Interaction of Human Macrophage C-type Lectin with
O-Linked N-Acetylgalactosamine Residues on
Mucin Glycopeptides*
Shin-ichiro
Iida,
Kazuo
Yamamoto, and
Tatsuro
Irimura
From the Laboratory of Cancer Biology and Molecular Immunology,
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
 |
ABSTRACT |
A fluorescein-labeled synthetic peptide,
PTTTPITTTTK, was converted into O-glycosylated
glycopeptides with various numbers of attached
N-acetyl-D-galactosamines (GalNAcs) by in
vitro glycosylation with UDP-GalNAc and a microsomal fraction
of LS174T human colon carcinoma cells. Glycopeptides with 1, 3, 5, and
6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes
were confirmed by matrix-assisted laser desorption ionization
time-of-flight mass spectrometry. Their sequences were determined by a
peptide sequencer to be PTTTGalNAcPITTTTK for G1,
PTGalNAcTTPITGalNAcTGalNAcTTK for G3,
PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK
for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK
for G6. A calcium-type human macrophage lectin (HML) was prepared in a
recombinant form, and its interaction with these glycopeptides was
investigated by surface plasmon resonance (SPR) spectroscopy and
fluorescence polarization. The affinity of recombinant HML (rHML) for
immobilized glycopeptides increased, as revealed by SPR, in
parallel with the number of GalNAc. The highest affinity was obtained
when the G6-peptide was immobilized at high density. Fluorescence
polarization equilibrium-binding assays also revealed that the
affinity of rHML for soluble gly-copeptides increased, depending on the
number of attached GalNAcs. Carbohydrate recognition domain
(CRD) fragments of HML were prepared, and their affinity for these four
glycopeptides was also determined, this affinity was
apparently lower than that of rHML. Affinity constants of rHML for the
G3- and G5-peptides were 11- and 38-fold higher, respectively, than for
the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold
higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylated
O-linked glycopeptides should be due to the trimer
formation of rHML.
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INTRODUCTION |
Calcium-dependent animal lectins (C-type
lectins)1 (1) comprise a
large family of recognition molecules in the immune system. Their
specificity and potential regulatory functions demand a perspective far
beyond the traditional concept of lectins (1, 2). A C-type lectin
specific for galactose (Gal) and
N-acetyl-D-galactosamine (GalNAc) as a
monosaccharide has been found in histiocytic macrophages in mice (3). A
very similar lectin is also expressed in human macrophages as
preferentially bound glycopeptides containing consecutive serine (Ser)
and threonine (Thr) residues with attached GalNAc, which is well known
as a carcinoma-associated Tn antigen (4). While these C-type lectins
are assumed to play roles in the recognition of malignant cells, their
immunological roles are actually more diverse (5-7). In rats, a lectin
homologous to these molecules was shown to be up-regulated in the area
of chronic rejection of heart transplants in rats (8). It has long been
known that other C-type lectins produced by hepatocytes are involved in
asialoglycoprotein uptake from the circulation; macrophage C-type
lectins from rodents seem to function in the same fashion (5, 9).
Because the interactions of some lectins with simple monosaccharides
are sometimes weak, the direct binding of these lectins with a
monosaccharide may be difficult to determine. If the cooperative effects of polyvalent carbohydrate ligands are present, however, and
the lectin has multivalency, a dramatic increase in the affinity could
prevail (2, 10, 11). Members of the C-type lectin family often form
oligomeric structures through the stalk regions containing the
-helical domain; this provides an advantage in polyvalent binding
(12, 13). A similar observation has been made in the binding of
carbohydrate-specific antibodies (14). For example, synthetic
neoglycoproteins and saccharide-derivatized polyacrylamide polymers
have been used to represent a multivalent ligand that has a high
affinity for lectins (15, 16). In other studies, the clustering effects
of carbohydrate chains were evaluated in detail through the use of
trivalent glycosides (17-19). Although the multivalency of
carbohydrate ligands is seen in a variety of native glycoproteins,
particularly O-linked glycoproteins, its functions in
carbohydrate-recognition pathogenic processes are not well known.
Furthermore, differences in the kinetics of protein-carbohydrate
interactions between monosaccharide and multivalent ligands were
unknown until recently. The first step toward determining the
significance of HML in the pathogenesis of a variety of diseases was
the cDNA cloning and sequencing of human macrophage lectin (HML),
described in a previous report (4). The next step should be to
elucidate the nature of natural ligand for this lectin. In the previous
study, HML was demonstrated to preferentially bind clusters of
N-acetylgalactosamine linked to Ser or Thr in a
carcinoma-associated mucin-like configuration (4).
