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INTRODUCTION |
Ameloblasts, specialized cells of the inner enamel epithelium,
synthesize and secrete amelogenins, the principal enamel matrix protein
(>90%) on to a premineralized collagenous dentine at the dentine-enamel junction
(DEJ)1 forming a layer of
extracellular matrix that eventually becomes mineralized with a calcium
hydroxyapatite creating the dental enamel (1-3). Other enamel matrix
components include the glycosylated and phosphorylated
"non-amelogenin" proteins (enamelins, tuftelins, and
ameloblastins). However, little is known about the interaction of
amelogenins with these other enamel proteins or with dentine matrix
proteins (collagens and sialophosphoproteins) (4-6). It has been
suggested that the EMGs2 may
bind to amelogenins in addition to interacting with the growing mineral
crystals (6); however, the molecular nature of any such interactions is unclear.
Amelogenins (5-28 kDa) are phosphorylated at position Ser-16 but are
not glycosylated (7, 8). Three domains of amelogenin primary structure
have been described: (a) a highly conserved NH2-terminal hydrophobic sequence of 44-45 amino acids,
containing 6 tyrosine residues (a region referred to as the
"tyrosine-rich amelogenin polypeptide" or TRAP) with one of the
tyrosyl-containing motifs consisting of three tyrosine residues with a
spacing as: XYXXYXYX
(PYPSYGYE); (b) a central hydrophobic core motif of 100-130 residues enriched in proline, leucine, glutamine, and histidine; and
(c) the amelogenin carboxyl-terminal peptide (ACP), an
acidic hydrophilic sequence of 13 amino acids (see Table I).
The amino acid sequences of the amelogenins are highly conserved in the
seven mammalian species so far studied (9-13), with a near identity of
sequence being observed for the NH2-terminal TRAP region
and the C-terminal teleopeptide. The biological roles of these highly
conserved sequences are little understood. During maturation of the
enamel, the amelogenins are processed by proteinases within the matrix
(14-16), an event that appears to signal a change in the functional
role of the intact protein.
Since amelogenins are secreted on the pre-existing organic matrix of
the dentine, molecular interactions at this dentine-enamel interface
may be anticipated and may be functionally important to the stability
of the developing tooth structure. Dentine matrix contains both
collagenous and non-collagenous proteins; the latter class also
includes glycoproteins and phosphoproteins (6, 17-21). It has been
shown that the sugar residues of the glycoconjugates are capable of
inhibiting hydroxyapatite crystal formation (22-24). We hypothesize
that amelogenins may bind to the sugar residues of EMGs at the DEJ to
facilitate biomineralization. Alternatively, such interactions may be
important in amelogenin matrix structure. In this investigation, we
have, for the first time, identified specific interactions between
amelogenins and the GlcNAc residues of glycoconjugates. Further, we
have identified the glyco-binding locus of the amelogenin structure
with a conserved tyrosyl motif of the amelogenin TRAP sequence and have
shown this motif to have a striking structural similarity to a
secondary GlcNAc-binding site of wheat germ agglutinin (WGA).
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EXPERIMENTAL PROCEDURES |
Expression, Isolation, and Purification of Recombinant Mouse
Amelogenin Proteins rM179 and rM166--
Recombinant murine amelogenin
rM179 was prepared by expression in Escherichia coli and
isolated and purified by high performance liquid chromatography as
described previously (25). The protein was further purified by
reversed-phase HPLC (C4-214TP54 column; Vydac/The Separations Group,
Hesperia, CA) and the homogeneity of the protein was assessed by
SDS-polyacrylamide gel electrophoresis (26) carried out in the presence
of 0.1% SDS, using a 15% acrylamide gel. The gel was stained with
Coomassie Brilliant Blue. The amelogenin "rM179" is identical in
sequence to M180, the principal native murine amelogenin except for the
lack of the amino-terminal methionine residue (27) and a phosphorylated
serine at position 16 (7, 8). The availability of a source of a defined
and purified amelogenin protein has now permitted us to study the sugar
binding properties of this molecule at a level not previously possible for amelogenin proteins isolated from in vivo sources.
Porcine amelogenins (P173, P148) were extracted and purified following the protocol of Fincham et al. (9). "rM166," a construct
that lacks the hydrophilic carboxyl-terminal 13-residue segment, was also purified as described for the rM179 (25). To identify the sugar
binding site, we used rM179, rM166, synthetic murine amelogenin polypeptides, TRAP, LRAP (leucine-rich amelogenin polypeptide, identical to the full-length amelogenin at its two termini but lacking
the center portion of the protein (28), and ACP (amelogenin C-terminal
peptide) (Table I). In addition, we used "ATMP" (amelogenin trityrosyl motif peptide) and two altered ATMP peptides. In one of
these, the third proline is replaced by threonine (T-ATMP), and in the
other, all three tyrosine residues are replaced by phenylalanine
(F-ATMP) (Table I). All the polypeptides used in this investigation
were synthesized by the USC Microchemical Core Laboratory using an
Applied Biosystems model 430A one-column peptide synthesizer with the
modified Merrifield procedure (29). Peptides were purified by
reversed-phase HPLC (C4-214TP54 column or C18 column; Vydac/The
Separations Group, Hesperia, CA) with a shallow gradient of 40-50% B
in 60 min (buffer B was 60% v/v aqueous acetonitrile in 0.1% v/v
trifluoracetic acid (TFA) and buffer A was 0.1% TFA) at a flow rate of
1.0 or 0.5 ml/min for small peptides depending on their size.
