From the Department of Basic Sciences,
The University of Texas-Houston Health Science Center, Dental Branch,
Houston, Texas 77030 and ¶ Department of Immunology and
Protein Chemistry Core Laboratory, Baylor College of Medicine,
Houston, Texas 77030
Received for publication, July 14, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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Two acidic proteins, dentin sialoprotein (DSP)
and dentin phosphoprotein (DPP), are present in the extracellular
matrix of dentin but not in bone. These two proteins are expressed in
odontoblasts and preameloblasts as a single cDNA transcript coding
a large precursor protein termed dentin sialophosphoprotein (DSPP).
DSPP is specifically cleaved into two unique proteins, DSP and DPP. However, the cleavage site(s) of DSPP and the mechanisms for regulating the cleavages are unknown. To identify the specific site(s) of DSPP
that are cleaved when the initial translation product is converted to
DSP and DPP, we performed a detailed analysis (Edman degradation and
mass spectrometry) on selected tryptic peptides of a size originating
from the COOH-terminal region of rat DSP. After cleavage with trypsin,
the DSP fragments were separated by a two-dimensional method
(size-exclusion chromatography followed by reversed phase high
performance liquid chromatography). We characterized 13 peptides from
various regions of DSP. The analyses showed that peptide
Ile409-Tyr421 was the major COOH-terminal
fragment, ending at Tyr421 only 9 residues from the
NH2 terminus of DPP. Peptide
Gln385-His406 represented a second, minor
COOH-terminal peptide that terminated at His406. Both of
these residues are well beyond the COOH terminus predicted previously
by two independent studies estimating that rat DSP contained 360-370
amino acids. Careful studies on two peptides showed that, among 9 potential casein kinase II phosphorylation sites, 2 serines were
phosphorylated. We found that rat DSP was heterogeneous with respect to
phosphorylation, because this same peptide sequence eluted in two
discrete peaks, one with 2 phosphoserines and the other having 1. The
finding that 3 lysines just preceding the COOH termini were modified by
a 43-Da substituent (possibly a carbamoyl substituent) suggests that
the lysines in this region were particularly susceptible to attachment
of this substituent.
The dentin extracellular matrix is formed by highly specialized,
postmitotic cells termed odontoblasts. These cells secrete a unique set
of gene products similar to those expressed by osteoblasts in the
formation of bone. The noncollagenous proteins include osteonectin,
osteocalcin, osteopontin (OPN),
1 bone sialoprotein, and dentin
matrix protein 1 (Dmp-1), found in bone and dentin, and dentin
phosphoprotein (DPP) and dentin sialoprotein (DSP), occurring in dentin
but not in bone (1-5). Because these noncollagenous proteins are very
acidic and are secreted into the extracellular matrix during the
formation and mineralization of these tissues, it is generally accepted
that they play key biological roles in the formation of dentin and bone
(6); however, details concerning their precise functions are unknown.
Type I collagen is the most abundant organic constituent of dentin
extracellular matrix, forming a fibrillar lattice for mineral deposition. DPP is the second most plentiful protein, accounting for as
much as 50% of dentin noncollagenous proteins. The most unusual
feature of DPP is the occurrence of large amounts of Asp and
phosphoserine (7-10). Many of these residues are present in repeating
sequences of (Asp-phosphoserine-phosphoserine)n and (Asp-phosphoserine)n. Energy minimization modeling
techniques (8) indicate that these repeating sequences of phosphoserine and aspartic acid assume extended backbone structures with relatively long ridges of carboxylate and phosphate groups on each side of the
peptide backbone. These structures fit well with the purported function of DPP in the nucleation and modulation of hydroxyapatite crystal formation (6).
DSP, a sialic acid-rich glycoprotein first discovered in our laboratory
(11, 12), accounts for 5-8% of the dentin noncollagenous proteins.
This protein shares overall characteristics with other sialoproteins
(OPN, bone sialoprotein, and Dmp-1) from bone and dentin, but the
levels of sequence similarities are low. Nevertheless, the fact that
the genes for DSP, Dmp-1, bone sialoprotein, and OPN are found at a
similar chromosomal location (i.e. on human chromosome
4q21-4q23) suggests some type of ancestral relationship (13-16). DSP
is a glycoprotein with 29.6% carbohydrate, including 9% sialic acid
(12). The molecular mass for DSP, determined by analytical
ultracentrifugation, was 52,570 Da. When the 29.6% carbohydrate
content was taken into account, the molecular mass for the core protein
was calculated to be 37,009 Da (12). From this molecular mass and the
average residue mass, the number of amino acid residues was calculated
to be 359 (12). With Edman degradation, the NH2-terminal
sequence was shown to be IPVPQLVPL (17).
