Identification of a High-Molecular-Weight Glycoconjugate from a Uruguayan Plant that Binds to the Tumor-Associated Tn Antigen

Abstract

The Tn determinant (GalNAc-O.-Ser/Thr), one of the most specific human carcinoma–associated structures, can be identified using both monoclonal antibodies and lectins. In this work we have explored, for the first time, the presence of Tn-binding proteins in aqueous extracts of plants collected in Uruguay. A total of 21 extracts from plants belonging to 12 Phanerogam families were screened by an ELISA assay (competitive inhibition of anti-Tn antibody binding) and by hemagglutination (HAG) activity. Five of the extracts displayed binding to Tn residues, although only those extracts prepared from fruit and leaf from Myrsine coriacea. (Sw.) R. Br. ex Roem & Schult caused total inhibition of the binding activity of anti-Tn MAb 83D4. Considering that Myrsine coriacea. extracts did not display HAG activity against several types of normal red blood cells, we characterized the material with Tn-binding activity from these extracts. We purified this material from Myrsine coriacea. leaf extracts using perchloric acid treatment followed by affinity chromatography and reverse-phase high performance liquid chromatography (HPLC), conserving its Tn-binding activity. We found a high-molecular-weight glycoconjugate that has an apparent molecular mass of approximately 670 kDa, as judged by SDS-PAGE.

Keywords:

• High-molecular-weight glycoconjugates
• plant lectins
• Tn antigen
• Tn-binding proteins
• Uruguayan plants

Introduction

First discovered by Dausset et al. (1959) on the surface of red blood cells from a patient with hemolytic anemia, the Tn antigen (GalNAc-O.-Ser/Thr) is responsible for the polyagglutination of erythrocytes caused by anti-Tn antibodies commonly present in human sera (Tn syndrome). It is recognized that this antigen, expressed in an unmasked form in about 90% of human carcinomas, is one of the most specific human cancer–associated structures (Hakomori, 1989). Tn is detected early in transformed breast cells (Babino et al., 2000), and a direct correlation has been shown between carcinoma aggressiveness and the density of this antigen (Springer, 1995). The Tn determinant has also been implicated in organotropic metastasis of tumor cells (Schlepper-Schafer & Springer, 1989). In addition, Tn has been found in the envelope glycoprotein gp120 of different HIV isolates where it functions as an immunoneutralization epitope (Hansen et al., 1991), thus emphasizing the relevance of the characterization of interactions between the Tn antigen and Tn-binding proteins.

Lectins are carbohydrate binding proteins other than immunoglobulins that display no enzymatic activity toward the recognized sugars. Plant lectins have served as structural models for the analysis of protein-carbohydrate interactions and have received considerable attention as biochemical tools to detect subtle variations in carbohydrate structures. Among anti-Tn plant lectins, the best known are isolectin B4 from Vicia villosa. seeds (VVLB4; Puri et al., 1992), the lectin from Salvia sclarea. (Medeiros et al., 2000), and isolectin A4 from Griffonia simplicifolia. lectin-I (Wu et al., 1999). Tn-binding lectins show a unique specificity among the plant lectins because of their high preference for GalNAc O.-linked to one amino acid (serine or threonine residues) rather than GalNAc at the reducing end of the saccharide chain. We have reported the amino acid sequence and the crystal structure of VVLB4 (Osinaga et al., 1997) and characterized the binding site of VVLB4 in complex with Tn antigen (Babino et al., 2003).

Lectins find application in a variety of biomedical domains, including blood group typing and erythrocyte polyagglutination studies, mitogenic stimulation of lymphocyte subpopulations, and histochemical recognition of malignant cells (Wang et al., 2000). The high economic potential of these proteins and their biological activities encourages the search for new sources that may reveal interesting new proteins with improved properties.

The Phanerogamic flora of Uruguay comprises approximately 2600 species belonging to 850 genera and 150 families (Muñoz et al., 1993). This flora includes species that are widespread in the world, some others with a more restricted distribution area, and there are also a few other species that are endemic only to Uruguay. In this work, we have explored for the first time the presence of Tn-binding proteins in aqueous extracts prepared from plants belonging to the Phanerogam families of Uruguay. As a result of this screening, we identified and purified a high-molecular-weight glycoconjugate from Myrsine coriacea. (common name: canelón.) that displays a strong Tn binding activity.