Homogeneity of the recombinant amelogenins rM179 (20.16 kDa) and rM166
(18.4 kDa), and the synthetic polypeptides TRAP (5.20 kDa) and LRAP
(6.82 kDa), was confirmed by SDS-polyacrylamide gel electrophoresis.
The estimated molecular mass of ATMP, T-ATMP, and F-ATMP ranges from
1.45 to 1.50 kDa.
Hemagglutination (HA) Assay--
The activity of the amelogenins
was assayed by measuring their ability to agglutinate erythrocytes. HA
assays were performed in 8 × 12 microtiter plates, with
U-bottomed wells, at 30 °C by 2-fold dilution of the protein and
visual estimation of erythrocyte agglutination on microtiter plates
1 h after adding the cells (30, 31). The reagents used include
Tris-buffered saline (TBS): 50 mM Tris, 100 mM
NaCl, pH 6.3 and 7.2; and modified Alsevier's medium containing
antibiotics (AMAB): 30 mM sodium citrate, pH 6.1, 70 mM NaCl, 114 mM glucose, 100 µg/ml neomycin
sulfate, 330 µg/ml chloramphenicol, 0.04% TFA. Erythrocyte
suspension from mouse, rat, horse, bovine, swine, and sheep (purchased
from Crane Laboratories, Inc., Syracuse, NY) and human A, B, and O
blood groups (from Interstate Blood Bank, Inc., Memphis, TN) were used. The volume of cells were measured by hematocrit; suspension was in AMAB
for up to 1 week at 4 °C. In the HA assays, the cell suspension was
diluted to 1.5% in TBS, pH 7.2. All buffers and erythrocyte suspensions were warmed to 30 °C for 15 min. Purified native and recombinant amelogenins and synthetic polypeptides (1 mg/ml) were suspended in 0.04% TFA, in which they were readily soluble. No difference in HA activity of amelogenins when tested in a medium containing diluted TFA (TFA (0.04%) + TBS, pH 6.3) or TBS (pH 6.3 or
7.2) was observed. To each well containing 12.5 µl of TBS (pH 6.3),
12.50, 6.25, 3.12, 1.56, 0.78, and 0.34 µl of amelogenin was added.
The final volume was adjusted to 25 µl with 0.04% TFA. After tapping
the wells gently, 25 µl of a 1.5% suspension of erythrocytes (washed
three times in TBS, pH 7.2) was added. The microtiter plate was covered
with Parafilm "M" (American National Can, Greenwich, CT) and gently
agitated on low speed vortex for 10 s. The degree of agglutination
was scored after 1 h at 30 °C. Hemagglutination titers were
reported as the reciprocal of the highest dilution of protein giving
complete agglutination after 60 min.
Hemagglutination Inhibition Assay--
The hemagglutination
inhibition (HAI) assays were also carried out in a microtiter system
(31, 32). The following carbohydrates (purchased from Sigma) and
GlcNAc- and/or NeuAc-containing oligosaccharides (purchased from
Glycotech) and glycoproteins (from Sigma) were tested for their ability
to inhibit the agglutination of 1.5% suspension of fresh mouse
erythrocytes at a fixed concentration of rM179. D(+)-glucose,
D(+)-galactose, D(+)-glucosamine, D(+)-galactosamine, N-acetyl-D-glucosamine (GlcNAc),
N-acetyl-D-galactosamine (GalNAc), N-acetylneuraminic acid (NeuAc),
N-glycolylneuraminic acid (NeuGc), D (+) lactose,
N-acetyllactosamine (LacNAc),
3'-N-acetylneuraminyllactose, 6'-N-acetylneuraminyllactose,
6'-N-acetylneuraminyl-N-acetyllactosamine, N-acetyl-neuraminyllactoneotetrose, chitobiose,
chitotetraose, and the glycoproteins, fetuin and submaxillary mucin,
representing sialoglyoproteins and ovalbumin (a protein with terminal
GlcNAc) (33). All free sugars were diluted (1/10 in TBS, pH 6.3) in Eppendorf tubes and were warmed to 30 °C before use. To each well, 12.5 µl of sugar solutions were added. The final concentration of
sugars/well was adjusted to 1 mM, 100 µM, 10 µM, 1 µM, 100 nM, 10 nM, 1 nM, and 100 pM. To these
wells 12.5 µl of amelogenin suspension capable of giving two-well
agglutination (6 µg/well) was added and incubated for 1 h at
30OC. After incubation, 25 µl of a 1.5% suspension of
mouse erythrocytes was added to all the wells. The plates were covered
with parafilm and subjected to gentle low speed vortex for 10 s
and incubated for 1 h at 30 °C and scoring done after 2 h.
The HAI titers were reported as the reciprocal of the lowest
concentration of inhibitors giving complete HAI after 2 h.
