(Received for publication, October 17, 1995)
From the
In the present study, we enumerate and characterize the proteins
that comprise the integral spicule matrix of the Strongylocentrotus
purpuratus embryo. Two-dimensional gel electrophoresis of
[S]methionine radiolabeled spicule matrix
proteins reveals that there are 12 strongly radiolabeled spicule matrix
proteins and approximately three dozen less strongly radiolabeled
spicule matrix proteins. The majority of the proteins have acidic
isoelectric points; however, there are several spicule matrix proteins
that have more alkaline isoelectric points. Western blotting analysis
indicates that SM50 is the spicule matrix protein with the most
alkaline isoelectric point. In addition, two distinct SM30 proteins are
identified in embryonic spicules, and they have apparent molecular
masses of approximately 43 and 46 kDa. Comparisons between embryonic
spicule matrix proteins and adult spine integral matrix proteins
suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform
of SM30. An adult 49-kDa spine matrix protein is also identified as a
possible adult isoform of SM30. Analysis of the SM30 amino acid
sequences indicates that a portion of SM30 proteins is very similar to
the carbohydrate recognition domain of C-type lectin proteins.
During the course of its development a sea urchin embryo constructs a pair of calcareous endoskeletal spicules. These spicules are rod shaped, mineralized structures which are calcitic assemblages of calcium carbonate (95%) and magnesium carbonate (5%) with an occluded proteinaceous integral matrix. The spicules are synthesized by a well characterized single tissue type, the primary mesenchyme cells. The calcite of each spicule rod is aligned along a single crystal axis, appearing as if each spicule is composed of one crystal of calcite. However, the spicule has greater flexural strength than a single crystal of calcite and it fractures as if it is made up of many microcrystals(1, 2, 3) . Persuasive biophysical evidence indicates that the proteins embedded within the mineral phase of the spicules, the integral spicule matrix proteins, cause these interesting physical characteristics(1, 4, 5, 6, 7) . It is believed that these proteins interact with specific faces of the calcite crystal when occluded within the mineral, and it is through these interactions that control of spicule growth occurs. However, the precise molecular mechanisms underlying interactions with noncollagenous integral matrix proteins that control the formation and the physical properties of mineralized tissues remain unknown. A question basic to our understanding of these mechanisms in mineralizing tissues is what is the nature of the noncollagenous integral matrix proteins that are intimately associated with the mineral portion of these tissues.
Many proteins have been identified and characterized as noncollagenous integral matrix proteins of hard tissues from various vertebrate and invertebrate organisms (most numerously from vertebrate bone and teeth). These types of proteins usually share the general properties of being soluble and acidic(8, 9) . However, it has proven difficult to ascribe to these types of proteins precise functions within the cell that synthesize them. In addition, while many noncollagenous integral matrix proteins have been identified, it has also proven difficult to determine with much certainty what particular integral matrix proteins are contained within a given mineral phase at a given stage of development. Many of these difficulties are due to the complex structures and dynamics of the mineralized tissues most widely studied, i.e. vertebrate bone and teeth.
Sea urchin spicule formation, on the other hand, is
particularly well suited to ask these sorts of basic questions. The
spicules are synthesized by a single well characterized tissue type and
they are relatively simple mineralized structures that do not have the
complex dynamics of vertebrate bones or teeth. The sea urchin embryo is
also very amenable to biochemical and molecular experimental analysis.
Much is known about the cell and developmental biology of sea urchin
embryos and particularly about the differentiation of the cell lineage
which synthesizes the spicules (for some reviews, see (10, 11, 12, 13, 14) ).
Benson et al.(15) reported that the spicule matrix
within the mineralized spicules is arranged in concentric sleeves of
irregular fibrillar proteinaceous material with some interconnections
between the layers of matrix material. The concentric layers of the
spicule matrix are also reflected in the concentric layered
architecture of the mineralized sea urchin spicules(2) . Benson et al.(16) and Venkatesan and Simpson (17) also identified 8-10 different proteins as
comprising the integral spicule matrix. In these studies,
one-dimensional SDS-PAGE ()was used to resolve the spicule
matrix proteins. In addition, both reports demonstrated that most of
these detected proteins are N-linked glycoproteins. Total
amino acid analysis revealed Strongylocentrotus purpuratus spicule matrix proteins are rich in acidic amino
acids(16) . This amino acid composition is similar to that of
other integral matrix proteins closely associated with the mineral
portion of other mineralized tissues(8, 9) .
Two
different cDNAs that encode two different spicule matrix proteins have
also been isolated from S. purpuratus cDNA expression
libraries. The first cDNA isolated encodes a protein designated SM50
which has a deduced amino acid sequence with a molecular mass of
approximately 50
kDa(18, 19, 20, 21) . The second
cDNA cloned encodes a protein designated SM30 which has a derived amino
acid sequence with a molecular mass of approximately 30
kDa(22) . The predicted chemical characteristics of the deduced
amino acid sequences of SM50 and SM30 cDNAs are somewhat different. The
cloned SM50 cDNA encodes a protein with an alkaline pI without any
consensus N-linked glycosylation site(21) . The cloned
SM30 cDNA encodes an acidic protein that contains a consensus N-linked glycosylation site(22) . Both SM50 and SM30
transcripts are expressed exclusively in the primary mesenchyme
cells(19, 22) . Sucov et al.(18) have shown that SM50 is a single copy gene in S.
purpuratus. Alternatively, there is experimental evidence that
there is a small family of SM30 protein genes. Akasaka et al.(23) presented Southern blotting analysis indicating that
there are between two and four copies of SM30 genes present in the S. purpuratus haploid genome. Akasaka et al.(23) further demonstrated that an isolated S. purpuratus genomic clone contains two different SM30 genes that are arranged
tandemly. These two SM30 genes were designated SM30- and SM30-
. Initial characterization of the genomic regulatory
regions of the SM50 gene and the SM30-
gene have
also been done(23, 24, 25, 26) .
