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Two jacalin-related lectins from seeds of the African breadfruit (Treculia africana L.) a

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Michiko Shimokawa , Shadrack Makuta Nsimba-Lubaki , Namiko Hayashi , Yuji Minami , a

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Fumio Yagi , Keiko Hiemori , Hiroaki Tateno & Jun Hirabayashi

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Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan b

Département de Biologie et Techniques Appliquées, Institut Supérieur Pédagogique de la Gombe, Kinshasa, R.D. Congo c

Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan Published online: 26 Aug 2014.

To cite this article: Michiko Shimokawa, Shadrack Makuta Nsimba-Lubaki, Namiko Hayashi, Yuji Minami, Fumio Yagi, Keiko Hiemori, Hiroaki Tateno & Jun Hirabayashi (2014): Two jacalin-related lectins from seeds of the African breadfruit (Treculia africana L.), Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.948376 To link to this article: http://dx.doi.org/10.1080/09168451.2014.948376

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Bioscience, Biotechnology, and Biochemistry, 2014

Two jacalin-related lectins from seeds of the African breadfruit (Treculia africana L.) Michiko Shimokawa1, Shadrack Makuta Nsimba-Lubaki2, Namiko Hayashi1, Yuji Minami1, Fumio Yagi1,*, Keiko Hiemori3, Hiroaki Tateno3 and Jun Hirabayashi3 1

Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan; Département de Biologie et Techniques Appliquées, Institut Supérieur Pédagogique de la Gombe, Kinshasa, R.D. Congo; 3Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan 2

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Received May 19, 2014; accepted July 7, 2014 http://dx.doi.org/10.1080/09168451.2014.948376

Two jacalin-related lectins (JRLs) were purified by mannose-agarose and melibiose-agarose from seeds of Treculia africana. One is galactose-recognizing JRL (gJRL), named T. africana agglutinin-G (TAA-G), and another one is mannose-recognizing JRL (mJRL), TAA-M. The yields of the two lectins from the seed flour were approximately 7.0 mg/g for gJRL and 7.2 mg/g for mJRL. The primary structure of TAA-G was determined by protein sequencing of lysyl endopeptic peptides and chymotryptic peptides. The sequence identity of TAA-G to other gJRLs was around 70%. Two-residue insertion was found around the sugar-binding sites, compared with the sequences of other gJRLs. Crystallographic studies on other gJRLs have shown that the primary sugar-binding site of gJRLs can accommodate Gal, GalNAc, and GalNAc residue of T-antigen (Galβ1-3GalNAcα-). However, hemagglutination inhibition and glycan array showed that TAA-G did not recognize GalNAc itself and T-antigen. TAA-G preferred melibiose and core 3 O-glycan. Key words:

jacalin-related lectin; galactose-binding; core 3 O-glycan; Moraceae; Treculia africana

Jacalin-related lectins (JRLs) have been found in plant kingdom including Angiosperm, Gymnosperm, and true fern.1–3) Two kinds of JRLs, galactose-recognizing and mannose-recognizing have been reported. Galactose-recognizing lectins (gJRLs) have been found only in Moraceae, whereas mannose-recognizing jacalin-related lectins (mJRLs) are widely distributed.1) They are different in sugar-binding specificity, structure, and subcellular localization.4) gJRLs are synthesized as preproproteins, which undergo co- and

post-translational processing, and built up of cleaved protomers, short β-chain and long α-chain.5–7) On the contrary, mJRLs are not cleaved and have a single polypeptide chains. gJRLs are produced by the cleavage of a loop in mJRL homologs, resulting that gJRLs show the preference to galactose and N-acetyl galactosamine. Pratap et al.8) reported the two differences between the carbohydrate binding sites of Artocarpin (mJRL) and Jacalin (gJRL). One is the loop cleavage produced by the post-translational proteolysis, and other one is the presence of four aromatic residues in the binding site of Jacalin. Stacking interactions of the aromatic residues with galactose are important for Jacalin, whilst no aromatic residue is found in the binding site of Artocarpin. Van Damme et al.9) reported that the bark of black mulberry (Morus nigra) contained a plenty of the two kinds of JRLs, Morniga G and Morniga M. Further, it has been known that jackfruit (Artocarpus integrifolia) contains the two kinds of lectins, Jacalin and Artocarpin,10) in the seeds. As gJRLs, Maclura pomifera agglutinin (MPA),5,11) Morniga G,5) and several Artocarpus lectins,12–16) including Jacalin, have been reported. gJRLs have been known to recognize GalNAc, T-antigen, and O-glycans.11,15,17–22) Several sugar-binding analysis of these gJRLs including frontal-affinity chromatography and glycan array revealed that these lectins could recognize core 3 O-glycan or other sugars.23–25) Synthesis of core 3 O-glycan is related to the regulation of tumor formation and metastasis.26,27) Therefore, the recognition of this glycan by gJRL is very important in biochemical and biomedical study of tumors. Kabir 28) reported the application of Jacalin in many immunological researches, including IgA nephropathy and the analysis of O-linked glycoproteins. Bies et al.29) described the use of MPA in drug delivery system. Recently, the use of gJRLs has been presented in