Oligosaccharides/glycoproteins (1.25 or 2.5 µg/12.5 µl) in TBS were
added individually to microtiter wells and mixed with rM179 (12.5 µl)
previously adjusted to 8 HA units. After 60 min of incubation at
30 °C, 25 µl of 1.5% suspension of mouse erythrocytes (TBS, pH
7.2) was added to each microtiter well and mixed. The inhibition of HA
titer was determined after a 2-h incubation at 30 °C.
HA after Sialidase Treatment of Erythrocytes--
The following
sialidases were used because of their differential ability to cleave
different
-ketosidic linkages of sialic acid (
2,3;
2,6;
2,8). A reaction mixture (total 1.0 ml) containing 10% washed mouse
erythrocytes in PBS, 0.05% BSA (pH 7.0), and 140 milliunits of
sialidase from Vibrio cholerae (Sigma) (cleaves NeuAc and
NeuGc at
2,3-,
2,6-, and
2,8-ketosidic linkages) or
Arthobacter ureafaciens (Sigma) (cleaves
2,3 and
2,6)
or Clostridium perfringens (Sigma type X) (cleaves
2,3
and
2,6 with preference for
2,3) were incubated at 37 °C for
4 h (32, 34). The control erythrocytes, were treated similarly
without sialidase. Sialidase-treated cells were washed with PBS-BSA
three times and pelleted by low speed centrifugation. The removal of sialic acids from the erythrocyte cell surface was monitored by performing a hemagglutination assay with Limax flavus (slug)
lectin (EY Laboratories), specific for NeuAc and NeuGc, against the
treated erythrocytes (31, 32, 34, 35). After ensuring that >95% of
the sialic acids were removed from erythrocytes using Limax lectin, the cells were further washed in TBS once and pelleted by low
speed centrifugation. Hemagglutination assays were performed against
these desialylated erythrocytes using purified amelogenins; WGA from
Triticum vulgaris, a lectin that binds preferentially to
GlcNAc and partially to NeuAc (36, 37), and Datura
stramonium, a GlcNAc-binding lectin (Sigma) were used as control
(38, 39).
Dose-dependent Binding of [14C]GlcNAc
to rM179--
One hundred microliters of known amount of
[14C]GlcNAc (2 × 104 cpm/100 µl of
TBS, pH 7.2) (purchased from Amersham International, Buckinghamshire,
United Kingdom) was added to polypropylene Eppendorf tubes containing
100 µl of increasing amounts of purified rM179 in 0.04% TFA, and
incubated under constant agitation for 90 min at 30 °C. The proteins
were precipitated with 1 ml of cold ethanol (ethyl alcohol, 200 proof)
or 10% trichloroacetic acid (TCA) or 150 µl of 30% polyethylene
glycol (w/v) (PEG 6000; purchased from Accurate Chemical Co., Westbury,
NY). When the proteins were partitioned with PEG, the mixture was
incubated for 15 min at 4 °C to permit precipitation. The tubes were
then centrifuged for 15 min at 10,000 × g in a Beckman
Microfuge 12, and the supernatant was removed. The tip of the tube
containing the pellet was cut off and transferred to a scintillation
vial and the radioactivity was measured 15 min after adding 3 ml of
scintillation fluid (Safety-solve or Bio-safe 11, Research Products
International Corp, Mount Prospect, IL) in a
counter. The proteins
precipitated with ethanol or TCA were then centrifuged for 10 min at
10,000 × g, and the supernatant was removed. The
unbound radioactive GlcNAc, if any, was removed completely by repeated
vortex mixing and washing (three times) with ethanol or TCA. Washing
with these protein precipitants did not affect the bound sugar. The
final protein pellets were dissolved in 50 µl of 1 N
NaOH, and the bound radioactivity was measured as described above. WGA
was used as a positive control and BSA as a negative control in all the
experiments. All samples (in triplicates) were treated in the same way
and counted for bound radioactivity.
Binding of [14C]GlcNAc as a Function of Increasing
Concentration of GlcNAc--
The total binding of
[14C]GlcNAc to rM179 was determined in duplicates using
increasing concentrations of [14C]GlcNAc (20-720 pmol)
to 6 nmol of rM179. The nonspecific binding of
[14C]GlcNAc was determined in triplicate in the presence
of 10 µM of cold GlcNAc, and it was subtracted from the
total binding to obtain the specific binding. A Scatchard plot analysis
of this specific binding was carried out.
Competitive [14C]GlcNAc Binding Assay with Cold
GlcNAc and NeuAc--
50 µl of unlabeled GlcNAc or NeuAc with
increasing concentrations were prepared in duplicates in Eppendorf
tubes. To each concentration of unlabeled sugar, 100 µl of
[14C]GlcNAc (2 × 104 cpm) was added and
mixed. 100 µl containing 7.5 nmol of rM179 was added to the mixture
and incubated for 90 min at 30 °C on a rotator. After incubation,
the proteins were precipitated with ethanol and washed as described
above. WGA and BSA were used as positive and negative controls, respectively.
