From the Osteoadherin is a recently described bone
proteoglycan containing keratan sulfate. It promotes integrin
( Connective tissues are dominated by an extensive extracellular
matrix. In bone, the matrix is dominated by mineral in the form of
hydroxyapatite. The mineral crystals are aligned along the fibrils of
collagen I (1), which is the predominant organic constituent (2). The
extracellular matrix of bone also contains small chondroitin sulfate
proteoglycans like decorin and biglycan, as well as other
non-collagenous proteins (for review, see Ref. 3). The role of decorin
and biglycan in the bone tissue is still unclear. Decorin has high
affinity for type I collagen (4) and has been proposed to be involved
in mineralization. In histochemical studies, it has been shown that
decorin disappears from the collagen fibrils in bone before or during
the mineralization of the tissue (5). However, in a recently described
inactivation of the decorin gene in mice, no skeletal changes could be
detected (6). Biglycan has a different distribution pattern than
decorin and does not appear associated with fibrils of collagen I. Biglycan can be found in the osteoid (7) and in preosteogenic cells,
suggesting a function in early bone formation.
More than a decade ago it was shown that decorin (8, 9) contains
distinct leucine-rich repeats
(LRRs).1 Since then, the
number of LRR proteins in connective tissues with identified primary
sequences has increased. Currently, this group of extracellular matrix
molecules with LRRs includes biglycan (10), decorin, fibromodulin (11),
lumican (12), PRELP (13), keratocan (14), chondroadherin (15),
proteoglycan-Lb (16), and osteoglycin (17). Except for chondroadherin
and PRELP, they are usually substituted with one or a few
glycosaminoglycan chains. Several of these LRR proteins have been shown
to bind components of the extracellular matrix, e.g.
collagen as mentioned above (18) but also fibronectin (19), growth
factors (20, 21), and cells (22, 23).
Osteoadherin has been isolated as a minor, leucine- and aspartic
acid-rich keratan sulfate proteoglycan found in the mineralized matrix
of bone (24). The protein is rather acidic and binds well to
hydroxyapatite. Interestingly, the protein can, in a
cation-dependent mechanism, bind osteoblasts via the
Protein Purification--
Osteoadherin was isolated from the
mineral compartment of bovine bone as described by Wendel et
al. (24).
Peptide Isolation, Amino Acid Sequencing, and N-terminal
Sequencing--
Peptides were isolated from digests in polyacrylamide
gel of purified protein with sequencing grade trypsin (Promega) as
originally described by Rosenfeld (25) and further developed by Hellman et al. (26). Peptides were separated by reversed-phase HPLC on a µRPC C2/C18 column by the use of a SMART system (Amersham Pharmacia Biotech) with a gradient of 0-40% acetonitrile over 160 min. Peptide peaks were collected and sequenced on an Applied Biosystems 477A automated sequencer with on-line analysis of
phenylthiohydantoin-derivative on an Applied Biosystems 120A microbore
HPLC. N-terminal sequencing of osteoadherin was performed on intact
protein by standard methods.
RNA Extraction--
Primary bovine osteoblasts were prepared
using the method of Robey and Termine (27) and total RNA extracted with
guanidine isothiocyanate essentially according to Adams et
al. (28). Chondrocytes from bovine tracheal cartilage were
isolated by collagenase digestion (29). Total RNA from these cells were
extracted by the same method. Various tissues from an approximately
2-trimester-old bovine fetus and calvaria from 5-day-old rats were
homogenized, and total RNA was extracted similarly.