*Corresponding author. Email: [email protected] Abbreviations: AHL, Artocarpus hirsuta lectin; BSM, Bovine submaxillary mucin; CCA, Castanea crenata agglutinin; CRLL, Cycas revoluta leaf lectin; ELISA, Enzyme-linked immunosorbent assay; MPA, Maclura pomifera agglutinin; Morniga G, Galactose-recognizing agglutinin from Morus nigra; TAA, Treculia africana agglutinin. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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lectin microarrays. Thus, gJRLs have been known as useful tools in the recognition of O-linked glycans. Treculia africana, known as African bread tree, belongs to Moraceae. Previously, one lectin with mannose-recognizing specificity has been reported from this plant.32) However, differing from the report, in this study, we isolated gJRL and mJRL (TAA-G and TAA-M) from seeds of T. africana and determined the primary structure of TAA-G. Furthermore, we discussed the sugar-binding property of this gJRL.

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Materials and methods Materials. Seeds of T. africana were collected in Democratic Republic of the Congo. Melibiose-agarose and mannose-agarose were purchased from Sigma Co. (St Louis, MO, USA). Sheep anti-rabbit IgG (Fab′)2, conjugated with peroxidase was from Wako Pure Chemical industries (Osaka, Japan). IgGs against Castanea crenata agglutinin (CCA) was obtained as previously described.33) Chymotrypsin and lysyl endopeptidase were purchased from Wako Pure Chemical Industries. GlcNAcβ1-3GalNAc-α-PNP (core 3 O-glycan) and Galβ1-3[GlcNAcβ1-6]GalNAc-α-PNP (core 2 O-glycan) were purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Quail albumin and quail ovomucoid were of our laboratory collections. Human IgA was purchased from Oriental Yeast Co. (Tokyo, Japan). Other saccharides, oligosaccharides, and glycoproteins were from Sigma Co. All other reagents were commercially available. Asialoglycoproteins were prepared by desialylation with 0.1 M H2SO4 at 80 °C. β-N-acetylglucosaminidase treatment of asialo-bovine submaxillary mucin (asialoBSM) was conducted with a final 0.5 unit/mL of β-Nacetylglucosaminidase (Canavalia ensiformis, Sigma Co.) in 0.1 M sodium acetate buffer, pH 5.0 for 18 h at 37 °C. Isolation of lectins (TAA-G and -M) from seeds of T. africana. All steps of purification were done at 4 °C. Hemagglutinating activity was measured throughout all the purification procedures with rabbit erythrocytes. A total of 2 g of seeds was homogenized and extracted with 100 mL of PBS at 4 °C for 1 h, and the homogenate was filtered through two layers of gauze and centrifuged at 12,000 × g. The solution was centrifuged to remove insoluble material. The supernatant solution was saturated with 90% ammonium sulfate. Protein precipitated was collected by centrifugation at 8000 × g and dissolved in 20 mL of phosphatebuffered saline, pH 7.2 (PBS). After centrifugation, the supernatant was applied onto a column of mannoseagarose (ϕ 9.0 × 32 mm) previously equilibrated with PBS. After washing the column with PBS, adsorbed protein was eluted with 150 mM methyl-α-mannoside. The flow through fraction was further applied onto a column of melibiose-agarose. After washing with PBS, hemagglutinating activity was eluted with 60 mM melibiose in PBS. On the other hand, the adsorbed fraction eluted with methyl-α-mannoside was dialyzed against PBS, and purified in a similar manner with the flow