Epithelial mucins are characterized by their tandem repeat domains,
which contain many Ser and Thr residues (20, 21). The tandem repeat
portion of the MUC2 mucin consists of 23 amino acids, including 14 Thr
residues; these Thr residues are reported to be glycosylated up to 78%
in the LS174T colon carcinoma cell line (22). Although the tandem
repeat domain of MUC2 mucin in other cells is also thought to be highly
O-glycosylated, whether the glycosylation patterns in
different cells and tissues are unique is not yet understood. Mucin
architecture represents an ideal framework for a variety of multiple
ligand arrangements that should serve as a recognition unit. In the
present study, we applied the surface plasmon resonance (SPR) biosensor
and fluorescence polarization (FP) spectroscopy to analyze
ligand-density dependence in carbohydrate-protein interactions.
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EXPERIMENTAL PROCEDURES |
Preparation of rHML and Its CRD Fragments--
The gene encoding
HML lacking the transmembrane domain was prepared by polymerase chain
reaction and constructed into an expression vector pET-8c to yield the
plasmid pET-8c-HML as described previously (4). The constructed plasmid
was introduced into Escherichia coli strain BL21(DE3) cells
and expressed. Soluble recombinant HML (rHML) was purified by
galactose-Sepharose 4B affinity chromatography. For the preparation of
its CRD portion, rHML (2 mg) was digested with 100 µg/ml of
N-tosylphenylchloromethyl ketone-treated trypsin (Sigma) in
10 mM HEPES buffer (pH 7.5) containing 2 mM
CaCl2 and 0.15 M NaCl (Ca-HBS) at 37 °C for
3 h. CRD fragments were separated by dialysis from digested
proteins. The purity of both the rHML and the CRD fragments was
confirmed by polyacrylamide gel electrophoresis (PAGE) on 12.5% gels,
as single bands of 29 and 19 kDa, respectively.
Determination of Oligomeric Structures of rHML through Chemical
Cross-linking--
rHML in aqueous solutions was cross-linked with
homobifunctional cross-linkers. Disuccinimidyl suberate (DSS; Pierce),
which potentially cross-links two amino groups located 1.14 nm apart, was used at a concentration of 1 mM in 100 µl of Ca-HBS
at room temperature for 3 h. Ethylene
glycolbis(succinimidylsuccinate) (EGS; Pierce), which potentially
cross-links two amino groups located 1.61 nm apart, was used at a
concentration of 1 mM under the same conditions. Aliquots
of the incubation mixtures were analyzed by 10% SDS-PAGE and
visualized by Coomassie Blue staining. Their molecular weights were
calculated according to the migration distance of molecular weight
markers (Marker II, Daiichi Pure Chemicals Co., Ltd. Tokyo, Japan).
Products of cross-linking reactions were also analyzed by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF MS) on Voyager Elite (Nihon PerSeptive Ltd.,
Tokyo, Japan) using
-cyano-4-hydroxycinnamic acid as a matrix.
Preparation of Glycosylated Peptides--
The
fluorescein-labeled peptide (FITC-PTTTPITTTTK) corresponding to the
MUC2 mucin tandem repeat domain (23) was prepared. The peptide was
purified by reversed-phase high performance liquid chromatography
(HPLC) (Jasco, Tokyo, Japan) on 6 × 150-mm column of Puresil
(C18, Nihon Waters, Tokyo, Japan). The fidelity and molecular mass of
the peptide was confirmed on an amino acid sequencer (PSQ-1, Shimadzu,
Kyoto, Japan) and MALDI-TOF MS. These peptides were labeled with
fluorescein isothiocyanate at its N-terminal amino acid at
pH 7.5 adjusted with 100 mM HEPES buffer. The lysine residue was not substituted with FITC as revealed by carboxypeptidase digestions. The peptide was glycosylated in vitro with crude
UDP-GalNAc:polypeptide
-GalNAc-transferases (EC 2.4.1.41) derived
from LS174T colon carcinoma cell lines, as described in detail
elsewhere.2 The glycosylated
peptides were fractionated by reversed-phase HPLC equipped with a
10 × 250-mm column of Cosmosil (5C18-AR, Nacarai
Tesque, Kyoto, Japan) or a Puresil column as above. The columns were
eluted with gradients of solvent A (0.05% trifluoroacetic acid in
water) and solvent B (0.05% trifluoroacetic acid and 70% propanol in
acetonitrile). A linear gradient of 0-50% solvent B in solvent A was
used for Cosmosil, and a linear gradient of 10-30% solvent B in
solvent A was used for Puresil, at a flow rate of 2 ml/min for 30 min.
Elutions were monitored by fluorescence intensity at an emission
wavelength of 520 nm, using excitation at 492 nm. The number of GalNAc
attached to these peptides was determined by the mass number according
to MALDI-TOF MS using
-cyano-4-hydroxycinnamic acid as a
matrix, as described elsewhere.2 The linear mode with
delayed extraction setting was applied. For the biotinylation of
glycopeptides, the glycopeptides were incubated with 1 mg of either
sulfosuccinimide-biotin (SNHS-biotin) or
sulfosuccinimidyl-6-(biotinamido)hexanoate (SNHS-LC-biotin) (Pierce) in 100 mM HEPES buffer (pH 8.0) at room
temperature for 1 h. The biotinylated glycopeptides were purified
by reversed-phase HPLC equipped with C18 columns; structures were
confirmed by MALDI-TOF MS analysis.