In addition to these two genes that have been shown directly to encode two spicule matrix proteins, a recent report by Harkey et al.(27) characterizes a gene encoding a nonglycosylated 27-kDa protein, designated PM27, that is closely associated with growing sea urchin spicules. While they did not show directly that the PM27 protein is an integral spicule matrix protein, they do show PM27 expression and biochemistry are similar to what one might expect from a spicule matrix protein. In addition they show that PM27 has some sequence similarity to SM50; Harkey et al.(27) point out that portions of PM27, SM50, and the Lytechinus pictus homologue of SM50 (designated LSM34 by Livingston et al.(28) ) also have some similarity to the carbohydrate recognition domain (CRD) of a number of C-type lectin proteins.
The
present paper enumerates more accurately the complexity of the S.
purpuratus spicule matrix proteins and more fully characterizes
these proteins. These studies provide a biochemical foundation
important for the study of the noncollagenous integral matrices of
mineralized tissues. Our findings also complement the previously
mentioned biophysical studies that examined occluded matrix proteins of
sea urchin embryonic and adult mineralized structures and their roles
in regulating mineralized tissue formation and structure (1, 4, 5, 6, 7) . The
studies in the present paper reveal that there are 12 spicule matrix
proteins that radiolabel intensely with
[S]methionine and approximately three dozen
other spicule matrix proteins that are less highly radiolabeled. The
majority of the spicule matrix proteins have an acidic pI, while
several other moderately radiolabeled to less radiolabeled spicule
matrix proteins have a more alkaline pI. Polyclonal antisera that react
specifically with the proteins encoded by the previously cloned SM50
and SM30 spicule matrix cDNAs were generated. Western blotting analysis
using these antisera identify the SM50 and SM30 proteins. In addition,
comparisons are made between the embryonic spicule matrix proteins and
the adult spine integral matrix proteins. Further analysis of the
protein encoded by SM30-
reveals that a portion of the
SM30 proteins is similar to the CRD of the C-type lectin family of
proteins.
Radiolabeled S. purpuratus spicule
matrix protein was isolated from micromeres cultured in seawater with
4% horse serum and [S]methionine. The isolation
and culture of micromeres was done essentially as described by Benson et al.(30) . The micromeres isolated from about 2
10
16-cell embryos were cultured in four 100-mm
Petri plates, each containing 10 ml of seawater containing 4% horse
serum that had been dialyzed against seawater. Two hundred µCi of
[
S]methionine (1000 Ci/mmol; Amersham) were
added to each plate just prior to the onset of spiculogenesis and left
in the medium until the time of harvest (24-72 h). At the
conclusion of labeling, carrier spicules from whole embryos were added
to the cultures, and the adherent spicules of the culture were scraped
from the Petri plates. The spicules were washed with and then placed
into 3.5% sodium hypochlorite overnight at room temperature. They were
then washed with 5-7 changes of dH
O. After the final
wash, the spicules were suspended in 1 ml of dH
O. An
aliquot was removed to quantitate the amount of radioactivity
incorporated into spicule matrix. To prepare the radiolabeled spicule
matrix protein for each two-dimensional gel, 5.0
10
dpm of the radiolabeled spicule sample was trichloroacetic acid
precipitated with 10 µg of cytochrome c carrier; this
procedure dissolves the calcite and precipitates the spicule matrix
protein. The pellet was then washed with acetone to remove residual
trichloroacetic acid. The pellet was dried and dissolved in the
appropriate sample buffer.
Isolated spicule matrix proteins were labeled in vitro with biotin following the protocol described by Meier et al.(31) using the biotinylation agent NHS-CC-biotin purchased from Pierce. The labeled proteins were localized using an enhanced chemiluminescence protocol described by Nesbitt and Horton (32) with the exception that the blocking agent used was 0.1% fish gelatin (Amersham), 0.8% bovine serum albumin, 0.02% Tween 80, 10 mM Tris, pH 8.0, 100 mM NaCl, and the dilution of strepavidin-horseradish peroxidase (Amersham) used was 1:3000.
Glycosidase treatment of spicule matrix protein was carried out at 37 °C overnight using endoglycosidase F/N-glycosidase F purchased from Boehringer Mannheim following the protocol provided by the manufacturer.
Mild alkaline hydolysis
-elimination of O-linked carbohydrate moieties on spicule
matrix proteins was done essentially as described by Florman and
Wassarman(33) . Spicule matrix proteins were incubated in 5
mM NaOH for 24 h at 37 °C, neutralized with HCl, and then
analyzed by Western blotting analysis as described below. Serine and
threonine O-glycosidic linkages are known to be labile in
alkaline conditions(34) .
Figure 2:
Two-dimensional gel electrophoresis of
radiolabeled sea urchin spicule matrix proteins.
[S]Methionine radiolabeled spicule matrix
proteins were separated by two-dimensional gel electrophoresis. The
first dimension separates the proteins on the basis of their pI using
different conditions for each of the three gels. The second dimension
of all three gels is a 10% SDS-PAGE gel. A, the first
dimension of this gel was a nonequilibrium pH gradient tube gel using
ampholytes with a pH range of 2.5 to 6.5. The gel was run in the acidic
direction for 20 min before placing it on the second dimension. B, the first dimension of this gel is an isoelectric focusing
gel using ampholytes with a pH range of 2.5 to 6.5. The gel was run in
the acidic direction until the proteins came to equilibrium at their pI
(about 2 h), before placing it on the second dimension. C, the
first dimension of this gel was a nonequilibrium pH gradient gel using
ampholytes with a pH range of 3 to 10. The gel was run in the basic
direction for 20 min before placing it on the second dimension. After
all the gels were run in both dimensions, the gels were processed for
fluorography and exposed to x-ray film. I, II, and III labels indicate the three spicule matrix proteins that were used
to align the proteins that were common among the gels in A-C. NEPHGE, non-equilibrium pH gradient gel
electrophoresis.