through fraction. Affinity-purified preparations were used for the following analyses except for amino-acid sequencing. Hemagglutination assay. Hemagglutination activity was measured in microtiter plates, in a final volume of 70 µL PBS. Each well contained 50 µL of lectin solution and 20 µL of a 4% (v/v) suspension of rabbit erythrocytes. Agglutination was assessed after incubation for 1 h at room temperature, and hemagglutinating activity was expressed as titer, namely the reciprocal of the highest dilution that gave a positive result. The specific hemagglutinating activity was defined as titer (mg lectin)−1. Quantitation of protein and carbohydrate. Protein was quantified by the method of Lowry et al.34) with bovine serum albumin as standard, and carbohydrate was quantified by the phenol-sulfuric acid method of Dubois et al.35) with D-mannose as standard. Two milligrams of TAA-G purified on melibiose-agarose was used for carbohydrate analysis.

Electrophoresis and molecular mass determination of TAA-G and TAA-M. The molecular masses of TAAs were measured by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOFMS, Bruker Daltonics, Bremen, Germany). The molecular masses of the lectins were also measured by gel filtration on a column of TSK-gel SWXL (ϕ 7.5 × 300 mm). The solvent used was 0.1 M sodium phosphate buffer, pH 7.0, at a flow rate of 0.4 mL min−1. Polyacrylamide gel electrophoresis (PAGE) was carried out by the method of Davis 36) at pH 8.9. Tricine SDS-PAGE was performed in 4% stacking gel and 16.5% resolving gel as described by Schagger and von Jagow. 37) The gel was stained with Coomassie Brilliant Blue R-250.

Amino-acid sequence. TAA-G was separated into α-chain and β-chain by HPLC separation on COSMOSIL Protein-R column (ϕ 4.6 × 250 mm; Nacalai Tesque, Kyoto, Japan) with a linear gradient of 0–60% acetonitrile in 0.1%(v/v) trifluoroacetic acid. HPLC showed the heterogeneity of α-chain and β-chain, and the main peaks of both chains were separately collected and used for sequencing. The β-chain was sequenced without proteolytic digestion. The sequence of α-chain was constructed after sequencing from the N-terminus and sequencing of fragment peptides obtained with two proteolytic digestions. Chymotryptic digestion of TAA-G (10 nmol) was carried out in 50 mM sodium bicarbonate (100 μL, pH 8.0) with enzyme: substrate, 1:100, mol/mol. Lysyl endopeptic digestion of TAA-G (10 nmol) was performed in 50 mM Tris–HCl buffer (100 μL, pH 9.0) with enzyme: substrate, 1:20, mol/mol. After the enzyme reactions for 4 h at 37 °C, fragment peptides were separated by reverse-phase HPLC on COSMOSIL 5 C18 MS-II column (ϕ 4.6×250 mm; Nacalai Tesque) with a

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Two JRLs from seeds of the African breadfruit

linear gradient of 0–80% acetonitrile in 0.1%(v/v) trifluoroacetic acid. Amino-acid sequencing was performed with an Applied Biosystems Procise 492 protein sequencer. 38) Enzyme-linked immunosorbent assay (ELISA). The wells in flat-bottomed titer plates were coated with solution of lectins in PBS overnight at 4 °C. Then the wells were washed twice with PBS containing 0.05% Tween 20 (PBS-T). The IgGs against CCA was diluted 4000-fold with PBS, and wells were incubated with diluted solution of the antibody for 2 h at room temperature. The plates were emptied again and washed twice with PBS-T. The wells were further incubated with the secondary antibody [sheep anti-rabbit IgG (Fab′)2, conjugated with peroxidase, diluted 2000-fold with PBS] for 1.5 h at room temperature. The wells were washed twice with PBS-T. Peroxidase reaction was performed with ELISA POD substrate TMB kit (Nacalai Tesque). The reaction was monitored at 620 nm with a microplate reader. Cycas revoluta leaf lectin (CRLL, mJRL 2)) was used as a positive control. Reactivities of lectins were expressed as % relative to the value of CRLL (A620 = 0.125). The blank value was measured without primary antibodies. Glycan array. Sugar-binding profiling of TAA-G was determined as described by Tateno et al.39). Glycoproteins and glycoside-polyacrylamide used for glycan array are the same described in the previous paper for Marasmius oreades lectin.40) Table 1 shows the glycans used for the array.

