Sugar Binding Properties of the Two Lectin Domains of the Tandem Repeat-type Galectin LEC-1 (N32) of Caenorhabditis elegans

DETAILED ANALYSIS BY AN IMPROVED FRONTAL AFFINITY CHROMATOGRAPHY METHOD*

Yoichiro ArataDagger, Jun Hirabayashi, and Ken-ichi Kasai

From the Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamika, Kanagawa, 199-0195, Japan

Received for publication, September 20, 2000, and in revised form, October 31, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14-kDa-type galectins. To elucidate the biological meaning of this unique structure containing two probable sugar binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containing N-linked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2-5-fold stronger binding toward all complex-type sugars compared with Nh. Both Nh and Ch preferred Galbeta 1-3GlcNAc to Galbeta 1-4GlcNAc. Because the Fucalpha 1-2Galbeta 1-3GlcNAc (H antigen) structure was found to interact with all immobilized protein columns significantly, the Kd value of pentasaccharide Fucalpha 1-2Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc-PA for each column was determined by analyzing the concentration dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 × 10-5, 1.3 × 10-4, and 6.5 × 10-5 M, respectively. The most significant difference between Nh and Ch was in their affinity for GalNAcalpha 1-3(Fucalpha 1-2)Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc-PA, which contains the blood group A antigen; the Kd value for immobilized Nh was 4.8 × 10-5 M, and that for Ch was 8.1 × 10-4 M. The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Galectins form a group of animal lectins characterized by their specificity for beta -galactosides (1-3). Although biological phenomena in which galectins are involved are extremely diverse, all members of the galectin family must meet two criteria: specific affinity for beta -galactosides and an evolutionally conserved sequence motif in the carbohydrate binding site. Galectins are widely distributed in the animal kingdom from humans to sponges. At least 11 galectins (galectins 1-11) have been found in mammals, and 11 also have been found in the nematode Caenorhabditis elegans.

Galectins can be classified into three types in terms of molecular architecture, i.e. proto-type, chimera-type, and tandem repeat-type (4). LEC-1 (or N32), the 32-kDa galectin of C. elegans, was the first example of the tandem repeat-type galectin composed of two homologous regions (5, 6). Multiple galectins belonging to this type were also found recently in mammals, e.g. galectin-4 (7, 8), galectin-6 (9), and galectin-8 (10). Because LEC-1 has a strong hemagglutination activity, even though LEC-1 itself seems to exist as a monomer, both of its two homologous regions appear to have carbohydrate binding ability (11).

In our previous studies, we prepared recombinant proteins of the whole molecule (LEC-1), the N-terminal lectin domain (Nh),1 and the C-terminal lectin domain (Ch) and compared their binding affinity for asialofetuin-Sepharose by frontal affinity chromatography (11). We found the ratio of their binding strengths to be 100:1.14:14.6, and this result demonstrates that both of the two domains have sugar binding ability but have different binding properties. A recent x-ray crystallographic study demonstrated that both domains of LEC-1 contain similar beta -sandwich motifs as those of proto-type galectins (12). LEC-1 was found to be localized most abundantly in the adult cuticle of C. elegans by immunohistochemical studies (13). Therefore, it may be an essential component of the adult cuticular matrix, serving as a cross-linker with two different sugar binding sites for the construction of the tough and durable outer barrier of the worm's body.

Although the binding strength of Nh for asialofetuin-Sepharose was relatively weak compared with that of Ch or the whole molecule LEC-1, some sugar structure strongly recognized by Nh may exist. If we can demonstrate that the sugar binding ability of Nh is comparable with that of Ch but that the specificity is somewhat different, the biological significance of the existence of such a cross-linker may be made clearer. Therefore, we decided to analyze their binding ability in more detail. For this purpose, Kd values for 35 different fluorescently labeled oligosaccharides (pyridylaminated (PA) sugars; Ref. 14) were determined by use of an improved frontal affinity chromatography method (15, 16).

Frontal affinity chromatography (17) proved to have many advantages from both theoretical and experimental viewpoints in comparison with zonal chromatography as an analytical tool for molecular interactions, such as enzyme-substrate analog (18, 19) and lectin oligosaccharides (11, 20). In frontal affinity chromatography, an excess volume of an analyte solution is continuously applied to the column packed with an affinity adsorbent. The theory is very simple, because we can describe this system in terms of a simple equilibrium. The procedure is also very easy to perform, and the results are reproducible and reliable. From a contemporary viewpoint, however, frontal affinity chromatography has a few drawbacks; e.g. it is time consuming, requires a relatively large amount of analyte, and has not been automated. In a previous study, for example, >100 ml of a protein solution (5 µg/ml) was applied to a relatively large volume column (bed volume, 2 ml), and one run took several hours.