Materials and Methods

Materials

Prepacked PD-10 columns (Sephadex G-25) and CNBr-activated Sepharose 4B were from Amersham Pharmacia (Uppsala, Sweden). Polyvinylpolypirrolidone (PVPP) and all the sugars assayed were from Sigma (St. Louis, MO, USA). Bicinchoninic acid (BCA) Protein Assay Kit was purchased from Pierce (Rockford, IL, USA).

The monoclonal antibody (Mab) 83D4 (IgM), which binds specifically to the Tn antigen (Osinaga et al., 2000), was produced from a mouse immunized with cell suspensions obtained from formalin-fixed paraffin-embedded sections of a human breast carcinoma (Pancino et al., 1990). MAb was precipitated from ascitic fluids by dialysis against demineralized water at 4°C, dissolved in a small volume of phosphate-buffered saline (PBS) supplemented with 0.5 M NaCl, applied to a Sephacryl S-200 gel column (2.5 × 85 cm), and eluted with PBS buffer. The IgM was excluded from the gel and recovered in the breakthrough. Purified ovine submaxillary mucin (OSM) was obtained according to Tettamanti and Pigman (1968). Asialo-OSM (aOSM), a natural source of Tn antigen, was obtained treating OSM with 1 N HCl at 80°C for 1 h.

Plant material

All screened plants were collected in rural places in Uruguay and identified and a sample registered and deposited at the Herbarium of the Facultad de Química, MVFQ, Montevideo, and the Herbarium of Dr. Eduardo Alonso Paz, EAP, Montevideo, Uruguay.

Preparation of the plant extracts

The fruits, flowers, stems, and tubers were washed with distilled water, processed using a blender mixer, and extracted with 50 mM sodium phosphate buffer pH 7.4 containing 0.15 M NaCl (PBS buffer) for 2 h at 4°C. The leaves were washed with distilled water and treated in a mortar by abrasion with sand (acid- and heat-treated and washed with distilled water). All the mixtures were filtered through cheesecloth and then centrifuged at 9200 × g for 30 min at 4°C. The supernatants were fractionated by ammonium sulfate precipitation (50% saturation), left overnight, and then centrifuged at 9200 × g for 20 min at 4°C. The precipitates were collected and resuspended in PBS buffer (extract).

The seeds were dried at room temperature, washed with distilled water, and ground into meal using a mill. The meal was suspended with 50 mM acetate buffer pH 6.0 (acetate buffer), processed using a blender mixer, and extracted for 2 h at 4°C. The suspension was treated as indicated above. When extracts were dark in color, they were treated with PVPP, then the mixture was centrifuged as before, and the PVPP treatment was repeated. In some cases, the clear supernatants were concentrated by precipitation with 50% saturation ammonium sulfate; the mixture was left overnight and then centrifuged at 9200 × g for 20 min at 4°C. The precipitate was collected and resuspended in acetate buffer.

Protein was determined by the bicinchoninic acid (BCA) method (Smith et al., 1985) according to the manufacturer's protocol at 60°C and an incubation time of 15 min.

Hemagglutination (HAG) activity

Rabbit and mouse red cells were obtained from fresh blood collected in Alsever's medium. The erythrocytes were washed four-times with PBS buffer by centrifugation for 3 min at 1500 × g, and they were diluted to give a suspension of 4% red cells. The trypsination of the red cells and HAG assays were done essentially according to Nowak et al. (1976). Red cells from rabbit and mouse (with and without trypsin treatment) were used to test HAG.

Sugar specificity

The N.-acetylgalactosamine (Gal-NAc) specificity of the lectin binding to erythrocytes was determined by inhibiting agglutination with 100 mM sugar solutions in 0.15 M NaCl. Galactose, GalNAc, 1-amino-1deoxy-β-d-galactose (Gal₁-NH₂), and 2-amino-2-deoxy-β-d-galactose (Gal₂-NH₂) were used for the analysis. The lectin dilution used for the end point was the highest dilution able to cause 50% HAG. To determine the minimum concentrations required for HAG inhibition by GalNAc and galactose related sugars, a two-fold serial dilution of the sugar solutions was prepared. The contents of the wells were mixed by gently shaking, the plates were covered with plastic wrap, and after 30 min of incubation the extent of HAG was detected visually. These inhibition studies were performed with the extracts that were able to cause visible HAG under the conditions described.

