Novel carbohydrate specificity of the 16-kDa galectin from Caenorhabditis elegans: binding to blood group precursor oligosaccharides (type 1, type 2, T{alpha}, and Tß) and gangliosides

Hafiz Ahmed2, Mario A. Bianchet3, L. Mario Amzel3, Jun Hirabayashi4, Ken-ichi Kasai4, Yuko Giga-Hama5, Hideki Tohda5 and Gerardo R. Vasta1,2

2 Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore, MD 21202, USA; 3 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA; 4 Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan; and 5 Research Center, Asahi Glass Co. Ltd., Yokohama-shi, Kanagawa 221, Japan

Received on December 7, 2001; revised on February 26, 2002; accepted on February 27, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Galectins, a family of soluble ß-galactosyl-binding lectins, are believed to mediate cell–cell and cell–extracellular matrix interactions during development, inflammation, apoptosis, and tumor metastasis. However, neither the detailed mechanisms of their function(s) nor the identities of their natural ligands have been unequivocally elucidated. Of the several galectins present in the nematode Caenorhabditis elegans, the 16-kDa "proto" type and the 32-kDa "tandem-repeat" type are the best characterized so far, but their carbohydrate specificities have not been examined in detail. Here, we report the carbohydrate-binding specificity of the recombinant C. elegans 16-kDa galectin and the structural analysis of its binding site by homology modeling. Our results indicate that unlike the galectins characterized so far, the C. elegans 16-kDa galectin interacts with most blood group precursor oligosaccharides (type 1, Galß1,3GlcNAc, and type 2, Galß1,4GlcNAc; T{alpha}, Galß1,3GalNAc{alpha}; Tß, Galß1,3GalNAcß) and gangliosides containing the Tß structure. Homology modeling of the C. elegans 16-kDa galectin CRD revealed that a shorter loop containing residues 66–69, which enables interactions of Glu67 with both axial and equatorial -OH at C-3 of GlcNAc (in Galß1,4GlcNAc) or at C-4 of GalNAc (in Galß1,3GalNAc), provides the structural basis for this novel carbohydrate specificity.

Key words: 16-kDa galectin/C. elegans/gangliosides/specificity/Type 1, Type 2, T{alpha}, Tß blood group


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Galectins, formerly known as S-type lectins or galaptins, are a family of ß-galactosyl-binding lectins characterized by a typical motif of conserved amino acids in their carbohydrate recognition domain(s) (CRDs) (Hirabayashi and Kasai, 1993Go; Barondes et al., 1994Go; Hirabayashi, 1996Go; Leffler, 1997Go). Based on their primary structure and subunit architecture, galectins have been classified as "proto" (subunit molecular mass 14.5–16 kDa), "chimera" (29–35 kDa), and "tandem repeat" (32–36 kDa) types (Hirabayashi and Kasai, 1993Go) or galectins 1–12 (Barondes et al., 1994Go; Leffler, 1997Go). Most galectins require a reducing environment but not divalent cations for their binding activity (Hirabayashi and Kasai, 1993Go; Barondes et al., 1994Go). Although initially described in vertebrate taxa, their presence has now been documented in protochordates, invertebrates, and fungi (Hirabayashi et al., 1992aGo,b, 1996; Pfeifer et al., 1993Go; Klion and Donelson, 1994Go; Cooper et al., 1997Go; Newton et al., 1997Go), suggesting that they are widely distributed in nature. Through studies carried out primarily with mammalian models, diverse biological roles for galectins have been proposed, ranging from regulatory functions to the cell–cell and cell–extracellular matrix interactions that take place during development, inflammation, apoptosis, and tumor metastasis (Raz et al., 1990Go; Cooper et al., 1991Go; Liu, 1993Go; Perillo et al., 1995Go).

Although all galectins bind lactose, a considerable diversity in their carbohydrate-binding specificities has been recognized (Leffler and Barondes, 1986Go; Oda et al., 1993Go; Ahmed and Vasta, 1994Go). It has been proposed that these subtle differences in carbohydrate-binding patterns may reflect the different endogenous ligands recognized by the various galectin types and, ultimately, their distinct biological roles. For example, laminin is the most likely endogenous ligand for galectin-1, a lectin proposed to mediate muscle development (Cooper et al., 1991Go) and induce apoptosis of activated T cells by binding with CD45 glycoprotein (Perillo et al., 1995Go). In contrast, mucin, IgE, fibronectin, cytokeratin, and laminin have been shown to interact in vitro with galectin-3 (Sato and Hughes, 1992Go; Bresalier et al., 1996Go), a lectin proposed to inhibit apoptosis (Yang et al., 1996Go).

The molecular properties, structural aspects and detailed carbohydrate specificity of galectins in both homeotherm and ectotherm vertebrates have been relatively well characterized in their specificity and structural aspects (Hirabayashi, 1996Go). The galectins identified in the few invertebrate species examined, however, lag far behind in this regard. Because these invertebrates appear to exhibit galectin "repertoires" with similar levels of diversity and complexity as those from vertebrates, they may be useful model organisms to understand the evolution of this protein family. Furthermore, the simpler body plans and tissue architecture of some invertebrate species represents an attractive feature for assessing the biological role(s) of galectins. Among the invertebrates, the nematode Caenorhabditis elegans constitutes an ideal model for studies related to overall differentiation, development, and genetics, because it is a transparent protostome with a relatively small number of cells, simple tissue organization, and a 3-day life cycle, for which the complete genome sequence has been accomplished (The C. elegans Sequencing Consortium, 1998).

