippedwith a 30-m DB5 capillary column and connected to a FinniganGCQ ion-trap mass spectrometer running in the electron-impactmode.

Structural characterization of oligosaccharides byelectrospray-ionization tandem mass spectrometry

A Finnigan MAT TSQ 700 triple quadrupole mass spectrometerequipped with an electrospray ion source (Finnigan MAT Corp.,San Jose, CA) was used for electrospray ionization MS.Reduced and permethylated samples (concentrations approxi-mately 10 pmol´mL21) were dissolved in acetonitrile saturatedwith NaCl and injected at a flow rate of 1 mL´min21. A voltageof +5.5 kV was applied to the electrospray needle. Forcollision-induced dissociation experiments, argon with a kineticenergy set at around 260 eV was used as the collision gas.

Synthetic Tn-glycopeptides

Peptide syntheses were performed manually using thestandard Fmoc chemistry protocol on a polystyrene resinactivated with p-benzyloxybenzyl alcohol (Wang resin) andesterified with a glycine residue. N a-Fmoc amino acids andthe glycosylated building blocks (3 mol equivalents) wereincorporated into the peptide chain using TBTU/HOBT/DIEA1 : 1 : 1.7 as activating agent and dimethylformamide assolvent [27]. Couplings were monitored by the Kaiser test[28] and were usually completed within 20 min. Fmoc cleavage

1436 A. Medeiros et al. (Eur. J. Biochem. 267) q FEBS 2000

was carried out with 20% piperidine in dimethylformamide.After each deprotection step, the resin was successively rinsedwith dimethylformamide, CH2Cl2, and dimethylformamide. Atthe end of the synthesis, the resin was extensively washed withdimethylformamide and CH2Cl2, dried, and added to 95%trifluoroacetic acid for 2 h. After filtration, the peptide solutionwas concentrated, precipitated with diethyl ether, filtered,dissolved in water and lyophilized. Peptides were purified byreversed-phase HPLC and obtained with a yield of about 25%.The acetyl groups of the sugar moiety were removed bydropwise addition of MeONa (pH 10.8) to a final concentrationof 1%. After 8 h, methanol was evaporated and the pH ofthe solution neutralized. The following peptides werepurified by HPLC on a reversed-phase column (C18) usinga gradient of 0.1% trifluoroacetic acid in water (solution A)and acetonitrile (solution B), and characterized by massspectrometry and amino-acid analysis: Tn0, Lys-(Gly)4-Ser-Thr-Thr-(Gly)3 (834.4 Da); Tn1, Lys-(Gly)4-Ser(a-GalNAc)-Thr-Thr-(Gly)3 (1037.6 Da); Tn2, Lys-(Gly)4-Ser(a-GalNAc)-Thr(a-GalNAc)-Thr-(Gly)3 (1240.4 Da); and Tn3, Lys-(Gly)4-Ser(a-GalNAc)-Thr(a-GalNAc)-Thr(a-GalNAc)-(Gly)3

(1443.9 Da).

Labelling of Tn-binding proteins

Proteins (SSL, VVLB4 and mAb 83D4) were labelled with 125I,purchased from CIS (Paris, France), using a modification of theN-chloro-p-toluene sulfonamide sodium salt (Chloramine-T)method [29]. Briefly, 5 mg protein at pH 7.4 were reacted with530 mCi (19.6 MBq) of a 125I solution (3.7 GBq´mL21) in thepresence of 5.3 nmol (5.3 � 1024 m) of Chloramine-T for1 min with vortex mixing. The efficiency of iodine incor-poration was controlled by precipitation with 20% trichloro-acetic acid of a small aliquot of labelled protein mixed with100 mL of BSA solution (50 mg´mL21). Labelled proteinswere freed from unbound 125I by gel-filtration chromatography

on a PS10 Sephadex G-25 column (Pharmacia). The functionalintegrity of the labelled proteins was evaluated by bindingassays on immobilized aOSM. To this end, samples of 200 mLof labelled protein adjusted to 180 000 c.p.m. were incubated intubes previously coated overnight with 200 mL of aOSM(10 mg´mL21 in carbonate buffer 0.1 m, pH 9.4). After over-night incubation at room temperature, the contents of the tubeswere removed by aspiration and the tubes were washed twicewith 1 mL of NaCl/Pi containing 0.05% Tween 20, and boundradioactivity was counted in a well-type gamma counter with70% efficiency for 125I (Compac 120, Northford, CT, USA).

