Báo cáo khoa học: Comparative analysis of carbohydrate-binding properties of two tandem repeat-type Jacalin-related lectins, Castanea crenata agglutinin and Cycas revoluta leaf lectin docx

Comparative analysis of carbohydrate-binding properties of two tandem repeat-type Jacalin-related lectins, Castanea crenata agglutinin and Cycas revoluta leaf lectin Sachiko Nakamura1, Fumio Yagi2, Kiichiro Totani3, Yukishige Ito3 and Jun Hirabayashi1 Glycostructure Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Japan Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Japan RIKEN (The Institute of Physical and Chemical Research), Saitama, Japan Keywords carbohydrate binding specificity; frontal affinity chromatography; Jacalin-related lectin family; lectin Correspondence J Hirabayashi, Glycostructure Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 2, 1-1-1, Umezono, Tsukuba, Ibaraki 305-8568, Japan Fax: +81 29 861 3125 Tel: +81 29 861 3124 E-mail: jun-hirabayashi@aist.go.jp (Received 27 December 2004, revised 23 February 2005, accepted April 2005) doi:10.1111/j.1742-4658.2005.04698.x Lectins belonging to the jacalin-related lectin family are distributed widely in the plant kingdom Recently, two mannose-specific lectins having tandem repeat-type structures were discovered in Castanea crenata (angiosperm) and Cycas revoluta (gymnosperm) The occurrence of such similar molecules in taxonomically less related plants suggests their importance in the plant body To obtain clues to understand their physiological roles, we performed detailed analysis of their sugar-binding specificity For this purpose, we compared the dissociation constants (Kd) of Castanea crenata agglutinin (CCA) and Cycas revoluta leaf lectin (CRLL) by using 102 pyridylaminated and 13 p-nitrophenyl oligosaccharides with a recently developed automated system for frontal affinity chromatography As a result, we found that the basic carbohydrate-binding properties of CCA and CRLL were similar, but differed in their preference for larger N-linked glycans (e.g Man7–9 glycans) While the affinity of CCA decreased with an increase in the number of extended a1–2 mannose residues, CRLL could recognize these Man7–9 glycans with much enhanced affinity Notably, both lectins also preserved considerable affinity for mono-antennary, complex type N-linked glycans, though the specificity was much broader for CCA The information obtained here should be helpful for understanding their functions in vivo as well as for development of useful probes for animal cells This is the first systematic approach to elucidate the fine specificities of plant lectins by means of high-throughput, automated frontal affinity chromatography Lectins are carbohydrate-binding proteins distributed in all of the organisms characterized so far A large number of plant lectins have been isolated and characterized [1,2] Van Damme et al classified them into seven families based on their molecular structures and carbohydrate-binding specificities [3] Members belonging to each family share some common properties Compared with legume and monocot lectin families, jacalin-related lectins (JRLs) were originally assumed to form a relatively small family [4] During the last decade, however, many new members belonging to this family were discovered, and some novel features became evident As a result, JRLs can now be classified into two subgroups in terms of their carbohydrate binding specificities, i.e galactose-binding-type JRLs (gJRLs) and mannose-binding-type JRLs (mJRLs) [5] Abbreviations CCA, Castanea crenata agglutinin; CRLL, lectin from leaves of Cycas revoluta; CRD, carbohydrate-recognition domain; FAC, frontal affinity chromatography; gJRLs, galactose-binding-type Jacalin-related lectins; JRLs, Jacalin-related lectins; mJRLs, mannose-binding-type Jacalinrelated lectins; M2M2M3Mb, Mana1–2Mana1–2Mana1–3Manb; M2M3M6Mb, Mana1–2Mana1–3Mana1–6Manb; MTX, methotrexate; M3GN2, Man3GlcNAc2; PA, pyridylaminated; pNP, p-nitrophenyl 2784 FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS S Nakamura et al gJRLs are represented by Jacalin, known as a useful probe for IgA [6], and also by Maclura pomifera agglutinin [4] and Morus nigra agglutinin (Morniga G) [3] All of these lectins, which come from Moraceae plants, have common structural features: they are composed of four protomers consisting of a short b-chain (2 kDa) and a long a-chain (13 kDa) as a result of proteolytic cleavage of a precursor polypeptide [3,7] On the other hand, the mJRL subgroup comprises many more members, such as Calsepa isolated from Calystegia sepium [8], Artocarpin from Artocarpus integrifolia [9], Heltuba from Helianthus tuberosus [10], BanLec from Musa acuminate [11], Morniga M from M nigra [7], Orysata from Oryza sativa [12,13] and PAL from Phlebodium aureum [14] Although these mJRLs were proved to form a b-prism fold I structure consisting of one carbohydrate-recognition domain (CRD) similar to Jacalin [15–17], no proteolytic modification of mJRLs occurs There is a view that the lack of proteolysis of them may result in preservation of their mannose ⁄ glucose specificity [18] Although all of the mJRLs mentioned above are of the single CRD type (15–16 kDa), new members of much larger molecular size (% 33 kDa) were discovered recently from Castanea crenata (angiosperm) and Cycas revoluta (gymnosperm) [19,20] Sequence analysis of these large mJRLs, i.e C crenata agglutinin (CCA) and C revoluta leave lectin (CRLL) revealed that they form a tandem-repeat structure composed of two jacalin-type CRDs [21,22] Therefore, mJRLs are now thought to be distributed more widely in the plant kingdom with more structural diversity than had ever been previously thought Both CCA and CRLL showed a similar extent of homology (30–40% in amino acid identity) to other JRLs Although the complete amino acid sequence has not yet been determined for CRLL, a high extent of intramolecular homology, i.e between N-terminal and C-terminal CRDs, is also evident for both CCA and CRLL (> 35%) [19,21] Since Greek key motif 3, a region assumed to form a carbohydrate-binding site, is highly conserved in both N-terminal and C-terminal CRDs in CCA and CRLL, all of these CRDs are likely to maintain a sugar-binding function Thus, distribution of a similar type of molecule in taxonomically