Figure 6: Two-dimensional Western blots of spicule matrix proteins using the anti-SM30 and anti-SM50 antiserum. A, 0.5 µg of unlabeled spicule matrix protein was separated in the first dimension on an isoelectric focusing gel using ampholytes with a pH range of 2.5 to 6.5 as described under ``Experimental Procedures.'' The second dimension was a 10% SDS-PAGE gel. The proteins were subjected to Western blotting using the anti-SM30 antiserum. B, 0.5 µg of unlabeled spicule matrix protein was separated in the first dimension on a nonequilibrium pH gradient gel run in the basic direction. The pH range of ampholytes used was 3 to 10. The second dimension was a 10% SDS-PAGE gel. The proteins were subjected to Western blotting analysis using the anti-SM50 antiserum. The immunoreactive proteins in these two blots were visualized using chemiluminescence. NEPHGE, non-equilibrium pH gradient gel electrophoresis.
A rabbit polyclonal antiserum raised against all of the spicule matrix proteins was generated following the procedure described by Benson et al.(16) and using spicule matrix protein isolated from embryonic spicules as immunogen.
Western blotting of one- and two-dimensional gels was done as described by Towbin et al.(40) . Chemiluminescent detection of immunoreactive proteins was done following the directions of the manufacturer (Amersham) except that 0.1% fish gelatin (Amersham), 0.8% bovine serum albumin, 0.02% Tween 80, 10 mM Tris, pH 8.0, 100 mM NaCl was used as the blocking solution. The anti-SM30 antiserum was used at a 1:2000 dilution and the anti-SM50 antiserum was used at a 1:1000 dilution for the chemiluminesence blots.
Detection of immunoreactive proteins using alkaline phosphatase-conjugated secondary antibody and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride (BCIP/NBT) as substrate was done as described by Richardson et al.(29) . The anti-total spicule matrix antiserum was used at a 1:1000 dilution, the anti-SM30 antiserum was used at a 1:250 dilution, and the anti-SM50 antiserum was used at a 1:100 dilution for these blots.
The
microinjection of the synthesized RNA into Xenopus oocytes and
the collection of [S]methionine-labeled secreted
proteins was done as described in Livingston et al.(28) . The immunoprecipitation of
[
S]methionine-labeled proteins using the
anti-SM30 antiserum or its preimmune serum at a dilution of 1:100 was
also done as described by Livingston et al.(28) . SM30
RNA was translated by reticulocyte lysate using the Ambion Reticulocyte
Lysate kit following the protocol provided by Ambion.
We decided, alternatively, that radiolabeling the
proteins with [S]methionine was a better way to
visualize these proteins. We found culturing isolated micromeres in the
presence of [
S]methionine to be the most
effective method of labeling sea urchin spicule matrix proteins. Harkey
and Whiteley (43) demonstrated that the time course of spicule
formation in S. purpuratus micromere cultures closely matches
the time course of events in whole embryos and that the two-dimensional
gel pattern of proteins synthesized by cells from micromere cultures
closely matched that of primary mesenchyme cells in intact sea urchin
embryos.
The radioactively labeled spicule matrix proteins
synthesized in culture were isolated and then fractionated by gel
electrophoresis. A typical one-dimensional gel fractionation of these
radiolabeled proteins is displayed in Fig. 1A. This
pattern is very similar to the pattern we observed for spicule matrix
proteins isolated from whole embryo spicules that were labeled in
vitro with biotin. The pattern of biotinylated proteins is
displayed in Fig. 1B. The most noticeable differences
in the radiolabeled and biotinylated spicule matrix patterns is that a
band at approximately 27 kDa is more prevalent on the radiolabeled
protein gel and there is strongly labeled material at 200 kDa on
the biotinylated protein gel. The one-dimensional SDS-PAGE patterns
observed here are very similar to the patterns shown by one-dimensional
SDS-PAGE silver staining and radiolabeling spicule matrix preparations
of whole embryos that were observed by Benson et al.(16) and Venkatesan and Simpson(17) . These
previously published one-dimensional SDS-PAGE patterns did not have the
prominent bands at
200 kDa that is present on the biotinylated
spicule matrix SDS-PAGE pattern. Taken together these results indicate
that radiolabeling spicule matrix proteins from micromere cultures
generates a labeling pattern similar to patterns obtained with stains.
Furthermore, given its greater sensitivity and clarity, we found
radioactive labeling the spicule matrix proteins better for further
two-dimensional gel analysis.
Figure 1:
One-dimensional SDS-PAGE of
radiolabeled and biotinylated spicule matrix protein. A,
[S]methionine radiolabeled spicule matrix
proteins were separated by a 10% SDS-PAGE gel. The radiolabeled
proteins were then localized using fluorography. B,
biotinylated spicule matrix proteins were separated on a 10% SDS-PAGE
gel and then electroblotted to nitrocellulose membrane. The
biotinylated proteins were then localized on the membrane using an
enhanced chemiluminescence protocol described under ``Experimental
Procedures.''
The results of two-dimensional gel analysis of the radiolabeled spicule matrix proteins are shown in Fig. 2. In Fig. 2A, the spicule matrix proteins are separated in the first dimension on the basis of their pI using a non-equilibrium pH gradient gel in which the proteins migrate in the acidic direction for a short time (about 20 min). The pH range of the ampholytes used in this gel was between 2.5 and 6.5. In Fig. 2B, the spicule matrix proteins were again separated in the first dimension on the basis of their pI, using the same pH range of ampholytes as in Fig. 2A. However, in this gel, the proteins were allowed to migrate to their equilibrium pI. In Fig. 2C, the proteins were also separated on the basis of their pI in the first dimension. The pH range of the ampholytes used here, however, was 3 to 10, and the first dimension was run in the opposite, basic direction for only short time (about 20 min), which does not allow the proteins to migrate to their equilibrium pI. If these proteins were allowed to migrate to their equilibrium pI, the most alkaline proteins would migrate off the alkaline end of the gel.