Screening of cDNA Library and DNA Sequencing--
Initially,
a rat calvaria library was extensively screened with affinity-purified
antibodies to osteoadherin, but no positive clones were obtained. A
unidirectional Northern Blots--
Ten µg of total RNA from various tissues
and species were electrophoresed on a 1% agarose/formaldehyde gel and
RNA transferred to nitrocellulose filter (NitroPure, Micron Separations
Inc.) by standard procedures. An 800-base pair bovine cDNA fragment (AccI/SacI) from the original clone was random
primer-labeled (Random Primed DNA labeling kit, Boehringer Mannheim)
with [ In Situ Hybridization--
A 50-mer oligonucleotide probe
(5'-GTA TAT GAG GGA AGC AGA GGA AAA TGT ATG AGC TTA TTG GCG CTT TCA
GC-3') complementary to bases 1149-1198 in the cDNA was
end-labeled with [ cDNA Cloning and Sequencing--
A unidirectional Department of Cellular and Molecular
Biology,
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
v
3)-mediated cell binding (Wendel,
M., Sommarin, Y., and Heinegård, D. (1998) J. Cell
Biol. 141, 839-847). The primary structure of bovine
osteoadherin has now been determined by nucleotide sequencing of a
cDNA clone from a primary bovine osteoblast expression library. The
entire translated primary sequence corresponds to a 49,116-Da protein with a calculated isoelectric point for the mature protein of 5.2. The
dominating feature is a central region consisting of 11 B-type,
leucine-rich repeats ranging in length from 20 to 30 residues. The
full, primary sequence contains four putative sites for tyrosine
sulfation, three of which are at the N-terminal end of the molecule.
There are six potential sites for N-linked glycosylation present. Osteoadherin shows highest sequence identity, 42%, to bovine
keratocan and 37-38% identity to bovine fibromodulin, lumican, and
human PRELP. Unique to osteoadherin is the presence of a large and very
acidic C-terminal domain. The distribution of cysteine residues
resembles that of other leucine-rich repeat proteins except for two
centrally located cysteines. Northern blot analysis of RNA samples from
various bovine tissues showed a 4.5-kilobase pair message for
osteoadherin to be expressed in bone only. Osteoadherin mRNA was
detected by in situ hybridization in mature osteoblasts located superficially on trabecular bone.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
v
3 integrin. In order to further
characterize osteoadherin, we have determined its primary structure.
This reveals that osteoadherin belongs to the LRR family of connective
tissue proteins. Osteoadherin is primarily expressed by mature
osteoblasts, as shown in studies of its expression by in
situ hybridization.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Zap (Stratagene) cDNA library was therefore made
from RNA isolated from primary bovine osteoblast cultures according to
instructions in the ZAP-cDNA® synthesis kit
(Stratagene). Approximately 500,000 plaque-forming recombinants were
screened using affinity-purified rabbit antiserum, and one clearly
positive clone was found. The pBluescript® SK(+) plasmid
containing the 2.1-kbp cDNA insert was rescued from the ZAP vector
by the use of in vivo excision. The cDNA was digested to
various lengths for sequencing by the use of the
Erase-a-Base® system (Promega). The cDNA was sequenced
in both directions by the standard, double-stranded dideoxy termination
method using T3, T7, and synthetic, internal primers. Two additional
clones were identified in the library by the use of a cDNA probe
corresponding to bases 1-225 of the original clone. These clones of
approximately 4.5 kbp were partially sequenced from the ends by the use
of T3 and T7 primers and five internal sequencing primers producing sequence covering most of the coding region.
-32P]dCTP (RedivueTM, Amersham
Pharmacia Biotech) and allowed to hybridize to the blotted mRNA
from the tissues. Nonhybridized probe was removed by a final wash with
0.2× SSC, 0.1% SDS at 55 °C, prior to detection of radiolabel with
a Fujix BAS2000 bio-imaging analyzer.
-35S]dATP (Amersham Pharmacia
Biotech, Solna, Sweden) using terminal deoxynucleotidyltransferase (NEN
Life Science Products, Boston, MA). The in situ
hybridization procedure used was similar to that previously described
by Sandell et al. (30) and Shen et al. (31). With
control sections, RNase digestion was performed before hybridization.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
Zap
cDNA expression library was prepared from cultured primary bovine
osteoblasts. The library was screened with an affinity-purified rabbit
polyclonal antiserum. From an initial screen of some 500,000 recombinants, one antibody-positive clone of 2,111 base pairs was
selected. This clone was sequenced completely in both directions. The
nucleotide and translated amino acid sequences are shown in Fig.