unrelated plants raises a basic question about the biological significance of tandem repeat-type mJRLs In this context, there are some lines of evidence that certain mJRLs are induced by treatment with methyl jasmonate or by salt stress [12,23] This suggests that mJRLs have some defensive roles in the plant body However, there is no clear evidence for this hypothesis or no such report for CCA and CRLL To understand their functions in plants, it is essential to elucidate FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS Oligosaccharide specificity of Jacalin-related lectins their biochemical properties in terms of carbohydratebinding specificities For this purpose, we recently developed an automated frontal affinity chromatography (FAC) system, which enables us to analyse lectin–oligosaccharide interactions in a high-throughput manner [24] Significant advantages of FAC include high sensitivity and reproducibility In addition, the method is well suited for determination of dissociation constants (Kds) for relatively low-affinity binding (e.g Kd > 10)6 m), thus making it practically advantageous for analysis of lectin–oligosaccharide interactions FAC was originally developed by Kasai et al [25], reinforced by Hirabayashi et al [26], and proved to be an effective alternative successfully applied to comparative analysis of animal lectins with a set of fluorescently labelled glycans [27] Other investigators also used an FAC system equipped with an MS detector for analysis of mushroom lectins, and demonstrated its efficiency [28,29] In this present study we applied two tandem repeattype mJRLs, CCA and CRLL, to this automated FAC system As a result, both conserved and divergent features of these taxonomically unrelated mJRLs became evident Results Evaluation of the lectin columns CCA and CRLL were purified by affinity chromatography on asialofetuin and mannose–agarose columns, respectively, as previously described [19,20] The thus purified proteins were immobilized on NHS-activated Sepharose 4FF, and the resulting resins were packed into miniature columns (inner diameter, mm; length, 10 mm; bed volume, 31.4 lL) as described under Experimental procedures The amounts of immobilized CCA and CRLL were determined to be 2.0 and 1.1 mgỈmL)1 gel, respectively For evaluation of the prepared columns, it was necessary to determine the effective ligand content (Bt) based on the so-called ‘concentration-dependence analysis’ [26,27] For this purpose, however, none of the commercially available p-nitrophenyl (pNP) derivatives of simple saccharides tested, i.e Man-a, Man-b, Glc-a, Gal-a, Gal-b, GalNAc-a, GalNAc-b, Fuc-a, Galb1–4Glc-b, Galb1– 4GlcNAc-b, Galb1–3GalNAc-a, Glca1–4Glc-a or (Glca1–4)5-a, showed any significant affinity for these columns Since these lectins are known to show high affinity for the mannotriose structure, Mana1– 3(Mana1–6)Man [19,20], we tested methotrexate (MTX)-derivatized Man3GlcNAc2 (M3GN2-MTX, Fig 1A) previously synthesized successfully [30] to see 2785 Oligosaccharide specificity of Jacalin-related lectins S Nakamura et al Fig Determination of Bt values (A) Structural formula of MTX-derivatized Man3GlcNAc2 (M3GN2-MTX), which was used for concentration-dependence analysis For determination of Bt values for the immobilized CCA (B) and CRLL (C), M3GN2-MTX was diluted to various concentrations (5–20 lM) and applied to each column The solid lines and dotted lines indicate elution profiles of M3GN2-MTX and control sugar (pNP-Lactose), respectively (left) Woolf–Hofstee-type plots were made by using V–V0 values (right) For details, see text if it would be appropriate for the above concentrationdependence analysis M3GN2-MTX showed strong retardation when it was applied to these lectin columns at the concentration of lm, whereas it showed no significant binding to a BSA–agarose column (2.6 mgỈmL)1, data not shown) Hence, concentrationdependence analysis was performed with M3GN2MTX at various concentrations ranging from to 20 lm (Fig 1B,C) As a result, Bt and Kd values were determined to be 1.49 nmol and 1.2 · 10)5 m, respectively, for the CCA column, and 0.81 nmol and 2.0 · 10)6 m, respectively, for the CRLL column Based on these data, availability of the CCA and CRLL columns was calculated to be 39 and 40%, respectively Specifications obtained for these columns are summarized in Table As regards the detection limit of low-affinity binding, such as those for the pNP-sugars described above, we found approximately lL of experimental error in the V–V0 value in the present FAC system considering the data collection interval (1 s) and the flow rate (0.125 mLỈmin)1) Under such conditions, low-affinity saccharides having Kd values > 7.5 · 10)4 m and > 4.1 · 10)4 m for the above CCA and CRLL columns, respectively, cannot be precisely characterized On the other hand, the maximum V–V0 value measurable in the present system is at least 120 lL, which corresponds to Kd values of 1.3 · 10)5 m and 6.8 · 10)6 m for the CCA and CRLL columns, respectively Thus, a dynamic range of 60-fold is achieved by using these columns with no change in ligand concentrations Table Specifications of CCA- and CRLL-immobilized columns used in this study Lectin name Origin Immobilized (mg ⁄ mL) Bt (nmol) Availability (%) r2 CCA CRLL Castanea crenata Cycas revoluta 2.0 1.1 1.49 0.81 39 40 1.00 0.97 a a M3GN2-MTX Kd (M) 1.2 · 10)5 2.0 · 10)6 Reliability of lines obtained as a result of Woolf–Hofstee-type plot in each concentration-dependence analysis 2786 FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS S Nakamura et al Overall features of oligosaccharide specificities of CCA and CRLL In order to briefly profile oligosaccharide specificities of CCA and CRLL from a global viewpoint, we prepared a panel of 102 pyridylaminated (PA) glycans including 55 N-linked glycans and 38 glycolipid-type glycans (Fig 2) For the determination of Kd, retardation of the elution front relative to that of PA-lactose, i.e V–V0, was measured for each analyte solution diluted to either 2.5 or 5.0 nm The amount of glycan required for determination of reliable Kd value was < pmol, which is much smaller (< 10)3) compared with other methods Since the concentrations are much lower than Kd values assumed for the present lectin columns as described (> % 10)5 m), Kd values could