It is apparent most of the proteins are acidic, with molecular masses ranging from about 20 to over 100 kDa. The 8-10 bands previously identified by Benson et al.(16) and Venkatesan and Simpson (17) as S. purpuratus spicule matrix proteins have apparent molecular masses similar to many of the proteins separated by the two-dimensional gels presented here. All of the previously identified bands can be accounted for by proteins fractionated on the gels presented in Fig. 2. Fig. 2, A and B, show that the majority of spicule matrix proteins, as well as all of the most highly radiolabeled proteins, are indeed acidic. Fig. 2A reveals a bit more complexity of the matrix proteins than seen in Fig. 2B, especially for the proteins migrating between apparent molecular masses of 30 and 69 kDa. This result is probably because the most heavily radiolabeled proteins on Fig. 2A do not streak out as much before they reach their isoelectric point as they do in Fig. 2B. Since most of the spicule matrix proteins are glycoproteins, one expects to see streaking on equilibrium gels(41) . This streaking presumably occurs because of the heterogeneity in the charge provided by the carbohydrate moiety. Since the first dimension of the gel in Fig. 2B has been run to equilibrium, it provides a more accurate relative comparison of the pI values of the various spicule matrix proteins.
The gel shown in Fig. 2C resolves spicule matrix proteins that have a
more alkaline pI. There are a number of spicule matrix proteins in the
more acidic portion of the gel in Fig. 2C that also
resolved in the more alkaline portion of the gels in Fig. 2, A and B. The proteins labeled I, II, and III ( Fig. 2and Fig. 3) were used as landmarks to orient the
proteins in all three of the gels. Fig. 2C demonstrates
that there are several spicule matrix proteins, with medium to low
radiolabeling, which have a somewhat more alkaline pI. The drawing in Fig. 3is a representation of the distribution of the various
spicule matrix proteins combining the results from the three kinds of
gels presented in Fig. 2. While the designation of relative
abundance is problematic with radiolabeled proteins, it is apparent
from the three gels shown in Fig. 2that there are 12 spicule
matrix proteins that are more highly radiolabeled by
[S]methionine than the rest. These proteins are
designated in Fig. 3with stars. Given that the most
highly radiolabeled spicule matrix proteins have molecular weights
coincident with the most highly staining bands of the one-dimensional
SDS-PAGE patterns of spicule matrix proteins shown in Fig. 1B and in Benson et al.(16) , and
Venkatesan and Simpson(17) , this suggests that the highly
radiolabeled spicule matrix proteins may actually be some of the more
prominent spicule matrix proteins.
Figure 3: Diagram of the S. purpuratus spicule matrix proteins. A diagram was drawn summarizing the distribution of the various spicule matrix proteins separated in gels presented in Fig. 2, A-C. Asterisk (*) indicates strongly radiolabeled spicule matrix protein; I, II, and III labels indicate the three spicule matrix proteins that were used to align the proteins that were common among the gels in Fig. 2, A-C. The identity SM30-A, SM30-B, and SM50 spicule matrix proteins are also designated. NEPHGE, non-equilibrium pH gradient gel electrophoresis.
Fig. 2, A-C, also shows approximately three dozen spicule matrix proteins that radiolabel less prominently and they are also represented in Fig. 3. We believe that these less highly radiolabeled proteins are not contaminants since we get very similar patterns on two-dimensional gels with many different batches of radiolabeled spicule matrix proteins. The only differences between batches that we see regularly are slight variations in the relative signals among the various proteins. There is other evidence that the radiolabeled proteins we have identified are not contaminants of non-spicule matrix proteins. Benson et al.(16) using scanning electron microscopy saw that bleach-treated spicule are completely free of embryonic and blastocoelic contaminants. In addition, we found that the two-dimensional pattern of radiolabeled spicule matrix proteins isolated from spicules that are extensively treated with proteinase K subsequent to washing with bleach and prior to demineralization appear identical to radiolabeled proteins isolated from untreated spicules (data not shown).
Fig. 4A is a Western blot of a one-dimensional SDS-PAGE separating untreated spicule matrix protein and endoglycosidase F/N-glycosidase F-treated spicule matrix protein. This blot was reacted with the anti-SM50 antiserum, and there is a prominent band of about 48 kDa that is detected with this antiserum. This apparent molecular mass is close to the deduced molecular mass of the mature processed protein encoded by the cloned SM50 cDNA(21) . This observation is the same result that Richardson et al.(29) reported. Western blots using the preimmune serum resulted in no staining (data not shown). It is also apparent from the results in Fig. 4A that the molecular mass does not shift after treatment with endoglycosidase F/N-glycosidase F, indicating that SM50 is not N-glycosylated. Since there is no consensus N-glycosylation site in the deduced SM50 amino acid sequence, this result is expected. There is an additional band visible, other than SM50, in each lane at about 69 kDa in Fig. 4A. This band is not present in other blots using this same antiserum with different spicule matrix preps (e.g.Fig. 6B and Fig. 7B). Also, given the apparent molecular mass of this band, we postulate that this antiserum is reacting with human keratin, a cross-reaction often seen with rabbit polyclonal antisera.