1. The overall nucleotide composition is
unusual in that it contains 61% A or T nucleotides. The osteoadherin cDNA sequence corresponds to a 422-residue protein. The identity of
the clone with isolated osteoadherin was unambiguously confirmed by
amino acid sequencing of the N terminus and several internal tryptic
peptide fragments distributed along the protein, as indicated in Fig.
1.
View larger version (64K):
[in a new window]
Fig. 1.
Complete cDNA and translated amino acid
sequence of the original clone. Peptide sequences obtained from
amino acid sequencing of bovine osteoadherin are underlined.
A potential signal peptide cleavage site is indicated by an
arrowhead. Potential N-glycosylation sites are
indicated by an asterisk. Putative tyrosine sulfation sites
are indicated by a filled diamond.
Protein Structure--
Examination of the deduced protein sequence
indicate a signal peptide cleavage site between 20 and 21 residues from
the first methionine (see Fig. 1). This site conforms well to the
1,
3 signal peptide cleavage site rule of von Heijne (34) and is preceded by a hydrophobic peptide. N-terminal sequence analysis of
isolated osteoadherin gave a clear DEDYDQEP sequence, indicating that
the protein most likely is first synthesized with a 7-amino acid
propeptide. The calculated molecular mass of the complete preprotein is
49,116 Da, whereas with and without propeptide the protein is 46,874 and 45,889, respectively. These sizes correspond well with the size of
isolated osteoadherin after removal of N-linked oligosaccharides (24). The calculated, theoretical isoelectric point of
the mature protein without propeptide is 5.2.
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Similarity to Other Proteins-- Similarity searches against GenBank and SwissProt data bases indicate similarity to several other LRR proteins. Comparison of primary sequences of mature LRR proteins shows that osteoadherin is most closely related to a group consisting of fibromodulin, lumican, keratocan, and PRELP (see Fig. 2). Construction of a dendrogram indicates that osteoadherin is not strongly related to any previously described protein forming a distinct subgroup within the family. Highest overall similarity, 42% identical residues, was with bovine keratocan followed by 37-38% identity with bovine lumican, fibromodulin, and human PRELP. An alignment against these proteins is shown in Fig. 3. On the other hand, it should be noted that the cysteines in the N-terminal region are separated by 3, 1, and 9 residues. This pattern is identical to the other proteins in the alignment. Thus, they all belong to the class II subfamily (37).
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Expression of Osteoadherin-- Northern blot analysis of total RNA from various tissues and cultured cells showed a 4.5-kilobase mRNA in preparations from trabecular bone and a very strong signal in the cultured primary bovine osteoblasts, Fig. 4. The hybridization signal detected is rather diffuse. This could be a result of degradation of the mRNA. However, the quality of the primary osteoblast RNA preparation was high. This preparation was used to construct the expression cDNA library used in this work, from which we could isolate several clones of 4.5 kbp. Through a number of experiments, we have, however, not been able to obtain well defined hybridization signals for bovine osteoadherin with several different preparations of bone tissue RNAs or by using alternative probes. One possible explanation is that there is considerable alternative or inefficient splicing of the osteoadherin pre-mRNA.
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Localization of Expression by in Situ Hybridization-- In situ hybridization analysis for osteoadherin mRNA in sections from the growth plate of bovine fetal fetlock joints showed that the protein is primarily expressed in osteoblasts on trabecular bone surfaces (Fig. 5). Strongest signal was seen over osteoblasts in well developed trabecular bone (see Fig. 5C), indicating high expression in mature osteoblasts.