Functional evaluation

The influence of the number of adjacent Tn structures in therecognition by Tn-binding proteins was evaluated by com-petition of the binding of the Tn-specific proteins toimmobilized aOSM by glycopeptides expressing Tn. Eachpeptide (Tn0, Tn1; Tn2 and Tn3) was serially diluted from0.75 mm to 2.5 � 1024 mm. Competition studies were per-formed by incubating 50 mL of each dilution of the peptideswith 150 mL 125I-labelled protein (180 000 c.p.m.) in aOSMcoated tubes. Bound radioactivity was measured as above.Assays were carried out in duplicate using 0.2% BSA inNaCl/Pi as controls.

R E S U LT S A N D D I S C U S S I O N

SSL is a disulfide-linked dimer

SSL was purified using ethanol precipitation followed byaffinity chromatograpy. SDS/PAGE showed that the lectinmigrated as a single band of apparent molecular mass of72 kDa under nonreducing conditions and as a 32-kDa bandafter reduction with 2-mercaptoethanol (Fig. 1, left panel),suggesting that SSL might consist of a disulfide-bonded

Fig. 1. Electrophoretic analysis of purified SSL. (Left panel) Silver-stained SDS/PAGE (12.5%) of reduced (A) and nonreduced (B) SSL. (Right panel)

Two-dimensional (NEPHGE/SDS/PAGE) electrophoretic analysis of reduced SSL.

q FEBS 2000 Characterization of Salvia sclarea lectin. (Eur. J. Biochem. 267) 1437

dimeric protein. The amino-acid composition of purified SSL isshown in Table 1 in comparison with results reported by Pilleret al. [14]. SSL is rich in glycine, acidic (Asx, Glx) andhydroxylic (Ser, Thr) amino acids. Two-dimensional gelelectrophoretic analysis (Fig. 1B) showed that SSL is anheterogeneous protein, which migrated as a smeared bandshowing a main spot at pI 5.5, suggesting that SSL could be amixture of (glycosylated) isoforms. The amino-acid compo-sition of SSL is in agreement with the acidic pI (5.5)determined by two-dimensional (NEPHGE/SDS/PAGE) elec-trophoretic analysis (Fig. 1, right panel) but is in contrast tothe previously reported basic pI for SSL [14].

Amino-acid analysis of the native lectin and of denatured,nonreduced lectin incubated with iodoacetamide both yielded0.2±0.3 mol% of cysteine. This value corresponds to 1.2±1.6cysteinyl residues per SSL molecule. In addition, cysteine was

quantitatively recovered as carbamidomethylcysteine in theamino-acid analysis of reduced and alkylated SSL. As a whole,these results indicated the existence of a single cysteine residueper SSL monomer and that this residue is engaged in theformation of an intermolecular disulfide bridge in the SSLdimer. Moreover, the molecular mass of SSL was determinedby MALDI-TOF MS. The intact lectin yielded a broad M + H+

ion peak with a centroid at about 60±61 kDa (Fig. 2A). This isin agreement with the observed pI heterogeneity of SSL (Fig. 1,right panel) and further indicated the existence of polypeptideand/or glycosylation heterogeneity. The molecular mass of thelectin did not change when the purified lectin was incubatedwith iodoacetamide under denaturing but nonreducing con-ditions (Fig. 2B), but it decreased to 29.5±30 kDa afterreduction and carbamidomethylation (Fig. 2C), confirmingthat native SSL is composed of two disulfide-linked subunits.

Fig. 2. Determination of the molecular

mass of SSL. The molecular masses of:

(A) native SSL; (B) nonreduced, denatured

SSL incubated with a fivefold molar excess of

iodoacetamide over cysteine residues;

and (C) reduced and carbamidomethylated

SSL were determined by MALDI-TOF mass

spectrometry. Insert, apparent molecular

masses of SSL in buffers of different pH

values determined by analytical

ultracentrifugation sedimentation equilibrium.