Because we keenly believed in the importance of reinforcing this method, we sought to improve it in the following ways: (i) use of mechanically stable chromatographic media for packing; for this purpose, HiTrap N-hydroxysuccinimide-activated Sepharose was used; (ii) adoption of a fluorescence-based detection for increasing the sensitivity; in the present study, fluorescence-labeled sugars (PA-oligosaccharides; 320 nm for excitation and 400 nm for emission; Ref. 14) were used; (iii) design of a semiautomated system; for this purpose, an ordinary high performance liquid chromatography system was used, and the analyte solution was loaded into a relatively large sample loop (1-2 ml) and injected into a miniature column (4.0 mm inner diameter × 10 mm; bed volume, 0.126 ml); and (iv) development of a simple and efficient data-processing procedure that enables the determination of very small differences in retardation with precision. These reinforcements were successful, and reliable data for binding ability were obtained.

Comparison of the Kd values for 35 different sugar chains obtained for LEC-1, Nh, and Ch clearly showed that LEC-1 has two comparably potent sugar binding sites with different affinities.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Thirty-three PA-oligosaccharides (for structures; see Tables I and II; the numbers denote commercially assigned ones and will be used throughout this and future works), PA-rhamnose, and PA-mannose were purchased from Takara Biomedicals. HiTrap NHS-activated columns (activated agarose gel) were purchased from Amersham Pharmacia Biotech. Stainless steel empty columns (4.0 × 10 mm; bed volume, 0.126 ml) were obtained from GL Sciences, Inc. polyetheretherketone sample loops (2 and 1 ml) were from Rheodyne. All other chemical reagents were analytical grade.

Production of Recombinant Galectins-- Expression and purification of recombinant nematode proteins were performed as described before (11). Briefly, DNA fragments encoding 32-kDa galectin (LEC-1 or N32), its N-terminal half-domain (Nh), and its C-terminal half-domain (Ch) were amplified by polymerase chain reaction using cloned cDNA as a template. The amplified fragments were ligated into digested pET21a (Novagen) and used to transform Escherichia coli BL21(DE3) cells. Production of recombinant proteins was induced by the addition of 1 mM isopropyl beta -D-thiogalactopyranoside. LEC-1 and Ch were purified by affinity chromatography on asialofetuin-Sepharose 4B, and Nh was purified by chromatography on DEAE Toyopearl 650S (Tosoh).

Preparation of Affinity Adsorbents-- The recombinant proteins were dissolved in 0.2 M NaHCO3, pH 8.3, containing 0.5 M NaCl and 0.1 M lactose and coupled to HiTrap NHS-activated columns following the manufacturer's instructions. After washing and deactivation of excess active groups by ethanolamine, the lectin-immobilized agarose beads were taken out from the cartridge. Each adsorbent was suspended in EDTA-PBS (1 mM EDTA, 20 mM Na-phosphate, pH 7.2, 150 mM NaCl), and the slurry was packed into a stainless steel column (4.0 × 10 mm). The amount of immobilized proteins was determined by measuring the amount of uncoupled protein in the washing solutions by the method of Bradford (21).

Principle for Determination of Kd-- The basic equation of frontal affinity chromatography, Equation 1, has been described before (17).


V<SUB>f</SUB>−V<SUB>0</SUB>=<FR><NU>B<SUB>t</SUB></NU><DE>K<SUB>d</SUB>+[A]<SUB>0</SUB></DE></FR> (Eq. 1)
Analyte and immobilized ligand are denoted A and B, respectively. Kd is the dissociation constant between interacting biomolecules, Bt is the total amount of immobilized ligand, [A]0 is the initial concentration of the analyte, A; Vf is the elution volume of A (see Fig. 1A, curve I); and V0 is that of a substance that has no specific interaction with the immobilized ligand (e.g. PA-rhamnose, p-aminophenyl-alpha -D-mannopyranoside; see Fig. 1A, curve II).



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Fig. 1.   A, elution profiles in frontal affinity chromatography. Curve I, elution profile of an analyte that specifically interacts with the immobilized lectin (elution volume = Vf). Curve II, elution profile of the analyte with no interaction. (elution volume = V0). B, to calculate the value Vf, the area under the curve was calculated first. The fluorescence intensity was measured every 2 s, and the small area (Delta Si) with Delta V (eluted volume at flow rate 0.25 ml/min for 2 s, i.e. 8.33 µl) for the base and [A]i for height was summed (Sigma Delta Si). The value of the base of the rectangle that makes the same area value of the added area (Sigma Delta Si) was calculated (i.e. Sigma Delta Si /[A]i). C, when [A]i reaches the plateau [A]0, the value Vi - Sigma Delta Si /[A]i will converge to Vf, which makes the two area-under-the-curve values equal.