Figure 4: One-dimensional Western blots of spicule matrix proteins using the anti-SM50 and anti-SM30 antisera. A, 0.5 µg of spicule matrix protein and 0.5 µg of endoglycosidase F/N-glycosidase F-treated spicule matrix protein were separated on a 10% SDS-PAGE gel. The gel was then subjected to Western blotting and reacted with the anti-SM50 antiserum. B, 0.5 µg of spicule matrix protein, 0.5 µg of endoglycosidase F/N-glycosidase F-treated spicule matrix protein, and 0.5 µg of alkali-treated spicule matrix protein were separated on a 10% SDS-PAGE gel. The gel was then subjected to Western blotting and was reacted with the anti-SM30 antiserum. The blots in A and B both used alkaline phosphatase-conjugated secondary antibody and BCIP/NBT as substrate to visualize the immunoreactive proteins.
Figure 7: Comparison of embryonic spicule matrix proteins and adult spine integral matrix proteins. A, embryonic spicule matrix protein and adult spine matrix protein were separated on a 10% SDS-PAGE gel and subjected to Western blotting analysis using a polyclonal antiserum raised against all of the spicule matrix proteins. B, embryonic spicule matrix protein and adult spine matrix protein were separated on a 10% SDS-PAGE gel and subjected to Western blotting analysis using the anti-SM50 antiserum. C, embryonic spicule matrix protein and adult spine matrix protein were separated on a 10% SDS-PAGE gel and subjected to Western blotting analysis using the anti-SM30 antiserum. D, endoglycosidase F/N-glycosidase F-treated embryonic spicule matrix protein and adult spine matrix protein were separated on a 10% SDS-PAGE gel and subjected to Western blotting analysis using the anti-SM30 antiserum. Alkaline phosphatase-conjugated secondary antibody and BCIP/NBT as substrate were used to visualize the immunoreactive proteins in these four blots.
Fig. 4B is a one-dimensional Western blot
using the anti-SM30 antiserum. Akasaka et al.(23) reported that there are between 2 and 4 copies of SM30 genes per haploid genome and that at least two different SM30 genes, designated SM30- and SM30-
, are arranged tandemly in the genome. It was
therefore not surprising that a doublet with approximate apparent
molecular masses of 43 and 46 kDa reacts with this anti-SM30 antiserum.
It also appears that the molecular mass of each doublet member
decreases approximately 3-4 kDa after treatment with
endoglycosidase F/N-glycosidase F indicating that the two SM30
proteins are N-glycosylated. This glycosidase treatment result
is also expected since the deduced amino acid sequence of the SM30-
gene and the SM30 pNG7 cDNA each contain one
consensus N-glycosylation site(22, 23) .
Western blotting using the preimmune serum for the anti-SM30 antiserum
resulted in no immunostaining at all (data not shown).
The apparent molecular mass of these anti-SM30 reactive proteins, however, is significantly higher than was expected based on the molecular mass of 30.6 kDa from the SM30 cDNA pNG7 deduced amino acid sequence(22) . The difference in the observed apparent molecular mass and the derived molecular mass cannot be explained solely by N-glycosylation since the removal of N-linked carbohydrate moieties only shifts the molecular mass of the SM30 doublet 3-4 kDa. To determine if O-glycosylation of the SM30 proteins is occurring and contributing to the larger than expected molecular mass, we treated spicule matrix proteins under alkali condition at 37 °C overnight. These conditions should remove any O-glycosylations(33, 34) . The third lane of the Western blot in Fig. 4B shows anti-SM30 antiserum reacted with alkaline-treated spicule matrix proteins. There is no visible alteration of the apparent molecular mass of the SM30 proteins. These results indicate that these anti-SM30 reactive proteins are not O-glycosylated.
Since glycosylation is probably not
the reason for the anomalous apparent molecular weight of the SM30
proteins, we devised an experiment to help us determine whether: 1) the
SM30 proteins are migrating with a larger apparent molecular mass
because of the inherent chemistry of these proteins, 2) the SM30
proteins are being modified further by post-translational modifications
other than glycosylation, or 3) the antibody is reacting to other
larger spicule matrix proteins that share epitopes with the protein
encoded by the SM30 cDNA. To address these issues, capped RNA
transcripts were synthesized in vitro using the pNG7 cDNA as
template. The pNG7 cDNA encodes a full-length SM30
protein(22) . This SM30 RNA and
[S]methionine were then co-microinjected into Xenopus oocytes. Radiolabeled proteins secreted by SM30 RNA
injected and control oocytes were collected, and the secreted proteins
were then subjected to immunoprecipitation using the anti-SM30
antiserum. The immunoprecipitates were then separated by SDS-PAGE. The
results of this experiment are shown in Fig. 5A. From
this gel, it is apparent that a 46-kDa secreted protein is synthesized
by the oocytes injected with pNG7 SM30 mRNA. Fig. 5A also shows that the SM30 protein synthesized by the Xenopus oocyte decreases in apparent molecular mass by 3-4 kDa when
it is treated with endoglycosidase F/N-glycosidase F.
Figure 5:
In vitro translation of SM30 RNA
by Xenopus oocytes and reticulocyte lysate. SM30 RNA was
synthesized in vitro using the SM30 cDNA clone pNG7 (22) as the template. A, Xenopus oocytes were
microinjected with in vitro synthesized SM30 RNA and
[S]methionine. The supernatant of the SM30 RNA
injected oocytes which contained radiolabeled, secreted proteins were
collected 2 days later (control). A portion of this supernantant was
treated with endoglycosidase F/N-glycosidase F (glycosidase treated). The untreated and glycosidase-treated
supernatants were then subjected to immunoprecipitation using the
preimmune and anti-SM30 antisera. The resulting precipitates were
separated on a 10% SDS-PAGE gel, processed for fluorography, and
exposed to x-ray film. The anti-SM30 antiserum specifically
immunoprecipitates a broad 46-kDa band from the untreated oocyte
supernantant (indicated as untreated SM30). This band decreases in
apparent molecular mass approximately 3-4 kDa when the oocyte
supernantant is glycosidase treated (indicated as treated SM30). B, rabbit reticulocyte lysate was used to translate in
vitro SM30 RNA, control RNA (Xenopus elongation factor
1), and no added RNA in the presence of
[
S]methionine. Equal portions of the lysates
that contained the various radiolabeled protein products were then
separated on a 10% SDS-PAGE gel and processed for fluorography. The
protein product synthesized using the SM30 RNA has an approximately
32-kDa apparent molecular mass.
