From the Department of Cell and Molecular Biology,
Section for Connective Tissue Biology, Lund University, BMC plan
C12, SE-221 84 Lund, Sweden, the ¶ Royal Veterinary College, Royal
College Street, London NW1 0TU, United Kingdom, and the
Shriner's Hospital for Children, Tampa, Florida 33612
Received for publication, December 4, 2000, and in revised form, December 27, 2000
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ABSTRACT |
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Asporin, a novel member of the leucine-rich
repeat family of proteins, was partially purified from human articular
cartilage and meniscus. Cloning of human and mouse asporin cDNAs
revealed that the protein is closely related to decorin and biglycan.
It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to
decorin and biglycan, asporin is not a proteoglycan. Instead, asporin
contains a unique stretch of aspartic acid residues in its
amino-terminal region. A polymorphism was identified in that the number
of consecutive aspartate residues varied from 11 to 15. The 8 exons of
the human asporin gene span 26 kilobases on chromosome
9q31.1-32, and the putative promoter region lacks TATA consensus
sequences. The asporin mRNA is expressed in a variety of human
tissues with higher levels in osteoarthritic articular cartilage,
aorta, uterus, heart, and liver. The deduced amino acid sequence of
asporin was confirmed by mass spectrometry of the isolated protein
resulting in 84% sequence coverage. The protein contains an
N-glycosylation site at Asn281 with a
heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser54. The name asporin
reflects the aspartate-rich amino terminus and the overall similarity
to decorin.
Cartilage matrix consists of fibrillar networks, primarily of
collagen II and highly negatively charged molecules of aggrecan. There
are also a number of noncollagenous glycoproteins that apparently contribute to the regulation of tissue assembly and properties. Among
them is the family of the leucine-rich repeat (LRR)1
proteins, which contains several members found
in the extracellular matrix. There are
currently 11 known members of this family. These molecules share a
common structure with a central stretch of LRRs. This LRR domain is
flanked by disulfide bridged loops, with 4 cysteine residues preceding
the LRR domain and 2 on its C-terminal side. Apart from chondroadherin,
these proteins also contain divergent amino-terminal extensions with
features unique for the different proteins. Based on amino acid
sequence and gene organization the family can be divided into four
distinct groups.
Decorin (1) and biglycan (2) constitute the first group (class I).
These proteins have 10 LRRs and carry one and two chondroitin or
dermatan sulfate chains, respectively. The glycosaminoglycan chains are
linked to serine residues in the amino terminus. The molecules in this
group are secreted with a propeptide.
The second group (class II) consists of fibromodulin (3), lumican (4),
keratocan (5), PRELP (6), and osteoadherin (7). Like the class I
proteins they consist of 10 LRRs. With the exception of PRELP, they all
carry polylactosamine or keratan sulfate chains linked to the LRR
region and sulfated tyrosine residues in the amino-terminal extension.
In contrast, the amino terminus of PRELP has a cluster of positively
charged amino acid residues that mediates binding to heparan sulfate
(8). Unlike all other family members, osteoadherin contains a
COOH-terminal extension (7).
Epiphycan/PG-Lb/DSPG3 (9-11), mimecan/osteoglycin (12, 13),
and opticin/oculoglycan (14-16) form the third group (class III).
These are smaller molecules with only 6 LRRs and all contain sulfated
tyrosine residues in the amino-terminal extension. In addition,
epiphycan carries chondroitin sulfate, other O-linked oligosaccharides, and a cluster of glutamate residues in this region
(9). The amino-terminal extension of opticin carries O-linked oligosaccharides (14), and contains a
heparin-binding consensus sequence (17).
Chondroadherin (18) forms the fourth branch on the extracellular matrix
LRR protein family tree (class IV). This protein contains 10 LRRs, but
lacks both amino- and COOH-terminal extensions outside the cysteine
motifs. Nyctalopin, a recently published glycosylphosphatidylinositol-anchored LRR protein may also be a member
of this subfamily (19, 20).
As is evident from the summary above, the subdivision of LRR proteins
into classes based on sequence does not reflect the functions of the
molecules. For example, decorin, biglycan, and epiphycan are
chondroitin or dermatan sulfate proteoglycans, and may as such be more
functionally related than, e.g. the different class II LRR
proteins. A major functional property that is shared between most of
the class I, II, and IV LRR proteins is a capacity to bind to
collagen via the LRR domain. This is a high affinity binding with
Kd in the nanomolar range. The different NH2-terminal extensions offer a variety of opportunities
for interactions with other matrix constituents, including other fibers
of collagen, thereby providing cross-linking and stabilization of the
fibrillar network.
Several of these molecules appear to have roles in modulating the
assembly of collagen fibrils as is indicated by experiments in
vitro (21-24) as well as by gene inactivation studies (25-27). Invariably, these studies show altered collagen fiber dimensions when
the abundance of the LRR protein is changed.