1438 A. Medeiros et al. (Eur. J. Biochem. 267) q FEBS 2000

Many leguminous plant lectins display pH-dependentoligomeric structures. The molecular mass of SSL in buffersof different pH was investigated by analytical centrifugationequilibrium sedimentation. The apparent molecular mass was59 ^ 9 kDa and this value did not change in the pH range of3.5±8.5 (Fig. 2A, insert), clearly indicating that native SSLis a dimeric protein and that its quaternary structure is notpH-dependent.

Structure of the N-linked oligosaccharides of SSL

Amino-acid analysis of SSL showed the presence of 5±6 molof glucosamine per mol lectin, clearly indicating that SSL is anN-glycoprotein bearing 2±3 oligosaccharide chains. Mono-saccharide analysis showed that the carbohydrate chains of SSLare composed of GlcNAc, Man, Xyl, and Fuc in approximatelymolar proportions of 2 : 3 : 1 : 1. The structure of reduced andpermethylated N-glycans, released from SSL by automatedhydrazinolysis, was determined by a combination of methy-lation analysis and collision-induced mass spectrometry.Electrospray ionization mass spectrometric analysis of reduced(with NaBH4) and permethylated total oligosaccharidesreleased by hydrazinolysis from SSL yielded molecular ionsat m/z 1522.4 [Hex3-Pent-HexNAc-dHex-HexNAc-ol + Na](40%) and 1318.5 [Hex2-Pent-HexNAc-dHex-HexNAc-ol +Na] (60%) suggested the presence of a proximally fucosylatedtrimannosyl- or dimannosyl common core structure bearingb1,2-linked xylose at the inner mannose residue. Suchcarbohydrate structures are characteristic for plant N-glycans.These assumptions were confirmed by collision-induceddissociation experiments. Figure 3A shows the deducedoligosaccharide at m/z 1522.4. An intense fragment ion at m/z489.9 [dHex-HexNAc-ol-1D + Na] together with the comple-mentary ion [M-(dHex-HexNAc-ol-1D) + Na] at m/z 1054.6indicated the presence of proximal Fuc. This residue must belinked to O-3 of the reduced GlcNAc as a secondary fragmention at m/z 284.1 is generated by elimination of Fuc from theprimary fragment ion. This is characteristic for 3-linkedsubstituents of GlcNAc residues and is not observed withproximal Fuc 1,6-linked. The latter linkage is characteristic ofmammalian oligosaccharides. Additional fragment ions of

lower intensity can be explained by the cleavage of otherglycosidic bonds.

The component at m/z 1318.5 (Fig. 3B) yielded a daughterion spectrum analogous to that of m/z 1522.4, compatiblewith a proximally 1±3 fucosylated dimannosyl structurebearing an additional xylose residue. Methylation analysisshowing derivatives characteristic for terminal xylose and2,3,6-trisubstituted mannose confirmed the presence of xylose1,2-linked to the common core. Similarly, the linkage offucose to O-3 of the proximal GlcNAc was confirmed. Thesingle terminal mannose residue was found to be attached toO-6 of the inner mannose residue, as small amounts of 2±6-disubstituted mannose were detected and O-2 was occupied bythe xylose residue.

The hexasaccharide of SSL is identical to the carbohydratechains of Robinia pseudoacacia, Erythrina variegata andVatairea macrocarpa seed lectins [30]. N-linked oligo-saccharides having a b1,2-linked xylose attached to theb-linked mannose of the core structure appear to be a commoncharacteristic of different allergenic proteins, and regulate thetargeting of these proteins to various organelles such asstorage bodies [31]. b1,2-xylotransferase, the enzyme thattransfers d-xylose from UDP-xylose to the b-linked mannose ofplant N-linked oligosaccharides, acts on biantennary glycanacceptors having b1,2GlcNAc residues on the Mana1±3 andMana1±6 arms. This strongly suggests that the oligosaccharideattached to SSL (and R. pseudoacacia, V. macrocarpa andE. variegata lectins) may be derived from a larger glycan byglycosidase processing.

Reactivity of SSL towards synthetic Tn glycopeptides.

The reactivity of SSL on synthetic Tn glycopeptides wascompared with that of other two Tn-binding proteins, VVLB4and monoclonal antibody 83D4. As a first step for the assay we

Table 1. Amino-acid composition of SSL (mol residue per 100 mol

total amino acids).