Among the several galectins present in C. elegans, the 16- and 32-kDa species, which belong to the "proto" and "tandem repeat" types, respectively, are the best characterized (Hirabayashi et al., 1992aGo,b, 1996, 1997; Arata et al., 1997Go). In its native form, the 16-kDa galectin is a noncovalently bound dimer containing one CRD per subunit (Hirabayashi et al., 1996Go), whereas the 32-kDa galectin is a monomeric protein that contains two similar CRDs (Hirabayashi et al., 1992aGo; Arata et al., 1997Go) analogous to mammalian galectin-4 (Oda et al., 1993Go; Barondes et al., 1994Go). For the aforementioned C. elegans galectins to constitute useful models for gaining insight in galectin structure–function relationships and evolution, it is critical that the detailed analysis of their sugar-binding properties and the identification and structural characterization of their putative endogenous ligands be carried out. We report herein the in vitro expression of the C. elegans 16-kDa galectin in the yeast Schizosaccharomyces pombe and the characterization of its novel carbohydrate specificity. We further discuss the structural basis for the carbohydrate specificity of the C. elegans 16-kDa galectin in the context of the structures of the galectin-ligand complexes described so far.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Production and characterization of the recombinant C. elegans 16-kDa galectin
The recombinant C. elegans 16-kDa galectin, obtained from the expression of the pTL2 nematode 16-kDa galectin (N16) construct in S. pombe and purified by affinity chromatography on asialofetuin (ASF)-agarose (Figure 1a), yielded 43–51 mg of purified protein per 1,000 ml culture. The purified recombinant C. elegans 16-kDa galectin showed a single protein band in sodium dodecyl sulfate–polyarcrylamide gel electrophoresis (SDS–PAGE) using 14% gel under reducing conditions, with electrophoretic mobility corresponding to approximately 16 kDa (Figure 1b, lane II), and exhibited a free amino-terminal amino acid (Ile) by direct protein sequencing analysis. Gel-permeation chromatography of the purified protein in all concentrations tested on a TSK G-2000SW-XL column under nondenaturing conditions (PBS-1 [0.02 M sodium phosphate/0.15 M NaCl (pH 7.2)]/ethylenediamine tetra-acetic acid [EDTA] at a flow rate of 1.0 ml/min) yielded a single peak (Figure 1c). Its retention time corresponded to the size of the dimer (calculated Mr: 34,300 Da), as reported for the native C. elegans 16-kDa galectin (Hirabayashi et al., 1996Go). Consistent with the observation that the recombinant C. elegans 16-kDa galectin is a dimeric species, it showed hemagglutinating activity toward trypsinized rabbit erythrocytes, requiring a minimum concentration for hemagglutination that ranged from 1 to 2 µg/ml.



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Fig. 1. (a) Purification of the C. elegans recombinant 16-kDa galectin on ASF-agarose column (bed volume 10 ml). Following adsorption of the cell extract, the column was washed with PBS- 1/EDTA/ME (indicated by an arrow) until the absorbance reached the baseline, and then the bound protein was eluted with 0.02 M lactose in PBS-1/EDTA/ME (indicated by an arrow) as described in Materials and methods. (b) SDS–PAGE of crude (I) and purified galectin (II) under reducing conditions. Each sample (2 µg crude extract; 50 ng purified galectin) was loaded on a 14% PAA gel, electrophoresed on a discontinuous buffer system, and the gel silver-stained. (c) Elution profile of variable amount of the C. elegans recombinant 16-kDa galectin on TSK G 2000 SW-XL (0.75 x 30 cm) in PBS- 1/EDTA at a flow rate of 1 ml/min. The calibration markers are shown in the elution profile for 0.1 mg/ml of galectin.

 
Preparation of the C. elegans recombinant 16-kDa galectin–horseradish peroxidase (HRP) conjugate and optimization of the solid phase assay
The conjugation of the C. elegans recombinant 16-kDa galectin with HRP appeared to be more effective than the one-step conjugation with glutaraldehyde (Ahmed et al., 1996aGo,b). After conjugation, approximately 100% of the total protein was recovered from lactosyl-Sepharose. Subsequently, gel permeation chromatography allowed the separation of the conjugate from unreacted galectin (Figure 2). The conjugate (peak A, approximately 70 kDa) represents an equimolar cross-linking of galectin (MW 34 kDa) and HRP (approximate MW 44 kDa), and exhibits both peroxidase and hemagglutinating activity (yield 80%). For optimization of solid phase assay, the wells were coated with varying concentrations (0.06–8.0 µg/well) of ASF, followed by fixing and extensive washing, and finally, the addition of varying concentrations (5–40 ng/100 µl per well) of galectin–HRP conjugate.



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Fig. 2. Elution profile of the C. elegans 16 kDa galectin-peroxidase conjugate on Superose 6 (1 x 30 cm) in PBS-2 (azide free)/0.25 M NaCl/0.01 M lactose, pH 7.5 (flow rate 0.4 ml/min). Point A, the conjugate; point B, unreacted galectin. The calibration markers were 1: BSA (66 kDa); 2: ovalbumin (43 kDa); 3: carbonic anhydrase (29 kDa); 4: ribonuclease A (13.7 kDa). Inset: Estimation of molecular weight of the conjugate (point A) and unreacted galectin (point B).

 
For all concentrations tested, the binding was approximately linear up to 0.5 µg/well of ASF, with absorbance values reaching a plateau beyond that concentration (Figure 3a). The binding of the conjugate to ASF, at various concentrations and as a function of time, was approximately linear during incubation times up to 28 min (Figure 3b). From these preliminary studies, the optimal concentration of ASF for coating the plates was established as 0.5 µg/100 µl per well, and the optimal concentration of galectin–HRP conjugate to be used for the binding and binding-inhibition experiments as 10 ng/100 µl per well. The optimal substrate incubation time was determined to be 28 min. Under the optimal conditions established, increasing concentrations of lactose inhibited the binding of galectin–HRP conjugate to ASF in an approximately sigmoid profile, which was reproducible in several experiments (Figure 3c). The lactose concentration required for 50% inhibition of the galectin–HRP conjugate binding to ASF varied from 0.19 to 0.30 mM. The binding activity of the C. elegans recombinant 16-kDa galectin to ASF was optimum at pH 6.5–7.5 (Figure 3d), with a dramatic decline observed below pH 6.5. All further experiments were carried out at pH 7.2.