be calculated according to Eqn (2) by using observed V–V0 values in a manner independent of [A]0 For the sake of comparison, bar graph representation was made in terms of affinity constant (Ka) in Fig Obviously, both CCA and CRLL showed affinity for a wide range of N-linked glycans, but not at all for glycolipid-type glycans, which lack mannose This observation is reasonable, because these lectins were characterized as mannose-specific lectins Thus, global features of CCA and CRLL are apparently similar, but detailed features are different as described below Comparison of fine specificities High-mannose-type N-glycans Chromatograms obtained for N-linked type glycans are shown in Fig In the case of CCA (Fig 4A), the strongest affinity was observed for 005 (designated M5, Kd ¼ 1.0 · 10)5 m), followed by 007 (M6, 1.2 · 10)5 m) and 003 (M3, 1.4 · 10)5 m) On the other hand, CCA showed no affinity toward 001 (M2) These results indicate that the Mana1–3Manb structure is essential for CCA binding and that the removal of a1–3Man abolished the affinity for CCA Because CCA could recognize 004 (M4, 2.6 · 10)5 m), which lacks the depicted Mana1–3Manb structure, it proved to have significant affinity to Mana1–3Mana, too In contrast, removal of a1–6Man from M3 (compare 002 and 003) had rather a small effect on affinity (78% relative to 003) This observation agreed with pervious analysis by means of haemagglutination inhibition assay [20,22], isothermal titration caloriemetry and enzyme-linked lectin assay (unpublished data) toward simple saccharides The binding affinity toward CCA was much reduced, when the nonreducing terminal Man of the Mana1–3Manb structure was modified by a1–2Man (008–014, corresponding to M7–9) The FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS Oligosaccharide specificity of Jacalin-related lectins tendency was confirmed by comparison of M6 isomers, 006 and 007; i.e their affinities relative to that of 005 were 36% and 83%, respectively CRLL also showed significant affinity for relatively small high-mannose type glycans, except for 001 and 004 (Figs and 4B) CRLL showed moderate affinity for an M2 saccharide, 002 (Kd ¼ 4.7 · 10)5 m), but not at all for its isomer, 001 Moreover, CRLL did not bind to 004, which lacks the Mana1–3Manb structure These results indicate that the core Mana1–3Manb structure forms an essential unit for CRLL recognition Considering the inability of CRLL to bind to 004, the terminal Mana1–3Mana cannot substitute for the core Mana1–3Manb structure, unlike the case for CCA However, CRLL showed somewhat (30%) higher affinity for 005 (Kd ¼ 3.0 · 10)5 m) than for 003 (3.9 · 10)5 m) By comparison between 002 (M2, 4.7 · 10)5 m) and 003 (3.9 · 10)5 m), it is clear that addition of the Mana1–6Manb branch had only a small effect, if any, on CRLL–glycan interaction CRLL also showed a significant difference in affinity for M6 isomers (006 and 007) When compared with their parental molecule, M5 (005, Kd ¼ 3.0 · 10)5 m), the addition of a1–2Man to the Mana1–3Manb branch resulted in almost complete loss of affinity; whereas that to the Mana1–3Mana1–6Manb branch still enhanced the affinity (2.4 · 10)5 m) The tendency observed here is essentially the same as that observed for CCA However, the effect of the a1–2Man addition makes a clear contrast between the two lectins Therefore, the addition of a1–2Man to the core Mana1– 3Manb had rather a destructive effect on CRLL recognition The most distinguishing feature of CRLL is its highly enhanced affinity for relatively large high-mannose-type glycans, i.e M8–9 As a matter of fact, CRLL showed the strongest affinity for 013 (M8, Kd ¼ 1.1 · 10)5 m) and 014 (M9, 9.5 · 10)6 m), whereas CCA could not bind to these large saccharides at all Binding ability of CRLL to large high-mannose-type glycans was consistent with the results obtained by haemagglutination inhibition assay using glycopeptides [19] Since these saccharides share two common structures, i.e Mana1– 2Mana1–2Mana1–3Manb (M2M2M3Mb) and Mana1– 2Mana1–3Mana1–6Manb (M2M3M6Mb), coincidence of these extended structural units may contribute to the observed high affinity in CRLL In this regard, CRLL showed 1.9-times stronger affinity for an M7 saccharide, 009, containing the M2M2M3Mb unit (Kd ¼ 4.1 · 10)5 m) than for its M7 isomer, 010, containing the M2M3M6Mb unit (7.9 · 10)5 m) Similar results were observed for M8 isomers, 012 (Kd ¼ 3.0 · 10)5 m) containing the M2M2M3Mb unit and 011 (4.9 · 10)5 m) 2787 Oligosaccharide specificity of Jacalin-related lectins S Nakamura et al Fig Schematic representation of oligosaccharide structures Note that the reducing terminal is pyridylaminated for FAC analysis Symbols used to represent pyranose rings of monosaccharides are shown in the box at the bottom of the figure Anomeric carbon, i.e position 1, is placed at the right side, and 2, 3, 4… are placed clockwise Thin and thick bars represent a and b linkage, respectively 2788 FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS S Nakamura et al Oligosaccharide specificity of Jacalin-related lectins Fig Bar graph representation of affinity constants (Ka) of CCA (left) and CRLL (right) toward N-linked glycans The small Arabic figures in the centre correspond to sugar numbers indicated in Fig 2; whereas large Roman figures on the left side of graphs represent types of glycans: high-mannosetype (I), agalacto-type (II), galactosylatedtype (III) and sialylated-type (IV) N-linked glycans, glycolipid-type glycans (V), and others (VI) containing the M2M3M6Mb unit In contrast, the increase in affinity was relatively small, when a1–2Man was added to the M6M6Mb unit; i.e compare M8 isomers, 011 (4.9 · 10)5 m) and 012 (3.0 · 10)5 m) with their parental M7 isomers, 010 (7.9 · 10)5 m) and 090 (4.1 · 10)5 m), respectively Since the increase was FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS approximately 1.5-fold in these cases, the addition of the terminal a1–2Man to the M6M6Mb unit still made some contribution to affinity enhancement In this context, the strongest contribution of a1–2Man was observed when it was added to the M2M3Mb unit: compare 010 with 013, and 011 with 014 (affinity 2789 Oligosaccharide specificity of Jacalin-related lectins S Nakamura et al Fig Elution profiles of N-linked glycans obtained with CCA (A) and CRLL (B) columns Chromatograms of N-linked glycans are shown in the order of sugar numbers together with retardation volumes (upper, V–V0, lL) and dissociation constants (lower, Kd, M) For the sake of convenience, the elution pattern of each saccharide is overlaid with that of PA-lactose, which has no affinity for either lectin, i.e the negative control 2790 FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS S Nakamura et al Oligosaccharide specificity of Jacalin-related lectins Fig (Continued) enhanced by 7.2 and 5.2 times, respectively) In the remaining case of the a1–2Man addition, i.e to the M3M6Mb unit, the effect was rather intermediate (approximately 3.5 times), when the affinity of 013 and FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS 014 were compared with that of their parent saccharides, 009 and 012, respectively These results indicate that the presence of nonreducing end mannose in the M2M2M3Mb unit plays a dominant role in the strong 2791 Oligosaccharide specificity of Jacalin-related lectins interaction between CRLL and large high-mannosetype glycans, whereas M2M3M6Mb and M2M6M6Mb contribute to lesser extents (i.e M2M3M6Mb > M2M6M6Mb) From a practical viewpoint, glycans having both M2M2M3Mb and M2M3M6Mb units show the highest affinity for CRLL Complex-type glycans In the present study, both CCA and CRLL were found to bind to several complex-type N-linked glycans, too As these lectins were previously characterized as mannose-specific lectins [19,20], possible reasons for this discrepancy should be given In the case of CCA, it showed significant affinity for 101 (Kd ¼ 2.2 · 10)5 m), 201 (2.8 · 10)5 m), 301 (1.6 · 10)5 m), and 401 (2.3 · 10)5 m); whereas it showed lower affinity for their position isomers, i.e 102 (3.5 · 10)5 m), 302 (3.7 · 10)5 m), and 402 (5.5 · 10)5 m) relative to 101, 301 and 401, respectively This observation indicates that CCA prefers mono-antennary, complex-type N-linked glycans, which have a1–6 branch, or nonsubstituted a1–3 Man Such a feature is consistent with the idea that Mana1–3Manb forms the core recognition unit On the other hand, we also found that CCA showed significant affinity for bi-antennary glycans, i.e 103 (Kd ¼ 2.9 · 10)5 m), 202 (5.2 · 10)5 m), 304 (3.4 · 10)5 m), 307 (3.7 · 10)5 m), 403 (5.0 · 10)5 m), 404 (5.4 · 10)5 m), and 405 (5.3 · 10)5 m) Their affinities for CCA were similar (3–5 · 10)5 m), but somewhat (40–80%) reduced in comparison with those for the respective parental mono-antennary glycans, i.e 101, 201, 301, and 401 These results indicate that modification of a1–3Man is never detrimental This may explain why CCA could bind to asialofetuinagarose [20], because this glycoprotein contains biantennary, complex-type glycans [31] On the other hand, tri-antennary glycans showed no affinity for CCA Therefore, C4-OH group of a1–3Man is essential for CCA recognition Similar to CCA, CRLL also showed significant binding to mono-antennary, complex-type N-linked glycans having the a1–6 branch, i.e 101 (Kd ¼ 3.9 · 10)5 m), 201 (2.7 · 10)5 m), 301 (4.1 · 10)5 m), and 401 (2.4 · 10)5 m) Unlike CCA, however, CRLL did not show affinity at all for their position isomers (102, 302, and 402) Moreover, CRLL had no affinity for bi-antennary glycans (e.g 103, 104, 202, 203, 303, 304, 307, 308, 403, 404, 405, and 406) Neither triantennary nor tetra-antennary N-linked glycans were targets for CRLL, either Therefore, CRLL is stricter than CCA in that the former never permits substitution of a1–3Man in complex-type glycans 2792 S Nakamura et al In the present study, the influence of a1–6 (core) fucosylation on lectin–glycan interaction could also be examined by comparison between 003 and 015, 101 and 201, 103 and 202, 104 and 203, 301 and 401, 302 and 402, 304 and 403, and 307 and 405 (Figs and 4) In the case of CCA, a1–6 (core) fucosylation slightly reduced the affinity; for example, compare 101 (Kd ¼ 2.2 · 10)5 m) with 201 (2.8 · 10)5 m) With CRLL, however, this type of fucosylation enhanced the affinity by 1.2–1.7 times; compare 101 (Kd ¼ 3.9 · 10)5 m) with 201 (2.7 · 10)5 m) The effect of a1–6 fucosylation was significant, but not very drastic in both cases So, the presence of a1–6Fuc does not have any essential role for the recognition of these mJRLs Oligosaccharides 501–505 represent sialylated glycans Among them, bi-antennary glycans (501, 502, and 503) were recognized by CCA to some degree, whereas none of them showed significant affinity for CRLL In the case of CCA, it is clear that the effect of sialylation of bi-antennary glycans was different between mono-sialylated isomers i.e 501 (Kd ¼ 1.1 · 10)4 m) and 502 (4.5 · 10)5 m) By comparison with nonsialylated glycan, 307 (3.7 · 10)5 m), the inhibitory effect of sialylation was more drastic on the a1–3 branch (affinity reduced to 34%) than on the a1–6 branch (to 82%) Again, this result is consistent with the above observation that Mana1–3Manb is an essential unit for CCA recognition Summary of FAC analysis of CCA and CRLL The sugar-binding properties of CCA described above may be summarized as follows: (a) CCA shows affinity for mannose-containing N-linked glycans, but not for glycolipid-type glycans; (b) it binds to relatively small high-mannose-type glycans, which contain a nonsubstituted a1–3Man residue in the tri-mannosyl core structure; (c) the binding is greatly diminished by the addition of a1–2Man residue(s) to the a1–3Man; (d) CCA can also bind to mono-, bi-antennary N-liked glycans with varied affinities, but not to tri- and tetraantennary ones On the other hand, CRLL has the following features: (a) similar to CCA, CRLL shows affinity only for N-linked glycans but not for glycolipid-type ones; (b) unlike CCA, however, CRLL binds to relatively large high-mannose-type glycans with much increased affinity; (c) a1–2Man extension in the M2M2M3Mb unit makes the strongest contribution to such high affinity for the large high-mannose type glycans; (d) affinity enhancement is also supported by a1–2Man extension in the other units M2M3M6Mb and M2M6M6Mb; (e) CRLL can bind only to a1–6 FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS S Nakamura et al branched mono-antennary, complex-type N-linked glycans, but never to a1–3 branched mono-, bi-, tri- and tetra-antennary glycans Discussion By using our