Kd, Bt and V0 are constant for a given column. Vf varies depending on [A]0 in the appropriate concentration range. Equation 1 can be rearranged as follows so that a plot of [A]0(Vf - V0) versus (Vf - V0) should be linear:
[A]<SUB>0</SUB> · (V<SUB>f</SUB>−V<SUB>0</SUB>)=B<SUB>t</SUB>−K<SUB>d</SUB> · (V<SUB>f</SUB>−V<SUB>0</SUB>) (Eq. 2)
This equation is homologous to the Woolf-Hofstee equation, which is used for enzyme kinetics. The value of Kd can be determined from the slope, which corresponds to -Kd. The value of Bt can be obtained from the intercept on the ordinate.

If [A]0 is negligibly small compared with Kd, Vf approaches the maximum value, Vm, which is independent of [A]0, and the following equation is obtained:
V<SUB>m</SUB>−V<SUB>0</SUB>=<FR><NU>B<SUB>t</SUB></NU><DE>K<SUB>d</SUB></DE></FR> (Eq. 3)
This means that the value of Vm - V0 is proportional to the affinity of a PA-sugar for an immobilized lectin. Therefore, we can compare quantitatively the binding strength of different PA-sugars by measuring the value of Vm - V0 at a concentration that satisfies [A]0 Kd provided that Bt is given.

Determination of the Elution Volume of PA-Sugars-- Vf can be considered the volume at which the hypothetical boundary of the analyte solution would appear if the boundary were not disturbed at all. Therefore, Vf is the point at which the area under the elution curve is equal to the area of the rectangle ([A]0 for height and (Sigma Delta Si)/[A]0 for base) shown in Fig. 1C. If fluorescence intensity data are collected periodically, the area under the elution curve becomes the sum of the areas of small rectangles with Delta V for base and [A]i for height; i.e. Delta Si = Delta V × [A]i (Fig. 1B). If Delta V is small enough, Sigma Delta Si (Fig. 1B, shaded) is close enough to the actual area. If the column i is located at the plateau, division of Sigma Delta Si by [A]i gives the length of the base of the large rectangle. Subtracting this value from Vi (i.e. Vi - (Sigma Delta Si)/[A]i) gives the point at which the area below the elution curve and the rectangle becomes equal. If we instruct the computer to calculate this value Vi - (Sigma Delta Si)/[A]i continuously, and when the elution curve reaches a plateau ([A]0), this value converges to a certain figure that should be Vf, the point at which the hypothetical boundary of the analyte appears (Fig. 1C). By this procedure, reliable values can be obtained even if the elution curve is not symmetrical.

The calculation procedure was as follows. Collected data were saved as a text file by using ChromData (Dynamax Compare Modules software; Rainin Instrument Company, Inc.), transferred to an Excel format, and calculated automatically. When Vf converged to a certain value, we considered it to be the true Vf. We made a homemade Excel template to obtain the Vf data easily (22). Multiple text data files were processed automatically by programs written in AppleScript. Although the Vf value includes the volume of the tubing from the outlet of the column to the fluorescence detector, this can be compensated, because we always consider values relative to V0, which is the elution volume of a protein without specific interaction with the affinity adsorbent (in this paper, PA-rhamnose or p-aminophenyl-alpha -D-mannopyranoside).

Operation of Frontal Affinity Chromatography-- PA-oligosaccharide was dissolved in EDTA-PBS and applied to the column through a 2-ml sample loop connected to the Rheodyne 7725 injector. The flow rate was controlled by a Shimadzu LC-10ADvp pump at 0.25 ml/min. The sample loop and the column were immersed in a 20 °C water bath. Elution of PA-oligosaccharide from the column was monitored by a Shimadzu RF10AxL fluorescence detector at 400 nm (excitation at 320 nm). The signal from the detector was sent to a computer (Power Macintosh 7300/180) through a control interface module (Varian Chromatography Systems) at 2-s intervals, and the collected data were processed by Dynamax Compare Module software and by a table-calculating software (Microsoft Excel). The outline of the system is shown in Fig. 2. For the determination of V0, PA-rhamnose, which has no affinity to galectins, was used.