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Fig. 3. Binding of variable concentrations of C. elegans 16-kDa galectin–HRP conjugate (5–40 ng/100 µl/well) to (a) variable amount of ASF (0.06–8.0 µg/100 µl/well) for 28 min of development by substrate, and (b) fixed amount of ASF (0.5 µg/100 µl/well) for a variable time of development by substrate; squares, 5 ng; circles, 10 ng; triangles, 20 ng; and diamonds, 40 ng. (c) Reproducibility of lactose inhibition curves (n = 16) for galectin–HRP conjugate. Error bars represent the inhibition values for n determinations. Each determination represents the average of two wells. Inhibition of galectin–HRP conjugate binding to ASF by various concentrations of lactose was determined by solid phase assay as described in Materials and methods. In (d) the binding of galectin–HRP conjugate to ASF was carried out at various pHs as described in Materials and methods.

 
Carbohydrate specificity of the C. elegans recombinant 16-kDa galectin
The carbohydrate-binding specificity of C. elegans recombinant 16-kDa galectin was determined by analyzing the binding of the galectin–HRP conjugate to ASF in the presence of several saccharides through the solid phase assay. Our previous studies (Ahmed et al., 1996aGo,b) have demonstrated that the inhibition data obtained through solid phase methodology are comparable to those estimated through other well-established methods (Leffler and Barondes, 1986Go; Ahmed et al., 1990Go; Lee et al., 1990Go).

For the solid phase inhibition studies, a complete inhibition curve was determined for each test saccharide. The molar concentrations that inhibited the binding of the lectin conjugate to ASF by 50% (I50) were calculated and normalized with respect to lactose, included in each plate as a standard (see Table I). The inhibitory activities of Galß1,4GlcNAc (4) and Galß1,4GlcNAcß1,6Gal (5) for the binding of C. elegans recombinant 16-kDa galectin to ASF were about sixfold higher than that of lactose (1). Galß1,3GlcNAc (6) and its derivative (7), and lacto-N-tetraose (8) had two- to fivefold higher inhibitory activity than lactose. The inhibitory activity of the human blood group A tetrasaccharide (23) was weaker than lactose, whereas the T disaccharide (Galß1,3GalNAc) (13), its {alpha} and ß derivatives (14–17), and asialo GM1 tetrasaccharide (18) were almost equal or better inhibitors than lactose. Cellobiose (25), 6'-sialyllactose (24), GlcNAcß1,6Galß1,4Glc (32), and 3-fucosyllactose (33) were inactive at the concentrations tested.


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Table I. Inhibition of the C. elegans 16-kDa galectin binding to asialofetuin by a series of saccharides expressed as I50 of each compound relative to lactose I50a
 
Table II shows the relative inhibitory activities of galactose and its derivatives for the galectin. Galactose (34) and its ß-derivative (methyl or p-nitrophenyl) (36, 38) and melibiose (39) exhibited a similar degree of inhibitory activity for the galectin, which was 53-fold lower than that of lactose. Gal{alpha}-OPhNO2 (37) and Gal{alpha}-OMe (35) had 8.5- and 1.4-fold higher activities than their corresponding ß-counterparts (36 and 38). Table III shows the relative inhibitory activity of several glycoconjugates as I50 relative to lactose I50, with all concentrations expressed as mg/ml. Asialolaminin was a 533-fold better inhibitor (based on dry weight) than lactose. Asialo-afuco porcine submaxillary mucin (PSM) was almost twice a better inhibitor than asialoPSM, and they were 100- and 57-fold, respectively, more effective than lactose. The binding-inhibition profile of the preferred saccharides for the C. elegans recombinant 16-kDa galectin was as follows: LacNAc > Galß1,3GlcNAc > Tß > Lac >(=) T{alpha}. This unique inhibition pattern suggests not only a carbohydrate specificity different to that of galectins with "conserved" CRDs but one that has not been described in any galectin with "variable" CRD characterized so far (Ahmed and Vasta, 1994Go).


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Table II. Inhibition of the C. elegans 16-kDa galectin binding to asialofetuin by a series of mono- and dissaccharides expressed as I50 of each compound relative to galactose I50a
 

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Table III. Inhibition of the C. elegans 16-kDa galectin binding to asialofetuin by a series of glycoconjugates expressed as I50 of each eompound relative to lactose I50
 
The relative inhibitory activities of three selected saccharides (Lac, Galß1,4Glc; LacNAc, Galß1,4GlcNAc; and Tß-PAA, Galß1,3GalNAcß-PAA) for the C. elegans 16 kDa galectin were compared with those for the bovine spleen and B. arenarum galectins (Figure 4), and analyzed against the backdrop of the primary structures of their CRDs. The inhibition profile of the three sugars for the B. arenarum galectin is similar to that for the bovine galectin because all amino acids critical for ligand binding are present in the correct positions (H44, N46, R48, H52, D54, N61, W68, E71, and R73 [the amino acid residue numbers correspond to those in the bovine spleen galectin sequence; see Figure 5]; Ahmed et al., 1996aGo,b). Furthermore, the protein-ligand interactions have been confirmed from the 3D structures of the B. arenarum galectin complexed with LacNAc and thiodigalactoside (Bianchet et al., 2000Go). The most dramatic difference in carbohydrate specificity of the C. elegans 16-kDa galectin, as compared to the bovine and B. arenarum galectins is the affinity of the former toward Gal ß1,3GalNAc and its {alpha} derivatives. In the C. elegans 16-kDa galectin, H44 (60), N46 (62), R48 (64), N61 (73), W68 (80), E71(83), and R73 (85) are also present, but H52 is deleted and D54 is replaced by E(67) (the amino acid residue numbers in parentheses correspond to those in the C. elegans 16-kDa galectin sequence; Hirabayashi et al., 1996Go).