Cloning and sequence determination of a rat DSP cDNA were used to
deduce the amino acid sequence (17). With the known NH2 terminus, this analysis indicated that DSP contained 366 amino acids.
The molecular mass calculated from the predicated 366 residues, when
added to that for the carbohydrate mass, was ~53 kDa, identical to
that determined by analytical ultracentrifugation (12, 17). The
calculated amino acid composition for the predicted sequence was
identical to that determined for the protein (12, 17). Thus, the length
of DSP was determined by two independent methods, and the conclusions
in each case of ~360 amino acids were in close agreement. It was
shown later that a single-base mistake in the original rat DSP cDNA
sequencing created a frame shift leading to an early stop codon
following the coding sequence for residue 366 (10, 18). The corrected
sequence creates an open reading frame and a sequence containing a
5'-DSP sequence and a 3'-DPP sequence (see below).
MacDougall et al. (10) and Feng et al. (13)
discovered that the nucleotide sequences for mouse DSP and DPP reside
on the same gene coding for a single cDNA transcript. This
transcript would result in a translational protein product termed
dentin sialophosphoprotein (DSPP) that would be specifically cleaved into two proteins, DSP and DPP, with unique physical and chemical characteristics. Analysis of the full-length cDNA revealed a
934-amino acid open reading frame corresponding to the mouse DSPP,
including a 17-amino acid signal peptide. The signal peptide and the
deduced sequence of the NH2-terminal portion (amino acids
1-360 numbered from the NH2 terminus of the secreted
protein) of the mouse DSPP were 75% homologous to the sequence of rat
DSP.
The NH2-terminal sequence of rat DPP determined by Edman
degradation was Asp-Asp-Pro-Asn for rat HP2 (a highly phosphorylated DPP; Ref. 19), and in human it was shown to be Asp-Asp-Pro (20). The
beginning of the DPP portion of mouse DSPP (10) was established from
the rat NH2-terminal sequence Asp-Asp-Pro-Asn (see Fig. 1). Beginning with the mouse Asp-Asp-Pro sequence, mouse DPP was shown to
contain 483 amino acids. The amino acid sequence of this portion was
highly similar to that of the rat DPP cDNA reported by Ritchie and
Wang (7) and by George et al. (9). Thus, the 5' region of
this mouse DSPP clone is identical to that of rat DSP cDNA (17),
and the 3' region is the same as that of the rat DPP (7, 9). In
situ hybridization with riboprobes specifically designed for DSP,
DPP, and the "linker" (dsp-dpp) regions of the mouse DSPP
nucleotide sequence showed a strict coexpression of the three probes in
odontoblasts and preameloblasts (21). The fact that the dsp-dpp linker
sequence remains identically codistributed with DSP and DPP sequences
suggests that formation of DSP and DPP molecules takes place after
translation of a single transcript (21). Recently Gu et al.
(22) cloned a human DSPP gene with genomic organization very similar to
that of mouse DSPP. All these data support the hypothesis that DSP and
DPP represent specific cleavage products of a large precursor protein.
However, the cleavage site(s) for the precursor protein DSPP and the
mechanism for conversion to DSP and DPP are unknown. The
NH2-terminal sequences for rat (7, 9, 19) and mouse DPP
(10, 13) establish that the cleavage sites precede (i.e. are
NH2-terminal to) residue 431 in rat or 435 in mouse (see
Fig. 1). As stated earlier, in two independent studies, the rat DSP was
estimated to contain ~360 amino acids, and the cleavage site of the
DSPP precursor was predicted to be very near residue 366 (6, 12, 17,
18). On the basis of this conclusion and the site of the
NH2-terminal residue of DPP, Asp431 (in rat),
it was postulated that an intervening sequence of ~64 amino acids
between DSP and DPP was cleaved out before or after secretion of DSPP
(6).
Our primary objective in this study was to identify the specific
sites of DSPP that are cleaved when the initial translation product is
converted to DSP and DPP. DSP tryptic peptides were purified by a
two-dimensional method, size-exclusion chromatography followed by
reversed phase high performance liquid chromatography (HPLC). With the
former method, we selected peptides that were 10-25 residues in
length, a size expected of most of the tryptic peptides of rat DSPP
between residues 366 and 431 (see Fig. 1). Fortuitously, two of these
peptides were COOH-terminal ends, as indicated by COOH-terminal amino
acids other than lysine or arginine. These two tryptic peptides,
originating from residues Gln385-His406 and
Ile409-Tyr421 (as shown by cDNA deduced
sequences), are those representing minor and major COOH-terminal ends,
respectively. We also performed a detailed analysis on other tryptic
peptides. In addition to sequences from the NH2-terminal
and central regions of DSP, our investigation led to a complete
sequence analysis of the COOH-terminal region beyond residue 366 and to
the identification of two phosphoserines.