Results Purification of TAAs Two JRLs were purified by two affinity chromatographies from seed flour of T. africana. Fig. 1 shows the Table 1.

Glycans used for glycan array.

Trivial name

Glycans

Melibiose Galα1-4LN Galα1-3LN Galα1-3Lac Galα1-3Gal Galα1-2Gal aGal GP BSM Siaα2-6Core 1 ST STn (Gc) STn aGP aBSM Galb-Core3 [3′S]Core1 Core8 Core6 Forssman Core4 Core3 Core2 Core1 Tn

Galα1-6Glcβ1-PAA Galα1-4Galβ1-4GlcNAcβ1-PAA Galα1-3Galβ1-4GlcNAcβ1-PAA Galα1-3Galβ1-4Glcβ1-PAA Galα1-3Galβ1-PAA Galα1-2Galβ1-PAA Galα1-PAA Human glycophorin (Disialyl T and sialyl Tn) Bovine submaxillary mucin (Sialyl Tn) Galβ1-3(Neu5Acα2-6)GalNAcα1-PAA Neu5Acα2-3Galβ1-3GalNAcα1-PAA Neu5Gcα2-6GalNAcα1-PAA Neu5Acα2-6GalNAcα1-PAA Asialo human glycophorin MN (T) Asialo bovine submaxillary mucin (Tn) Galβ1-4GlcNAcβ1-3GalNAcα1-PAA (3OSO3)Galβ1-3GalNAcα1-PAA Galα1-3GalNAcα1-PAA GlcNAcβ1-6GalNAcα1-PAA GalNAcα1-3GalNAcβ1-PAA GlcNAcβ1-3(GlcNAcβ1-6)GalNAcα1-PAA GlcNAcβ1-3GalNAcα1-PAA Galβ1-3(GlcNAcβ1-6)GalNAcα1-PAA Galβ1-3GalNAcα1-PAA GalNAcα1-PAA

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Fig. 1. Affinity chromatographic purification of the two fractions separated on mannose-agarose. Notes: A, the flow through fraction on mannose-agarose was purified on melibiose-agarose (2 mL); B, the adsorbed fraction on mannose-agarose (2 mL) was purified on melibiose-agarose. The arrow indicated the starting position of elution with 60 mM melibiose. Protein amount corresponding to 0.35 g seeds was purified on the column. Collected volumes were 2.0 mL for fraction number 1–15, and 1.0 mL for fraction number 16~. Circle, hemagglutination activity and broken line, absorbance at 280 nm.

second affinity chromatography on melibiose-agarose of the two fractions separated by mannose-agarose. The fraction eluted from melibiose-agarose was named T. africana agglutinin-G (TAA-G) and the fraction bound to only mannose-agarose was named TAA-M. Table 2 summarizes the purification of two lectins. The amounts of TAA-G and TAA-M were estimated to be 7.0 mg and 7.2 mg/g seeds. Specific activity of the two lectins were different, 64,000 titer/mg for TAA-G and 3200 titer/mg for TAA-M. The two TAA-G fractions in Table 2 gave the same results on SDS-PAGE, MALDITOFMS, and hemagglutination inhibition. Fig. 2 shows SDS-PAGE and native PAGE of TAA-G and TAA-M. The molecular masses of TAA-G and TAA-M were 15 and 17 kDa, respectively. The molecular mass of TAA-G corresponds to α-chain. Native PAGE shows the heterogeneity of both preparations. MALDI-TOFMS showed several peaks and broad peaks close to the main peaks of TAA-G and TAA-M, though the main peaks were estimated to be m/z 2100 for β-chain of TAA-G and m/z 14,830 for α-chain of TAA-G, and m/ z 16,120 for TAA-M, respectively (data not shown). HPLC gel filtration shows the same molecular masses of the two lectins, 64 kDa. Therefore, TAA-G was estimated to be composed of four small subunits and four large subunits, and TAA-M was composed of four subunits. Fig. 3 shows the reactivity of TAA-G and -M against anti-CCA antibodies by ELISA. TAA-G was found to be a gJRL, from its primary structure, but the sequence of TAA-M was not analyzed. Instead, its

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Table 2. Purification of two Jacalin-related lectins from T. africana seeds. Hemagglutination activity was measured with native rabbit erythrocytes. Yield was calculated on the basis of protein amount. Details, see Materials and methods.