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Fig. 4. Relative inhibitory activities of lactose, LacNAc, and Galß1,3GalNAcß-PAA (Tß-PAA) for C. elegans 16-kDa galectin (N16), bovine galectin-1 (BS), and B. arenarum galectin (BA). The interacting amino acids of each carbohydrate binding site are shown in Figure 5.

 


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Fig. 5. Sequence alignment showing partial amino acid sequence identities of BS (galectin-1 from bovine spleen, Ahmed et al., 1996a), BA (galectin from Bufo arenarum, Ahmed et al., 1996bGo), and N16 (C. elegans 16-kDa galectin, Hirabayashi et al., 1996Go). Deletions introduced for maximum homology are indicated by dashes. The critical amino acids for ligand binding are numbered (corresponding to those in the bovine galectin sequence).

 
Binding of the C. elegans 16-kDa galectin with commercial gangliosides
The binding of the C. elegans 16 kDa galectin to gangliosides (see Figure 6 for structures) was compared to that of galectins with "conserved" CRDs (Ahmed and Vasta, 1994Go), such as B. arenarum galectin (Ahmed et al., 1996bGo) and bovine galectin-1 (Ahmed et al., 1996aGo). For each galectin, the appropriate HRP conjugate amounts for the solid phase assay were chosen based on their binding to lactosyl ceramide (LacCer) (Figure 7). The galectin–HRP conjugates tested bound LacCer in a concentration-dependent manner, with the highest absorbance (in the range of 0.5–0.6) at 40 µg/well. Therefore, the amounts of C. elegans galectin–HRP, B. arenarum galectin–HRP, and bovine galectin-1–HRP to be used for the following experiments were 40 ng/well, 20 ng/well, and 7.5 ng/well, respectively. The binding of the three galectin–HRP conjugates to commercial gangliosides is shown in Figure 8. The C. elegans galectin–HRP showed the strongest binding to GD1a, GT1b and asialoGM1, with absorbance in the range of 0.8–1.1. GD3 and GM1 were bound moderately, and GM3 bound weakly (Figure 8). Negligible or no binding was observed with GM2 and GQ1b (data not shown). For all gangliosides tested, negligible or no binding was observed with B. arenarum galectin–HRP or bovine galectin-1–HRP (Figure 8).



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Fig. 6. Structures of gangliosides used in this study.

 


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Fig. 7. Binding of C. elegans galectin–HRP, B. arenarum galectin–HRP, and bovine galectin-1–HRP conjugates to immobilized lactosyl ceramide (LacCer). A fixed amount of galectin-HRP from (diamonds) C. elegans (40 ng/100 µl/well); (squares) B. arenarum (20 ng/100 µl/well, 31); and (triangles) bovine (7.5 ng/100 µl/well, 33) were allowed to bind variable amount of LacCer (0.08–20 µg/100 µl/well).

 


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Fig. 8. Binding of C. elegans galectin–HRP, B. arenarum galectin–HRP, and bovine galectin-1–HRP conjugates to gangliosides. (a) GD1a, (b) GT1b, (c) GD3, (d) asialoGM1, (e) GM1, and (f) GM3. A fixed amount of galectin–HRP from (diamonds) C. elegans (40 ng/100 µl/well); (squares) B. arenarum (20 ng/100 µl/well, 31); and (triangles) bovine (7.5 ng/100 µl/well, 33) were allowed to bind variable amount of several gangliosides (0.08–5 µg/100 µl/well).

 
3D model of the C. elegans 16-kDa galectin CRD
The final model of the C. elegans 16-kDa galectin dimer (not shown) is, as expected, highly similar to those of the templates (RMS difference for the C{alpha} atoms are 1.1 Å and 1.5 Å with respect to the bovine spleen galectin-1 and human galectin-2, respectively). The predicted structure of the CRD (Figure 9) is also close to those of the bovine and human galectins. However, amino acid substitutions at positions 46 and 48 (29 and 31 in bovine galectin) suggest that unlike in bovine galectin-1, these positions (strand 3) are involved in sugar binding. R46 (S29 in the bovine galectin sequence) interacts with 3'- and 4'-OH of the Gal residue. Also in strand 3 of the C. elegans 16-kDa galectin, H48 replaces a hydrophobic residue, L31 of bovine galectin. H48 may participate in sugar binding, although its interactions with the sugar may be hampered by steric hindrance. A shorter loop 2 (containing residues 66–69 between strand 4 and 5, Figure 9) allows E67 to interact with equatorial -OH at C-3 of GlcNAc (in Galß1,4GlcNAc) as well as axial -OH at C-4 of GalNAc (in Galß1,3GalNAc). The model of the binding site of the C. elegans 16-kDa galectin presented here also provides a rationale for the binding to {alpha} and ß-derivatives of Galß1,3GalNAc.



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Fig. 9. Overlap of the sugar-binding site region of the bovine spleen galectin-1 and the homology model of the C. elegans 16-kDa galectin with LacNAc. The residues belonging to the C. elegans 16-kDa galectin and sugar atoms are colored as follows: white, carbon; red, oxygen; blue, nitrogen. The R46 and R85 of the C. elegans 16-kDa galectin corresponds to S29 and R73, respectively, in the bovine galectin sequence as shown in Figure 5. The shorter loop of C. elegans 16-kDa galectin is indicated by an arrow.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The molecular characterization and activity of the purified C. elegans recombinant 16-kDa galectin through SDS–PAGE, gel permeation chromatography, and hemagglutination assay suggest that the recombinant lectin processed in S. pombe is identical to the authentic protein (Hirabayashi et al., 1996Go). Gel permeation chromatography of various amounts of the recombinant galectin suggested that, unlike galectin-1, the C. elegans 16-kDa galectin does not exhibit concentration-dependent monomer–dimer equilibrium (Cho and Cummings, 1995Go; Ahmed et al., 1996bGo). The nature of the authentic C. elegans 16-kDa galectin has not been tested in this regard (Hirabayashi et al., 1996Go). In the present study we have identified and characterized a novel galectin carbohydrate specificity. Modeling of the protein based on its primary sequence, revealed structural features in the binding site that have enabled us to explain the differences in specificity of the C. elegans 16-kDa galectin with other known galectins.