SM30 RNA synthesized from the pNG7 cDNA was also translated in vitro using rabbit reticulocyte lysate, which does not post-translationally modify translation products. Fig. 5B shows that the reticulocyte lysate synthesizes an approximately 32-kDa product which is the molecular masss of the deduced amino acid sequence of the pNG7 SM30 cDNA that contains its signal sequence. Given these results, it appears that one or more post-translation modifications other than N-glycosylation produces the anomalous electrophoretic migration of the SM30 proteins. Since the Xenopus oocyte seems to be modifying the pNG7 SM30 protein correctly, a convenient in vitro assay exists to determine the nature of the post-translational modifications occurring to SM30 proteins. We are presently attempting to identify the as of yet unknown post-translational modification(s) of the SM30 proteins with this system.
To further determine if the observed SM30 doublet was real or apparent, we also ran gels with spicule matrix proteins subjected to higher amounts of reducing agent and higher SDS concentrations. However, these treatments did not alter the migration or presence of the anti-SM30 reactive doublet (data not shown). Since the 43- and 46-kDa anti-SM30 reactive proteins appear to encode genuine SM30 proteins, these proteins are designated SM30-A and SM30-B, respectively.
To determine which of the spots on the two-dimensional gels of the spicule matrix proteins are actually the SM30 and SM50 proteins, Western blots of two-dimensional gels were done. Fig. 6A is a two-dimensional Western blot using the anti-SM30 antiserum. The first dimension of this gel was an equilibrium isoelectric focusing gel using ampholytes with a range of 2.5-6.5 (similar to the first dimension of the gel in Fig. 2B). From this blot it is apparent that the anti-SM30 antiserum reacts with two closely migrating spots at the acidic end of the gel with the same apparent molecular weight as the SM30-A and SM30-B proteins in Fig. 4B. When this gel is compared to a gel separating labeled spicule matrix proteins that was run in parallel, it is apparent that SM30-A and SM30-B are two of the more acidic and highly radiolabeled spicule matrix proteins. The identity of the SM30-A and SM30-B proteins are marked in Fig. 3.
Fig. 6B is a two-dimensional Western blot using the anti-SM50 antiserum. The first dimension of this blot was a non-equilibrium pH gradient gel using ampholytes with a pH range of 3-10 and the gel run in the basic direction (similar to the first dimension of the gel in Fig. 2C). From this blot it is apparent that the anti-SM50 antiserum reacts with a single protein that migrates with an apparent molecular mass of 48 kDa at the most alkaline portion of the gel. When compared to a gel separating labeled spicule matrix protein that was run in parallel, it is apparent that SM50 is the spicule matrix protein with the most alkaline isoelectric point. This observation is consistent with the pI of the deduced amino acid sequence of the SM50 cDNA being approximately 12(21) . The identity of the SM50 protein is marked in Fig. 3.
Comparison of one-dimensional Western blots of embryonic spicule matrix protein and adult spine integral matrix protein, using an antiserum raised against all of the sea urchin embryonic spicule matrix proteins, is shown in Fig. 7A. This figure reveals extensive cross-reactivity between the two tissues. Qualitatively, the most immunoreactive proteins in adult spine matrix seem to have apparent molecular masses larger than those from the spicule matrix. One exception is a band at approximately 120 kDa which has a similar sized counterpart in the spicule matrix protein. We know, however, that the embryonic spicule matrix band at 120 kDa is composed of a few different proteins of that same size in the spicule matrix (see Fig. 2and Fig. 3).
A Western blot using anti-SM50 antiserum (Fig. 7B) reveals that SM50 has the same apparent molecular mass in spine matrix as it does in spicule matrix, an observation seen by Richardson et al.(29) using a different anti-SM50 antiserum. One-dimensional blot analysis of spicule and spine matrix proteins using the anti-SM30 antiserum reveals differences between these two tissues. This result is shown in Fig. 7, C and D. While anti-SM30 reacts with spicule matrix bands with apparent molecular masses of 43 and 46 kDa, this antiserum reacts with a doublet of 46 and 49 kDa in spine matrix (see Fig. 7C). These two sets of SM30 doublets in Fig. 7C are located at exactly the same apparent molecular mass as two prominent immunoreactive doublets in the spicule and spine lanes in Fig. 7A that reacted with the anti-total spicule matrix antiserum. To determine whether the apparent size difference in the doublets that react with anti-SM30 is caused by differential glycosylation, glycosidase-treated spicule and spine matrix protein were analyzed by Western blotting. As can be seen in Fig. 7D, both spicule and spine doublets have reduced apparent molecular masses of about 3 kDa each after glycosidase treatment. However, a size difference between the SM30 doublets present in spicule and spine matrix persists. This finding suggests that the difference in apparent molecular weight is not because of differential N-glycosylation. Rather, it suggests that the 49-kDa protein is a different form of SM30 and/or it is post-translationally modified (other than N-glycosylation) differently than SM30-A or SM30-B. We designate this apparent adult isoform of SM30 as SM30-C. The results in Fig. 7, C and D, also suggest that the 43-kDa SM30-A protein is an embryonic isoform of SM30 since there is no protein of that size in the adult spine matrix lane. The 46-kDa protein, SM30-B, is apparently expressed in both spicules and adult spines. Of course, at this point, we cannot rule out the possibility that the 46-kDa proteins in the embryonic spicule matrix and the adult spine matrix are encoded by different genes. Akasaka et al.(23) have shown that there are up to four different SM30 genes. But, at this time, we have no reason to invoke that possibility.