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Fig. 2.   System diagram of the improved frontal affinity chromatography system. A PA-oligosaccharide solution (2 ml) is injected into a column through a 2-ml sample loop. Elution of PA-oligosaccharide is monitored by measuring the fluorescence (emission (Em.)) at 400 nm (excitation (Ex.) at 320 nm). The fluorescence signals are collected at 2-s intervals and sent to a computer through a control interface module, and the data are processed by Microsoft Excel.

Determination of Bt and Kd-- PA-labeled lacto-N-fucopentaose I (PA-043) was used to determine the concentration dependence of the Vf value. PA-043 solutions of various concentrations were applied to the column through a 1-ml sample loop connected to the Rheodyne 7725 injector. For immobilized LEC-1 and Ch, the retardation Vf - V0 was measured at 50, 20, 10, 5, and 2 µM PA-043. For immobilized Nh, which showed weaker affinity for PA-043, the concentrations of PA-43 were 100, 50, 25, 10, and 5 µM. Elution patterns were monitored by UV absorption at 300 nm using Shimadzu SPD-10Avp UV monitor, because the concentrations used were too high for fluorescence monitoring. For the determination of V0, p-aminophenyl-alpha -D-mannopyranoside was used.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Analysis of Binding Properties of the Two Domains of LEC-1-- The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) of the 32-kDa galectin of the nematode C. elegans were expressed in E. coli by using the expression vector pET21a. Purified recombinant proteins were immobilized on HiTrap NHS-activated columns (Amersham Pharmacia Biotech). After the reaction had been terminated, the plastic cartridge was broken, and the lectin-immobilized gel was packed into a stainless steel column (4.0 × 10 mm). The amounts of immobilized proteins for LEC-1-, Nh-, and Ch-immobilized gels were 5.9, 6.6, and 1.9 mg/ml gel, respectively.

The outline of the system for the improved frontal affinity chromatography is shown in Fig. 2. One of the PA-sugars dissolved in EDTA-PBS at a concentration of 10 nM was applied continuously to a column containing its counterpart galectin.

Elution profiles of PA-sugars from LEC-1-, Nh-, and Ch-immobilized columns are collectively shown in Fig. 3, A-C, respectively. Elution pattern of each PA-sugar was overlaid with that of a control sugar (PA-rhamnose), which does not show any affinity for LEC-1, Nh, or Ch (data not shown). PA-sugar numbers (for structure, see Tables I and II) are given at the upper left of each profile, and the extent of retardation (Vf - V0) in milliliters is indicated in the middle of each elution pattern (when no retardation was observed, the value was omitted). Because the concentration of every PA-sugar was 10 nM and adequately small in comparison with Kd, the retardation volume Vf - V0 was proportional to the strength of binding to the immobilized lectin (as described under "Experimental Procedures").




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Fig. 3.   Elution profiles of various PA-oligosaccharides after application to immobilized LEC-1-, Nh-, and Ch-columns. PA-oligosaccharides were dissolved in EDTA-PBS at a concentration of 10 nM, and 2 ml of each solution was applied to the column (10 × 4.0 mm, 0.126 ml) through a 2-ml loop (inner diameter, 0.75 mm) at a flow rate of 0.25 ml/min at 20 °C. Each elution pattern of PA-oligosaccharide was superimposed on that of PA-rhamnose so that the retardation could be seen. Large numbers at top left of each elution pattern (001~050) correspond to the reference numbers of PA-oligosaccharides described in the catalogue of Takara Shuzo Corporation (see Tables I and II). Man corresponds to PA-mannose, and Rha corresponds to PA-rhamnose. Small numbers indicated in the elution patterns are detected retardation volumes (Vf - V0, in ml) for each PA-oligosaccharide. A, LEC-1; B, Nh; C, Ch.


                              
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Table I
Structures of PA-oligosaccharides from N-glycans used in this study and the obtained Kd values for LEC-1, Nh, and Ch immobilized on HiTrap columns


                              
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Table II
Structures of PA-oligosaccharides from glycolipid glycans used in this study and the obtained Kd values for LEC-1, Nh, and Ch immobilized on HiTrap columns

The elution profiles of 001, 002, and 004 indicate that N-acetyllactosamine-containing, N-linked, complex-type sugar chains interacted with both Nh and Ch and also with LEC-1 and that the binding was stronger for more branched complex-type sugar chains (001 < 002). This was predictable because there would be more chances for interaction. The reason why the affinity of the tetraantennary oligosaccharide (004) was almost the same as that of the triantennary oligosaccharide (002) is not known, but it may be attributable to steric hindrance. We did not obtain the Vf - V0 for a single branch complex-type sugar, because its PA derivative was not commercially available. The affinity for the glycolipid-type disaccharide Galbeta 1-4Glc-PA (026) was very weak, and retardation was not detectable, probably because the reducing end of the Glc moiety becomes an opened chain structure because of the pyridylamination process. We will probably be able to determine the affinity for conventional galectin-recognized disaccharides such as lactose and N-acetyllactosamine by measuring the inhibition of retardation of strongly recognized PA-sugars (e.g. PA-043) in the near future.