Amino Acid Sequencing--
Pulsed liquid Edman degradation amino
acid sequencing of glycopeptides was performed on an Applied Biosystems
490 Procise protein sequencing system (Perkin-Elmer). With this system,
a phenylthiohydantoin (PTH) derivative of GalNAc-Thr was identified as
a pair of peaks eluted near the positions of PTH-Ser and PTH-Thr (24).
Amino acid sequencing of the fully glycosylated peptide (PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTGalNAcK)
confirmed these eluting positions (data not shown).
Binding of Biotinylated Carbohydrate-Polyacrylamide
Polymers to Immobilized rHML--
rHML (2 µg/ml) was coated on each
well of 96-well microtiter plates (Greiner, Frickenhausen, Germany) in
100 mM sodium bicarbonate buffer (pH 9.5) containing 10 mM CaCl2 overnight at 4 °C. After blocking
with Ca-HBS containing 3% bovine serum albumin at room temperature for
2 h, various concentration of biotinylated
saccharide-polyacrylamide polymers (saccharide-BPP) was added. The
plates were then incubated at room temperature for 2 h. After
washing with Ca-HBS three times, horseradish peroxidase-conjugated
streptavidin (diluted 1:1000, Zymed Laboratories Inc.,
South San Francisco, CA) was incubated at room temperature for 2 h. The wells were rinsed several times with Ca-HBS and developed with a
1 mM solution of 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
containing H2O2 in 0.1 M citrate
buffer (pH 4.2). Absorbance at 405 nm was measured on a Bio-Rad
microplate reader (model 550). Eight different saccharide-BPPs were
used: GalNAc
-BPP, GalNAc
-BPP, Gal
-BPP, GlcNAc
-BPP,
Man
-BPP, GalNAc
1-3GalNAc
-BPP (Forssman disaccharide),
GalNAc
1-3(Fuc
1-2)Gal
-BPP (blood group A-type trisaccharide),
and Gal
1-3(Fuc
1-2)Gal
-BPP (blood group B-type
trisaccharide antigen) (15); all of the saccharide-BPPs were purchased
from Seikagaku Kogyo Ltd. (Tokyo, Japan). According to the instruction
provided by the distributor, these saccharide-BPPs have approximate
Mr of 30,000 and con tain 20% (mole)
carbohydrates. The carbohydrate biotin ratio was approximately 4:1, and
approximately every fifth amide side chain of polyacrylamide was
thought to be substituted with a carbohydrate chain. In the case of
competitive enzyme-linked immunosorbent assays (ELISAs), each of the
five monosaccharides (GalNAc, Gal, methyl-
-Gal, methyl-
-Gal, and GlcNAc) was mixed with GalNAc
-BPP (1 nM) at various
concentrations and added to the wells.
Interaction of rHML with Immobilized Glycopeptides--
The
interaction of rHML with immobilized glycopeptides was determined with
BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden), in which the
measurement is based on the SPR (25). Each biotinylated glycopeptide
was introduced onto the surface of a streptavidin-coated sensor chip
(SA5) at a flow rate of 10 µl/min. Based on the saturated response of
anti-FITC monoclonal antibody (mAb) (clone FL-D6, Sigma) at 2500-3000
response units, the amount of each glycopeptide immobilized onto
different sensor chips was confirmed to be almost identical. rHML at
various concentrations (0.1-10 µM) dissolved in 30 µl
of Ca-HBS was applied over each glycopeptide-immobilized surface at
25 °C. Anti-trimeric Tn mAb (clone B231.5, kindly donated by Dr.
Mark Reddish, Biomira Inc., Alberta, Canada) (26) was also used. To
minimize an effect of the mass transport limit, flow rate was
maintained at 10 µl/min (27). The surface was regenerated with
HEPES-buffered saline containing 10 mM EDTA for the binding
of rHML, and with 200 mM glycine-HCl buffer (pH 2.7) for
the binding of anti-trimeric Tn mAb. Association and dissociation rate
constants (ka and kd) were
calculated using BIAevaluation 2.0 software (Pharmacia Biosensor AB).
Determination of Interaction of rHML and Its CRD Fragment with
Soluble Glycopeptides--
To determine the interaction of rHML with
soluble glycopeptides, FP equilibrium-binding assays were performed.
The principal advantage of this method is that the assay is done in
fluid phase. In addition, no separation of bound glycopeptide from free
glycopeptide is required (28). To reach an equilibrium,
fluorescence-labeled glycopeptides (0.1 pmol; total intensity of 200)
were incubated with the indicated concentration of rHML or CRD
fragments in 100 µl of Ca-HBS at room temperature for 90 min. FP
values were measured by Beacon 2000 (Takara Shuzo, Shiga, Japan) at
25 °C.
 |
RESULTS |
Properties of rHML and CRD Fragments--
rHML cDNA lacking
its transmembrane domain was expressed in E. coli. Soluble rHML protein was purified by affinity
chromatography, as described previously (4). Its CRD fragments were
obtained by digestion with N-tosylphenylchloromethyl
ketone-treated trypsin. As shown in Fig.