Residue This work Piller et al. [14]

Cys 0�.2 1�.9

Asx 11�.24 9�.8

Thr 11�.16 9�.5

Ser 10�.62 9�.2

Glx 6�.72 5�.8

Gly 12�.06 10�.6

Ala 8�.99 9�.2

Val 7�.26 8�.4

Met 0�.28 0�.53

Ile 3�.88 4�.5

Leu 5�.09 5�.49

Tyr 3�.38 3�.66

Phe 8�.97 8�.54

His 2�.14 2�.8

Lys 1�.66 2�.32

Arg 3�.45 3�.4

Pro 2�.86 3�.7

Fig. 3. Characterization of SSL oligosaccharides. The structures of

reduced and permethylated oligosaccharides released from SSL by

hydrazinolysis was determined by a combination of methylation analysis

and collision-induced dissociation electrospray ionization MS. Structural

assignments of daughter ions obtained by collision-induced dissociation of

parent ions at m/z 1522.4 and 1318.5 are displayed.

q FEBS 2000 Characterization of Salvia sclarea lectin. (Eur. J. Biochem. 267) 1439

determined the optimal concentration of aOSM solution to beused for coating the tubes. Bound activity ranged from0.13 ^ 0.02% for tubes coated with 0.05 mg´mL21 aOSM to9.3 ^ 0.14% of total activity bound to the tubes incubated with

10 mg´mL21 aOSM. The latter condition was selected for thecompetition studies as well as for quality controls. The peptidewithout Tn structure (peptide Tn0) did not produce inhibition ofthe binding of proteins to immobilized aOSM. The binding ofSSL and VVLB4 to aOSM was inhibited by all Tn glyco-peptides (Tn1, Tn2 and Tn3) in a dose-dependent manner,indicating that both lectins recognized Tn-bearing glyco-peptides independently from the density of Tn structures(Fig. 4A±C). On the contrary, the antibody 83D4 did not reactwith the Tn1 glycopeptide, and was only reactive to glyco-peptides with two or three consecutive Tn residues. Theseresults and the values of glycopeptide concentration producing50% inhibition over the binding of labelled anti-Tn proteins(Table 2) indicate that the molecular structure identified by themonoclonal antibody 83D4 is not the same as that recognizedby the Tn-specific lectins, and suggests the involvement ofhigher density of GalNAc residues O-linked to serine/threoninein the formation of the epitope for 83D4. In this respect, it hasbeen reported that clusters of Tn structures are an essential partof the epitope recognized by other proteins, like the monoclonalantibody MLS128 [12] and the Tn-specific human macrophageC-type lectin [15], all of which react poorly or are unreactivewith glycopeptides bearing Tn structures that are notorganized into clusters. The fact that the Tn-binding activityexhibited by SSL and VVLB4 is not dependent on thedensity of Tn determinants may indicate nonhomologous Tnrecognition mechanisms of plant lectins and antibodies.Crystallographic studies of 83D4, SSL and VVLB4 complexedwith Tn-containing molecules are in progress in ourlaboratories, which may provide an atomic view of the distinctstructural determinants underlying the different Tn-bindingspecificity of antibodies and lectins.

A C K N O W L E D G E M E N T S

This work was partially supported by grants from the ComisioÂn Honoraria

de Lucha Contra el Cancer (Uruguay), DireccioÂn General de InvestigacioÂn

CientõÂfica y TeÂcnica (Spain), CSIC (Universidad de la Republica, Uruguay),

and ECOS France-Uruguay Program. We thank Drs Silvia Chifflet and

Alvaro Babino (Depto. BioquõÂmica, Facultad de Medicina, Montevideo,

Uruguay), and Ing. Jorge Arboleya (INIA, Las Brujas, Uruguay) for their

helpful contribution.

R E F E R E N C E S

1. Hounsell, E.F., Davies, M.J. & Renouf, D.V. (1996) O-linked protein

glycosylation structure and function. Glycoconjugate J. 13, 19±26.

2. Dausset, J., Moullec, J. & Bernard, J. (1959) Acquired hemolytic

anemia with poly-agglutinability of red blood cells due to a new

factor present in normal human serum (anti-Tn). Blood 14,

1079±1093.

3. Springer, G. (1984) T and Tn, general carcinoma autoantigens. Science

224, 1198±1206.

Table 2. Glycopeptide concentration producing 50% inhibition over the Tn binding activity of SSL, VVLB4 and mAb 83D4.