The fucose residue added to the chitobiose structure did not affect the affinity (compare 001 and 009, 002 and 010, and 004 and 011). Because the affinity was reduced when position 6 of nonreducing Gal was substituted by NeuAcalpha 2-6 (021, 022, and 023), position 6 of Gal proved to be important for recognition by both Nh and Ch. Ch showed 2-5-fold stronger binding ability toward all sugars except 047 in comparison with Nh (this will be discussed later). Furthermore, Nh and Ch preferred Galbeta 1-3GlcNAc to Galbeta 1-4GlcNAc. This can be seen by comparing the affinity between 002 and 003 (one of the antennae of the former is Galbeta 1-4GlcNAc and one of the latter is Galbeta 1-3GlcNAc) and that between 041 and 042.

When position 3 of GlcNAc of 041 was substituted by Fucalpha 1-3 (045), or when the position 4 of GlcNAc of 042 was substituted by Fucalpha 1-4 (044), the affinity was considerably reduced. These results suggest that the recognition meets the rules found for other vertebrate galectins, i.e. that position 3 of GlcNAc in the Galbeta 1-4GlcNAc structure or position 4 of GlcNAc in the Galbeta 1-3GlcNAc structure is important for recognition. When the Gal residue at the nonreducing end was modified by Fucalpha 1-2 (Fucalpha 1-2Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc-PA; 043, H Antigen), the affinity became much stronger for all immobilized columns. In this case, Galbeta 1-3GlcNAc was still the major structure for recognition, because the substitution of position 4 of GlcNAc with Fucalpha 1-4 (046) resulted in significant reduction in the affinity.

A significant difference between Nh and Ch was found. When position 3 of Gal at the nonreducing end of 043 was substituted with GalNAcalpha 1-3 (047; blood group A antigen), the affinity for immobilized Nh, but not for Ch, became 3-fold stronger (Vf - V0 increased to 0.384 from 0.143 ml). Such an effect was also reported in the case of mammalian galectin-3. Galbeta 1-3GlcNAc was still the principal recognition structure, because when position 4 of GlcNAc was substituted with Fucalpha 1-4 (048), the affinity was almost lost (Vf - V0 became 0.010 ml). For Ch, on the other hand, the substitution changing 043 to 047 resulted in significant reduction in the affinity (Vf - V0 was reduced to 0.010 from 0.124 ml). The affinity was recovered, but not completely, by further substitution at position 4 of GlcNAc by Fucalpha 1-4 (048; Vf - V0 was 0.047 ml). The reason is not explainable at this moment.

Although a galectin from a marine sponge was reported to have marked affinity for the blood group A-related Forssman antigen saccharide (GalNAcalpha 1-3GalNAcbeta 1-; 040; Ref. 23), neither immobilized Nh nor Ch from C. elegans showed any affinity for this structure.

Recent x-ray crystallographic analysis of LEC-1 demonstrated that both lectin domains are composed of a similar beta -sandwich motif found in mammalian proto-type galectins (12). Together with the present biochemical data, both of the domains function as sugar binding parts, but they have independent sugar recognition properties.

Determination of Ligand Content, Bt, and Kd-- We could compare the affinity of the various PA-sugars for the immobilized galectin by simply looking at the profile shown in Fig. 3, because Vf - V0 values are proportional to the affinities of PA-sugars for a given column. However, to compare the data obtained from different columns, we required Kd values instead of Vf - V0 values. For this purpose, we had to determine the content of immobilized lectin in a given column (Bt). Therefore, as shown in Fig. 4, we determined the Bt values for all immobilized galectin columns by analyzing the concentration dependence of retardation of PA-043 (PA-lacto-N-fucopentaose I), which proved to have relatively strong affinity for all immobilized lectin columns. Concentrations sufficiently higher than the dissociation constant (10-5 ~ 10-4 M in this case of PA-043) were needed for this purpose. Absorbance at 300 nm was used to monitor the elution of PA-043 instead of the fluorescence to avoid possible quenching caused by the relatively high concentration of the PA-sugar. p-Aminophenyl-alpha -D-mannopyranoside, which has no affinity for galectins but is detectable by its absorbance at 300 nm, was used to determine the V0 value. To minimize the consumption of PA-043, which is extremely expensive, we used a 1-ml sample loop.