The conjugation of the C. elegans 16-kDa galectin to HRP for the development of the solid phase assay presented a problem not encountered previously with other lectins. Because unlike most galectins from vertebrates, the C. elegans 16-kDa galectin contains only one lysine (Hirabayashi et al., 1996Go) that may not be accessible for glutaraldehyde-mediated coupling (Ahmed et al., 1996aGo,b), we used sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) for conjugating this protein with HRP. Sulfo-SMCC is a heterobifunctional cross-linker consisting of an NHS-ester and a maleimide group connected with a spacer arm that can react with sulfhydryl and amino groups. The conjugation was successful, and values of both the yield and specific activities (binding and enzymatic) of the product suggest that this two-step method is more efficient than the routinely used one-step glutaraldehyde conjugation method (Ahmed et al., 1996aGo,b). Once optimized, the solid phase assay described here was very reproducible, and could be carried out in less than 3 h when using ASF-precoated plates.

Results obtained with the solid phase assay for binding-inhibition by mono- and oligosaccharides and complex glycans indicate that the C. elegans 16-kDa galectin exhibits some binding properties common to all galectins, but also some features that make it unique among all galectin specificities characterized so far. Among the galactose derivatives with hydrophobic aglycon, the C. elegans 16-kDa galectin preferentially binds to the {alpha}-anomers [Gal{alpha}-OMe (35) and Gal{alpha}-OPhNO2 (37) were 1.4 and 7.7-fold, respectively better than Gal]. The ß-counterparts were as active as Gal, suggesting that additional hydrophobic interactions that may be facilitated in the {alpha}-derivatives, would not occur in the ß-derivatives. A similar inhibitory pattern was observed for the bovine spleen galectin-1 (Ahmed et al., 1996aGo). Like all galectins-1, the inhibitory activities of Galß1,3GlcNAc (6), Galß1S1ßGal (9), Galß1,3Ara (10), Galß1,4Man (11), and Galß1,4Fruf (12) were comparable or two- to fourfold higher than lactose (1) probably because of the presence of topologically equivalent hydroxyls (Leffler and Barondes, 1986Go; Ahmed et al., 1990Go; Lee et al., 1990Go). The inhibitory inactivity of cellobiose (25), 6'-sialyllactose (24), GlcNAcß1,6Galß1,4Glc (32), and 3-fucosyllactose (33) for the C. elegans 16-kDa galectin indicates that either the epimerization of 4'-OH (of the Gal residue in Galß1,3(4)Glc(NAc)) or the substitution of 6'-OH (of Gal ) and 3(4)-OH (of Glc/GlcNAc) in the carbohydrate ligand decreases the binding affinity, which is a property shared with all galectins described so far (Leffler and Barondes, 1986Go; Ahmed et al., 1990Go; Marschal et al., 1992Go; Oda et al., 1993Go; Ahmed and Vasta, 1994Go). However, because Galß1,3GalNAc and its derivatives show equal or better inhibitory activity than lactose, it can be concluded that the epimerization of 3(4)-OH, does not affect binding of the C. elegans 16-kDa galectin. The similar activity of free Galß1,3GalNAc and its {alpha}-aglycon is probably due to the fact that in solution, the -OH at C1 of GalNAc remains in the {alpha}-configuration (Flowers and Shapiro, 1965Go; Ahmed and Chatterjee, 1989Go). Galß1,3GalNAcß- polyacrylamide (Tß-PAA) was a fivefold better inhibitor than Galß1,3GalNAc{alpha}-PAA (T{alpha}-PAA), suggesting that the C. elegans 16-kDa galectin prefers the ß-derivative of the T-disaccharide (Tß) over the {alpha}-derivative (T{alpha}). Furthermore, the 3.8-fold higher activity of Galß1,3Galß-OMe (19) can be explained by the fact that this sugar is topologically similar to the Tß. The asialoGM1 tetrasaccharide (18) contains the Galß1,3GalNAcß structure and thus, its 2.8-fold higher inhibitory efficiency over lactose can be equally justified. The preferential binding to Tß relative to the T{alpha} was also observed in the sponge galectin (Hanisch et al., 1996Go), although the specificities of the two invertebrate galectins are substantially different.

Among the glycoconjugates, laminin (N-linked oligosaccharides), ASF (both N- and O-linked oligosaccharides), and the mucin-type glycoproteins (O-linked oligosaccharides) bind effectively to the C. elegans 16-kDa galectin most likely because of the presence of LacNAc and Galß1,3GalNAc{alpha} in their N- and O-linked oligosaccharides, respectively (van Halbeek et al., 1981Go; van den Eijnden et al., 1983Go; Bendiak et al., 1989Go; Knibbs et al., 1989Go). The relatively weaker binding with asialo ovine submaxillary mucin (OSM), as compared to asialoPSM, is probably due to the presence of lower content (4%) of Galß1,3GalNAc in its oligosaccharide chains (van den Eijnden et al., 1983Go). Interestingly, laminin, asialolaminin, and ASF showed a similar relative inhibitory profile with the B. arenarum galectin, but the inhibitory activities of asialoPSM and asialoOSM were very low (Ahmed and Vasta, unpublished data).