The present work started with a study of altered biosynthesis of
proteins in early human osteoarthritis. We found a number of proteins
to be up-regulated, one being a component with an apparent size of 39 kDa. This component appeared structurally related to fibromodulin since
it cofractionated in a variety of separation procedures. We now define
the primary structure including a putative polymorphism,
oligosaccharide side chain substituents, and tissue expression of the
protein. It represents a novel member of the LRR protein family
belonging to the decorin/biglycan group (class I). The protein is named
asporin based on the presence of a polyaspartate stretch in the
amino-terminal region and the similarity with decorin.
Tissue Extraction--
Normal human knee cartilage (18.5 g of
tissue wet weight, donor age 32 to 50 years) and menisci (3.5 g of
tissue wet weight) were obtained at surgery. The tissues were dissected
clean, sliced into fine pieces, and disrupted using a high speed
homogenizer (Polytron, Kinematica GmbH) in 12 volumes (v/w) of 4 M GdnHCl, 0.05 M sodium acetate, pH 5.8, containing protease inhibitors (5 mM benzamidine
hydrochloride, 0.1 M 6-aminohexanoic acid). After
extraction for 24 h at 4 °C the remaining insoluble material was removed by centrifugation at 20,000 × g at 4 °C
for 30 min.
Protein Purification--
Proteins in the cartilage extract were
separated from proteoglycans by CsCl density gradient centrifugation
with a starting density of 1.5 g/ml under dissociative conditions in 4 M GdnHCl as described elsewhere (28). The gradient tube was
divided into 4 equal fractions using a Beckman tube slicer, and the top
fraction (D4) was used for subsequent purification. The D4 fraction was then concentrated by ultrafiltration (PM-10 membrane, Amicon), followed
by diaflow against 4 M GdnHCl, 20 mM Tris-HCl,
pH 8, and applied to a Superose 6 column (2.2 × 100 cm) in 2.5-ml
aliquots. Fractions of 2.5 ml were collected, monitored for protein
content by measuring their absorbance at 280 nm, and analyzed by
SDS-PAGE after ethanol precipitation, as previously described (29).
The proteins from the extract were separated into two peaks, a larger
containing proteins of high molecular weight (fractions 25 to 45) and a
smaller containing the smaller proteins. The latter fractions (46 to
65) were pooled and concentrated by ultrafiltration followed by diaflow
against 7 M urea, 20 mM Tris-HCl, pH 8. The pooled material was then loaded onto a 30-ml bed volume column of
DEAE-cellulose (1.6 × 15 cm, DE52, Whatman) equilibrated in the
urea buffer. After sample loading, the column was washed with 5 bed
volumes of the equilibration buffer, and eluted with a 800-ml linear
gradient (27 bed volumes) of 0 to 1 M NaCl in the
equilibration buffer at a flow rate of 20 ml/h. Fractions of 10 ml were
collected, monitored for protein content by measuring their absorbance
at 280 nm, and analyzed by SDS-PAGE.
The fractions containing asporin were pooled, concentrated by
ultrafiltration followed by diaflow against 7 M urea, 10 mM HCOOH, pH 4.0, and chromatographed on a 20-ml bed volume
of Q-Sepharose Fast Flow (1.6 × 8.5 cm, Amersham Pharmacia
Biotech) anion exchange column equilibrated in urea buffer. The column
was washed with 5 bed volumes and the bound proteins were step eluted
at a flow rate of 20 ml/h with the equilibration buffer containing 1 M NaCl. Fractions of 2 ml were collected, monitored for
protein content by measuring their absorbance at 280 nm, and analyzed
by SDS-PAGE.
The fractions containing asporin were pooled and equilibrated by
diaflow to 7 M urea, 10 mM HCOOH, pH 4.0, and
applied to a Mono Q HR 5/5 column (Amersham Pharmacia Biotech). The
bound proteins were eluted with a 15-ml linear gradient (15 bed
volumes) from 0 to 1 M NaCl at a flow rate of 30 ml/h.
Fractions of 2 ml were collected, monitored for protein, and
analyzed by SDS-PAGE.
Initial characterization of the meniscus extract showed a low
content of high molecular weight proteoglycan. Therefore, the sample
was taken directly to chromatography, omitting cesium chloride gradient
centrifugation. Forty milliliters of the meniscus extract were
equilibrated to 7 M urea, 20 mM Tris-HCl, pH
8.0, by diaflow and directly chromatographed over Q-Sepharose Fast Flow
followed by chromatography over Mono Q, as described above except that the pH was kept at 8.0.