Isolation of DSP--
DSP was isolated from rat dentin by
standard procedures as described (11, 12). Briefly, rat incisor pieces
were extracted with 0.5 M EDTA in 4 M
guanidium-HCl (GdmCl; Acros Organics) containing protease inhibitors.
Next, EDTA extracts were subjected to gel chromatography on Sephacryl
S-200 in GdmCl. Using this procedure, the high molecular weight protein
fraction (ES1) was separated from osteocalcin, which eluted in an
included volume (ES2). ES1 was next chromatographed on
diethylaminoethyl-Sephacel, eluted with a linear gradient formed from
750 ml of each starting buffer (50 mM Tris-HCl, 6 M urea, pH 7.2) and 50 mM Tris-HCl, 6 M urea, pH 7.2, containing 0.7 M NaCl. DSP
eluted in a position in the gradient corresponding to ~0.2
M NaCl; this peak, referred to as fraction B (11, 12), was
further purified by gel filtration on a Biogel A1.5m in 4 M
GdmCl, Tris-HCl, pH 7.2. DSP eluted in one major peak, separated from
lower molecular weight proteins (see Fig. 1 in Ref. 12). The purity of
DSP was assessed by 5-15% SDS-polyacrylamide gel electrophoresis. The
samples of DSP displayed one major protein band at about
Mr 95,000. In addition, two minor bands at
Mr 75,000 and 65,000 were often observed (see
Ref. 12). Each of these minor DSP bands reacted with DSP antibodies on
Western immunoblots (data not shown), and we believe that they
represent fragments of DSP; their presence did not interfere with the
studies reported here. Note that the presence of either GdmCl or urea in each step of this preparative procedure prevented artifactual degradation of DSP.
Trypsin Digestion--
DSP was digested overnight at 37 °C
with trypsin (Roche Molecular Biochemicals) at an enzyme:substrate
ratio of 1:50 in 50 mM Tris-HCl, pH 8.0. The final
concentration of trypsin was 20 µg/ml. Before the addition of
trypsin, the chymotrypsin inhibitor L-1-tosylamido-2-phenylethyl chloromethyl ketone (Roche
Molecular Biochemicals) was added to a final concentration of 100 µg/ml to inhibit possible chymotrypsin activity.
Peptide Purification--
After trypsin digestion of DSP, HPLC
separation of peptides gave rise to a large number of overlapping
peptide peaks that would be less useful in our structure determination.
Therefore, peptides were separated using a two-dimensional approach to
produce a series of pure peptides that could be unambiguously
characterized. Another objective was to obtain peptides with sizes that
were likely to emanate from the COOH-terminal region of DSP. Inspection of the sequence (Fig. 1) showed that trypsin cleavage at lysyl and
arginyl bonds would result in several peptides of 11 to 22 amino acids
from that area. This approach did not ensure that a COOH-terminal
peptide would be in this size range. In the first phase, peptides were
separated according to size on Superdex 75 HR 10/30 (Amersham Pharmacia
Biotech) equilibrated and eluted at 0.5 ml/min in 4 M GdmCl
(SigmaUltra, pH 6.0). Using fast protein liquid chromatography, the
eluant was monitored at 280 nm. DSP peptides separated into six peaks,
and each peak was subdivided into three or four subfractions.
Subfractions containing peptides with desired sizes were selected for a
second phase HPLC separation. In the second phase, each selected
subfraction was subjected to HPLC with a 2.1 × 250-mm C18
reversed phase column (Vydac). For optimal separations, two separate
HPLC gradient systems were used, depending on the types of peptides to
be purified. The first approach used a 3-35% acetonitrile linear
gradient in 0.1% trifluoroacetic acid over 100 min at a flow rate of
300 µl/min; this gradient was used to separate most of the peptides.
The second elution approach was to initially wash with water (0%
acetonitrile) for 30 min before starting the 0-30% acetonitrile
gradient (0.1% trifluoroacetic acid, 300 µl/min over 100 min). This
latter gradient system was used to separate hydrophilic peptides
because of difficulties in achieving purity; using the first approach,
these peptides emerged from HPLC in short times and were of low purity.
For all HPLC separations, the eluant was monitored at 218 nm, and peaks were collected by hand.