Preparation of N.-acetylgalactosaminyl-Sepharose

This was done according to Franco Fraguas et al. (2003). Sepharose 4B was activated with epichlorohydrin, and the resulting epoxy-activated gel was suspended in 10% (w/v) GalNAc solution in 0.5 M sodium hydroxide and gently rotated end-over-end overnight at room temperature. The gel was washed on a glass filter with 0.5 M sodium hydroxide, and the remaining epoxy groups were blocked with β-mercaptoethanol for 2 h at room temperature. The gel derivative was washed with water until pH was neutral, equilibrated with 0.15 M NaCl solution, and kept at 4°C until used.

ELISA assay

Microtiter wells (Nunc, Roskilde, Denmark) were coated with 100 µl of aOSM (0.1 µg/ml in 0.1 M carbonate buffer, pH 9.6) by overnight incubation at room temperature. The wells were washed with 0.1% Tween 20 in PBS and incubated with 1% gelatin in PBS at 37°C for 60 min. After 3 washes, wells were incubated with 100 µl of samples diluted in PBS (1/1, 1/3, and 1/15) for 90 min at 37°C. After three washes, the wells were incubated with 100 µl of 2.5 µg/ml anti-Tn MAb 83D4 for 60 min at 37°C, and after three washes biotin-labeled anti-mouse IgM in 0.5% gelatin, 0.1% Tween 20 in PBS (PBS/TG) at 37°C for 60 min. Unbound material was then washed off, and 100 µl of 1/2000 avidin-peroxidase complex (Sigma) in PBS/TG was added for 60 min at 37°C. Peroxidase activity was demonstrated by incubation in 2,2-azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS) (3 mg) and 30% hydrogen peroxide (5 µl) in phosphate-citrate buffer (10 ml), pH 5.0. Reaction was allowed to proceed for 30 min at room temperature, and absorbance was read at 405 nm with an ELISA reader. VVLB4 (5 µg/ml) was used as positive inhibition control. Inhibition of binding was considered significant when higher than 50% inhibition.

Perchloric acid (PCA) treatment of the Myrsine coriacea. extracts

PCA precipitation of the samples was performed as previously described (Pancino et al., 1991). Each sample was precipitated with PCA to a final concentration 0.2 N and dialyzed against PBS at 4°C. Tn-binding activity of soluble and precipitated fractions were determined by ELISA.

Purification of Tn-binding material from Myrsine coriacea.

The leaf extract from Myrsine coriacea. was fractionated by 0.2 N PCA precipitation. The soluble material was dialyzed against PBS and applied to a column of GalNAc-Sepharose (5.0 ml of packed gel) equilibrated with PBS buffer. The unbound material was washed off with PBS buffer supplemented with 1 M NaCl. Elution was performed with 0.1 M glycine pH 2.5 and 0.1 M triethylamine pH 11.5. The eluted fractions were pooled, concentrated, and dialyzed against distilled water. The material with Tn binding activity desorbed from the affinity column by basic elution was then applied to Gilson HPLC system (Middleton, WI, USA) with a C₁₈ reverse-phase Lichrospher RP100 column (25 × 0.4 cm, 5 µm particle size) eluting at 1 ml/min with a mixture of 0.1% (v/v)trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B) employing the following chromatographic conditions: first gradient of 5–40% B for 35 min, followed by 40–70% B for 10 min, and isocratic at 70% B for 5 min. The eluate was monitored at 220 and 280 nm, and chromatographic fractions were collected manually. The eluted material was concentrated, dialyzed against distilled water, and used for Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), HAG, and inhibition sudies.

SDS-PAGE

SDS-PAGE was carried out as described (Laemmli, 1970) using homogeneous 7.5% and gradient 5–15% gels. SDS-PAGE analysis of samples under reducing and nonreducing conditions was performed, and the proteins were silver stained. Analysis was also performed using Phast-System equipment (Pharmacia, Uppsala, Sweden) with 8–25% gradient Phast Gels. SDS-PAGE under reducing conditions was performed, and proteins were silver and periodic acid–Schiff (PAS) stained according to the manufacturer's instructions.