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Fig. 4.   Determination of Kd of PA-lacto-N-fucopentaose I and Bt for the immobilized LEC-1, Nh, and Ch. PA-Lacto-N-fucopentaose I (PA-043) was dissolved in EDTA-PBS at various concentrations (2-100 µM), and 1 ml of each of the solutions was applied to the lectin column (column volume, 0.126 ml) through a 1-ml sample loop. The thick elution curve is that of PA-rhamnose. The value of Kd can be determined from the slope of the linear plot of [A]0(Vf - V0) versus (Vf - V0) according to Equation 2. The value of Bt can be obtained from the intercept on the ordinate of the same plot. A, LEC-1; B, Nh; C, Ch.

The obtained data were treated according to Equation 2. The value of Kd was calculated from the slope, which corresponds to -Kd. The value of Bt was obtained from the intercept on the ordinate. The linearity was found to be very good, and the results were reproducible. Almost the same values were obtained if we used a double-reciprocal plot (1/[A]0 (Vf - V0) versus 1/[A]0 resembles the Lineweaver-Burk plot).

Bt values of PA-043 were 1.1 × 10-2, 1.8 × 10-2, and 7.1 × 10-3 µmol for immobilized LEC-1, Nh, and Ch columns, respectively. Kd values of PA-043 for immobilized LEC-1, Nh, and Ch were 6.0 × 10-5, 1.3 × 10-4, and 6.5 × 10-5 M, respectively.

Once the value of Bt was obtained for a given column, we could calculate Kd values for all other PA-sugars according to Equation 3 if the concentration of PA-sugar was low enough to satisfy [A]0 Kd (we used 10 nM for this analysis, which is ~0.01% of Kd). Therefore, Vm - V0 is proportional to 1/Kd. Because an isocratic elution system was used throughout a series of runs, we could immediately apply another PA-sugar solution after complete elution of a previous PA-sugar solution, and there was no need for regeneration and reequilibration of the column. This enabled us to obtain the Kd values one after another.

Tables I and II list the values of Kd of all PA-sugars for immobilized LEC-1, Nh, and Ch. Table I is for branched N-linked oligosaccharides, and Table II is for sugar structures found in glycolipids. Kd values for PA-sugars that did not show any significant difference in retardation compared with PA-rhamnose (this PA-sugar has no affinity for galectins) are not presented. The data shown in Tables I and II also indicate that Nh and Ch have different sugar binding properties.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We demonstrated the different sugar binding properties of the two lectin domains in the tandem repeat-type galectin LEC-1 (or N32) of the nematode C. elegans by using improved frontal affinity chromatography. This method enabled us to determine very small differences in elution volumes of PA-sugars. Monitoring by a fluorescence detector and treatment of the data by commercially available software (Microsoft Excel) made the procedure extremely efficient. We also succeeded in determining Bt and Kd values. This improved frontal affinity chromatography system proved to be very useful and will surely contribute to the studies of lectin-sugar interactions as much as other methods recently developed, such as affinity capillary electrophoresis (24, 25) and a biosensor method based on surface plasmon resonance (26, 27). This system is rapid and sensitive, and the results are reproducible and reliable. Furthermore, no need for column regeneration makes it possible to carry out many runs of chromatography to obtain many Kd values in a short period (<20 min for each run). Only a slight modification of the high performance liquid chromatography system (such as addition of a control interface module) is required. Introduction of a highly expensive device (such as a surface plasmon resonance biosensor) is not necessary. The theory is very simple because we can describe this system in terms of a simple equilibrium problem. The system is also applicable to small molecules such as oligosaccharides. If the objective is to compare the binding affinity of analytes for a single column, we need not know the precise concentration of the analyte provided the concentration is low enough ([A]0 Kd), and we can monitor the elution profile. One of the greatest advantages of this system is that it is suitable for analyzing weak interactions. Almost all other procedures require a relatively high concentration of analyte (comparable with its Kd) to determine the Kd value. However, the present procedure does not require such a high concentration even if a very low affinity is to be measured. We can detect very slight retardation by analyzing the elution profile by commercially available software and can analyze very weak interactions. Therefore, this improved frontal affinity chromatography system can be easily constructed in laboratories of moderate economical conditions and can be flexibly modified depending on the researcher's demands.