Sequence Analysis and Determination of Mass--
Most purified
peptides of desired sizes from HPLC were first sequenced by Edman
degradation on Applied Biosystems ABI 473A and 477A sequencers using
standard techniques. Next, they were further analyzed for molecular
mass and sequence by mass spectrometry. For mass spectrometry, the
samples were dried under vacuum and redissolved in aqueous solution
containing ~50% methanol and 1% formic acid. Aliquots of the
solutions were deposited in metal coated glass nanoelectrospray
capillary tubes for analysis on a PE Sciex API 3000 triple quadrupole
mass spectrometer (Concord) equipped with a Protana nanoelectrospray
source (Odense). The samples were analyzed by one or more of the
following regimens. For determination of peptide molecular mass, full
scan mass spectra were recorded using Q1 as the resolving analyzer in
positive ion mode. Product ion spectra were aquired using Q1 to
transmit the precursor ion of interest to the radio
frequency-only collision cell Q2 under collisionally activated
decomposition conditions. Nitrogen was the collision gas, and collision
energies in the range of 20-50-electron volt product ions were
analyzed using Q3 to determine peptide sequence. Phosphorylated
peptides were detected by scanning Q1 for negative ion precursors of
the m/z 79.1 product ion, formed under collisionally
activated decomposition conditions in Q2 at a collision energy of 120 electron volts with Q3 as a mass filter to detect the product ion.
Rat DSPP cDNA Sequence--
Fig.
1 shows the cDNA deduced rat DSPP
sequence of the region from the NH2 terminus of secreted
DSP to the established NH2 terminus of DPP. Because a
full-length rat DSPP cDNA has never been reported, we based this
cDNA sequence on the published data from three studies. The first
two are by Ritchie et al. (17) and Ritchie and Wang (7), who
reported the cDNA sequence for rat DSP and DPP in two independent
papers. The third reference is by George et al. (9) who
published a cDNA sequence for rat Dmp3, representing the same
region as the mouse DSPP. In formulating this sequence we also referred
to the mouse DSPP sequence reported by MacDougall et al.
(10) for comparison. Amino acids are numbered from the NH2
terminus of secreted DSP, excluding the 17-amino acid signal
peptide.
Two-dimensional Separation of DSP Peptides--
Direct HPLC
analysis of peptides generated by digestion of DSP with trypsin
resulted in poor separations (results not shown) attributable to the
high degree of heterogeneity; using only this separation technique
would make sequencing of most peptides impossible. To improve the
separation, a trypsin digest of DSP was first applied to a Superdex 75 gel filtration column to separate peptides according to size. DSP
tryptic peptides were separated into six major peaks on Superdex 75 (results not shown). Because there were still too many peptides in each
major peak to obtain ideal purification and analysis, we subdivided
each peak into three or four subfractions. Totally, we obtained 22 subfractions, some of which were selected for the next dimension: HPLC
separation (Fig. 2). This two-dimensional separation technique enabled us to obtain a number of pure peptides suitable for sequencing by Edman degradation and mass
spectrometry (MS).
Structures of Peptides from DSP--
In our efforts to obtain
peptides from the COOH-terminal region of DSP, we used the predicted
cDNA sequence (Fig. 1) as a guide. We reasoned that, after
treatment of DSP with trypsin, we should obtain peptides comprising
~10-25 amino acids from residue 366 to the beginning of DPP (residue
431). To be more specific, we expected tryptic peptides of 22, 11, 13, 4, and 12 residues from the COOH-terminal region (see Fig. 1). From the
trypsin digest we selected peptides of ~10-25 amino acids by gel
filtration and purified them by HPLC. In fact, we characterized 13 peptides arising not only from the COOH terminus but also from the
NH2 terminus and the central regions of DSP (Figs. 1 and
3). These peptides, accounting for 167 amino acids, were sequenced by a combination of Edman microsequencing
and MS peptide sequencing. For confirmation of the structures of most
peptides, we compared the theoretical molecular mass with that
determined by MS. Fig. 3 shows the data for the 13 peptides, which form
the basis for our conclusions in this publication.
Identification of the COOH-terminal Peptides--
The
identification of COOH-terminal peptides, after tryptic digestion, was
made by searching for peptides with a COOH-terminal amino acid other
than lysine or arginine. Although we realized that such a peptide might
terminate at any point and might not occur in the selected size range,
we actually identified two COOH-terminal peptides, containing 13 and 22 amino acids. One of these peptides was peptide 13 (Fig. 3). This
peptide, Ile409-Tyr421 was first partially
sequenced by Edman degradation and then fully sequenced by tandem MS.