Figure 8:
The similarity between SM30- protein
and proteins containing a C-type lectin carbohydrate recognition
domain. A region of SM30-
protein is aligned with various proteins
containing a C-type lectin carbohydrate recognition domain. Amino acids
that are boxed in black indicate identity with the
SM30-
protein. Amino acids that are boxed in gray indicate a conservative amino acid substitution. Numbers in brackets next to protein names indicate the residue
numbers of the various proteins that are aligned in the figure. Dash(-) indicates gaps that were added to help align the
sequences. Numbers at the top of the alignment refer
to amino acid residue number for the SM30-
protein (accession
number P28163). Protein names are in Swiss Prot format. LITHRAT, rat lithostathine precursor (accession number
P10758); LITHHUMAN, human lithostathine precursor
(accession number P05451); MANRHUMAN, human
macrophage mannose receptor (accession number P22897); PAP1HUMAN, human pancreatitis-associated protein 1 precursor
(accession number Q06141); LITHBOVIN, bovine
lithostathine precursor (accession number P23132); PAP1RAT, rat pancreatitis-associated protein 1 precursor
(accession number P25031); PAP1MOUSE, mouse
pancreatitis-associated protein 1 precursor (accession number P35230); TETNHUMAN, human tetranectin precursor (accession
number P05452); LECAPLEWA, Pleuradeles waltii lectin precursor (accession number Q02988); LECEANTCR, Anthocidaris crassispina echinoidin
(accession number P06027). Arrows above the SM30-
protein
sequence indicate residues that are different in the protein encoded by
pNG7 SM30 cDNA. The different amino acid residues in the pNG7 protein
are indicated on top of the arrows. Symbols for the conserved structure and amino acid residues of C-type
lectins determined by Weis et al.(44) :
= aliphatic;
= aromatic;
= aliphatic
or aromatic; Z = E or Q; B = D or N; W = side
chain containing oxygen (D, N, or Q); W, D, P, and C = single
letter symbols of amino acids; 1, first Ca
binding
site; 2, second Ca
site; S, small hydrophobic core;
L, large hydrophobic core.
When SM30- was initially cloned and characterized, it was
noted there were nine amino acid differences between the protein
encoded by it and the protein encoded by the SM30 pNG7
cDNA(23) . It was unclear if these differences reflected the
well know polymorphisms of sea urchin genomic sequences or if it
reflected that SM30-
and pNG7 encode different SM30
proteins. It is interesting to note that seven of the nine amino acid
differences between the SM30-
protein and the pNG7 SM30 protein
occur over the approximately 40% portion of the SM30 proteins that is
similar to the CRD of C-type lectins. Arrows in Fig. 8indicate where these differences occur. Most of these
differences alter residues shown in the present study to be conserved
between SM30-
protein and C-type lectins or in residues shown by
Weis et al.(44) to be conserved among C-type lectins.
While it is premature to conclude too much from these differences,
these findings suggest that SM30-
and pNG7 SM30 may be different
forms of the SM30 protein.
Acidic integral matrix proteins isolated from calcitic adult sea urchin exoskeletal tissues have been shown to bind to specific faces of calcite crystal in vitro(4) . It has also been shown that the intercalation of the matrix proteins within the calcite is responsible for some of the physical properties of adult sea urchin skeletal elements(5, 6) . However, these studies did not characterize the integral matrix proteins other than determining partial amino acid composition. There is evidence that the integral matrix proteins of adult mineralized tissues and the integral matrix proteins of embryonic spicules control similar physical properties of these calcitic tissues (6) . In addition, transcripts complementary to the previously cloned spicule matrix SM50 and SM30 cDNAs are expressed in adult mineralized tissues(22, 29) . Therefore we decided to further characterize the embryonic spicule matrix proteins and compare them to adult integral matrix proteins.
Our studies presented here show that there are many more spicule matrix proteins than were detected in previous studies. A dozen prominently radiolabeled spicule matrix proteins and some three dozen less prominently radiolabeled spicule matrix proteins are detected. The apparent molecular mass of the proteins range from 20 kDa to over 100 kDa. Most of these proteins, including the more prominent ones, are acidic. We also show that there are several less intensely radiolabeled spicule matrix proteins that have more alkaline isoelectric points. The predicted chemistry of the spicule matrix proteins is consistent with that found for other integral matrix proteins of calcified structures for other organisms(8, 9) . The results presented in this article further indicate that the adult integral matrix proteins are similar to embryonic spicule matrix proteins since there is significant Western blot cross-reactivity with the anti-total spicule matrix antiserum. However, since the one-dimensional SDS-PAGE Western antibody staining pattern is different from spicule matrix protein's pattern, this suggests that there are several different integral matrix proteins, or at least several different forms of integral matrix protein, in the adult spine.
Western blot analysis of the previously cloned SM50 gene shows that the SM50 protein is the spicule matrix protein with the most alkaline isoelectric point and that it has an apparent molecular mass of approximately 48 kDa. This result is consistent with the observations of Livingston et al.(28) and Richardson et al.(29) . Livingston et al.(28) cloned the L. pictus homologue of SM50. This 34-kDa protein, designated LSM34, is also not glycosylated and has an alkaline pI as determined from its derived amino acid sequence. The glycosidase results of the present paper and Livingston et al.(28) are to be expected since there is no N-linked glycosylation consensus sequence in the derived amino acid sequence of the S. purpuratus SM50 cDNA (21) and the L. pictus LSM34 cDNA(28) . Richardson et al.(29) generated an anti-SM50 specific polyclonal antiserum and they observed that it reacted with a single band of approximately 48 kDa in S. purpuratus embryonic spicules and adult spines.