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DISCUSSION |
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Through this work, osteoadherin has been shown to be an additional
member of the family of leucine-rich repeat proteins in the
extracellular matrix. Although osteoadherin is rather similar to
lumican, fibromodulin, keratocan, and PRELP, it is not closely related
to these. Osteoadherin contains 11 clearly identifiable LLRs, with a
well conserved leucine repeat. In lumican, fibromodulin, keratocan, and
PRELP, the repeats are arranged in triplets consisting of two repeats
of 24-26 amino acids, followed by one shorter repeat of 20-21
residues. This kind of triplet pattern is also present in osteoadherin.
The significance of the triplet pattern is not understood. One
explanation could be that this enables the -sheets of the
20-26-amino acid repeats to be aligned in parallel in the same fashion
as in the known three-dimensional structure of ribonuclease inhibitor,
which almost entirely consists of equally long 28-residue repeats.
However, modeling studies (38) indicate that there appears to be room
for considerable flexibility in both the loop region connecting the
-sheets with the
-helix region and the
-helix region itself
allowing the
-sheets to be in register despite differing lengths of
the repeats. Indeed, modeling of the LRR region of decorin to the
ribonuclease inhibitor (39) shows that decorin can be fitted well
despite the shorter repeats. The decorin study suggests that all the
members of this family could have a structure in the central part
similar to ribonuclease inhibitor.
The relatively large and very acidic C-terminal peptide extension is a feature that distinguishes osteoadherin from the other LRR-containing proteins and proteoglycans. The C-terminal region after the last cysteine is thus considerably larger than in the related proteins, consisting of 69 amino acids compared with 9-10 in lumican, fibromodulin, keratocan, or PRELP. This C-terminal region in osteoadherin is extremely acidic, with 16 negatively charged aspartic or glutamic acids in the last 38 residues. Osteoadherin binds well to hydroxyapatite, a property utilized in the isolation of the protein from mineralized bone (24). Other hydroxyapatite-binding proteins in bone like bone sialoprotein have long stretches of acidic residues thought to mediate the binding to the mineral (40). A likely function for the very acidic and probably exposed C terminus could thus be to anchor the protein to the mineral. It should be noted that there are two arginines in position 361-362 just prior to the acidic residues. Exposed dibasic sequences are often recognized by proteases, e.g. of the furin family (41). This presents an interesting possibility for proteolytic processing of the C terminus. Indeed, it has been shown for chondroadherin, another LRR protein of the extracellular matrix, that at least two forms of the protein exists differing by 9 C-terminal amino acids (15, 42). This processing is the result of proteolytic processing as this region of chondroadherin is encoded by one exon in the mouse chondroadherin gene (43). However, in contrast to osteoadherin the peptide that is removed in chondroadherin is basic. Interestingly, we have in extracts of mineralized bone found no lower Mr protein of dimensions expected after removal of the C-terminal peptide. Thus, either this may not happen or the major part of the protein released from this putative anchor to the mineral may be rapidly lost from the tissue.
Another unusual feature of osteoadherin is the presence of two, presumably closely situated cysteines in LRR 5 and 6 (see Table I). In view of their close proximity, it is likely that these residues are disulfide-bonded. No such pairs of cysteines in the central LRR region are found in the other members of this family of extracellular matrix proteins. In chondroadherin, one single cysteine is found in repeat four. This residue appeared not to be free but to be involved in stabilizing the structure of the repeat as it could not be chemically derivatized (15). Another interesting possibility is that the cysteines could participate in disulfide exchange with other matrix proteins in bone. One example where this may occur is to osteonectin, inasmuch as an essential step in the complete separation of osteoadherin from osteonectin is reduction of disulfide bonds prior to the final purification step (16).
The N-terminal region of osteoadherin has similarities to lumican and fibromodulin in that it has potential sites for tyrosine sulfation. This fits well with our previous finding that removal of radioactive sulfate-labeled keratan sulfate from osteoadherin still leaves a molecule with radiosulfate attached (24). The functional significance of tyrosine sulfation is not known, but it has been suggested to be of importance for intracellular transport (44). The first potential site is found in the propeptide. It is therefore tempting to speculate that this region is involved in intracellular processing of the protein. A propeptide is also found in decorin (8). It has been suggested that this propeptide is of importance for intracellular transport as its deletion leads to an increase in intracellular retention time during synthesis and secretion of the proteoglycan (45).