Recently, Hindsgaul's group reported a frontal affinity chromatography system applicable to screening specific compounds synthesized by combinatorial chemistry (28). They introduced an electrospray mass spectrometer as an on-line detector, making it possible to monitor multiple analytes in one run of frontal affinity chromatography. This system seems extensively versatile, although it will be rather difficult to become commonly used because of the cost.

Both Nh and Ch had affinity for N-acetyllactosamine-containing, N-linked, complex-type sugar chains, and the binding ability was stronger for the more branched complex-type sugar chains. Ch showed 2-5-fold stronger binding ability toward every complex-type sugar chain compared with Nh. Furthermore, Nh and Ch preferred Galbeta 1-3GlcNAc to Galbeta 1-4GlcNAc. Positions 4 and 6 of Gal were critical determinants for recognition by both domains. Fucalpha 1-2Galbeta 1-3GlcNAc (H Antigen, 043) showed stronger affinity for all immobilized columns. Nh showed much stronger affinity for the GalNAcalpha 1-3-modified sugar structure than Ch for GalNAcalpha 1-3(Fucalpha 1-2)Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc-PA (047), or blood group A antigen.

Recent x-ray crystallographic analysis results for LEC-1 demonstrated that both two domains of LEC-1 contain a beta -sandwich motif similar to that of proto-type galectins (12). Our present data provide evidence for independent sugar binding of these two domains. If LEC-1 does actually have two independent sugar binding sites, the apparent binding constant Ka of PA-043 for immobilized LEC-1 for PA-043 will be the average of binding constants for immobilized Nh, Ka1, and immobilized Ch, Ka2 (Equation 4).
K<SUB>a</SUB>=1/2(K<SUB>a1</SUB>+K<SUB>a2</SUB>) (Eq. 4)
Therefore, the apparent Kd of PA-043 for immobilized LEC-1 is as follows:
K<SUB>d</SUB>=<FR><NU>1</NU><DE>K<SUB>a</SUB></DE></FR>=<FR><NU>2</NU><DE>K<SUB>a1</SUB>+K<SUB>a2</SUB></DE></FR>=<FR><NU>2</NU><DE><FR><NU>1</NU><DE>K<SUB>dNh</SUB></DE></FR>+<FR><NU>1</NU><DE>K<SUB>dCh</SUB></DE></FR></DE></FR> (Eq. 5)
Then, Kd can be calculated as 8.7 × 10-5 M. The actually measured Kd for LEC-1 was 6.0 × 10-5 M (Table II), ~30% stronger than expected. The reason for this difference cannot be explained yet, but it may be the result of the orientation of the immobilized proteins. For example, Nh might be more susceptible to damage by the immobilization process; e.g. the sugar binding site might have been facing the supporting matrix. The numbers of Lys residues (containing NH2) in the two domains (11 for Nh and 8 for Ch) may result in a difference in the amount of available immobilized protein. The two domains of LEC-1 may not equally contribute to binding when immobilized. The development of other procedures that do not include an immobilization step, such as capillary affinity electrophoresis, should avoid such differences.

The protein contents of the adsorbent of LEC-1-, Nh-, and Ch-immobilized resins were determined as 5.9, 6.6, and 1.9 mg/ml gel, respectively. This means that the amount of proteins immobilized on the gel packed in the 4.0 × 10-mm column (bed volume, 0.126 ml) were 2.3 × 10-2, 5.1 × 10-2, and 1.6 × 10-2 µmol, respectively. The Bt values obtained from the plot of [A]0(Vf - V0) versus (Vf - V0) were 1.1 × 10-2, 1.8 × 10-2, and 7.1 × 10-3 µmol, respectively. The calculated available binding sites (Bt) for the immobilized LEC-1 represented 24% of the theoretical maximum binding sites. For Nh and Ch, those figures were 35 and 45%, respectively. If the recombinant proteins are immobilized randomly, 50% of the immobilized proteins are expected to have available binding sites. If the proportion is <50%, the binding site may have a tendency to face the supporting matrix and be unavailable. Existence of multiple Lys residues near the sugar binding site may result in fewer available sites. Because the two binding sites exist in one molecule, it is possible that these two sites are not equally available after immobilization.