1, the purity of rHML and its CRD
fragments was confirmed by SDS-PAGE on 12.5% gels and the apparent
relative molecular mass values were 29 and 19 kDa, respectively. The
oligomeric status of these preparations in aqueous solution was
examined by chemical cross-linking. Following treatment with either DSS or EGS, the resultant proteins were analyzed by SDS-PAGE (Fig. 1). rHML
was cross-linked with DSS and with EGS, as shown by bands migrating at
positions corresponding to a molecular mass of 58 and 87 kDa,
respectively. These sizes suggested that the proteins are dimeric and
trimeric forms of rHML. Such cross-linked products were not observed,
however, when DSS- or EGS-treated CRD fragments were analyzed by
SDS-PAGE, indicating that CRD fragments were present predominantly in
monomeric form (Fig. 1). The major cross-linked product was an 87-kDa
molecule, even after a prolonged incubation for 20 h and
incubation with high concentrations of cross-linking agents (data not
shown).

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Fig. 1.
Profiles of SDS-PAGE on 12.5% gels of
chemically cross-linked rHML and its CRD fragments under reducing
conditions. rHML (29 kDa) and its CRD (19 kDa) were treated with
Me2SO alone (solvent) (N), or 1 mM DSS (D), or with 1 mM EGS
(E). A mixture of molecular weight marker
proteins (myosin, -galactosidase, albumin, aldorase, carbonic
anhydrase, and myoglobin) was used; their Mr are
indicated on the left side of the panel. The gels were
stained with Coomassie Blue.
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MALDI-TOF MS analysis indicated that the average molecular masses of
rHML and CRD fragments were 25,334 and 17,780, respectively; these
findings are consistent with predicted masses of 25,322 and 17,732, respectively. Following the cross-linking of rHML, signals of 26,673, 53,163, and 79,845 were obtained (data not shown). For the evaluation
of the binding kinetics of HML and CRD, the predicted mass figures of
Mr 25,322 and 17,732 were used.
Carbohydrate Specificity of rHML toward Polyvalent Oligosaccharide
Derivatives--
As subjects in an initial screening of the
specificity of rHML, as shown in Fig. 2,
we chose polyvalent oligosaccharide derivatives constructed on a
polyacrylamide backbone (15). Results of ELISA indicated that
GalNAc
- and GalNAc
-BPPs strongly bind immobilized rHML in a
calcium-dependent manner; as no binding of other
monosaccharide BPPs, including Gal
-, GlcNAc
-, and Man
-BPPs,
were observed at concentrations up to 30 nM (Fig.
2A). Two BPPs closely related to GalNAc
-BPP, a Forssman
disaccharide derivative (GalNAc
1-3GalNAc
-BPP) and a blood group
A-type trisaccharide derivative
(GalNAc
1-3[Fuc
1-2]Gal
-BPP), showed no affinity for rHML.
Competitive inhibition experiments with various monosaccharides
demonstrated that GalNAc inhibits the interaction of GalNAc
-BPP with
rHML and that Gal does not. When 2-5 mM GalNAc were added
to the binding assay in which 1 nM rHML was used, the
binding of GalNAc
-BPP dropped to half of the maximum level (Fig.
2B). Even though rHML was purified by affinity
chromatography on Gal-Sepharose, neither the binding of Gal-BPP to rHML
nor the inhibition of GalNAc-BPP binding by Gal was detected under the
conditions used.

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Fig. 2.
Binding of rHML to GalNAc analogs and
derivatives as determined by ELISA. A, immobilized rHML
(2 µg/ml) on a 96-well plate were incubated first with
saccharide-BPPs at concentrations indicated on the panel at room
temperature for 1 h, and then with horseradish
peroxidase-conjugated streptavidin. Binding was measured as absorbance
at 405 nm following colorimetric reaction with
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium. The
following saccharide-BPPs were used; GalNAc -BPP (open
squares), GalNAc -BPP plus 10 mM EDTA
(closed squares), GalNAc -BPP (open
diamonds), GalNAc 1-3GalNAc -BPP (Forssman
disaccharide) (closed diamonds),
GalNAc 1-3(Fuc 1-2)Gal -BPP (blood group A-type
trisaccharide) (open triangles),
Gal 1-3(Fuc 1-2)Gal -BPP (blood group B-type trisaccharide
antigen) (closed triangles), Gal -BPP
(open circles), GlcNAc -BPP
(closed circles), and Man -BPP (no
symbol). B, the following five monosaccharides
were tested for their inhibitory activity against rHML binding to
GalNAc -BPP (1 nM): GalNAc (closed
squares), Gal (open squares),
-Me-Gal (closed triangles), -Me-Gal
(open triangles), and GlcNAc (closed
circles).