recently developed FAC system, we analysed detailed sugar-binding specificities of CCA and CRLL The advantage of FAC in sensitivity, economy and reliability enabled us to reveal fine specificity of these lectins Based on the results, both conserved and divergent properties were reveled for these two mJRLs For relatively small high-mannose-type glycans, CCA and CRLL showed similar sugar-binding profiles: they bound to Mana1–3Manb as a main recognition unit, and the affinity was reduced by the addition of an a1–2Man residue to the Mana1–3Manb structure However, according to the X-ray crystallographic study on Heltuba, another mJRL, in complex Oligosaccharide specificity of Jacalin-related lectins with a1–3 mannobiose, no direct hydrogen bond was found at the O2 atom of mannose [5] Thus, substitution of 2-OH with a1–2Man seems to have no destructive effect on interactions On the other hand, a modelling study on Artocarpin, another mJRL, with ManNAc also revealed that severe steric hindrance occurs between the N-acetyl group of ManNAc and the residues in the loops b1-b2 and b11-b12, which construct the primary-binding site [32] At a glance, the amino acid sequences around the region critical for sugar-binding function are tightly conserved among mJRLs (Fig 5) Therefore, the reason why the affinity was diminished by the addition of a1–2Man residue(s) to the core Mana1–3Manb unit can probably be attributed to significant steric hindrance by the residue As regards the relatively large high-mannose-type glycans (M7–M9), which showed much enhanced affinity only for CRLL, considerable steric hindrance will Fig Sequence alignment of mJRLs Individual CRDs of CCA (N- and C-domains) and partial amino acid sequences reported for CRLL [19] are aligned with other representative mJRLs Residues conserved in all mJRLs are indicated in bold letters Secondary structure elements (b-sheet) and a1–3 mannobiose-binding residues reported for Heltuba are indicated by arrows and filled circles, respectively [5] FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS 2793 Oligosaccharide specificity of Jacalin-related lectins occur, if they interact in the same manner as the mannobiose discussed above Therefore, CRLL may create an alternative binding mode specifically for such large high-mannose-type glycans In Heltuba, three loops (b1–b2, b7–b8 and b11–b12) construct the sugarbinding site, and another loop (b5–b6) affects protein– sugar interactions Among these loops, CRLL, but not CCA, has two large deletions corresponding to b5 and loop b7–b8 (Fig 5, T Haraguchi & F Yagi, unpublished data) These deletions should affect the overall conformation of these loops and thus leave space, which results in a change in the binding mode In this context, the interaction between CRLL and large highmannose-type glycans was drastically enhanced by the coexistence of M2M2M3Mb and M2M3M6Mb units This observation implies multiple binding via either inter- or intramolecular interactions In fact, an X-ray crystallographic study demonstrated that octamer assembly enables Heltuba to cross-link with mannose moieties belonging to different antennae of a single N-glycan [5] Because CRLL exists as a monomer [19], such an effect may be compensated by its tandem-repeat type architecture At present we don’t know whether such assumed cross-linkage actually occurs, and whether CRLL is composed of two homologous but distinct domains in terms of sugar-binding properties or not To clarify these points, it will be necessary to analyse sugarbinding profiles of individual domains for both lectins In addition to having affinity for high-mannosetype glycans, CCA showed significant affinity for mono- and bi-antennary, complex-type N-linked glycans In other words, the addition of b1–2GlcNAc to Mana1–3Manb is acceptable for CCA This fact suggests that steric hindrance is more significant when a ‘double axial’ linkage is formed, e.g by C1-a-OH of nonreducing terminal mannose and C2-OH of reducing terminal mannose in Mana1–2Man, than in the case of an ‘equatorial–axial’ linkage, e.g when GlcNAcb1–2Man is formed On the other hand, because CRLL had no affinity for a1–3 branched (a1–3Man substituted), mono-antennary N-linked glycans, it permits neither kind of linkage In contrast, a1–6 branched (a1–6Man substituted), mono-antennary N-linked glycans showed affinity for both CCA and CRLL In the Artocarpin–mannotriose complex, it was revealed that a1–6Man was exposed to solvent and had no significant interaction with the protein [32] Therefore, the observed affinity of CCA and CRLL for a series of a1–6 branched, mono-antennary N-linked glycans is consistent with the assumption that nonmodified Mana1–3Manb is the basic recognition unit and a1–6Man has essentially no interference with the recognition 2794 S Nakamura et al There are various lines of evidence indicating that some mJRLs are specifically induced by treatment with jasmonate [23], by salt stress [12] or by microbial (e.g Xanthomonas campestris) infection [13] Thus, one possible role for them is protection against environmental stress and foreign enemies In this study, we demonstrated that CCA and CRLL bound to highmannose-type glycans, which are widely found in fungi, insects and animals, while at the same time each of them showed respective characteristic sugar-binding properties Therefore, it is possible that they are involved in nonself recognition targeting of pathogens or herbivorous animals in respective contexts Although the relationship between plant lectins and host glycans remains to be elucidated, the wealth of information obtained in the present study may give us clues to understand better the physiological functions of lectins in the plant body From an evolutionary viewpoint, it is of interest to argue the issue of origin of sugar-binding specificities of JRLs, in the particular context of biological significance of tandem-repeat type structures At the moment, however, there have been only few data of fine oligosaccharide specificities of JRLs for systematic comparison To this approach, more comprehensive analysis of other JRLs is in progress Experimental procedures Materials NHS-activated Sepharose was purchased from Amersham Pharmacia Biotech (Little Chalfont, Bucks, UK) All chemical reagents used in this study were of