The theoretical molecular mass (1325.4 Da) agreed with that determined
by MS (1325.5 Da). The yield of peptide
Ile409-Tyr421 was high, as indicated by peak
areas and by the phenylthiohydantoin (PTH)-derivative yield
during Edman degradation. The peak area ratio between peptide
Ile409-Tyr421 and another tyrosine-containing
peptide, Gln53-Arg62 originating from the
NH2-terminal region, is 0.9 (Fig. 2, compare peptide
13 with peptide 2).
A second COOH-terminal peptide, Gln385-His406
(Fig. 3, peptide 12), was longer than expected from the
predicted sequence if all lysines were fully cleaved. Because peptide
12 contained a modification of Lys397, the lysyl bond was
uncleaved, and we obtained a fragment 9 residues longer than expected.
Edman microsequencing of peptide 12 identified 20 amino acids (Fig. 3).
Tandem MS indicated that it had 22 amino acids, ending at
His406 and not at Arg408, as we expected from
trypsin cleavage. The yield of peptide 12 was considerably less than
that of peptide 13, as indicated by peak areas and by the
PTH-derivative yield during Edman degradation. From these observations
we estimate that the ratio of peptide 13 to peptide 12 was
~3-7:1.
Search for Other COOH-terminal Peptides--
It is possible that a
portion of DSP ends at residue 430, representing only one proteolytic
cut when DSPP is processed. We have ruled out this possibility by
searching for a fragment after trypsin cleavage representing
Ile409-Gly 430 with 22 amino acids including 5 aspartic acids. Such a peptide would be hydrophilic and would elute
earlier from the C18 column than other less acidic peptides of similar sizes.
To be able to select peptides of known size, we studied the Superdex 75 elution position of different-sized DSP peptides, as well as those from
OPN,2 so that we knew the
size range of each subfraction. We observed that an individual peptide
eluted in three continuous subfractions of Superdex 75 but was
predominantly in one of them. By comparing the peaks of an unknown
peptide with the same elution time overlapped in three adjacent HPLC
runs (from three Superdex 75 subfractions), we were able to predict the
size of the peptide. For example, peptide Gly270-Arg
291 (Fig. 3, peptide 8) was eluted in three
continuous subfractions. It was moderately enriched in subfraction 13 (containing peptides with >22 amino acids), most abundant in
subfraction 14 (with peptides of 17-25 amino acids), and almost absent
in subfraction 15 (containing peptides with <22 amino acids). Because
of its elution profile, we predicted that peptide 8 contains 22-25
residues. Edman degradation and MS confirmed that peptide 8 was
Gly270-Arg 291 in which Ser 281 is
phosphorylated, making this peptide equivalent to 23 amino acids in size.
Using these approaches with respect to size and hydrophilicity, we
sequenced eight candidate peptides with a hydrophilic nature ranging in
size from 10 to 25 amino acids. However, we did not detect any peptides
that extend beyond Tyr421. Therefore, we concluded that the
major COOH-terminal amino acid is Tyr421. Additionally, we
did select for two phosphorylated fragments (Fig. 3, peptides
7 and 8) containing 22 amino acids; as predicted the hydrophilic nature led to early elution from HPLC. Sequence and MS
data confirmed the fact that they were highly negatively charged.
Posttranslational Modifications--
We found two types of
posttranslational modifications, phosphorylated serines and modified
lysines. Two phosphoserines were present in the amino acid sequence
Gly270-Arg291 (Fig. 3, peptides 7 and 8). Phosphoserines were detected by a combination of
Edman degradation and MS. Edman degradation of peptides containing
phosphorylated serines resulted in gaps or disproportionately low
yields of the PTH-Ser residues. With tandem MS, the phosphorylated
serines showed a molecular mass of 167 Da (serine plus one phosphate).
Additionally, the molecular mass of the peptides containing one or two
phosphates increased in mass by 80 or 160 Da, respectively, compared
with the nominal mass of the cDNA deduced sequence. The sequence
Gly270-Arg291 showed heterogeneity with respect
to phosphorylation. In peptide 7, both Ser275 and
Ser281 were phosphorylated, as shown by MS sequencing and
molecular mass (Fig. 3), whereas in peptide 8 only Ser281
was phophorylated. The phosphorylation of Ser281 resulted
in peptides longer than expected, presumably because the phosphate
moiety converted the adjacent Lys282-Glu283
into a resistant bond.