Relative GlcNAc Binding Efficiency of Recombinant and Synthetic
Amelogenin Polypeptides--
The binding assays were carried out in
96-Remova-well strips in a microtiter plate (Dynatech Labs. Inc.,
Chantilly, VA). rM179, rM166, and synthetic polypeptides (TRAP, LRAP,
ACP, ATMP, T-ATMP, and F-ATMP) were suspended in carbonate-bicarbonate
buffer, pH 9.6 (40). At this pH amelogenins are readily soluble (41). The polypeptides (5 nmol/100 µl) were added to wells in triplicate kept at 4 °C overnight. WGA and BSA were used as positive and negative controls, respectively. The plates were washed with TBS (pH
7.2). 100 µl of 14C-labeled GlcNAc (2 × 104 cpm) in TBS (pH 7.2) was added to each well and
incubated for 90 min at 30 °C. After incubation the unbound
radiolabeled GlcNAc was removed and washed (three times) with TBS (pH
7.2). The Remova wells were then transferred to scintillation vials.
The bound [14C]GlcNAc was released into 3 ml of
scintillation fluid (Safety-Solve, Research Products International
Corp.) by shaking the vials on a shaker for 5 min and then by vortex
mixing. The solid-matrix assay was carried out in triplicates at least
on two different occasions following the International Committee of
Harmonization of Technical Requirements guidelines.
Statistical Analysis--
Descriptive statistics were carried
out throughout. In addition, Mann-Whitney sample test and regression
analyses (linear and polynomial curve fit, r2,
and test of significance of slope) were carried out using Origins software (Microcal Software Inc.). Two-tailed p-values were
obtained to assess the level of significance.
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RESULTS |
Homogeneity of Purified Recombinant Amelogenins--
A typical
reverse phase HPLC profile of rM179 used in this study is shown in Fig.
1A. Fig. 1B shows
electrophoretic homogeneity of rM179, rM166, and the synthetic peptides
TRAP and LRAP used in this study. The purified preparations (rM166)
were devoid of or had negligible contamination with vector proteins.

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Fig. 1.
Electrophoretic homogeneity of recombinant
amelogenins (rM179 and rM166) and the synthetic polypeptides
representing TRAP and LRAP regions of amelogenin, purified by
analytical reverse phase HPLC. The recombinant murine amelogenin
rM179 was prepared by expression in E. coli and isolated and
purified by high performance liquid chromatography. The protein was
further purified by analytical reversed phase HPLC (A). The
homogeneity of rM179, rM166 and other polypeptides were assessed by
polyacrylamide gel electrophoresis using 15% acrylamide gels in the
presence of 0.1% SDS (B). The 15% gels were stained with
Coomassie Blue. The molecular masses of these proteins are as follows:
rM179, 20.16 kDa; rM166, 18.60 kDa; TRAP, 5.20 kDa; LRAP, 6.82 kDa; and
WGA, 18.5 kDa.
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Hemagglutination of Amelogenins--
Purified and homogenous
native (porcine) and recombinant amelogenins agglutinated of human A,
B, and O, sheep, rat, mouse, pig, and horse erythrocytes but not bovine
erythrocytes. The highest HA titer of native and recombinant
amelogenins (3 µg/well) was 16 with mouse erythrocytes. HA titer with
other erythrocytes ranged from 2 to 4. Since mouse erythrocytes were
agglutinated better than other erythrocytes by rM179 and native porcine
amelogenin, these erythrocytes were used for all hemagglutination
inhibition experiments. Parallel experiments with the protein extracts
of E. coli used for preparation of rM179/rM166 did not
agglutinate any of the erythrocytes.
Inhibition of Hemagglutination of Recombinant Amelogenin by Sugars,
Oligosaccharides, and Glycoproteins--
The carbohydrate-binding
specificity of purified rM179 was probed by sugar inhibition of the
hemagglutination. Table II shows that the nature of sugars that
inhibited hemagglutination of amelogenin. Of the various sugars tested,
only GlcNAc and NeuAc inhibited the hemagglutination of rM179. GlcNAc
inhibited the hemagglutination at 10 nM, whereas 100 nM NeuAc was required to cause such inhibition. Thus the
inhibitory potency of GlcNAc is 10-fold greater than that of NeuAc.
None of the other sugars (GalNAc, NeuGc, and LacNAc) tested inhibited
hemagglutination, even at concentrations as high as 100 µM. Results presented in Table III confirm that
amelogenin recognizes GlcNAc in chitobiose, chitotetraose,
and ovalbumin.
In striking contrast to free NeuAc, sialyloligosaccharides with varying
-ketosidic linkages failed to inhibit the HA titer of amelogenin,
suggesting that the part of NeuAc recognized by the amelogenin is not
accessible when NeuAc is linked to other sugars. The above contention
is also supported by HA titers observed with sialidase-treated
erythrocytes. Table IV shows that HA titers of rM179 and TRAP molecules
are unaffected by sialidase treatment, while hemagglutination of
desialylated erythrocytes was abolished by Limax lectin. HA
titers of both WGA and D. stramonium lectins also are
unaffected by sialidase treatment, suggesting that hemagglutination of
these lectins as well as rM179 and TRAP molecules is due to GlcNAc
residues but not due to NeuAc residues.
