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
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ABSTRACT |
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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 Gal Galectins form a group of animal lectins characterized by their
specificity for 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 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.
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
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).
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
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:
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
(
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- 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.
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 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
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
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 NeuAc
When position 3 of GlcNAc of 041 was substituted by Fuc
A significant difference between Nh and Ch was found. When position 3 of Gal at the nonreducing end of 043 was substituted with GalNAc
Although a galectin from a marine sponge was reported to have marked
affinity for the blood group A-related Forssman antigen saccharide
(GalNAc
Recent x-ray crystallographic analysis of LEC-1 demonstrated that both
lectin domains are composed of a similar 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
The obtained data were treated according to Equation 2. The value of
Kd was calculated from the slope, which corresponds to
Bt values of PA-043 were 1.1 × 10
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
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.
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 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 Gal Recent x-ray crystallographic analysis results for LEC-1 demonstrated
that both two domains of LEC-1 contain a 1-3GlcNAc to Gal
1-4GlcNAc. Because the
Fuc
1-2Gal
1-3GlcNAc (H antigen) structure was found to interact
with all immobilized protein columns significantly, the
Kd value of pentasaccharide
Fuc
1-2Gal
1-3GlcNAc
1-3Gal
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
GalNAc
1-3(Fuc
1-2)Gal
1-3GlcNAc
1-3Gal
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
-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
-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.
-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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
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-
(Eq. 1)
-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 ( Si) with
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
(
Si). The value of the base of the
rectangle that makes the same area value of the added area
(
Si) was calculated (i.e.
Si /[A]i). C,
when [A]i reaches the plateau
[A]0, the value Vi
Si /[A]i will converge to
Vf, which makes the two area-under-the-curve values
equal.
V0) versus (Vf
V0) should be linear:
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
(Eq. 2)
Kd. The value of Bt can be
obtained from the intercept on the ordinate.
This means that the value of Vm
(Eq. 3)
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.
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
V for base and
[A]i for height; i.e.
Si =
V × [A]i (Fig. 1B). If
V is small enough,
Si (Fig.
1B, shaded) is close enough to the actual area. If the
column i is located at the plateau, division of
Si by [A]i gives the
length of the base of the large rectangle. Subtracting this value from
Vi (i.e. Vi
(
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
(
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.
-D-mannopyranoside).
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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.
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-
-D-mannopyranoside was used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
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
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
V0 for a single branch
complex-type sugar, because its PA derivative was not commercially
available. The affinity for the glycolipid-type disaccharide
Gal
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.
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 Gal
1-3GlcNAc to Gal
1-4GlcNAc. This can be seen by
comparing the affinity between 002 and 003 (one of the antennae of the
former is Gal
1-4GlcNAc and one of the latter is Gal
1-3GlcNAc)
and that between 041 and 042.
1-3 (045),
or when the position 4 of GlcNAc of 042 was substituted by Fuc
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
Gal
1-4GlcNAc structure or position 4 of GlcNAc in the
Gal
1-3GlcNAc structure is important for recognition. When the Gal
residue at the nonreducing end was modified by Fuc
1-2
(Fuc
1-2Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA; 043, H Antigen),
the affinity became much stronger for all immobilized columns. In this
case, Gal
1-3GlcNAc was still the major structure for recognition,
because the substitution of position 4 of GlcNAc with Fuc
1-4 (046)
resulted in significant reduction in the affinity.
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.
Gal
1-3GlcNAc was still the principal recognition structure, because
when position 4 of GlcNAc was substituted with Fuc
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
Fuc
1-4 (048; Vf
V0 was
0.047 ml). The reason is not explainable at this moment.
1-3GalNAc
1
; 040; Ref. 23), neither immobilized Nh nor
Ch from C. elegans showed any affinity for this structure.
-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.
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-
-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.
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).
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.
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
1-3GlcNAc to Gal
1-4GlcNAc.
Positions 4 and 6 of Gal were critical determinants for recognition by
both domains. Fuc
1-2Gal
1-3GlcNAc (H Antigen, 043) showed
stronger affinity for all immobilized columns. Nh showed much stronger
affinity for the GalNAc
1-3-modified sugar structure than Ch for
GalNAc
1-3(Fuc
1-2)Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA (047),
or blood group A antigen.
-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).
Therefore, the apparent Kd of PA-043 for
immobilized LEC-1 is as follows:
(Eq. 4)
Then, Kd can be calculated as 8.7 × 10
(Eq. 5)
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 × 102, 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 Gal1-4GlcNAc 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 Gal1-4GlcNAc 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
Gal1-4GlcNAc and Gal
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.
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FOOTNOTES |
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* 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.
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
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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.
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