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Crude extract Mannose-agarose A, flow-through B, 150 mM Me-α-Man Melibiose-agarose A, 60 mM melibiose (TAA-G) B, flow-through (TAA-M) 60 mM melibiose (TAA-G)

Protein (mg)

Hemagglutination activity (titer)

Specific activity (titer/mg protein)

Yield (%)

58.0 41.7 16.0 12.7 14.4 1.3

960,000 800,000 130,000 814,000 46,000 83,200

16,550 19,180 8,125 64,000 3,200 64,000

100 71.9 27.6 21.9 24.8 2.2

Fig. 2. Electrophoreses of TAAs. Notes: A, Tricine-SDS-PAGE. B, Native PAGE at 12.5% gel and pH 8.9. Lanes M, 1, and 2, molecular mass markers, TAA-G and TAA-M, respectively. Molecular mass markers for SDS-PAGE: lysozyme 14.3 kDa, trypsin inhibitor 20.1 kDa, carbonic anhydrase 29 kDa, lactate dehydrogenase 36.5 kDa, glutamate dehydrogenase 55 kDa, bovine serum albumin 69 kDa, and phosphorylase b 97.4 kDa (wide range; Technical Frontier, Tokyo).

Fig. 3. Reactivities of TAAs against anti-CCA antibodies in comparison with CRLL. Notes: The reactivities of lectin (2 µg) against anti-CCA antibodies were compared. All the values were average of three determinations.

antigenicity was examined with anti-CCA antibodies. CCA is an mJRL with tandem-repeat structure. CRLL is also an mJRL with tandem repeat structure. TAA-M shows the higher antigenicity than TAA-G. It is apparent that TAA-M is an mJRL.

Amino-acid sequence of TAA-G In this study, only the sequence of TAA-G was determined as shown in Fig. 4. The longer one corresponds to α-chain of gJRLs, and the shorter one, β-chain. The β-chain consisted of 22 amino-acid residues, and full length of the sequence (residues Asn1Ser22) was determined with the protein sequencer. Molecular mass, 2100, calculated from the sequence was compatible with the value estimated by MALDITOFMS. On the other hand, α-chain consisted of 134 amino-acid residues. Chymotryptic hydrolysis and lysyl endopeptic hydrolysis gave 13 and 4 peptides, respectively. By overlapping the amino-terminal sequence (residues Gly1- Tyr64) and these proteolytic peptides, full length of the α-chain was constructed. The calculated mass of α-chain also coincided with the value analyzed by MALDI-TOFMS. Fig. 5 shows the comparison of six gJRLs, and Jacalin, Artocarpus hirsuta lectin (AHL) and Frutalin (Artocarpus incisa lectin) of them belong to the genus Artocarpus. Identity of TAA-G toward Jacalin, MPA, and Morniga G was relatively high; 69, 71 and 75%, respectively. Differing from other gJRLs, TAA-G had two-residue insertion at residues 79 and 80, and one residue was lacking at C-terminus. Fig. 6 shows the phylogenetic tree of gJRLs from primary structure. TAA-G was close to Morniga G but distant from Artocarpus gJRLs.

Sugar-binding specificity of TAAs Table 3 shows the hemagglutination inhibition of TAAs by various sugars and glycoproteins. The inhibition of TAA-G was completely different from that of TAA-M. Of the saccharides, Gal-α-Me and melibiose were inhibitory toward TAA-G. However, GalNAc-α-PNP, T-antigen (Galβ1-3GalNAc) and Galβ1-3GalNAc-α-PNP were not inhibitory. GlcNAcβ1-3GalNAc-α-PNP (core 3 glycan) was the most potent inhibitor but core 2 of O-glycan was not inhibitory. Of the glycoproteins tested, asialo-BSM was the most potent inhibitor, but BSM was not as effective as asialo-BSM. β-N-acetylglucosaminidase treatment of asialo-BSM reduced the inhibitory potency of asialoBSM. This means that core 3 glycan structure is important for the recognition of asialoBSM with TAA-G. On the other hand, TAA-M was inhibited by mannose, methyl-α-mannoside, oligo-mannosides, and glycoproteins with high mannose sugar chains. Of the glycoproteins, asialothyroglobulin (bovine) was the most potent inhibitor.