Direct Binding of [14C]GlcNAc to Amelogenin--
The
binding of [14C]GlcNAc to amelogenin increased with the
concentration of the protein in a dose-dependent manner
(Fig. 2). The recovery of bound GlcNAc
was higher after PEG precipitation. However, ethanolic precipitates
showed a consistent polynomial fit as evidenced by significant slope
(p < 0.001) and r2 (0.83). The
positive controls, WGA, a GlcNAc/NeuAc-binding lectin showed similar
dose-dependent binding. However, dose-dependent binding of rM179 with PEG paralleled with that of WGA. Furthermore, there is no significant difference in the slope between WGA and rM179
in ethanol. The binding was minimal and insignificant with the negative
control (BSA).

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Fig. 2.
Dosimetry of [14C]GlcNAc
binding with the purified recombinant amelogenin M179. To
polypropylene Eppendorf tubes containing 100 µl of GlcNAc (2 × 104 cpm or 300 pmol), 100 µl of increasing amounts of
protein (circle, rM179; solid square,
WGA, positive control; open square, BSA, negative
control) was added and incubated for 90 min at 30 °C. After
precipitation or filtration with EtOH/TCA/PEG and washing, the bound
radioactivity was assessed. The mean values of triplicate analyses for
each concentration were plotted on a logarithmic scale to evaluate
dosimetric binding of [14C]GlcNAc and assessed for
polynomial regression. The polynomial curve fit (slope) is restricted
to the ethanolic precipitate of rM179. r2 and
significance of the slope are indicated. The mean values of triplicate
analysis at each concentration of other preparations suggest a similar
trend (curve fit not shown).
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Specific Binding of [14C]GlcNAc to
Amelogenin--
Fig. 3 shows the
specific binding of [14C]GlcNAc to amelogenin as a
function of increasing concentration GlcNAc. The nonspecific binding
was measured with cold GlcNAc and subtracted to obtain specific GlcNAc
binding. A Scatchard plot of the binding of
[14C] GlcNAc to rM179 indicates that the GlcNAc binding
site is homogeneous with respect to the association constant.

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Fig. 3.
Specific binding of [14C]GlcNAc
to rM179 and Scatchard plot. Specific and nonspecific
binding of [14C]GlcNAc to purified amelogenin (6 nmol)
as a function of increasing concentration of GlcNAc. The
saturation binding of [14C]GlcNAc to rM179 was determined
using increasing concentrations of GlcNAc (20-720 pmol). The
nonspecific binding was determined in the presence of 10 µM of cold GlcNAc, and it was subtracted from the total
binding to obtain specific binding. The Scatchard analysis of this
specific binding is shown as an inset. Each point represents
the average of triplicate determinations; Regression
analysis showed that the r2 is
0.988.
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Competitive Binding of Radiolabeled and Unlabeled GlcNAc and NeuAc
to Amelogenin--
Competitive binding studies confirmed the HAI
observations made earlier. The unlabeled GlcNAc dosimetrically
inhibited binding of [14C]GlcNAc to rM179 (Fig.
4A). The inhibition slope is
significant (p < 0.001), and r2
is 0.998. Similarly unlabeled NeuAc also inhibited dosimetrically the
binding of [14C]GlcNAc to rM179 (Fig. 4B) and
resulted in an excellent fit (r2 = 0.999).
Comparison of the slopes of unlabeled GlcNAc and NeuAc revealed
somewhat similar binding affinity of the two sugars to amelogenins. The
positive control WGA was used as reference. The unlabeled GlcNAc also
inhibited dosimetrically the binding of [14C]GlcNAc to
WGA (data not presented).

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Fig. 4.
Competitive binding of radiolabeled
N-acetylglucosamine. A mixture of 100 µl of
14C-labeled GlcNAc (2 × 104 cpm) and 50 µl of varying concentrations of unlabeled GlcNAc (A) or
NeuAc (B) was added to known amounts of amelogenin (7.5 nmol) in Eppendorf tubes. The reaction mixture was incubated for 90 min
at 30 °C on a shaker. After precipitating the protein with ethanol,
unbound GlcNAc was removed and the radioactivity was measured. Each
value represents the mean of duplicates at each concentration. WGA and
BSA were used as positive and negative control. The cpm of bound
radiolabeled GlcNAc in the absence of cold GlcNAc (A)/NeuAc
(B) is 1335 (*).
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Identification of the Sugar Binding Motif in Amelogenin--
In
order to identify the domain of the polypeptide that may recognize
GlcNAc, we have used different fragments of amelogenins to assess their
hemagglutination. The structural domains of amelogenin and their
synthetic counterparts used for analysis are presented in Table
I. The full-length rM179 agglutinated
mouse erythrocytes efficiently (Table V). The recombinant amelogenin
rM166, lacking the 13 C-terminal hydrophilic residues (ACP), also
agglutinated equally, suggesting that the ACP may not be required for
binding. The ACP alone failed to cause hemagglutination, confirming
that this region is not a necessary requisite for binding. The
NH2-terminal TRAP region of 45 amino acids also
agglutinated effectively (Table V). LRAP shares the
NH2-terminal amino acid sequence found in TRAP in addition
to 26 amino acids constituting the carboxyl-terminal end. Despite
sharing 33 NH2-terminal amino acids of TRAP, LRAP failed to
agglutinate the erythrocytes. Since LRAP is devoid of the tyrosine-rich
portion of the TRAP domain, we suspected that the ATMP consisting of 13 amino acids might be necessary for hemagglutination. The 13 amino acids
constituting ATMP are PYPSYGYEPMGGW. Using radiolabeled GlcNAc, we have
further confirmed the role of ATMP in binding to GlcNAc (Fig.