Results and Discussion

Evaluation of Tn-binding activity of the extracts

The botanical details, as well as the common names and popular uses of the Uruguayan plants used in this screening, are detailed in Table 1. The binding to Tn of the extracts assayed by ELISA is shown in Figure 1. Five of the 21 extracts investigated produced some inhibition of MAb 83D4 binding activity. Only two extracts, those prepared from fruit and leaf extracts from Myrsine coriacea., caused complete inhibition of MAb 83D4 binding. We also found that the fruit and leaf extracts from Sebastiana schottiana. both caused some inhibition, as well as the fruit extract from Pavonia sepium..

Table 1.. Plant species used in the current screening.

Plant name	Family	Herbarium number	Common names	Popular medicine uses
Bidens laevis.	Asteraceae	EAP S/N MVFQ 4164	—	—
Cayaponia martiana.	Cucurbitaceae	EAP 2872	Tayuyá	Laxative
Sebastiana brasiliensis.	Euphorbiaceae	EAP 2877	Blanquillo	—
Sebastiana schottiana.	Euphorbiaceae	EAP 2878	Sarandí negro	—
Vigna luteola.	Fabaceae	EAP S/N MVFQ 4166	Porotillo	—
Pavonia sepium.	Malvaceae	EAP 2874	Malvavisco de cerco	Emollient; treatment of respiratory catarrh
Myrsine coriacea.	Myrcinaceae	EAP 2875	Canelón	—
Salix humboldtiana.	Salicaceae	EAP 2876	Sauce criollo	Antipyretic, digestive aid
Datura ferox.	Solanaceae	EAP S/N MVFQ 4163	Chamico	Antiparasitic, antiasthmatic
Solanum commersonii.	Solanaceae	EAP S/N MVFQ 4218	Batatilla purgante	Laxative
Daphnopsis racemosa.	Thymelaceae	EAP 2873	Envira	—

Figure 1. Tn-binding activity in 21 Uruguayan plant extracts. The assay was performed by competitive ELISA on wells coated with aOSM, evaluating the inhibition of binding activity of the anti-Tn MAb 83D4. The study was performed several times in duplicate; mean results of 1/1 dilution are shown. The extract numbers correspond to those in Table 2. Bar 22 corresponds to the reactivity of MAb 83D4 without inhibitor.

Table 2.. Hemagglutination (HAG) activity and effect of galactose-related sugars in the extracts prepared from Uruguayan plants on 4% suspensions of red cells (native and trypsin-treated).

Extract number	Plant name	Part used	Protein (mg/ml)	HAG^a.	Sugar specificity
1	B. laevis.	Flowers	0.4	—	—
2	B. laevis.	Leaves	0.4	—	—
3	C. martiana.	Leaves	0.6	R	Gal
4	C. martiana.	Fruits	4.5	R
5	C. martiana.	Stems	0.2	—	—
6	S. brasiliensis.	Fruits	2.2	R,^TR	Gal1-NH2, lactose
7	S. schottiana.	Fruits	4.9	—	—
8	S. schottiana.	Leaves	6.7	—	—
9	V. luteola.	Beans	12.3	R	Gal, Gal1-NH2
10	P. sepium.	Leaves	0.3	R	Gal1-NH2, lactose
11	P. sepium.	Fruits	0.9	—	—
12	M. coriacea.	Leaves	2.4	—	—
13	M. coriacea.	Fruits	4.0	^TR	Lactose
14	S. humboldtiana.	Flowers	0.3	R,^TR	—
15	S. humboldtiana.	Leaves	42.0	M,^TM	Mellibiose
16	D. ferox.	Fruits	1.7	R	Fetuin
17	D. ferox.	Seeds	1.0	R	—
18	S. commersonii.	Tubers	4.0	R
19	S. commersonii.	Leaves	2.0	R
20	D. racemosa.	Fruits	4.5	R,^TR
21	D. racemosa.	Leaves	1.2	—	—

^a.R, rabbit red cells; ^TR, trypsin-treated rabbit red cells; M, mouse red cells; ^TM, trypsin-treated mouse red cells.