Two JRLs from seeds of the African breadfruit

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Fig. 4. Sequence of TAA-G. Notes: α-Chain and β-chain of TAA-G were sequenced after HPLC separation by a Protein-R column. The sequence of α-chain was constructed based on the sequence from N-terminus and peptide sequences obtained after enzyme digestion. Solid line, sequence from N-terminus; dashed line, lysyl endopeptic peptide; and long-dashed dotted line, chymotryptic peptide.

Fig. 5. Comparison of the sequences of galactose-binding Jacalin-related lectins. Notes: TAA-G sequence is aligned using CLUSTAL W with other gJRLs, AHL: Artocarpus hirsuta lectin,41) Frutalin,42) Jacalin,43) MPA,43) Morniga G.9) Shaded letters denote the residues found in the binding with T-antigen.44–46) Asterisks indicate identical residues, and dashes or colons, similar residues. Arrows above sequence show β-strands of Jacalin (PBD code 1ugw).

Fig. 7 shows the glycan array of TAA-G. TAA-G recognized neither Tn (GalNAc) nor the core 1 structure (T-antigen structure), though TAA-G recognized asialoBSM and asialoglycophorin strongly. Core 3 and core 8 O-glycans were also good ligands for TAA-G. Core 3 O-glycan was a good inhibitor for the hemagglutination inhibition as described above. Sugar recognition of TAA-G is limited, compared with other gJRLs, and TAA-G prefers disaccharides containing galactose and galactoside to other sugars.

Discussion Fig. 3 shows the reactivity of CRLL, and two lectins from seeds of T. africana against anti-CCA antibodies. The reactivities of CRLL, TAA-M, and TAA-G were 100, 37.6 and 18.4%, respectively, suggesting that the two lectins from seeds of T. africana are JRLs. Lim et al.47) reported the contents of two types of JRLs in seeds of two Artocarpus species, and Van Damme et al.9) reported them in bark and seeds of M. nigra.

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Fig. 6. A phylogenetic tree of gJRLs. Notes: The phylogenetic tree based on Clustal W (ver. 1.83) was drawn by Phylodendron (D. G. Gilbert, http://iubio.bio.indiana.edu/ soft/molbio/java/apps/trees/)

The two types of lectins are abundant in seeds of Artocarpus integer and in bark of M. nigra. The content of the two lectins is more than 50% of total protein in the two materials. On the other hand, their content is low in seeds of M. nigra. In seeds of A. integliforia, the amount of Jacalin is very high but KM+(Artocarpin, mJRL) is very low. We have shown that seeds of T. africana contain both JRLs, corresponding to approximately 50% of total soluble protein. Native PAGE and MALDI-TOFMS show that TAA-G and TAA-M are heterogeneous. Heterogeneity (isolectins and N-glycosylation) is widely known in JRLs.5–7,48) Sugar content of TAA-G was estimated to be less than 2.5 µg/mg protein (0.24 sugar residue/mole protein) by phenol-sulfuric method. Furthermore, no Asn-X-Ser (Thr) sequence was found in TAA-G. Accordingly, the

heterogeneity of TAA-G seemed to be derived from isolectins. Several variants were found as follows: N-terminus defect in β-chain, and Gln at residue 42, Ser at residue 62, Lys at residue 74, Gly at residue 94, and Lys at residue 102 in α-chain. TAA-M strongly recognized trimannoside and mannopentaose as found for Morniga M and Artocarpin.49) Sugar-binding profile of TAA-G was different from those of gJRLs reported so far. Firstly, TAA-G did not bind N-acetylgalactosamine and T-antigen, though gJRLs were reported as GalNAcα- and T-antigen recognizing lectins. Fig. 7 shows that TAA-G does not bind T-antigen itself and several compounds related to T-antigen. Furthermore, GalNAc-α-PNP and GalNAc-α-Me did not inhibit hemagglutination of TAA-G. Secondly, it has been known that galactosides with hydrophobic aglycones such as p-nitrophenyl and 4-methylumberyferyl showed the enhanced affinity toward gJRLs,15,21,22,50) compared with Gal-α-Me. However, such an effect was not found for TAA-G. Gal-α-Me inhibited the hemagglutination of TAA-G at 25 mM, but Gal-α-PNP did not at the higher concentration of 50 mM. Additionally, p-aminophenyl α-galactoside-agarose could not retain TAA-G (data not shown). Thirdly, melibiose bound to TAA-G more strongly than Gal-α-Me. On the contrary, this disaccharide was a very weak inhibitor for other gJRLs.21–23,25) Of the other glycosides, core 3 glycan is the best ligand to Jacalin,24) MPA,25), and TAA-G. Fig. 7 shows that TAA-G recognizes core 8 glycan. Similarly, MPA recognized core 8 glycan,25) whereas Jacalin recognized this glycan very poorly.23) Furthermore, in common with Jacalin and Morniga G,44,51,52) TAA-G can recognize mannose as Fig. 1(B) shows the adsorption of TAA-G to mannose-agarose. Fig. 6 shows that TAA-G is closest to Morniga G, distant from Artocarpus gJRLs. Sugar-binding pattern of Morniga G was close to those of other gJRLs, and did not show the characteristics for TAA-G described