It is particularly interesting that the
Western blotting analysis using the anti-SM30 antiserum reveals that
there are multiple forms of the SM30 protein in the sea urchin
embryonic spicule matrix and in the adult spine matrix. This result is
consistent with our assertion that adult spine integral matrix contains
some proteins that are different from embryonic spicule matrix. When
the SM30 cDNA was originally isolated, Northern blots revealed only one
band hybridizing with the SM30 cDNA probe(22) . However, recent
work (23) has demonstrated that SM30 is a small multigene
family with between two and four copies of the SM30 gene present per
haploid genome of S. purpuratus. A genomic clone was also
isolated and it was found that at least two SM30 genes are tandemly
arranged (designated SM30- and SM30-
).
Therefore, it is consistent, given these findings, to see a doublet of
SM30 proteins in the spicule matrix of 43 and 46 kDa (designated as
SM30-A and SM30-B, respectively) and a doublet of SM30 protein in adult
spine matrix of 46 and 49 kDa (designated SM30-B and SM30-C,
respectively).
Harkey et al.(27) reported that
SM50 and the PM27 proteins are similar to C-type lectin proteins and we
now report that SM30 is also similar to the CRD domain of C-type
lectins. The C-type lectins are a very heterogenous family of proteins
that are most often involved in recognition events outside cells (see
for reviews, (45, 46, 47) ). Usually these
recognition events are mediated through a Ca dependent binding to carbohydrate moieties. However, certain
C-type lectins have also been shown to bind proteins directly (48) , and to act as antifreeze molecules in coldwater
fish(49) .
It is interesting to note that two types of
C-type lectins that were found to be most similar to SM30- protein
in our studies here have previously been found to be involved in the
formation of mineralized structures within animals. One of these C-type
lectins is tetranectin which is a blood and extracellular matrix
component. Wewer et al.(50) reported localization of
tetranectin in mineralizing mouse bone osteoblastic cells that were
differentiating in vitro. However, the precise molecular role
tetranectin plays in osteogenesis remains unknown. The other C-type
lectin previously found to be involved in mineralized structures is
called lithostathine (formerly called pancreatic stone
protein(51) ). Lithostathine is present in pancreatic juices
and it is believed to bind calcium carbonate and prevent calcium
carbonate from precipitating and forming calcitic pancreatic stones
(see for review, (52) ). However, the 11 amino acid peptide
sequence at the amino end of lithostathine that is thought to interact
with calcium carbonate is not within the CRD of lithostathine and it is
not well conserved in SM30 or SM50 (data not shown). So whether there
is homology of function of SM30-
protein and lithostathine to go
along with the similarity of their sequences remains enigmatic. These
findings, however, raise several interesting questions. Could there be
other regions of these spicule matrix proteins that bind calcium
carbonate? Could the similarity to C-type lectins be reflective of the
SM30 and SM50 proteins binding the carbohydrate moieties of other
spicule matrix proteins? SM30-
protein has imperfect matching at
the calcium ion binding sites typically found in C-type lectins. Does
SM30-
protein bind Ca
? Many attempts in our
laboratory over the years by different techniques have failed to reveal
Ca
binding to SM30. Why are so many of the known
proteins which are closely associated with spicule formation similar to
C-type lectins? Experiments are underway to address these questions.
It remains undetermined which of the SM30 proteins identified in the
present paper are encoded by the SM30-, SM30-
, or
the pNG7 SM30 cDNA. Results presented in this paper point out that 7
out of 9 differences in the amino acid sequence between the protein
encoded by the pNG7 SM30 cDNA and the protein encoded by SM30-
occur over the approximately 40% portion of the SM30 proteins that
comprise the region similar to the CRD of C-type lectins. Most of these
differences alter conserved residues suggesting that they may encode
for different forms of the SM30 proteins. In addition to the studies
presented here, unpublished RNase protection studies show that SM30-
gene transcript is expressed in the sea urchin
embryo and not in the adult spine. (
)Since SM30-A protein
seems to be present in embryonic spicule and not in adult spines, this
unpublished finding suggests that SM30-A may be encoded by the SM30-
genomic sequence. The complete sequence of SM30-
is not yet known (23) and results presented
in the current paper suggest that there is at least a third SM30 gene that has not been cloned. Therefore, until the complete
sequence of all of the SM30 genomic sequences as well as their
expression patterns are known, we cannot be sure if SM30-
encodes SM30-A or if pNG7 SM30 cDNA encodes for a SM30 protein
different from the one encoded by SM30-
. Studies
addressing these issues are underway.
Our enumeration, characterization, and comparisons of spine and spicule matrix proteins complement studies of Berman et al.(4, 5, 6) . They observed that sea urchin integral matrix proteins are not as acidic as some other invertebrate integral matrix proteins. We have found that there is a range of acidic to alkaline pI values for the spicule matrix proteins, although the net pI of the proteins as a whole is acidic(16) . Berman et al.(4) showed that the integral matrix proteins from adult sea urchin mineralized tissues, but not the more acidic integral proteins from the mollusc Mytilus californus prismatic layer, were able to bind specific calcite crystal faces, even though the mollusc proteins are known nucleators of calcite when they are adsorbed on a rigid substrate. Berman et al.(6) also studied differences in texture of calcite crystals among calcitic tissues from different taxonomic groups, including analysis of sea urchin embryo spicules and adult spines. They showed that the manipulation of crystal structure is under biological control and that the integral matrix proteins probably play a role in this control. The studies presented here provide a fuller characterization of the embryo spicule and adult spine matrix proteins integral matrix proteins. Future studies looking at physical interactions of individual spicule matrix proteins with calcite crystals in vitro will be particularly informative.