If the binding constants of the two sugar binding sites are considerably different, the plot of [A]0(Vf - V0) versus (Vf - V0) described as Equation 2 will become biphasic with a bend in the middle. In our case, the binding properties of two sites were not so different, and a bend in the plot was not observable. The difference between two sites was ~2-fold and probably not large enough (such as 10-fold) for a bend to appear in the plot. We are planning to immobilize sugars having strong affinity for galectins and to analyze the elution profiles of recombinant galectins by improved frontal chromatography. Analysis by such an inverse system will be able to compensate for the problems caused by immobilization of lectin proteins.

In a previous study, when the glycoprotein asialofetuin, which contains a considerable amount of Galbeta 1-4GlcNAc structures, was immobilized on a Sehparose resin, the ratio of binding strength of LEC-1, Nh, and Ch was 100:1.14:14.6 (11). Therefore, both independent Nh and Ch proved to interact very weakly with the immobilized asialofetuin. The two binding sites of LEC-1 may have interacted simultaneously with different N-acetyllactosamine-containing sugar chains of the asialofetuin, and this might be the reason why the binding strength of LEC-1 was much stronger than the sum of those strengths of Nh and Ch.

We found that the two domains of LEC-1 showed strong affinity for blood group H antigen. Although we do not have evidence at present that C. elegans actually has such a sugar structure, this structure could be one of the physiological ligands for LEC-1. Because some PA-sugars such as blood group B chains are very expensive or not commercially available, we have not yet checked all the possible sugar structures and might have missed some sugars having stronger affinity. We are now searching for sugar structures present in C. elegans that have high affinity for galectins.

This is the first report showing that LEC-1 has two equally potent sugar binding sites with different sugar binding affinities. Discovery of a sugar structure that is strongly recognized by Nh clearly excluded the possibility that Nh has intrinsically only weak binding ability and that the sugar binding site of Nh had been seriously damaged because of expression as a single domain. The weak binding strength of Nh for asialofetuin-Sepharose may be explained by the fact that asialofetuin is rich in Galbeta 1-4GlcNAc structure but does not contain sugar structures preferred by Nh. Preferable recognition of the blood group A sugar structure (or related structure) by the first lectin (Nh) domain and that of N-acetyllactosamine-containing structures (or related structure) by the second lectin domain (Ch) suggests that LEC-1 is able to cross-link not only homologous but also different glycoconjugates.

Although the primary structures of both of the binding domains of LEC-1 are homologous to those of vertebrate galectins, some important residues, which had been elucidated by site-directed mutagenesis (29, 30) and x-ray crystallography (31-33), were found to have been replaced (6). In the Nh region the asparagine residue (Asn-61), conserved in almost all galectins and reported to form a hydrogen bond with the C-4 hydroxyl group of the galactose moiety in lactose, was substituted by serine (Ser-61). Conserved amino acids on both sides of the asparagine were also changed to valine (conserved sequence His-Phe-Asn-Pro-Arg-Phe was changed to His-Val-Ser-Val-Arg-Phe). The unique binding property of the Nh domain might be attributed to these substitutions. We are currently working on x-ray crystallographic structural studies of LEC-1 in complexes with sugars having different structures, and this could lead to a clear explanation of the difference in affinity between the two domains.

The nematode C. elegans has multiple tandem repeat-type galectins (34). Mammals also have multiple galectins belonging to this type. Different sugar binding properties of the two domains of rat galectin-4 have been reported by measuring the concentration of sugars causing 50% inhibition to binding to lactosyl-Sepharose (7). The first and second domains of this galectin showed preferential binding for Galbeta 1-4GlcNAc and Galbeta 1-3GlcNAc, respectively. The present report is the first to show the properties of the two binding sites of a tandem repeat-type galectin in terms of Kd values. Tandem repeat-type galectins should have different biological roles from proto-type galectins, which form a homodimer resulting in two identical sugar binding sites. For further characterization of the binding properties of the two domains of the tandem repeat-type galectin and investigation of the biological significance of cross-linking sugar structures with different affinities, the improved frontal affinity chromatography reported in this article will be a very useful tool. It will also greatly contribute to the studies of all other types of galectins.


    FOOTNOTES

* This study was supported in part by Grants-in-Aid for Scientific Research 11159212, 11680614, 11771453, and 10178102 from the Ministry of Education, Science, Sports, and Culture of Japan and by Mizutani Foundation for Glycoscience.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, 199-0195, Japan. Tel.: 81-426-85-3741; Fax: 81-426-85-3742; E-mail: y-arata@pharm.teikyo-u.ac.jp.

Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M008602200


    ABBREVIATIONS

The abbreviations used are: Nh, N-terminal lectin domain of LEC-1; Ch, C-terminal lectin domain of LEC-1; PA, pyridylaminated; PBS, phosphate-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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