The strong binding of C. elegans 16-kDa galectin with the Tß oligosaccharide led us to investigate its potential interactions with gangliosides. The binding of C. elegans galectin to the Tß blood group precursor oligosaccharide, and to asialoGM1, GM1, and GT1b suggests that the Tß structure is equally recognized by the C. elegans galectin in the free oligosacharide as well as in the glycolipid. This is particularly noteworthy because neither the bovine galectin-1 nor the B. arenarum galectin recognized those free or glycolipid-bound Tß structure (see Figure 8). Galectin-1 has been shown to be a major receptor for ganglioside GM1 on human neuroblastoma cells in culture (Kopitz et al., 1998Go). This result is in contrast to our binding studies on solid phase assay. The relatively lower binding of C. elegans galectin to GM1 compared to that of asialoGM1 indicates the probable steric hindrance due to the presence of sialic acid {alpha}2,3-linked to the Gal residue (of -Galß1,4Glcß1-Cer). The lower binding of GM3 and GD3 compared to LacCer is also consistent with the lower inhibitory activity of 3'-sialyllactose (22, Table I) compared to lactose (1, Table I).

To explain the structural basis of differences in the carbohydrate-binding specificity between the C. elegans 16-kDa galectin and the bovine and B. arenarum galectins, a homology modeling approach was implemented. The 3D model of the structure of the C. elegans 16-kDa galectin suggests that although its CRD is similar to those of the bovine and human galectins, differences in two amino acid residues may result in new interactions with the carbohydrate ligand. Particularly relevant is the interaction between E67 and the -OH (equatorial) at C-3,(4) of GlcNAc (in Galß1,3(4)GlcNAc) and the -OH (axial) at C-4 of GalNAc (in Galß1,3GalNAc). The model also supports the biochemical data on interactions of C. elegans 16-kDa galectin with Tß and T{alpha} oligosaccharide structures. Thus, shortening of the loop containing residues 66–69, provides the structural basis for the observed binding of the C. elegans 16-kDa galectin to Galß1,3GlcNAc, Galß1,4GlcNAc, and Galß1,3GalNAc and its {alpha} and ß derivatives. In the case of the galectins from bovine spleen and B. arenarum, interactions of the equivalent residue D54 with the axial -OH at C-4 of Galß1,3GalNAc are not possible because of steric hindrance (Liao et al., 1994Go; Bianchet et al., 2000Go). In summary, both the experimental evidence and the modeling studies indicate that the carbohydrate specificity of the C. elegans 16-kDa galectin is novel because unlike all galectins characterized so far, it interacts with most blood group precursor oligosaccharides (T{alpha}, Galß1,3GalNAc{alpha}; Tß, Galß1,3GalNAcß; type 1, Galß1,3GlcNAc, and type 2, Galß1,4GlcNAc) whereas galectins-1 interact only with type 1 and type 2 oligosaccharides.

Among galectins with variable CRDs, the RI36-I (galectin-4) preferentially binds to T{alpha} and type 1 oligosaccharides (Oda et al., 1993Go). Furthermore, the sponge (Geodia cydonium) galectin interacts preferentially with the Forssman pentasaccharide, and the disaccharide responsible for the binding is GalNAc{alpha}1,3GalNAcß. The order of inhibitory activities is as follows: GalNAc{alpha}1,3GalNAcß1,3 Gal{alpha}1,4 Galß1,4Glc > GalNAc{alpha}1,3GalNAcß > GalNAc{alpha}1,3(Fuc{alpha}1-2)Galß >> Galß1,3GlcNAcß > Galß1,4Glcß (Forssman pentasaccharide > Forssman disaccharide > A trisaccharide > T ß > lactose) (Hanisch et al., 1996Go), and is clearly different from the profile observed for the C. elegans 16-kDa galectin. A short loop similar in length—albeit not identical in amino acid sequence—to that in the C. elegans 16-kDa galectin is present in the CRDs of other galectins, such as galectin-2 (Gitt et al., 1992Go), galectin-5 (Gitt et al., 1995Go), domain 2 of galectin-9 (Tureci et al., 1997Go), both domains of C. elegans 32-kDa (Hirabayashi et al., 1992aGo), both domains of a parasitic nematode (Teladorsagia circumcincta) 32-kDa galectin (Newton et al., 1997Go), and both domains of the filaria (Onchocerca vulvulus) 32-kDa galectin (Klion and Donelson, 1994Go), suggesting the possibility that other variable type CRDs may interact with gangliosides carrying the Tß structure.

The C. elegans 32-kDa galectin is abundantly expressed in the epidermal layer of adult nematodes and has been proposed to be involved in maintaining the cuticular structure (Arata et al., 1996Go). However, the tissue localization of the C. elegans 16-kDa galectin as well as its biological function(s) are unknown. Several glycoproteins (30–300 kDa) from C. elegans cell membrane fractions were isolated by affinity chromatography on a column of C. elegans 16-kDa galectin immobilized on agarose (Hirabayashi et al., 1996Go), but the endogenous ligands for this protein are yet to be clearly identified. In C. elegans, a laminin-related protein encoded by the unc-6 gene has been shown to be important for the guidance of pioneer axons and migration of cells along the body wall (Ishii et al., 1992Go). Because of its binding to LacNAc, the C. elegans 16-kDa galectin would be the good candidate to interact with the endogenous laminin, a glycan with high content in polyl-N-acetyllactosamine chains (Knibbs et al., 1989Go). From the studies reported herein, however, it is clear that in addition to laminin and mucin-type glycoproteins, glycolipids containing T{alpha}/Tß structure may also serve as ligands for the C. elegans 16 kDa galectin.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Reagents
Unless stated otherwise, enzymes for recombinant DNA procedures were purchased from New England Biolabs (Beverly, MA) or Takara Shuzo (Kyoto, Japan). Plasmids were prepared in Escherichia coli strain DH5 (Toyobo, Tokyo). HRP and electrophoresis and chromatography protein standards were from Sigma Chemical (St. Louis, MO). The HRP substrate, diammonium 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). The cross-linker sulfo-SMCC was obtained from Pierce Chemical (Rockford, IL). Blood group A and asialoGM1 tetrasaccharides were purchased from Accurate Chemicals (Westbury, NY). The T-disaccharides {alpha}/ß-linked to PAA (Galß1,3GalNAc{alpha}-PAA, T{alpha}-PAA, and Galß1,3GalNAcß-PAA, Tß-PAA, each 30 kDa containing approximately 20% mol carbohydrate) were obtained from Glycotech (Rockville, MD), whereas the rest of the inhibitory sugars were from Sigma. Lactosyl ceramide and gangliosides were purchased from Matreya (Pleasant Gap, PA). Fetuin, laminin, PSM, and OSM were desialylated with 0.025 N sulfuric acid at 80°C for 1 h (Vasta and Marchalonis, 1986Go). For desialylation and defucosylation PSM was treated with 0.1 M trifluoroacetic acid at 100°C for 1 h (Manzi and Varki, 1993Go).