Tn1 Tn2 Tn3

Glycopeptide

(mm)

Glycopeptide/

protein (m/m)

Glycopeptide

(mm)

Glycopeptide/

protein (m/m)

Glycopeptide

(mm)

Glycopeptide/

protein (m/m)

SSL 0�.093 1�.5 � 107 0�.032 5�.1 � 106 0�.028 4�.5 � 106

VVLB4 0�.022 4�.5 � 106 0�.008 1�.6 � 106 0�.006 1�.2 � 106

mAb 83D4 ± ± 0�.066 2�.6 � 107 0�.022 8�.5 � 106

Fig. 4. Comparison of theTn-binding reactivity of SSL, VVLB4 and

mAb 83D4. The Tn-binding characteristics of SSL, VVLB4 and mAb 83D4

were studied by competition assays of the binding of the proteins to

immobilized aOSM by mixing the 125I-labelled Tn-binding proteins

(180 000 c.p.m.) with serial dilutions of synthetic Tn-glycopeptides (A)

Tn1; (B) Tn2; and (C) Tn3.

1440 A. Medeiros et al. (Eur. J. Biochem. 267) q FEBS 2000

4. Hakomori, S. (1990) Biochemical basis of tumor-associated carbo-

hydrate antigens. Current trends, future perspectives, and clinical

applications. Immunol. Aller. Clin. N. Am. 10, 781±802.

5. Springer, G. (1995) T and Tn pancarcinoma markers: auto-antigenic

adhesion molecules in pathogenesis, pre-biopsy carcinoma detection

and long-term breast-carcinoma immunotherapy. Crit. Rev. Oncogen.

6, 57±85.

6. Singhal, A., Fohn, M. & Hakomori, S. (1991) Induction of N-acetyl-

galactosamine-O-serine/threonine (Tn) antigen-mediated cellular

immune response for active immunotherapy in mice. Cancer Res.

51, 1406±1411.

7. Jensen, T., Galli-Stampino, L., Mouritsen, S., Frische, K.,

Peters, S., Meldal, M. & Werdelin, O. (1996) T-cell recognition of

Tn-glycosylated peptide antigens. Eur. J. Immunol. 26, 1342±1349.

8. Lo-Man, R., Bay, S., Vichier-Guerre, DeÂriaud, E., CantacuzeÁne, D. &

Leclerc, C. (1999) A fully synthetic immunogen carrying a

carcinoma-associated carbohydrate for active specific immuno-

therapy. Cancer Res. 59, 1520±1524.

9. Schlepper-Schafer, J. & Springer, G. (1989) Carcinoma auto-antigens T

and Tn and their cleavage products interact with Gal/GalNAc-specific

receptors on rat Kupffer cells and hepatocytes. Biochem. Biophys.

Acta 1013, 266±272.

10. Hansen, J.E.S., Nielsen, C., Arendrup, M., Olofsson, S., Mathiesen, L.,

Nielsen, J. & Clausen, H. (1991) Broadly neutralizing antibodies

targeted to mucin-type carbohydrate epitopes of human immuno-

deficiency virus. J. Virology 65, 6461±6467.

11. Hirohashi, S., Clausen, H., Yamada, T., Shimosato, Y. & Hakomori, S.

(1985) Blood group A cross-reacting epitope defined by monoclonal

antibodies NCC-LU-35 and -81 expressed in cancer of blood group O

and B individuals: its identification as Tn antigen. Proc. Natl Acad.

Sci. USA 82, 7039±7043.

12. Nakada, H., Inoue, M., Numata, Y., Tanaka, N., Funakoshi, I., Fukui,

S., Mellors, A. & Yamashina, I. (1993) Epitopic structure of Tn

glycophorin A for an anti-Tn antibody (MLS 128). Proc. Natl Acad.

Sci. USA 90, 2495±2499.

13. Tollefsen, S. & Kornfeld, R. (1983) The B4 lectin from Vicia villosa

seeds interacts with N-acetylgalactosamine residues alpha-linked to

serine or threonine residues in cell surface glycoproteins. J. Biol.

Chem. 258, 5172±5176.

14. Piller, V., Piller, F. & Cartron, J.-P. (1986) Isolation and charac-

terization of an N-acetylgalactosamine specific lectin from Salvia

sclarea seeds. J. Biol. Chem. 261, 14069±14075.