analytical grade Oligosaccharides p-Nitrophenyl glycosides of Gal-a, Gal-b, GalNAc-a, Galb1–3GalNAc-a, Man-b, and Galb1–4GlcNAc-b, were from Sigma (St Louis, MO, USA); and Glc-a was from Calbiochem (San Diego, CA, USA) Other pNP-glycosides (GalNAc-b-, Galb1–4Glc-b-, Man-a-, Fuc-a-, Glca1–4Glca-, and (Glca1–4)5-a -pNP) were obtained from Funakoshi Co (Tokyo, Japan) Pyridylaminated oligosaccharides used in this study were listed in Fig PA-N-linked glycans numbered 001–014, 103, 105, 107, 108, 307, 313, 314, 323, 405, 410, 418, 419, 420, and 503 were from Takara Bio Inc (Kyoto, Japan); and the others were from Seikagaku Co (Tokyo, Japan) Glycolipid-type glycans numbered 701–703, 705–713, 715– 721, 724, 726, and 728–731 were obtained from Takara Bio The sources of nonlabelled glycans were as follow: FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS S Nakamura et al 727 from Funakoshi Co., 733, 734, and 908 from Dextra Laboratories, Ltd (Reading, UK); 725, 909, and 910 from Calbiochem; 906 and 907 from Seikagaku Co Oligo-lactosamines 901, 902, 903, and 905 and milk oligosaccharides 722, 723, 732, and 735–739 were generous gifts from K.-i Yoshida (Seikagaku Co.) and from T Urashima (Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan), respectively The nonlabelled glycans were pyridylaminated with GlycoTag (Takara Bio) before use Oligosaccharide specificity of Jacalin-related lectins 79.20, 79.46, 80.27, 80.74, 81.16, 100.27, 100.01, 101.93, 103.16, 113.15 · 2, 129.40, 129.52 · 2, 130.04, 130.10, 149.89, 150.02, 152.61, 163.86, 169.83, 173.13, 175.24 · 2, 175.31, 176.89 178.97 L; MS (MALDI-TOF) calcd for C56H82N2O30Na (M + Na)+ m ⁄ z 1425.5, found 1425.5 A Synthesis of MTX-derivatized Man3GlcNAc2 Methotrexate-derivatized Man3GlcNAc2 was synthesized as previously described [30] Man3GlcNAc2 (21.8 mg, 0.0239 mmol) was dissolved in saturated aqueous NH4HCO3 (2 mL) and stirred at 40 °C for 14 h, and then the mixture was concentrated and coevaporated with H2O in vacuo The residue was dissolved in dioxane/H2O (1 : 1; mL), after which NaHCO3 (4.8 mg, 0.057 mmol) and FmocGlyCl (9.1 mg, 0.029 mmol) were added at °C After the mixture had been stirred at room temperature for h, NaHCO3 (2.4 mg, 0.029 mmol) and FmocGlyCl (4.5 mg, 0.014 mmol) were added to it at °C After having been stirred at room temperature for h, the mixture was purified by gel filtration (Sephadex LH20, H2O ⁄ MeOH, : 3) to give FmocGly-possessing saccharide The FmocGly-possessing saccharide was dissolved in dimethylformamide (1 mL), and piperidine (0.2 mL) was added at °C After having been stirred at room temperature for h, the mixture was concentrated in vacuo The residue was purified by gel filtration (Sephadex G15, H20) to give 21.5 mg (93%) of Man3GlcNAc2NHGly To a cold ()18 °C) solution of Man3GlcNAc2NHGly (10.7 mg, 0.0111 mmol) and MTX (atBu; 8.5 mg, 0.017 mmol) in dimethylformamide (1 mL) was added 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (6.2 mg 0.019 mmol) The mixture was stirred at °C for 19 h, at room temperature for h, and then concentrated in vacuo The residue was purified by preparative thin-layer chromatography (PTLC) (H2O ⁄ i PrOH, : 3) to give the coupled product, which was then dissolved in CF3COOH at °C After having been stirred at room temperature for h, the mixture was concentrated and coevaporated with toluene in vacuo The residue was purified by PTLC (H2O ⁄ MeOH, : 5) to give 11.2 mg (72%) of Man3GlcNAc2-MTX: TLC, Rf 0.63 (H2O ⁄ iPrOH, : 2); 1H NMR (400 MHz, D2O) d 1.90 (s, H), 2.06 (s, H), 2.03–2.50 (m, H), 3.13 (m, H), 3.50–3.96 (m, 34 H), 4.06 (s, H), 4.25 (s, H), 4.36 (m, H), 4.59 (d, J ¼ 6.8 Hz, H), 4.60–4.85 (m, H), 4.90 (s, H), 5.09 (s, H), 6.79 (m, H), 7.64 (m, H), 8.52 (m, H); 13C NMR (125 MHz, D2O) d 22.55, 22.98, 28.38, 32.88, 39.42, 43.32, 44.06, 51.32, 54.15, 54.95, 55.54, 55.66, 60.50, 60.64, 61.62, 61.81, 63.18, 66.49, 67.46, 67.55, 70.56, 70.68, 70.81, 71.01, 71.08, 72.61, 73.13, 73.34, 74.10, 74.86, 75.06, 76.83, FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS B Fig Scheme of the FAC procedure (A) Analyte solution, the initial concentration of which is represented by [A]0, is continuously applied to a lectin-immobilized column The elution front of an analyte that does not interact with the immobilized lectin (Analyte I) is observed immediately, whereas that of an analyte that specifically interacts with the immobilized lectin (Analyte II) is retarded depending on the strength of the interaction (B) Curve I (solid line) indicates the elution profile of Analyte I, showing no interaction with the immobilized lectin; and Curve II (dotted line), that of Analyte II, showing significant affinity for the immobilized lectin The elution volumes of Analyte I and Analyte II are indicated as V0 and V, respectively The basic equation of FAC is shown as Eqn (1) For details about the principle of FAC, see Experimental procedures 2795 Oligosaccharide specificity of Jacalin-related lectins Purification of CCA and CRLL CCA was purified from Castanea crenata cotyledons as previously described [20] Briefly, 100 g of cotyledons were homogenized in 50 mm phosphate buffer pH 7.0, containing 0.9% (w ⁄ v) NaCl (NaCl ⁄ Pi), 10 mm 2-mercaptoethanol, mm EDTA, and 5% (w ⁄ w) polyvinylpolypyrrolidone After the extract had been stirred at °C for h, it was applied onto an asialo-fetuin Sepharose 4B column (10 mgỈmL)1) equilibrated with NaCl ⁄ Pi After the column had been washed with the same buffer, the lectin fraction was eluted with NaCl ⁄ Pi containing 0.2 m d-mannose The active fraction was further applied to a Mono Q column equilibrated with 50 mm Tris ⁄ HCl pH 8.0 Elution was carried out with a linear gradient of NaCl from to 0.5 m The lectin fraction was concentrated by salting with ammonium sulfate, and then applied on a Superose 12 column The mobile phase was NaCl ⁄ Pi and the flow rate was kept at 0.6 mLỈmin)1 Cycas revoluta leaf lectin (CRLL) was purified from leaves of Japanese cycad as previously described [19] Briefly, leaf powder was extracted with 10 vols 6.67 mm sodium phosphate buffer pH 7.2, containing 0.8% (w ⁄ v) NaCl, and the homogenate was filtered through two layers of gauze and centrifuged After salting out by ammonium S Nakamura et al sulfate, the precipitate was dissolved in the same buffer as used for the