Preparation of the C. elegans recombinant 16-kDa galectin
Construction of the expression plasmid for the C. elegans recombinant 16-kDa galectin.
Standard recombinant DNA procedures were carried out following Sambrook et al. (1989)Go. To construct the C. elegans 16 kDa galectin expression plasmid pTL2N16, the total open reading frame was inserted between the AflIII and HindIII sites of pTL2M1 expression vector (Tohda et al., 1994Go; Tohda, 1997Go). Primers 5'-GGGTC ATG ATC GGA GGA GGA ATC G-3' (initiation codon underscored) for AflIII-tag addition at the 5'-terminus and 5'-GGGAAGC TTA GTG AGA AAC ATG GGC GG-3' (termination codon uncerscored) for HindIII-tag addition at the 3'-terminus were used for the mutation of open reading frame. After polymerase chain reaction amplification by KOD DNA polymerase (Toyobo), the product was digested with AflIII and HindIII. After purification, the fragment was ligated with pTL2M1 treated with the same enzymes.

Expression and isolation of the
C. elegans recombinant 16-kDa galectin. The C. elegans recombinant 16-kDa galectin was produced in fission yeast S. pombe. The S. pombe strain (genotype: leu1-32 h, ATCC 38399) was transformed with the plasmids described, together with PstI-digested transducing vector pAL7, according to Okazaki et al. (1990)Go. Transformed cells were spread on MMA minimum culture plates without leucine, incubated at 32°C for 3 days, transferred to a YE-liquid medium containing 10 µg G418/ml (sulfate, Life Technologies), and cultured at the same temperature for 2 days. Transformed cells were grown in YPD medium (1% Bacto yeast extract [Difco, Detroit, MI]/2% Bacto peptone [Difco]/2% glucose) supplemented with the appropriate concentrations (200 µg/ml) of G418. For the production of C. elegans recombinant 16-kDa galectin, an appropriate yeast clone was grown at 32°C for 24 h in 5 ml of YE-liquid medium containing 10 µg/ml of G418, and the culture was further propagated at 32°C for 24 h in 500 ml of the medium containing the increased concentration of G418 (50 µg/ml) to maintain the plasmid copy number (Tohda et al., 1994Go; Tohda, 1997Go). When the OD660 reached 4.0, the cells (approximate total cell number: 1010, 4–5 g wet weight) were harvested by centrifugation (6,000 x g at 4°C for 15 min), and disrupted by sonication in 0.02 M sodium phosphate (pH 7.2)/PBS-1/EDTA/ME containing protease inhibitors (aprotinin [1 µg/ml, Sigma], phenylmethylsulphonyl fluoride [1 mM, Sigma], and p-aminobenzenesulfonic acid [0.1 mM, Wako Chemicals, Tokyo]), by using a Branson Sonifier. After centrifugation (20,000 x g, at 4°C for 25 min), the cell extract (containing 200–250 mg protein) was directly applied to an ASF-agarose column (bed volume, 10 ml) (Hirabayashi et al., 1992aGo). The column was extensively washed with PBS-1/EDTA/ME (500 ml), and the bound C. elegans recombinant 16 kDa galectin was eluted with PBS-1/EDTA/ME containing 0.02 M lactose.

Analytical procedures.
Protein concentrations were determined on 96-well flat-bottom plates with the Bio-Rad protein assay reagent using bovine serum albumin (BSA) as a standard as described elsewhere (Ahmed et al., 1996bGo). SDS–PAGE was carried out on 14 % (w/v) acrylamide gels under reducing conditions as reported elsewhere (Laemmli and Favre, 1973Go). The native molecular weight of the C. elegans recombinant 16-kDa galectin was estimated by gel permeation chromatography on a Tosoh TSK GS-2000 SW-XL column (0.75 x 30 cm) equilibrated with PBS-1/EDTA at a flow rate of 1 ml/min in a high-performance liquid chromatography (HPLC) system as described elsewhere (Hirabayashi et al., 1996Go). Gel permeation chromatography of the HRP-conjugated galectin was carried out in a Beckman HPLC system, using a Pharmacia Superose 6 (1 x 30 cm) column equilibrated with azide-free 0.01 M sodium phosphate (pH 7.2)/0.15 M NaCl (PBS-2) containing additional 0.25 M NaCl and 0.01 M lactose, as described elsewhere (Ahmed et al., 1996bGo).