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Characterization of Glycopeptide Prepared by Cell-free
Glycosylation of MUC2 Mucin Peptides--
To determine the effects of
the density of GalNAc residues in glycopeptides on their affinity with
rHML, undecapeptides corresponding to the tandem repeat portion of the
MUC2 mucin with various numbers of attached GalNAc residues were
generated in vitro as described elsewhere.2
Fluorescence-labeled peptides with one, three, five, or six GalNAc residues (G1-, G3-, G5-, and G6-peptides) were immobilized by biotinylation on streptavidin-coated sensor chips (see below). The
fidelity of the products was confirmed by MALDI-TOF MS analysis as
shown in Fig. 3. Mass data revealed that
all of the peptides contained 1 mol of FITC and biotin (or LC-biotin)
and the predicted number of GalNAc residues. Additional peaks observed
in the areas of lower mass numbers seemed to represent degradation
products generate during the mass analysis, because each glycopeptide
eluted as a single peak on the reversed phase HPLC under various
conditions. Furthermore, as shown in Fig.
4, a significant portion of the material
with a mass number of 2094.4 was identified as
PTTTGalNAcPITTTTK by peptide sequence analysis. When an
analytical C18 column was applied in place of a preparative
column and eluted with less steep gradient, material corresponding to
the peak with three attached GalNAc residues was separated into two
peaks. The second peak was found to be composed primarily of
PTGalNAcTTPITGalNAcTGalNAcTTK. It
was this glycopeptide, with three attached GalNAc residues, that was
examined to determine its interaction with HML. Sequence analysis of
the glycopeptide with five attached GalNAc residues was shown to
be
PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK
and that with six attached residues was shown to be
PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK.

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Fig. 3.
MALDI-TOF MS analysis of biotinylated
glycopeptides. Each biotinylated glycopeptide was mixed on a tip
with 10 mg/ml -cyano-4-hydroxycinnamic acid dissolved in 50%
ethanol containing 0.1% trifluoroacetic acid. Mass was measured in the
linear mode, in either the [M + H]+ form or the [M + Na]+ form. a, GalNAc residue free peptide
(predicted mass, 1777.0); b, one GalNAc-attached residue
(predicted mass, 2093.4); c, three GalNAc-attached residues
(predicted mass, 2386.6); d, five GalNAc-attached residues
(predicted mass, 2793.0); e, six GalNAc-attached residues
(predicted mass, 3109.4). Single asterisk denotes
obtained mass as [M + Na]+ form; double
asterisk denotes LC-biotinylated peptides.
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Fig. 4.
Profiles of amino acid sequencing
chromatograms of the oligopeptide and its derivatives with incorporated
GalNAc residues (G1-, G3-, G5-, and G6-peptides). Following
separation by reversed-phase HPLC, these glycopeptides were analyzed on
the Applied Biosystems 490 Procise protein sequencing system.
A, untreated FM2-1. B, FM2-1 with one
GalNAc residue. C, FM2-1 with three GalNAc residues.
D, FM2-1 with five GalNAc residues. E, FM2-1
with six GalNAc residues. Single asterisks and
double asterisks indicate putative peaks of PTH
derivatives of -GalNAc-Thr (TGalNAc).
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Interaction of rHML with Immobilized Glycopeptides as Determined by
SPR Spectroscopy--
Glycopeptides (0.1 pmol in 100 µl) were
introduced onto each flow cell. Approximately equal amounts of
glycopeptides were immobilized, as confirmed by a similar resonance
response following anti-FITC mAb binding (~3000 response units). For
the kinetic analysis, rHML was applied at concentrations of 0.15-10
µM to each flow cell; association and dissociation were
monitored in real time.
As can be seen in the representative sensorgraphs of the interactions
between rHML and the immobilized glycopeptides shown in Fig.
5, rHML bound G1-, G3-, G5-, and
G6-peptides in a dose-dependent manner (Fig. 5,
A-C and E), while no binding to the GalNAc-free peptides was not observed. As shown in Table
I, ka, kd, and KD constants were
calculated according to a single class receptor-ligand binding
equation. The affinity of rHML for glycopeptides, as well as increases
in ka and decreases in kd, were
dependent upon the number of bound GalNAc residues resulting in
apparent KD constants of 1.0 × 10
6, 4.4 × 10
7, 1.9 × 10
7, and 1.5 × 10
7 M for
G1-, G3-, G5-, and G6-peptide, respectively.

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Fig. 5.
Interaction of rHML with immobilized G1-,
G3-, G5-, and G6-peptides analyzed with SPR biosensor.
Glycopeptides were immobilized on streptavidin-coated sensor chips
through avidin-biotin binding, and different concentrations of rHML
were then introduced: a (10 µM), b
(5 µM), c (2.5 µM), d
(1.25 µM), e (0.6 µM),
f (0.3 µM), g (0.15 µM), h (0 µM), i (10 µM plus 5 mM EDTA), j (5 mM EDTA alone). A, G1-peptide was immobilized;
B, G3-peptide; C, G5-peptide; D,
GalNAc-free peptide; E, G6-peptide; F, dense
G6-peptide; G, effect of EDTA on binding of rHML (10 µM) to immobilized G5-peptide.