5). The binding of
[14C]GlcNAc to both TRAP and ATMP are significantly
higher than that bound to LRAP. The binding of
[14C]GlcNAc to the ATMP was not significantly different
from the binding of [14C]GlcNAc to TRAP. However,
substitution of the third proline by threonine (T-ATMP) and
substitution of all three tyrosine residues by phenylalanine (F-ATMP)
resulted in complete loss of binding of the peptide to GlcNAc (Fig.
5).
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Table I
Amino acid sequences of recombinant amelogenins (rM179 and rM166) and
synthetic polypeptides used in hemagglutination and binding studies
The sequence in bold is amelogenin tyrosyl motif.
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Fig. 5.
Relative GlcNAc-binding efficiency of
recombinant and synthetic amelogenin polypeptides. Remova-well
plates were coated in triplicate with 100 µl of 5 nmol of
protein/peptide in carbonate buffer (pH 9.6) overnight at 4 °C.
Unbound proteins were removed, and the plates were washed with TBS/PBS
(pH 7.2). Radiolabeled GlcNAc (100 µl, 2 × 104) was
added and incubated for 90 min at 30 °C. The unbound GlcNAc was
removed by washing the plates (three times) with the same buffer, and
the radioactivity was measured. WGA and BSA were used as positive and
negative controls, respectively. The background values (uncoated wells)
are also presented in the figure. Standard deviation and the
sample size for each test material are indicated in the figure.
Two-tailed p-values after non-parametric analyses are
indicated.
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DISCUSSION |
GlcNAc Specificity of Amelogenin--
Amelogenin, the major
extracellular protein constituting the developing enamel matrix,
possesses lectin-like activity. Both native and recombinant amelogenins
bind to acetyl esters of GlcNAc and NeuAc but not to that of GalNAc.
Amelogenin discriminates N-acetyl residue (NeuAc) from
N-glycolyl residue (NeuGc) of sialic acids. These
observations show that the specific sugar binding requires acetyl
esters and an additional group common to GlcNAc and NeuAc but not
GalNAc. The GlcNAc affinity of amelogenin was further confirmed by the
dose-dependent binding of amelogenin with
[14C]GlcNAc, specific binding in relation to varying
concentrations of GlcNAc, Scatchard plot analysis, and the competitive
inhibition studies with cold GlcNAc. The minimal GlcNAc affinity
constant of amelogenin (10 nM), is higher than that of
other lectins, which could be due to the self-assembly properties of
amelogenin (see below).
The ability of NeuAc to inhibit rM179 binding to
[14C]GlcNAc suggests that both these sugars may
compete for a single binding site. Yet another protein with identical
dual affinity is WGA (36, 37). The interaction of WGA with GlcNAc and
NeuAc is attributed to their structural similarities, namely the
superimposable conformation of amino and hydroxyl groups at C-5 and C-4
of the pyranose of NeuAc with C-2 and C-3 of GlcNAc (37, 42). The same
superimposable conformation of NeuAc and GlcNAc may be responsible for
recognition of NeuAc by amelogenin. It is interesting to note that only
free NeuAc, and not sialylated oligosaccharides (Table II) or sialoglycoproteins (Table
III), inhibited the hemagglutination of amelogenin. Furthermore, amelogenin agglutinated desialylated erythrocytes (Table IV). Probably, the
amino and hydroxyl groups required for amelogenin-sugar interaction are
not accessible when sialic acid is in a bound state. However,
amelogenin is able to recognize chitobiose and chitotetraose, as well
as ovalbumin, in which GlcNAc also occurs as a terminal sugar (33)
suggesting that C-2 and C-3 of the bound-GlcNAc is accessible to
amelogenin. In this respect, amelogenin resembles a GlcNAc-specific
lectin from the ascidian Didemnum ternatanum (43). The
reports that GlcNAc is present in enamelins and in other EMGs (44, 45) support our hypothesis that amelogenins may bind to these
GlcNAc-containing glycoproteins. Based on these observations, we are
undertaking a detailed immuno-and lectin histochemistry study of the
DEJ-related proteins (enamelins, tuftelins, and ameloblastins) to
identify and characterize the presence of such GlcNAc-containing
glycoconjugates.
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Table II
Inhibition of hemagglutination of rM179 by sugars
Purified sugars (12.5 µl) serially diluted in TBS, pH 6.3, were added
to microtiter wells and mixed with rM179 (12.5 µl) previously
adjusted to 8 HA units; after 60 min of incubation at 30 °C, 25 µl
of 1.5% suspension of mouse erythrocytes (TBS, pH 7.2) were added to
each microtiter well and mixed. The HA titer was determined after a 2-h
incubation at 30 °C. The relative inhibitory potency of each sugar
is indicated.