Hemagglutination activity

Some of the extracts used in the current study were previously screened for HAG activity and characterized in terms of their sugar specificity by analysis of the inhibition of HAG caused by different sugars (Plá et al., 2003). In this work, in parallel with the ELISA analysis, we also assayed the HAG activity of the extracts, and we explored the inhibition of HAG caused by different galactose-related sugars. The information from the two analyses together may help to differentiate Tn-binding lectins from GalNAc-binding lectins. Results of HAG and inhibition studies using galactose-related compounds are summarized in Table 2. Thirteen of the 21 analyzed extracts were positive for HAG activity when using the rabbit and mouse (treated and nontreated) red cells. Among these, only in six cases was the HAG inhibited by galactose-related sugars. It is interesting that in none of these extracts was HAG inhibited by GalNAc. For the case of the flower extract from Salix humboldtiana., the HAG was positive for the rabbit red cells (treated and nontreated), and it was not inhibited by any of the sugars assayed, indicating that HAG in this case was nonspecific. For the other extracts, HAG was inhibited by the other sugars different from galactose or galactose-related compounds.

Purification and partial characterization of Tn-binding material from Myrsine coriacea.

Based on the results from the ELISA and HAG assays, we attempted to isolate the lectin from Myrsine coriacea. leaf extract. We explored different approaches for the purification of the component responsible for the Tn-binding activity of the extracts. We concluded that treatment with 0.2 N PCA was most appropriate for the lectin purification, as the activity remained in the soluble fraction. The purification procedure included affinity chromatography on GalNAc-Sepharose column (data not shown). The material eluted from this column with 0.1 M triethylamine pH 11.5 displayed absorption at 280 nm and strong Tn-binding activity, whereas the breakthrough material did not bind Tn (data not shown). The native proteins eluted from affinity chromatography were applied to a RP-HPLC C₁₈ column, and two main components were detected (Fig. 2). We found Tn-binding activity in the fast-eluted material (at 5 min, data not shown). SDS-PAGE analysis of this fraction showed that it migrates as a smear, with its main component having an apparent molecular mass of approximately 670 kDa under nonreducing and reducing conditions (Fig. 3). As this smear was also identified with the PAS stain (data not shown), we presume it contains carbohydrate components. The very fast elution from RP-HPLC and its solubility in 0.2 N PCA suggests that it is a high-molecular-weight glycoconjugate.

Figure 2. Reverse-phase HPLC elution profile. The C₁₈ column was loaded with the fraction eluted from affinity chromatography using GalNAc-Sepharose. The arrow indicates elution time of the component with Tn-binding activity (5% solvent B). UA 280 nm: Units Absorbance at 280 nm.

Figure 3. SDS-PAGE of purified material from Myrsine coriacea. leaves. The 7.5% gel was silver stained. The material used was first treated with 0.2 N PCA, then subjected to affinity chromatography and finally to RP-HPLC (fast peak), as described in the text. Lanes: (a) reduced, (b) nonreduced.

In summary, this first evaluation using 21 extracts from Uruguayan plants has allowed us to identify Myrsine coriacea. as a source of a potent competitive inhibitor of the Tn-binding activity of MAb 83D4. The inhibitory effect was found in the extracts prepared from leaf and fruit, and it was also found in the purified material from the leaf extracts. To the best of our knowledge, this is the first Tn-binding molecule shown to exhibit a relative molecular mass of approximately 670 kDa as judged by SDS-PAGE. This work also contributes to increasing knowledge about our regional plants. Future work will be focused on determining the exact molecular nature of this Tn-binding glycoconjugate, which could be a high-molecular-weight lectin or a proteoglycan. In addition, we will evaluate whether this glycoconjugate could be a new tool to improve Tn antigen detection in clinical samples of patients with cancer.

Acknowledgments

The work was supported by the International Foundation for Science (IFS, Project F-2834-3), Sweden, by grants from the Comisión Honoraria de Lucha Contra el Cancer (Uruguay) and from Conicyt (Uruguay). We thank Dr. Valerie Dee for linguistic revision.