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Table I
KD evaluated by the interaction of rHML with immobilized
glycopeptides
Data were fit to association and dissociation models to obtain
ka and kd, and
KD was calculated from these parameters.
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Using the same flow cells, with the peptides incorporating different
residues, anti-trimeric Tn mAb (~30 µg/ml) was tested (Fig.
6). It did not react with the GalNAc-free
and the G1- and G3-peptides, but it did react with the G5- and
G6-peptides (over 1000 response units). These results confirmed that
G5- and G6-peptides contain trimeric-Tn within three and four
consecutive Thr residues.

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Fig. 6.
Interaction of mAb clone B231.5
(anti-trimeric Tn) with G1-, G3-, and G5-peptides analyzed with SPR
biosensor. Glycopeptides were immobilized on streptavidin-coated
sensor chips at 100 pmol (a, b, and d)
or 0.1 pmol (c, e, and f). The same
concentration (30 µg/ml) of mAb clone B231.5 was introduced.
Glycopeptide used were G5-peptide (a, c),
G3-peptide (b, e), and G1-peptide (d,
f).
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It should be noted that when greater than 10 pmol of peptides were
immobilized, anti-trimeric Tn mAb also reacted with G1-peptides (Fig.
6). rHML had very high affinity in flow cells for densely immobilized
G6-peptides, as shown in the bottom rows in Table I
(KD: 5.3 × 10
8 M).
However, because kd decreased drastically, the
affinity of rHML for G6-peptides immobilized in a more sparse density
was much lower (KD: 1.5 × 10
7
M). Binding of anti-trimeric Tn mAb was not intense to
G1-peptides immobilized at 0.1-10 pmol (100 µl, 10 µl/min) (Fig.
6). When rHML was tested for it binding to immobilized G1-peptides in
an even more sparse density, its affinity constant was calculated to be
lower (KD: 1.3 × 10
6
M;
: 0.2 × 10
6 M) (data
not shown). Specific binding of rHML to immobilized glycopeptides was
completely inhibited by the presence of EDTA, as shown in Fig. 5.
Interaction of rHML with Soluble Glycopeptide--
In the
measurement of the affinity constant, one of several ways to avoid the
effect of distance between each immobilized glycopeptide is to
determine the interaction of rHML and glycopeptides in solutions. This
can be achieved with the FP equilibrium-binding assay system (28).
Before assays were conducted, it was determined that the time required
to reach equilibrium was less than 5 min (data not shown). With the
assay system, as shown in Fig. 7,
fluorescence-labeled glycopeptides were incubated with the indicated
concentration of rHML at room temperature for 90 min. The binding
of rHML to glycopeptides, as indicated by changes in FP intensity,
depended upon its concentration (Fig. 7A) and calcium ion
(data not shown); no binding to GalNAc-free peptides was observed.
Affinity constants of rHML were obtained by Scatchard analysis (Fig.
7B), yielding apparent KD of 6.8 × 10
6, 6.4 × 10
7, and 1.8 × 10
7 for G1-, G3-, and G5-peptides, respectively. The
affinity constants for G3- and G5-peptides were 11- and 38-fold higher,
respectively, than the constant for G1-peptides. These findings were in
agreement with the results from the interaction of rHML with
immobilized glycopeptides except for the G1-peptide.

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Fig. 7.
Interaction of rHML with soluble G1-, G3-,
and G5-peptides analyzed with an FP detector. Four
fluorescein-labeled glycopeptides, G1-peptide (squares),
G3-peptide (circles), G5-peptide (triangles), and
GalNAc-free peptide (diamonds), were incubated with rHML at
concentrations indicated on the panel at room temperature for 90 min.
A, the results of fluorescence polarization; B,
affinity constants determined by plotting the data according to
Scatchard analysis.
|
|
Interaction of CRD Fragments with Soluble
Glycopeptides--
Interactions of CRD fragments from rHML with
glycopeptides were primarily evaluated with the FP equilibrium-binding
assay system (Fig. 8). The signals
revealed by SPR spectroscopy with CRD fragments were not high enough to
permit the calculation of affinity constants. Interactions were
detected, however, when relatively high doses of CRD fragments were
analyzed; the maximum response was observed in the interaction with
G5-peptides (~14 µM). Scatchard analysis (Fig.
8B) revealed that affinity of CRD is lower than that of
rHML. They were 8.3, 3.9, and 1.4 × 10
6
M for G1, G3, and G5-peptide
(Table II), respectively. The affinity of
CRD fragments was 2-fold greater for G3-peptides and 6-fold greater for
G5-peptides than for G1-peptide. As was found regarding the specific
binding of rHML to immobilized glycopeptides, the binding of CRD
fragments to each of the these four soluble glycopeptides was inhibited
by the presence of EDTA (data not shown).

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Fig. 8.