Totally, we identified 12 lysines, and 3 of them located in the region
directly preceding the COOH termini were modified with a substituent
showing a molecular mass of 43 Da. The modification of
Lys384 resulted in two unexpected peptides displaying the
same amino acid sequence corresponding to
Asn381-Lys397 (Fig. 3, peptides 10 and 11). In these two peptides, the
Lys384-Gln385 bond was uncleaved. In peptide
10, Lys384 was modified by the 43-Da substituent, whereas
in peptide 11, both Lys384 and Lys390 were
modified in a similar manner. The modification of Lys397 by
the 43-Da substituent resulted in another unexpected peptide, Gln385-His406, in which the
Lys397-Ser398 bond could not be cleaved by
trypsin. Note that peptides Asn381-Lys397 and
Gln385-His406 had an overlap of 13 amino acids.
After Edman degradation, the elution time for the PTH-derivative of
this substituted lysine was similar to that for a succinylated lysine;
however, tandem mass spectrometric analysis showed a mass of only 43 Da, differing from the 83-Da mass of a succinyl substituent. This mass
of 43 Da fits a carbamoyl group, a substance resulting from reaction of
the The two tooth-specific proteins, DPP and DSP, isolated from dentin
as distinct proteins with unique physical and chemical characteristics,
are considered important in dentinogenesis (1, 2, 6). DPP, extremely
rich in aspartic acid and phosphoserine, is believed to play key roles
in the nucleation of hydroxyapatite onto dentin matrix collagen and the
subsequent growth of the hydroxyapatite crystals (5, 6, 8), whereas the
function of DSP is unknown. It is now well accepted that DPP and DSP
are encoded by a single mRNA transcript and that the initial,
larger protein, DSPP, contains sequences for both DSP and DPP (10, 13,
21, 22). The occurrence of one gene transcribing a single mRNA
encoding both DSP and DPP must indicate that certain specific proteases
are required to cleave the primary translation product DSPP, giving
rise to the individual proteins (6). The identity of these proteases,
how their activities are controlled, and their localizations are
important directions for future studies on the structure and functions
of these two dentin-specific proteins. To study this process it is necessary to clearly define the COOH termini of DSP. In the present study, the first investigation on this question, we report the detailed
studies on the COOH-terminal region of rat DSP.
We have isolated and sequenced a total of 13 peptides after cleavage of
DSP with trypsin, accounting for 167 amino acids (Figs. 1 and 3). All
the sequences identified in this study are identical with, and confirm,
the cDNA deduced sequence of rat DSP, except for Asp57
in peptide 2, which is Asn in the cDNA deduced sequence.
Four peptides (Asp368-Arg 380,
Asn381-Lys397,
Gln385-His406, and
Ile409-Tyr421) originated from the COOH
terminal region (see Fig. 1). Peptide
Ile409-Tyr421, with a COOH terminus 9 amino
acids away from the established NH2 terminus of DPP
(Asp431), was fully analyzed by Edman degradation and
tandem MS (Fig. 3). The yield of peptide 13, as indicated by both peak
areas and the PTH-derivative yield during Edman degradation,
establishes this sequence as a major form in DSP. Thus, we conclude
that a majority of DSP molecules end at Tyr421. The fact
that the peak area of peptide Ile409-Tyr421 in
Fig. 2 is very close to that of the other tyrosine-containing peptide
(peptide 2, Gln53-Arg62) originating from the
NH2-terminal region further strengthens our conclusion that
Ile409-Tyr421 is a major COOH-terminal peptide.
Another COOH-terminal peptide Gln385-His406
(peptide 12), terminating 15 amino acids earlier than
Tyr421, is in a minor amount.
Assuming that the 22-amino acid peptide
Ile409-Gly430 or a peptide starting from
Ile409 and terminating between Tyr421 and
Gly430 might be present, we sequenced all of the peptides
ranging in size from 10 to 25 amino acids and having a hydrophilicity
similar to that of this putative COOH-terminal peptide. However, after sequencing eight candidates, we did not detect any peptides that extend
beyond Tyr421.
Tyrosyl peptide bonds are not trypsin cleavage sites; however, we
considered the possibility that the
Tyr421-Asp422 bond may have been cleaved by
chymotrypsin contaminating the trypsin preparation. According to the
manufacturer's data sheet, the highly pure, sequencing grade trypsin
used in the present study is free of chymotrypsin, a protease that
preferentially hydrolyzes peptide bonds at the COOH terminus of Trp,
Tyr, and Phe. Nevertheless, to avoid any problems arising from
contamination by this enzyme, we added the chymotrypsin inhibitor
L-1-tosylamido-2-phenylethyl chloromethyl ketone to our
trypsin digestions at a concentration of 100 µg/ml, five times as
high as that of trypsin (20 µg/ml). Another finding that refutes this
possibility is the presence of peptide
Gln53-Arg62 (QVHSDGGYER) in which
Tyr60 is not cleaved, suggesting again that the presence of
chymotrypsin is highly improbable. Taken together, we conclude that
peptide Ile409-Tyr421 is not an artifactual
product resulting from chymotrypsin contaminating the trypsin, but
rather that Tyr421 is one of the COOH termini of DSP.