extraction The supernatant was loaded onto a column of mannose-agarose (2 lmolỈmL)1) previously equilibrated with 50 mm Tris ⁄ HClpH 7.5 The column was washed with the same buffer, and the bound fraction was eluted with 0.2 m d-mannose Preparation of lectin columns Thus purified CCA and CRLL were dissolved in 10 mm phosphate buffer pH 7.4, containing 0.7% NaCl and coupled to NHS-activated Sepharose according to the manufacturer’s instructions After deactivation of excess NHS groups by m monoethanolamine followed by extensive washing, the lectin-Sepharose was suspended in 10 mm Tris ⁄ HCl pH 7.4, containing mm CaCl2 and mm MnCl2 The slurry was packed into a capsule-type miniature column (inner diameter, mm; length, 10 mm; bed volume, 31.4 lL) The amount of immobilized protein was determined by measuring the amount of uncoupled protein in the above wash fraction by the method of Bradford [33] The resulting lectin-columns were slotted into a stainless holder, and connected to the FAC-1 machine, which had been specially designed and manufactured by Shimadzu Co [24] Fig Scheme of the developed system for automated FAC (FAC-1) (A) The FAC-1 machine is equipped with two pumps, an autosampler, a column oven, and two miniature columns connected in parallel One of the pumps is used exclusively for analysis, and the other is for regeneration of the columns Analyte solutions are applied via the autosampler by pump A, and elution of analytes from the lectin-immobilized column is monitored by UV or fluorescence detectors In the figure, the bold line indicates the pathway when analysis is done with column (B) Outline of the dual column switching system The columns are used in ‘one after the other’ fashion to reduce by half the total analysis time 2796 FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS S Nakamura et al Oligosaccharide specificity of Jacalin-related lectins Principle of FAC The principle of FAC was previously described by Hirabayashi et al [26] Briefly, an excess volume (0.6–1.0 mL) of dilute PA-oligosaccharide solution is continuously applied to a lectin-immobilized column (Fig 6A) When the amount of the applied analyte molecules exceeds the ability of the column, leakage occurs and the concentration of oligosaccharides in the elution finally reaches a plateau, where the concentration is equal to that of the initial solution (Fig 6B) In the case of oligosaccharides having some affinity for the immobilized lectin (Fig 6A, right), they interact with and are retained by the immobilized lectin, and the elution is observed as in curve II (Fig 6B) The volume of the elution front (V) of each oligosaccharide is calculated essentially as described previously [26] Retardation of the elution front relative to that of an appropriate standard oligosaccharide (Fig 6A, left, and Fig 6B, curve I), i.e V–V0, is then determined Kd values for dissociation of lectin and oligosaccharide are obtained from V–V0 and Bt, according to the basic equation of FAC, Eqn (1), where Bt is the effective ligand content (expressed in mol), and [A]0 is the initial concentration of PA-oligosaccharide Equation (1) can be simplified to Eqn (2), where [A]0 (e.g < 10)8 m) is negligibly small compared with Kd (e.g > 10)6 m) Kd ¼ Bt =ðV À V0 ị ẵA0 Kd ẳ Bt =V V0 Þ; if Kd ) ½A0 Š ð2Þ ð3Þ FAC procedure Frontal affinity chromatography was performed by using a recently developed machine for automated FAC (FAC-1) [24] A scheme for the total procedure is illustrated in Fig Briefly, the system consists of an FAC-1 machine (Shimadzu) equipped with two isocratic pumps, an autosampler, and a couple of miniature columns (2.0 · 10 mm, 31.4 lL) connected in parallel to either a fluorescence detector (Shimadzu, RF10AXL) or UV detector (Shimadzu, SPD-10A VP) and a PC workstation loaded with ‘LCsolution’ software (Fig 7A) By use of the parallel column system, the time for analysis can be cut almost in half; i.e during analysis with one column, the other column is being washed (Fig 7B) Lectin columns were equilibrated with 10 mm Tris ⁄ HCl pH 7.4, containing 0.8% NaCl (TBS) The flow rate and the column temperature were kept at 0.125 mLỈmin)1 and 25 °C, respectively After equilibrium of the miniature col- FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS Evaluation of lectin columns For the determination of effective ligand content, Bt, concentration-dependence analysis and subsequent Woolf– Hofstee-type plot were performed as described previously [26] Here, various concentrations ([A]0) of MTX-derivatized Man3GlcNAc2 dissolved in TBS were applied to the miniature column, and the elution was monitored by absorbance at 304 nm Woolf–Hofstee-type plots, i.e (V-V0) vs (V-V0)[A]0, were made to determine Bt and Kd values from the intercept and the slope, respectively, of the fitted curve Acknowledgements This work was supported by NEDO (New Energy and Industrial Technology Organization) under the METI (The Ministry of Economy, Trade, and Industry, Japan) ð1Þ It is often favourable to discuss the matter of lectin–oligosaccharide interactions in terms of affinity constant (Ka) instead of Kd The two equilibrium constants are in the following relationship: Kd ¼ 1=Ka umns had been obtained, either PA- (2.5 nm) or pNP-oligosaccharides (5.0 lm) dissolved in TBS were successively injected into a pair of lectin-columns by the auto-sampling system Elution of PA-oligosaccharides was monitored by fluorescence (excitation and emission wavelengths of 310 and 380 nm, respectively), whereas that of pNP-glycosides was detected by UV (280 nm) References Iglesias JL, Halina L & Sharon N (1982) Purification and properties of a d-galactose ⁄ N-acetyl-d-galactosamine-specific lectin from Erythrina cristagalli Eur J Biochem 123, 247–252 Shibuya N, Goldstein IJ, Van Damme EJM & Peumans WJ (1988) Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb J Biol Chem 263, 728–734 ´ Van Damme EJM, Peumans WJ, Barre A & Rouge P (1998) Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles Crit Rev Plant Sci 17, 575– 692 Young NM, Johnston RA & Watson DC (1991) The amino acid sequences of jacalin and the Maclura pomifera agglutinin FEBS Lett 282, 382–384 Bourne Y, Zamboni V, Barre A, Peumans WJ, Van Damme EJM & Rouge P (1999) Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins Structure 7, 1473–1482 Loomes