Interaction of CRD with soluble G1-, G3-, and
G5-peptides determined with an FP detector. Four
fluorescence-labeled glycopeptides, G1-peptide (squares),
G3-peptide (circles), G5-peptide (triangles), and
GalNAc-free peptide (diamonds), were incubated with CRD
fragments at concentrations indicated on the panel at room temperature
for 90 min. A, the results of fluorescence polarization;
B, affinity constants determined by plotting the data
according to Scatchard analysis.
|
|
 |
DISCUSSION |
To examine the interaction of HML with GalNAc residues
arranged on peptides, we prepared short synthetic glycopeptides
corresponding to a portion of the tandem repeat domain of MUC2 mucin
with different numbers of attached GalNAc residues. Molecular sizes
were determined by MALDI-TOF MS, and the locations of the incorporated
residues were determined by peptide sequencing. Kinetics of the binding were examined in both the fluid-solid phase with SPR and the
fluid-fluid phase with FP; both equilibrium-binding assays of the
interaction of rHML with immobilized glycopeptides revealed that the
number of GalNAc residues does affect KD. Affinity
of the G3-peptide (PTGalNAcTTPITGalNAcTGalNAcTT[K])
for HML was 11-fold greater than that of the G1-peptide
(PTTTGalNAcPITTTTK) and that of the G5-peptide was 38-fold
greater; when KD values were normalized according to
the number of GalNAc residues, the increases were 3.5- and 7.6-fold, respectively.
Differences in affinity for CRD fragments of G3 and G5 relative to G1,
based on the molar amount of glycopeptide, were 2- and 6-fold,
respectively; differences based on the number of GalNAc residues were
0.7- and 1.2-fold, respectively. Therefore, rHML fragments but not CRD
fragments revealed a drastic increase in affinity that depended on the
number of GalNAc residues present. The differences among G1-, G3-, and
G5-peptides should be due to the increased avidity of polyvalent
ligands. This effect was more prominent with rHML than with CRD
fragments due to the oligomeric nature of rHML, whereas CRD fragments
apparently remained in monomeric in aqueous solutions (Fig. 1).
The G5- and G6-peptides were shown to contain three consecutive
Thr-linked GalNAc residues, i.e. Tn antigen. Anti-trimeric Tn mAb (26) was shown to bind these glycopeptides. It has already been
shown that rat hepatic lectin, a type of trimeric C-type lectin, also
bound preferentially to clustered GalNAc residues, as shown by using
synthetic cluster glycosides based on Tris (17, 18). Therefore, in
addition to the impact of increased number of GalNAc residues, three
consecutive Thr attached with GalNAc residues might also contribute to
the high binding affinity for HML.
Dense immobilization of all of the synthetic glycopeptides leads to
high affinity association. The difference between densely immobilized
and sparsely immobilized glycopeptides seemed to be due to the dramatic
decrease in the dissociation rates (Table I). Although the organization
of the densely immobilized glycopeptides on the membranes was unknown,
the binding of anti-trimetric Tn mAb strongly suggested that some
GalNAc residues from separate glycopeptides were located very close to
each other; thus, the clustering of GalNAc residues may well be one of
the more important factors in the high affinity of HML (Fig. 6).
Because the immobilization was done via biotin-streptavidin, it may be
unconceivable that the distance of GalNAc would be as close as to mimic
a single amino acid distance between them. Thus, the spacing among the carbohydrate chains may contribute to macromolecular ligand
interactions with HML, as observed in amebic lectin binding (16). There
may be another level at which clusters in macromolecular carbohydrate ligands contribute to high affinity (18). Simulation by x-ray crystallography (29) has revealed three binding sites of trimeric mannose binding protein spaced approximately 4.5 nm apart. If HML also
accommodates such a structure, GalNAc residues spaced more sparsely
than trimeric Tn might be required for viable ligand-carbohydrate interactions.
The results of FP equilibrium-binding assays, with one exception, were
consistent with the findings from the experiments using the SPR
biosensor, KD for G1-peptides from SPR (1.0 µM for rHML) was higher than KD based
on FP binding data (6.8 µM for rHML and 8.3 µM for CRD). The difference might be due to an increase
in conformational stability on solid surfaces. It is also probable that
the solid phase configuration was more appropriate for the formation of
the multiple attachment sites preferred by oligomeric lectin than are
glycopeptides in solutions. Collectively, slightly lower constants from
FP assays in fluid-fluid phases than those from SPR measurements seemed
to be reasonable. From FP assays conducted under ideally diluted
conditions, KD values between monovalent lectin and
GalNAc monosaccharide can be obtained by the Scatchard plot (Fig. 6).
All KD values reported here were based on the
monovalent ligand-monovalent receptor model. Such analysis was used
because the binding of multivalent lectins to multivalent glycopeptides
is inherently more complex than are antigen-antibody interactions, and
thus it is difficult to simulate the factors involved in a simple
equation (14). The results, however, fit well with the single-site
binding model. It is possible, then, that, regardless of the valency,
binding of rHML might be an "all-or-nothing" event with no
intermediate forms, as discussed by Adler and co-workers (16).