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Table III
Inhibition of hemagglutination of rM179 by GlcNAc-containing
oligosaccharides and glycoproteins
Oligosaccharides/glycoproteins (1.25 or 2.5 µg/12.5 µl) in TBS,
were added individually to microtiter wells and mixed with rM179 (12.5 µl) previously adjusted to 8 HA units; after 60 min of incubation at
30 °C, 25 µl of 1.5% suspension of mouse erythrocytes (TBS, pH
7.2) were added to each microtiter well and mixed. The inhibition of HA
titer was determined after a 2-h incubation at 30 °C. +,
inhibition; , no inhibition.
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Table IV
HA titers of recombinant amelogenin (rM179) and TRAP molecule before
and after sialidase treatments
Details of the treatments are described under "Experimental
Procedures." A reaction mixture (total 1.0 ml) containing 10% washed
mouse erythrocytes in PBS, 0.05% BSA (pH 7.0) and 140 milliunits of
sialidase of V. cholerae or C. perfringens or
A. ureafaciens were incubated at 37 °C for 4 h. The
control erythrocytes were treated similarly without sialidase
(Buffer-treated). Sialidase-treated cells were washed with PBS-BSA
three times and pelleted by low speed centrifugation. The concentration
of lectins or amelogenin or TRAP added to the first well of the titer
plate is 5 µg/25 µl. This amount was serially diluted for testing
HA titers.
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The ATMP Motif of TRAP Binds to GlcNAc--
The GlcNAc-binding
domain of amelogenins rM179 and rM166 is localized in the 45-amino acid
residue TRAP sequence, but not in the LRAP sequence (see Table
V and Fig. 5). Although LRAP shares the
NH2-terminal 33 amino acid residues with TRAP (Table I), it
failed to agglutinate the mouse erythrocytes or bind to [14C]GlcNAc. The same is true for the ACP, which is also
present in LRAP. Since [14C]GlcNAc bound to the 45-mer
TRAP molecule but not to the 33-residue region shared by TRAP and LRAP,
we inferred that the binding motif was located in the 13 amino acid
residues of ATMP domain of TRAP. This ATMP consists of 3 tyrosyl
residues, spaced as
XYXXYXYX in the sequence
PYPSYGYEPMGGW. Indeed, the synthetic ATMP bound to [14C]GlcNAc as efficiently as the complete TRAP molecule
(Fig. 5). Replacing the tyrosyl residues with phenylalanine (F-ATMP)
resulted in complete loss of GlcNAc affinity. Furthermore, and of
biological significance, we found that a mutation in the ATMP sequence
(substitution of proline-3 with threonine, as in T-ATMP), as has been
found in some cases of human X-linked amelogenesis
imperfecta (46), also resulted in failure of the peptide to
bind to [14C]GlcNAc. Dehydroxylation of tyrosyl residues
and removal of proline-3 abolishes GlcNAc affinity, thereby confirming
the importance of the tyrosyl motif in GlcNAc binding.
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Table V
HA titers of native, recombinant amelogenins and synthetic polypeptides
representing different regions of the amelogenin
HA assays were performed in a microtiter system at 30 °C. All
buffers and erythrocyte suspensions were warmed to 30 °C for 15 min.
Purified rM179 and other polypeptides (1 mg/ml) were suspended in
0.04% TFA. To each well containing 12.5 µl of TBS (pH 6.3), the
protein concentration was adjusted 12.5, 6.25, 3.12, 1.6, 0.8, and 0.4 µg/well. The final volume was adjusted to 25 µl with 0.04% TFA. 25 µl of 1.5% suspension of washed erythrocytes was added. The final pH
of the suspension is 6.3. The agglutination was scored after 1 h
at 30 °C.
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GlcNAc-binding Tyrosyl Motifs in Other Lectins--
The
GlcNAc-binding domains of WGA have already been defined (36). The WGA
has two different GlcNAc-binding sites, the secondary GlcNAc-binding
site of which shows a striking similarity with the tyrosyl motif of
TRAP.
There is almost 50% similarity in the sequence of the GlcNAc
binding motif of WGA and amelogenin. In WGA intrapeptide tyrosyl residues are also implicated in the sugar binding (36). The GlcNAc
binding motif of amelogenins also showed some similarity (Ser-Tyr
residues) to the GlcNAc binding motif of another lectin, UEA-II (47).
Most importantly, both these motifs carried tyrosine, proline, and glycine.
Lectin-like Property of Amelogenin--
In contrast to the
multiple sugar-binding sites of the lectins (WGA, UEA-II), the
amelogenin polypeptide has a single binding site, but its ability to
bind to sugar or agglutinate erythrocytes is postulated to be dependent
on the self-assembly properties of the amelogenins (48, 49). The number
of GlcNAc-binding sites of an amelogenin assembly may vary with the
size of the nanosphere and the microenvironment in which sugar-protein
interactions occur. There appears to be only one trityrosyl site per
molecule of amelogenin. The site is restricted to the C-terminal region of the TRAP sequence. Our studies confirm that this ATMP is the GlcNAc
binding domain. We postulate that the lectin-like activity of
amelogenins, namely hemagglutination of erythrocytes, results from
amelogenin self-assembly creating multivalent binding sites. Indeed,
works of Fincham et al. (48, 49) document these properties of amelogenins.