Fig. 4 shows the amino acid sequence
alignment of the region representing the DSP COOH-terminal portion and
DPP NH2-terminal portion of rat and human DSPP. The
cDNA sequence data for human DSPP are from Gu et al.
(22). The rat DSPP sequence is based on the studies by Ritchie et
al. (17), Ritchie and Wang (7), and George et al. (9).
It is worth noting that both Tyr421 and His406
are conserved between rat and human DSPP. It is also interesting to
note that the flanking region of Tyr421 shows a high level
of conservation between rat and human.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence deduced from rat DSPP
cDNA showing the region from the NH2 terminus of
secreted DSP to the NH2 terminus of DPP. Amino
acids are numbered from the NH2 terminus of
secreted DSP. The NH2-terminal sequence of secreted DSP
identified previously by protein microsequence analysis is underlined
with a single line, whereas that of DPP is underlined with a
double line. The shaded amino acid sequences are
those identified by peptide sequencing in the present study.
P connected to a serine by a vertical bar
represents phosphoserine. A dot over lysine represents the
lysine modified by a 43-Da substituent.
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Fig. 2.
Two-dimensional separation of a tryptic
digest of rat DSP. DSP (600 µg) was digested with 12 µg of
trypsin. Peptides were first separated into 22 subfractions by a
Superdex 75 column (results not shown), and then a subfraction
containing peptides ranging in size from 7 to 15 amino acids was
separated with a reversed phase C18 column, as shown here. The gradient
conditions were 3-35% acetonitrile in 0.1% trifluoroacetic acid over
100 min at a flow rate of 300 µl/min. Peaks are named according to
Fig. 3. AU, arbitrary units.
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Fig. 3.
Sequence analysis of peptides isolated from
DSP after treatment with trypsin. a, peptides we have
sequenced, numbered (P#) starting from the
NH2-terminal region (see Fig. 1). b, peptide
sequence deduced from rat DSPP cDNA (cDNASeq; see
Fig. 1). c, theoretical molecular mass
(TMM; Da) of peptide sequence deduced from cDNA.
d, peptide sequence determined by Edman degradation
(EDSeq). e, Molecular mass determined by MS
(MSMM). f, peptide sequence determined by
MS (MSSeq). g, not determined. h, D differs from
the cDNA-deduced amino acid N in peptide 2. I, indicates absence of a PTH-derivative during Edman degradation.
j, S represents phosphoserine. k,
s indicates a low yield of PTH-Ser. l,
? indicates an unidentified PTH-derivative after Edman
degradation. m, K indicates that a
lysine is modified by a 43-Da substituent.
-amino group with cyanate in urea. At present we have no data to
directly identify the 43-Da substituent.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 4.
Amino acid sequence alignment of the region
representing the DSP COOH-terminal portion and DPP
NH2-terminal portion of rat and human DSPP.
Amino acids are numbered in the right column
starting from the NH2 terminus of the secreted DSP. The
COOH termini of rat DSP are marked with vertical arrows. The
NH2-terminal sequence determined for DPP is underlined with
a double line. The RGD sequence is underlined with a
single line. Identical residues between the two species are
indicated by double dots. Apparent deleted amino acids are
indicated by dashes.
Rat DSP has nine potential casein kinase II and four potential casein kinase I phosphoryaltion sites (17). Two phosphoserines were identified in this study. Rat DSP is heterogeneous with respect to phosphorylation. Peptide Gly270-Arg291 was eluted in two separate peaks demonstrating the same amino acid sequence but differing in the number of phosphates (Fig. 3, peptides 7 and 8). Two phosphoserines were identified in peptide 7 in which both Ser275 and Ser281 were phosphorylated, whereas in peptide 8, only Ser281 was phophorylated. Phosphorylation of Ser281 resulted in an unexpected peptide, Gly270-Arg291, that was not cleaved at the COOH terminus of Lys282 by trypsin; this resistance to cleavage is undoubtedly attributable to the juxtaposed phosphoserine. Our previous studies have revealed that rat bone OPN, another sialic acid-rich protein, is very heterogeneous with respect to phosphorylation (23). We postulate that the other two sialic acid-rich proteins abundant in the mineralized tissue, bone sialoprotein and Dmp-1, will also be heterogeneous in posttranslational modifications.