In microtiter assays, rHML bound only GalNAc
- or
-monosaccharide-BPPs linked directly to the spacer arm on the
polymer backbone. A GalNAc residue located away from the polymer
backbone, such as those in the Forrsman disaccharide and the blood
group A-type trisaccharide (Fig. 2), did not have strong affinity with HML. On the contrary, a low albeit significant affinity for HML by
these oligosaccharides was detected through measurement on the SPR
biosensor (data not shown). Such a discrepancy might be due to
different steric hindrances in the access of this lectin to the binding
sites using these two methods. The preferred ligand structure for HML
binding was multiple GalNAc residues attached to linear backbones,
regardless of the specific synthetic polymers or oligopeptides. As
noted above, the Gal
residue attached to the same polymer neither
bound directly to the lectin nor inhibited the binding of GalNAc to
rHML. These results were consistent with our previous data, which
showed that rHML bound neither N-linked carbohydrate chains
with terminal Gal residues nor the N-terminal octapeptide of
human glycophorin A with three consecutive desialized O-linked carbohydrate chains (4). Greater specificity for
GalNAc than Gal was also observed with a human hepatic lectin (30), in
contrast to a mouse macrophage lectin that preferentially bound Gal
instead of GalNAc residues (31, 32). Using chimeric and mutagenized CRD
fragments of hepatic and macrophage lectin, amino acid residues likely
to be involved in the selective binding of GalNAc in the human hepatic
lectin were predicted (30) (His-256 and Asn-208). These amino acids
apparently correspond to His-260 and Tyr-212 in HML, which may be the
most important residue in the interaction between HML and GalNAc. HML
could serve as an useful model to further investigate the nature of
GalNAc-protein interactions.
When solutions of either rHML or CRD fragments were incubated in the
presence of cross-linking agents, a homotrimer of rHML was the only
major oligomeric lectin protein (Fig. 1). The CRD fragments lacking the
HML stalk region did not appear to form oligomers. Other members of the
C-type lectin family, including CD23, CD72, rat asialoglycoprotein
receptor, chicken hepatic receptor, Kupffer cell receptor, and
mannose-binding protein, are also known to form oligomers (12, 29). The
primary amino acid sequence of the stalk region of rHML has a structure
known to be involved in oligomer formation. When the amino acid
sequence of the stalk region (from Ile-87 to Glu-153) deduced from the
nucleotide sequence was arranged in order to accommodate two heptads
(a, b, c, d, e, f, and g), hydrophobic residues were arranged at
positions a and d in a way that should result in an
-helix
coiled-coil. Thus, HML forms a trimeric configuration and would have
high affinity for ligands with GalNAc residues such as in the case of
CD23 with IgE (13).
We have determined affinity constants between HML and peptides with a
single GalNAc residue using FP equilibrium-binding assays. A
carbohydrate density-dependent increase of affinity for the lectin was revealed, and the increase was more prominent with intact
rHML than with CRD fragments. In the present study, variations in the
distance between multiple GalNAc residues on a single peptide were not
systematically investigated. More dispersed arrangements of GalNAc
residues than those used in this study may generate more preferable
configurations as functional ligands for HML, and therefore this will
be an important subject for future studies.
 |
ACKNOWLEDGEMENTS |
We thank Dr. David M. Wildrick, University of
Texas M. D. Anderson Cancer Center, for editorial assistance and
Chizu Hiraiwa for assistance in preparing this manuscript.
 |
FOOTNOTES |
*
This work was performed as part of the Research and
Development Projects of the Industrial Science and Technology Frontier Program supported by the New Energy Development Organization (NEDO) and
PROBRAIN. This work was also supported by Grants-in-aid 05274101, 05557104, and 07407063 from the Ministry of Education, Science, Sports
and Culture of Japan, and grants from the Ministry of Health and
Welfare, the Japan Health Science Foundation, and the Research Association for Biotechnology.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. Tel.: 81-3-3812-2111 (ext. 4870); Fax: 81-3-3815-9344; E-mail:
irimura{at}mol.f.u-tokyo.ac.jp.
2
S. Iida, H. Takeuchi, K. Kato, K. Yamamoto, and
T. Irimura, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
C-type lectin, calcium-dependent animal lectin;
Ca-HBS, HEPES buffer
containing CaCl2 and NaCl;
CRD, carbohydrate recognition
domain;
DSS, disuccinimidyl suberate;
EGS, ethylene glycolbis(succinimidylsuccinate);
ELISA, enzyme-linked immunosorbent
assay;
FITC, fluorescein isothiocyanate;
FP, fluorescence polarization;
HML, human macrophage lectin;
HPLC, high performance liquid
chromatography;
mAb, monoclonal antibody;
MALDI-TOF MS, matrix-assisted
laser desorption ionization time-of-flight mass spectrometry;
Man
-BPP, mannose
-biotinylated polyacrylamide polymer;
rHML, recombinant human macrophage lectin;
PTH, phenylthiohydantoin;
saccharide-BPP, biotinylated saccharide-polyacrylamide polymers;
PAGE, polyacrylamide gel electrophoresis;
SPR, surface plasmon
resonance.
 |
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