LM, Stewart WW, Mazengera RL, Senior BW & Kerr MA (1991) Purification and characterization of human immunoglobulin IgA1 and IgA2 isotypes from serum J Immunol Methods 141, 209– 218 2797 Oligosaccharide specificity of Jacalin-related lectins Van Damme EJM, Hause B, Hu J, Barre A, Rouge P, Proost P & Peumans WJ (2002) Two distinct jacalinrelated lectins with a different specificity and subcellular location are major vegetative storage proteins in the bark of the black mulberry tree Plant Physiol 130, 757–769 Peumans WJ, Winter HC, Bemer V, Van Leuven F, Goldstein IJ, Truffa-Bachi P & Van Damme EJM (1997) Isolation of a novel plant lectin with an unusual specificity from Calystegia sepium Glycoconj J 14, 259– 265 Misquith S, Rani PG & Surolia A (1994) Carbohydrate binding specificity of the B-cell maturation mitogen from Artocarpus integrifolia seeds J Biol Chem 269, 30393–30401 10 Van Damme EJM, Barre A, Mazard AM, Verhaert P, Horman A, Debray H, Rouge P & Peumans WJ (1999) Characterization and molecular cloning of the lectin from Helianthus tuberosus Eur J Biochem 259, 135–142 11 Koshte VL, van Dijk W, van der Stelt ME & Aalberse RC (1990) Isolation and characterization of BanLec-I, a mannoside-binding lectin from Musa paradisiac (banana) Biochem J 272, 721–726 12 Zhang W, Peumans WJ, Barre A, Astoul CH, Rovira P, Rouge P, Proost P, Truffa-Bachi P, Jalali AA & Van Damme EJM (2000) Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants Planta 210, 970–978 13 Hirano K, Teraoka T, Yamanaka H, Harashima A, Kunisaki A, Takahashi H & Hosokawa D (2000) Novel mannose-binding rice lectin composed of some isolectins and its relation to a stress-inducible salT gene Plant Cell Physiol 41, 258–267 14 Tateno H, Winter HC, Petryniak J & Goldstein IJ (2003) Purification, characterization, molecular cloning, and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J Smith (Polypodiaceae) J Biol Chem 278, 10891– 10899 15 Sankaranarayanan R, Sekar K, Banerjee R, Sharma V, Surolia A & Vijayan M (1996) A novel mode of carbohydrate recognition in jacalin, a Moraceae plant lectin with a beta-prism fold Nat Struct Biol 3, 596– 603 16 Bourne Y, Roig-Zamboni V, Barre A, Peumans WJ, Astoul CH, Van Damme EJM & Rouge P (2004) The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold J Biol Chem 279, 527– 533 17 Pratap JV, Jeyaprakash AA, Rani PG, Sekar K, Surolia A & Vijayan M (2002) Crystal structures of artocarpin, a Moraceae lectin with mannose specificity, and its complex with methyl-alpha-D-mannose: implications to the generation of carbohydrate specificity J Mol Biol 317, 237–247 2798 S Nakamura et al 18 Peumans WJ, Van Damme EJM, Barre A & Rouge P (2001) Classification of plant lectins in families of structurally and evolutionary related proteins Adv Exp Med Biol 491, 27–54 19 Yagi F, Iwaya T, Haraguchi T & Goldstein IJ (2002) The lectin from leaves of Japanese cycad, Cycas revoluta Thunb (gymnosperm), is a member of the jacalinrelated family Eur J Biochem 269, 4335–4341 20 Nomura K, Ashida H, Uemura N, Kushibe S, Ozaki T & Yoshida M (1998) Purification and characterization of a mannose ⁄ glucose-specific lectin from Castanea crenata Phytochemistry 49, 667–673 21 Nomura K, Nakamura S, Fujitake M & Nakanishi T (2000) Complete amino acid sequence of Japanese chestnut agglutinin Biochem Biophys Res Commun 276, 23–28 22 Nakamura S, Ikegami A, Matsumura Y, Nakanishi T & Nomura K (2002) Molecular cloning and expression of the mannose ⁄ glucose specific lectin from Castanea crenata cotyledons J Biochem (Tokyo) 131 (2), 241–246 23 Peumans WJ, Zhang W, Barre A, Houles Astoul C, Balint-Kurti PJ, Rovira P, Rouge P, May GD, Van Leuven F, Truffa-Bachi P & Van Damme EJM (2000) Fruit-specific lectins from banana and plantain Planta 211, 546–554 24 Hirabayashi J (2004) Lectin-based structural glycomics: glycoproteomics and glycan profiling Glycoconj J 21, 35–40 25 Kasai K, Oda Y, Nishikawa M & Ishii S (1986) Frontal affinity chromatography: theory, for its application to studies on specific interaction of biomolecules J Chromatogr A 379, 33–47 26 Hirabayashi J, Arata Y & Kasai K (2003) Frontal affinity chromatography as a tool for elucidation of sugar recognition properties of lectins Methods Enzymol 362, 352–368 27 Hirabayashi J, Hashidate T, Arata Y, Nishi N, Nakamura T, Hirashima M, Urashima T, Oka T, Futai M, Muller WE, Yagi F & Kasai K (2002) Oligosaccharide specificity of galectins: a search by frontal affinity chromatography Biochem Biophys Acta 1572, 232–254 28 Zhang B, Palcic MM, Mo H, Goldstein IJ & Hindsgaul O (2001) Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry Glycobiology 11, 141– 147 29 Rempel BP, Winter HC, Goldstein IJ & Hindsgaul O (2002) Characterization of the recognition of blood group B trisaccharide derivatives by the lectin from Marasmius oreades using frontal affinity chromatography-mass spectrometry Glycoconj J 19, 175–180 30 Totani K, Matsuo I & Ito Y (2004) Tight binding ligand approach to oligosaccharide-grafted protein Bioorg Med Chem Lett 14, 2285–2289 FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS S Nakamura et al 31 Green ED, Adelt G, Baenziger JU, Wilson S & Van Halbeek H (1988) The asparagine-linked oligosaccharides on bovine fetuin Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy J Biol Chem 263, 18253–18268 32 Jeyaprakash AA, Srivastav A, Surolia A & Vijayan M (2004) Structural basis for the carbohydrate specificities FEBS Journal 272 (2005) 2784–2799 ª 2005 FEBS Oligosaccharide specificity of Jacalin-related lectins of artocarpin: variation in the length of a loop as a strategy for generating ligand specificity J Mol Biol 338, 757–770 33 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 2799 ... members of much larger molecular size (% 33 kDa) were discovered recently from Castanea crenata (angiosperm) and Cycas revoluta (gymnosperm) [19,20] Sequence analysis of these large mJRLs, i.e C crenata. .. to comparative analysis of animal lectins with a set of fluorescently labelled glycans [27] Other investigators also used an FAC system equipped with an MS detector for analysis of mushroom lectins,. .. influence of a1–6 (core) fucosylation on lectin? ??glycan interaction could also be examined by comparison between 003 and 015, 101 and 201, 103 and 202, 104 and 203, 301 and 401, 302 and 402, 304 and