Table 3. Inhibition by sugars and glycoproteins of hemagglutinating activity of TAAs. Minimum concentration required for the complete inhibition of titer 4 hemagglutinating activity. TAA-G

TAA-M (mM)

Mannose Man-α-Me Manα1-6(Manα1-3)Man-α-Me Mannopentaose Gal-α-Me Glc-α-Me Melibiose GlcNAcβ1-3GalNAc-α-PNP

– NI at 100 – – 25 NI at 100 12.5 1.56

BSM AsialoBSM AsialoBSM (treated with N-acetyl glucosaminidase) Thyroglobulin (bovine) Asialothyroglobulin (bovine) Asialofetuin IgA Ovomucoid (quail)

1.0 0.156 0.624 NI at 1000 250 62.5 30 250

25 25 5.0 10.0 NI at 100 NI at 100 NI at 100 – (µg/mL) – NI at 1000 – 125 15.6 500 – 62.5

Notes: NI, No inhibition; -, No experiment. The following sugars and glycoproteins were not inhibitory toward TAA-G: galactose at 200 mM; GalNAc, GlcNAc, glucosamine, D-glucose and lactose up to 100 mM; Gal-α-PNP at 50 mM; Gal-β-PNP, GalNAc-α- PNP, GalNAc-β-PNP, Galβ1-3GalNAc, Galβ1-3GalNAc-α-PNP and core 2-α-PNPat 25 mM; fetuin at 1 mg/mL.

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Two JRLs from seeds of the African breadfruit

7

Fig. 7. Glycan array of TAA-G. Notes: Cy3-labeled TAA-G was applied on the glycoconjugate array, and binding was detected by the scanner. Scan image of TAA-G was analyzed with the Array Pro analyzer ver. 4.5.

above. Crystallographic studies of gJRLs have showed that the sugar binding sites contain three loops (residues 46–52, 76–82, and 122–125) and Gly, the N-terminus of α-chain.41,43,53,54) The primary binding site of gJRLs could accommodate galactose and N-acetylgalactosamine residues. Notwithstanding TAA-G contained four residues common in the primary site of other gJRLs, Gly1, Tyr122, Trp123, and Asp125, TAA-G could not recognize T-antigen and a few α-glycosides of GalNAc. However, it is estimated that the primary binding site of TAA-G can accommodate GalNAcα- residue of core 3 and core 8 glycans. The two-residue insertion between residue 78 and 79 for other gJRLs was found in TAA-G. This insertion might alter the interaction of sugar and protein, because Tyr78 and Thr79 in MPA (Val79, Artocarpus gJRLs) were involved in sugar–protein interaction. Raval et al.43) reported the presence of a fourth loop around residues 20–23 which can also influence the conformation of some residues and hence the specificity in Jacalin.25) The residues in this loop and the following residues 24 and 25 present the interaction between Asn20-Gly50, Glu22-Arg82, Thr23-Arg82, Ala24Val75, and Ile25-Leu124 (residue numbers for Jacalin). In TAA-G, Glu22 and Gly50 were replaced to Lys and Asp, respectively. Furthermore, the two-residue insertion at residues 79 and 80 might shift the position of Arg82. For the further elucidation on the sugar specificity and protein structure, crystallographic study of TAA-G is necessary. TAA-G recognized core 3 and core 8 glycans, but neither T-antigen nor GalNAc. On the contrary, other

gJRLs have the broader sugar specificities. This narrow sugar specificity of TAA-G is advantageous for the detection of core 3 glycan, because the sugar detection by TAA-G is more specific. Taken together, TAA-G is expected to be a new sugar detection tool.

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