Some peptides that we purified contained lysyl bonds that were uncleaved by trypsin. Peptides 10 and 11 (Asn381-Lys397) and peptide 12 (Gln385-His406) were longer than expected because of lysine modification, which resulted in the refractory nature of the lysyl bonds. Peptide Asn381-Lys397 was also eluted in two discrete peaks (Fig. 3, peptides 10 and 11). In peptide 10, Lys384 was modified by a substituent with a mass of 43 Da, whereas in peptide 11, both Lys384 and Lys390 were modified by the 43-Da substituents. The modification of Lys384 resulted in lack of cleavage of peptide Asn381-Lys397 at Lys384 by trypsin. The third 43-Da substituent was found in peptide Gln385-His406 (Fig. 3) in which the modified Lys397 could not be cleaved by trypsin. It is worth noting that all three lysines modified in the same manner are found in a region just preceding the COOH termini. Totally, we have identified 12 lysines, but only the 3 lysines directly preceding the COOH termini are modified. If these modifications occur within the cells synthesizing DSPP (i.e. they are biological), they may play key roles in signaling for the cleavage of DSPP precursor. On the other hand, the mass of the 43-Da substituent corresponds to that of a carbamoyl moiety, a substance that results from reaction of amino groups with cyanates, formed from urea. Thus, these lysine modifications are likely to be artifacts, and their formation is probably related to the spatial structure of DSP, making the lysines in this region more accessible to modifications.
As stated earlier, DSP and DPP encoded by a single gene, DSPP, are found in dentin extracellular matrix as distinct proteins. Thus, the initial translation product must be proteolytically processed in a manner that has not been elucidated. The data presented here, along with the sequences deduced from cDNA, indicate that one of the major bonds proteolytically cleaved is Tyr421-Asp422, giving rise to the principal COOH terminus of DSP. A second major cleavage site is Gly430-Asp431, resulting in the NH2 terminus of DPP. A third minor area of proteolytic hydrolysis appears to be His406-Ser407. Although the proteinase(s) catalyzing these scissions is unknown at this time, we speculate that tissue-specific enzymes, designed to activate DSPP by cleaving it into DSP and DPP, are involved. One candidate is a tooth-specific proteinase, enamelysin (matrix metalloproteinase 20), which is expressed by odontoblasts and ameloblasts (24, 25). Experiments using in situ hybridization and immunohistochemistry show that enamelysin transcripts are expressed before the onset of mineralization in sites where DSPP and ameloblastin translation products could be immunodetected (25). Enamelysin may be involved in cleaving protein substrates, including DSPP and ameloblastin, derived from odontoblasts and young ameloblasts, converting them from inactive precursors into their biologically active forms (25). Recently, it was shown that recombinant bovine enamelysin (recombinant matrix metalloproteinase 20) cleaved tyrosine-rich bovine amelogenin peptide at a site between Trp and Leu and leucine-rich bovine amelogenin peptide between Pro and Ala (26). It was also shown that recombinant porcine enamelysin cleaves recombinant porcine amelogenin at virtually all of the possible sites that have previously been described (27, 28); the authors concluded that the substrate specificity of enamelysin is broad, and a consensus target sequence cannot be defined. Thus, enamelysin appears to have broad enough specificity to suggest that it could catalyze the proteolytic cleavages necessary to convert DSPP to DSP and DPP. It is likely that the spatial structure rather than the primary amino acid sequence determines the cleavage sites of DSPP.
We postulate that the conserved sequences around
Tyr421-Asp422 and
Gly430-Asp431 may form a structure readily
exposed and susceptible to the proteinase(s) involved in this
conversion. We envision that this area is open and accessible to the
proteinase(s) involved in this activity. Clearly, definite answers to
the questions concerning mechanisms involved in the proteolytic
processing of DSPP require a complete analysis of the three-dimensional
structures of DSPP, DSP, and DPP.
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ACKNOWLEDGEMENT |
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We thank Jan C. Brunn for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DE 05092 (to W. T. B.).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: Chunlin Qin, Department of Basic Sciences, The University of Texas-Houston Health Science Center, Dental Branch, 6516 John Freeman Ave., DBB, Rm. 4.133. Houston TX 77030. Tel.: 713-500-4583; Fax: 713-500-4568; E-mail: cqin@mail.db.uth.tmc.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M006271200
2 C. Qin, R. G. Cook, R. S. Orkiszewski, and W. T. Butler, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: OPN, osteopontin; Dmp-1, dentin matrix protein 1; DPP, dentin phosphoprotein; DSP, dentin sialoprotein; DSPP, dentin sialophosphoprotein; MS, mass spectrometry; HPLC, high performance liquid chromatography; GdmCl, guanidium-HCl; PTH, phenylthiohydantoin.
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REFERENCES |
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