15. Iida, S., Yamamoto, K. & Irimura, T. (1999) Interaction of human

macrophage C-type lectin with O-linked N-acetylgalactosamine

residues on mucin glycopeptides. J. Biol. Chem. 274, 10697±10705.

16. Van den Steen, P., Rudd, P., Dwek, R. & Opdenakker, G. (1998)

Concepts and principles of O-linked glycosylation. Crit. Rev.

Biochem. Mol. Biol. 33, 151±208.

17. Babino, A., Pritsch, O., Oppezzo, A., Du Pasquier, R., Roseto, A.,

Osinaga, E. & Alzari, P. (1997) Molecular cloning of a monoclonal

anti-tumor antibody specific for the Tn antigen and expression of an

active single-chain Fv fragment. Hybridoma 16, 317±324.

18. Osinaga, E., Tello, D., Batthyany, C., Bianchet, M., Tavares, G., Duran,

R., CervenÄansky, C., Camoin, L., Roseto, A. & Alzari, P. (1997)

Amino acid sequence and three-dimensional structure of the

Tn-specific isolectin B4 from. Vicia Villosa. FEBS Lett 412,

190±196.

19. Loris, R., Hamelryck, T.W., Bouckaert, J. & Wyns, L. (1998) Legume

lectin structure. Biochim. Biophys. Acta 1383, 9±36.

20. Hamelryck, T.W., Loris, R., Bouckaert, J., Dao-Thi, M.-H., Strecker,

G., Imberty, A., Fernandez, E., Wyns, L. & Etzler, M.E. (1999)

Carbohydrate binding, quaternary structure and a novel hydrophobic

binding site in two legume lectin oligomers from Dolichos

biflorus. J. Mol. Biol. 286, 1161±1177.

21. Piller, V., Piller, F. & Cartron, J.-P. (1990) Comparison of the

carbohydrate-binding specificities of seven N-acetyl-d-galacto-

samine-recognizing lectins. Eur. J. Biochem. 191, 461±466.

22. Osinaga, E., Pancino, G., Porchet, N., Mistro, D., Aubert, J.-P. &

Roseto, A. (1992) Analysis of the carcinoma-associated epitope

defined by monoclonal antibody 83D4. J. Tumor Marker Oncol. 7,

13±24.

23. Laemmli, U. (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227, 680±685.

24. O'Farrell, P., Goodman, H. & O'Farrell, P. (1977) High resolution

two-dimensional electrophoresis of basic as well as acidic proteins.

Cell 12, 1133±1142.

25. Chaplin, M.F. (1982) A rapid and sensitive method for the analysis

of carbohydrate components in glycoproteins using gas-liquid

chromatography. Anal. Biochem. 123, 336±341.

26. Nimtz, M., Noll, G., Paques, E.-P. & Conradt, H.S. (1990)

Carbohydrate structures of a human tissue plasminogen activator

variant expressed in recombinant Chinese hamster ovary cells.

FEBS Lett. 271, 14±18.

27. Fields, C.G., Llyod, D.H., Macdonald, R.L., Otteson, K.M. & Noble,

R.M. (1991) HBTU activation for automated Fmoc solid-phase

peptide synthesis. Peptide Res. 4, 95±101.

28. Kaiser, E., Colescott, R.L., Bossinger, C.D. & Cook, P.I. (1970) Color

test for detection of free terminal amino groups in the solid-phase

synthesis of peptides. Anal. Biochem. 34, 595±598.

29. Greenwood, F.C., Hunter, W.M. & Glover, J.S. (1963) The preparation

of 131I-labelled human growth hormone of high specific radioactivity.

Biochem. J. 89, 114±123.

30. Calvete, J.J., Santos, C.F., Mann, K., Grangeiro, T.B., Nimtz, M.,

Urbanke, C. & Sousa-Cavada, B. (1998) Amino acid sequence,

glycan structure, and proteolytic processing of the lectin of Vatairea

macrocarpa seeds. FEBS Lett. 425, 286±292.

31. Zeng, Y., Bannon, G., Thomas, V.H., Rice, K., Drake, R. & Elbein, A.

(1997) Purification and specificity of beta1,2-xylosyltransferase,

an enzyme that contributes to the allergenicity of some plant proteins.

J. Biol. Chem. 272, 31340±31347.