GlcNAc Is the Ligand for Amelogenin at the DEJ--
The molecular
microenvironment at the DEJ may contain a wide array of glycosylated
proteins secreted by ameloblasts or odontoblasts, although amelogenins
per se are not known to be glycosylated (7, 8, 50). The
total hexosamine content of enamel and of dentine-cementum has been
determined to be 1.2 and 3.0 µmol/g, respectively (51), while GlcNAc
has been specifically identified in protein fractions of enamelin (44,
45). During ontogeny of enamel, GlcNAc-bearing proteins are secreted
earlier than amelogenin (52, 53). Since the DEJ, during the
presecretory phase, contains proteins with GlcNAc-containing
oligosaccharides, the newly secreted amelogenin may be anchored to such
glycoproteins. While the amelogenins constitute about 90% of the total
mass of enamel matrix protein, their molecular mass (~20 kDa) is
smaller compared with the EMGs with masses up to 70 kDa. Recent studies
of the "enamelin" protein (54) have demonstrated both O-
and N-linked glycosylation sites to be present in this
matrix protein (54-56). We suggest that the GlcNAc residues of the
EMGs may constitute the ligand for amelogenin, anchoring the molecules
at the DEJ through the TRAP ATMP motif.
In this context, the observations of Fincham et al. (57, 58)
are striking in that they observed that the amino-terminal fragment of
amelogenin, namely TRAP, is the principal amelogenin that persists past
early enamel maturation stage. If the EMGs are implicated in the
biomineralization process, then the role of the tyrosyl motifs of the
TRAP region cannot be ignored because they may serve to mask sugar
residues, which could be inhibitory for nucleation of calcium
hydroxyapatite crystals (22-24). Indeed, Doi et al. (59)
demonstrated that the seeded growth of enamel apatite crystals in
vitro was inhibited by bovine "enamelin" preparations.
Significance of Amelogenin-GlcNAc Interaction in Enamel
Biomineralization--
It is now well established that, in
vivo, the amelogenins self-assemble to form supramolecular
structures (nanospheres), which comprise the bulk of the
secretory-stage enamel matrix (48, 49). It is presently unknown how
these (~20 nm diameter) structures become organized to form the
three-dimensional matrix. The specific lectin-like interactions with
glycosylated matrix proteins such as the enamelins or ameloblastins may
be postulated to mediate this process.
If the function of the tyrosyl motif of amelogenin is to bind to a
saccharide ligand at the DEJ, then one can reasonably expect that
protein-carbohydrate interaction of amelogenin and EMGs in the
formation of nanospheres associated with the developing apatite crystals. We envisage that the amelogenin-GlcNAc interaction may be
similar to arthropodin-chitin (a polymer of GlcNAc) interaction observed in the matrix for calcification in crustacean cuticle (60, 61)
and molluscan shells (62).
The microenvironment of amelogenin in the developing enamel includes
proteinases (7, 16, 63, 64). Indeed, amelogenin is fragmented by these
proteinases. The fragmentation commences at the COOH-terminal end (65)
but not at the NH2-terminal end. During enamel maturation,
fragmentation appears to extend to the ATMP region of TRAP. However,
the GlcNAc-bound TRAP region may be resistant to proteinase activity.
The isolated TRAP is 45 amino acid residues in length, resulting from a
cleavage between Trp-45 and Leu-46 of the parent amelogenin (10, 66).
However, a 43-mer TRAP also exists suggesting that a cleavage may also
occur between Gly-43 and Gly-44 (67). The formation of the 45-mer and
43-mer TRAPs may possibly depend on whether the protein is bound to
GlcNAc or not, since the COOH-terminal -MGGW may constitute the binding pocket of GlcNAc very similar to that found in WGA.
A number of investigators have observed ameloblast-derived proteins
along odontoblast cell surfaces (52, 68), and it is also known that the
odontoblast cell processes (tubular processes in the dentine) extend to
the dentine-enamel interface (69-71). These observations suggest that
amelogenins may also have interactions either with the extended
odontoblast cell surface or with the product released from dentine
tubular process at the dentine-enamel interface. Although the
functional significance of such interactions is far from clear, it may
be envisaged that amelogenins can function as cell adhesion proteins
very similar to the E-, L-, and P-selectins (72-73) in binding to
carbohydrate residues on the surface of odontoblasts. The specific
oligosaccharide ligands expressed on odontoblasts and in EMGs deserve a
more detailed investigation.
In conclusion, this study has shown a novel lectin-like function for
the amelogenins, the principal proteins secreted by the ameloblasts of
the developing tooth. The specificity of amelogenin for GlcNAc is
significant from the point of view of the probable location of such
sugars at the DEJ or within the matrix and the terminal domain of
mammalian cell surface oligosaccharide-receptors. The GlcNAc-binding
tyrosyl motif of amelogenin shares homology with WGA. Ser-Tyr motifs
appear to be a common characteristic feature of GlcNAc-binding lectins.
The ATMP, a highly conserved domain of amelogenin structure, has a
potential functional role in enamel protein-carbohydrate interactions.
A point mutation in the tyrosyl motif of amelogenin, as observed in
human X-linked amelogenesis imperfecta, results in the loss of GlcNAc
affinity of amelogenin. The significance of such amelogenin-GlcNAc
interactions in enamel biomineralization is presently unknown and
deserves further study.