Characterization of the carbohydrate specificity
Preparation of the galectin–HRP conjugate.
The purified freeze-dried C. elegans recombinant 16-kDa galectin was dissolved in 0.5 ml of PBS-2 containing 0.01 M ME, repurified on a lactosyl-Sepharose column (0.6 ml bed volume), and stored on a DEAE-Sepharose column (0.5 ml bed volume) (Ahmed et al., 1996bGo). To activate the HRP, 1 mg sulfo-SMCC was mixed with 4 mg HRP in 0.5 ml of PBS-2 (pH 7.2), and the mix was incubated with 37°C for 30 min and desalted on a desalting column (Isolab). For conjugation with the activated HRP, the DEAE-Sepharose column containing purified galectin was washed with azide free PBS-2 (diluted 1:10 in water) to remove lactose and mercaptoethanol, and the bound protein was eluted with 1.5 ml of PBS-2 (azide free)/0.5 M NaCl/0.1 M lactose. The eluted galectin (1.0 mg) was conjugated to activated HRP at 4°C overnight with continuous stirring; subsequently the conjugation mixture was diluted 50-fold with cold water and adsorbed onto DEAE-Sepharose (0.5 ml) preequilibrated with azide free PBS-2 (1:10). The column was washed to remove lactose, and the conjugate was eluted with 4 ml PBS-2 (azide free)/1 M NaCl and purified by affinity chromatography on lactosyl-Sepharose as indicated. Finally, the conjugate was separated from the unreacted galectin by gel permeation chromatography on a Superose 6 column as described (Ahmed et al., 1996bGo). The purified galectin–HRP conjugate was stored in 1% BSA–50% glycerol at –20°C. Each step of the preparation and purification of the galectin–HRP conjugate was monitored by assessing both its sugar-binding and enzyme activities.

Optimal pH for binding.
To determine the optimal pH for C. elegans recombinant 16-kDa galectin binding to ASF, 24 ng galectin–HRP conjugate in 60 µl water containing 0.1% Tween 20 were mixed with 60 µl of various buffers (0.2 M), and 100 µl of this mix was used in triplicate in the binding assay as described. The buffers used were citrate-phosphate, pH 4.0–6.0; phosphate, pH 6.5–8.0; and carbonate-phosphate, pH 8.5–9.5.

Solid phase binding-inhibition assay.
Binding of the N16 to ASF and its inhibition by sugars were determined following a procedure reported elsewhere (Ahmed et al., 1996aGo,b). Briefly, ASF (0.5 µg/100 µl/well) in 0.1 M Na2CO3/0.02% NaN3 (pH 9.6) was adsorbed onto the wells of microtiter plates and fixed with 2% formaldehyde in PBS-2 at 37°C for 30 min. After washing the plate three times with PBS-2 (azide free)/0.05% Tween 20, the wells were incubated with 100 µl of the galectin–HRP conjugate (for binding assays) or with 100 µl of preincubated mixture of conjugate and test ligands (for binding-inhibition assays) in duplicate. The conjugate-inhibitor mix was made with equal volume (60 µl each) of the conjugate (24 ng) and the test ligand at varying concentrations and incubated for 1 h at 4°C. After incubation for 1 h at 4°C, the wells were washed with ice-cold PBS-2 (azide free)/0.05% Tween 20, and the bound peroxidase activity was assayed with ABTS.

Solid phase binding of the
C. elegans 16-kDa galectin with lactosyl ceramide and gangliosides. Lactosyl ceramide and gangliosides were dissolved in ethanol and twofold serially diluted. One hundred microliters of each dilution was added to flat-bottom plastic microwells (Microtest III, Beckton Dickinson) and air-dried at room temperature. The wells were blocked with 200 µl azide-free PBS containing 1% gelatin at 37°C for 30 min. After removing the blocking buffer, galectin–HRP (100 µl/well) was added and incubated at 4°C for 1 h. The wells were washed two times with ice-cold blocking buffer and once with ice-cold azide-free PBS; the bound peroxidase activity was assayed with ABTS.

3D model of the
C. elegans 16-kDa galectin CRD. The sequence of the C. elegans 16-kDa galectin was aligned with those of the template molecules, the bovine spleen galectin-1 (Liao et al., 1994Go) and galectin-2 (Lobsanov et al., 1993Go), taking special care in the places of possible insertions and deletions. The initial 20 residues of the C. elegans 16-kDa galectin sequence are not present in the bovine galectin-1 and were not included in the model. Coordinates for atoms common to model and templates were obtained from the bovine spleen galectin-1 (Liao et al., 1994Go; PDB ID code 1slt). The noncommon atoms that result from mutations, insertions, and deletions were built using the program Quanta and their conformations optimized by visual inspection. The region spanning residues (66–69, DEGA) which is a much longer loop in the template molecules, was modeled maintaining the presence of the acidic group (E67 in N16) in the hydrogen bond network that stabilizes the sugar binding site, as is observed in the 3D structure of the bovine galectin-1 and human galectin-2. The model built with this procedure showed strain in some of the regions in which substitutions, insertions, and deletions were introduced. The structure was relaxed by energy minimization and a short molecular dynamics simulation at 300 K, keeping the positions of CRD atoms tethered to the original positions. CHARMM2.3 was used for regularization, energy minimization, and molecular dynamics simulations.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Supported by grant no. 95-31 from the Lucille P. Markey Trust Fund and grant MCB-00-77928 from the National Science Foundation to G.R.V. and grant 1P01GM51362 from the National Institute of General Medical Sciences to L.M.A. We thank Ms. Sachiko Yamamoto for expert technical assistance.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ABTS, diammonium 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate); ASF, asialofetuin; BSA, bovine serum albumin; CRD, carbohydrate recognition domain; EDTA, ethylenediamine tetra-acetic acid; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; ME, 2-mercaptoethanol N16, nematode 16 kDa galectin; OSM, ovine submaxillary mucin; PAA, polyacrylamide; PBS-1, 0.02 M sodium phosphate/0.15 M NaCl (pH 7.2); PBS-2, 0.01M sodium phosphate/0.15 M NaCl/0.01% NaN3 (pH 7.2); SDS–PAGE, sodium dodecyl sulfate–polyarcrylamide gel electrophoresis; PSM, porcine submaxillary mucin; sulfo-SMCC, sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: vasta@umbi.amd.edu Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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