Osteoadherin has been shown to be a cell attachment protein binding
primary osteoblasts in a cation-dependent interaction (24).
This interaction was inhibited by RGD-containing peptides but not by
RGE peptides, indicating the presence of a classical cell-binding
region as in fibronectin. However, no RGD sequence is found in the
primary sequence of osteoadherin. The best candidate region is an RID
sequence in the last LRR. This sequence is positioned at the end of the
presumed -sheet region. The isoleucine is therefore likely to be
oriented inward and the two charged residues exposed at the beginning
of the loop region. In fibronectin, the arginine and aspartic acid has
been shown to be exposed in a loop structure with the glycine pointing
inward (46). This opens the possibility that the RID sequence in
osteoadherin is a functional mimic of the RGD in fibronectin. Indeed,
an RLD sequence has been found to be part of the minimal active site in
fibrinogen responsible for binding to the
M
2 integrin (47). However, all of the LRR proteins in the alignment in Fig. 3 have an RLD sequence in the same
position as the osteoadherin RID sequence, but none of these proteins
has been shown to possess cell binding activity.
Osteoadherin appears to be rather specific for bone. By Northern blot analysis, it was found to be expressed only in bone cells. The expression seems to be restricted to mature osteoblasts as highest expression of mRNA for osteoadherin was detected by the use of in situ hybridization in osteoblasts located on the trabeculae at some distance from the growth plate. This pattern is similar to that found for osteopontin (48), whereas bone sialoprotein is expressed much earlier with some expression in late hypertrophic chondrocytes (49), but most prominently by osteoblasts at osteochondral junctions (50).
In primary cultures of bovine osteoblasts, strong osteoadherin expression was detected by Northern blot analysis. Samples of RNA from trabecular bone showed a weaker but easily detectable signal. The size of the mRNA was approximately 4.5 kilobases. Interestingly, with a probe covering the coding region, a rather diffuse band with a strong trailing was seen. However, if a probe corresponding to the 3'-UTR of the first clone was used, no detectable signal was found. In contrast, if a probe 3'-UTR from the second set of 4.5-kbp clones was used, a better defined 4.5-kbp band with less prominent trailing was found (data not shown). Thus, summarizing the presence of SINE/art2 sequences together with an absence of a classical polyadenylation signal in the first clone and presence of such a signal in the second two clones indicates that the first clone isolated could represent a not fully processed mRNA with remaining intron sequences. Alternatively, the first clone could represent an alternatively spliced form. In any case, it is rather unusual with alternative splicing in 3'-UTR. A similar, but not identical, situation exists in the mouse gene for chondroadherin, where an intron is present in the 3'-UTR in close proximity after the stop codon (43).
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ACKNOWLEDGEMENTS |
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We are very grateful to Ulrika Petterson and Charlotte Bratt for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Medical Research Council, the Folksam's Foundation, the King Gustav V 80th Birthday Foundation the Greta and Johan Kock Trust, the Alfred Österlund Trust, and the Medical Faculty, University of Lund (to D. H., M. W., and Y. S.) and a grant from the Ludvig Foundation (to U. H.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U67279.
§ To whom correspondence should be addressed: Lund University, Dept. of Cell and Molecular Biology, Section for Connective Tissue Biology, P. O. Box 94, S-221 00 Lund, Sweden. Tel.: 46-46-222-85-70; Fax: 46-46-211-34-17; E-mail: yngve.sommarin{at}medkem.lu.se.
1 The abbreviations used are: LRR, leucine-rich repeat; HPLC, high performance liquid chromatography; kbp, kilobase pair(s); UTR, untranslated region.
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REFERENCES |
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