After SDS-PAGE analysis, the fractions from the Mono Q chromatography
containing asporin were pooled and concentrated by ultrafiltration, followed by diaflow against 4 M GdnHCl, 50 mM
sodium acetate, pH 5.8. This material was further chromatographed on
two serially coupled columns of Superose 6 and Superdex 200 (Amersham
Pharmacia Biotech) equilibrated and eluted at 0.2 ml/min with 4 M GdnHCl, 50 mM sodium acetate, pH 5.8. Fractions of 0.5 ml were collected, monitored for protein content by
measuring their absorbance at 280 nm. Protein patterns were analyzed by
SDS-PAGE.
Protein Sequencing--
Proteolytic digestion with Lys-C (Roche
Molecular Biochemicals) was performed at enzyme to substrate ratios of
1:50 according to the manufacturer's instructions. Peptides were
separated by reversed phase HPLC on a Vydac C18 column (2.1 × 30 mm), eluted with a gradient of acetonitrile (0-70% over 45 min) in
0.1% trifluoroacetic acid at a flow rate of 0.2 ml/min. The effluent
was monitored at 220 nm. Peptides were sequenced on an Applied
Biosystems 477A automated sequencer with on-line analysis of
phenylthiohydantoin-derivatives on an Applied Biosystems 120A microbore
HPLC.
cDNA Cloning--
All the molecular biological procedures,
including agarose gel electrophoresis, restriction enzyme digestion,
ligation, bacterial transformation, and DNA sequencing, were performed
according to standard methods (30).
The amino acid sequences obtained from endoproteinase Lys-C-digested
asporin were used to search the GenBankTM data base with
the TBLASTN 2.1 program (31). The EST sequences identified from this
search were aligned and assembled. The resulting full-length sequence
was used for designing primers h39k-S (5'-CTTCTACACTAAGACACC-3') and
h39k-AS (5'-AAATGGACATTACCAATTAC-3').
Human osteoarthritic articular cartilage was obtained at surgery after
total hip replacement, kept in phosphate-buffered saline during
dissection, shaved and frozen in liquid nitrogen. Total RNA and
mRNA were purified as described previously (32). First strand
cDNA was primed with oligo-(dT)15 and reverse
transcribed with Superscript II reverse transcriptase (Life
Technologies). After digestion of the mRNA with RNase H, the
asporin cDNA was obtained using the polymerase chain reaction (PCR)
with primers h39k-S and h39k-AS and Pfu DNA polymerase.
After an initial denaturation step at 95 °C for 1 min, the DNA was
amplified for 30 cycles of 45 s at 95 °C, 45 s at
54 °C, and 2 min 40 s at 72 °C. The resulting 1.2-kilobase
product was isolated from an agarose gel, purified using the QiaQuick
kit (Qiagen), and ligated into the pCR-Script Amp SK(+) vector
(Stratagene). The PCR product and several of the resulting pCR-Script
clones were sequenced using the BigDye kit (ABI) and run on a ABI 310 DNA sequencer. In addition to primers T3, T7, h39k-S, and h39k-AS two
internal primers were used: h39k-IntS (5'-ATGAAAATAAAGTTAAGAAAATAC-3')
and h39k-Int AS (5'-AGGGTTTGCACTCATTTC-3'). The resulting sequence
tracings were assembled using the SeqMan II module of the LaserGene 99 software (DNAstar Inc).
A first draft full-length mouse asporin sequence was assembled from
sequences obtained through a BLASTN search of the mouse EST section of
GenBankTM with the human asporin sequence. Using this draft
sequence the primers m39k-S (5'-ACTTGTACACAGGCCAGC-3'), m39k-AS
(5'-TTTTATATTTAATGGATGTCATG-3'), m39k-IntS
(5'-GACCTTCAAAATAATAAAATC-3'), and m39k-IntAS
(5'-TGGTATATTAGCAAAAGTTC-3') were designed. Mouse aorta first strand
cDNA was prepared and asporin cDNA amplified from this by
reverse transcriptase PCR using primers m39k-S and m39k-AS, as
described above. The PCR product was cloned into pCR-Script and
sequenced using all four m39k primers, as well as T3 and T7 primers.
The human and mouse asporin cDNA sequences were deposited in
GenBankTM with the accession numbers AF316824 and AF316825, respectively.
Messenger RNA Expression Analysis--
For Northern blot
analysis 10 µg of total RNA isolated from human osteoarthritic
articular cartilage were electrophoresed on 1% formaldehyde-agarose
gel, and transferred to a nitrocellulose filter (NitroPure, Micron
Separation). Membranes of Multiple Tissue Northern blot and Human RNA
Master Blot were from CLONTECH. The membranes were
hybridized with a 463-base pair cDNA fragment (nucleotides 382-845
of the human sequence, Fig. 3) labeled with [ Enzymatic Deglycosylation--
Samples to be digested were
precipitated with ethanol, resuspended in 0.1 M Tris-HCl,
pH 6.8, containing 0.1% SDS, and incubated in a boiling water bath for
3 min. Then an equal volume of 0.125 M Tris-HCl, pH 6.8, was added, plus 5 µl of 0.5% Nonidet P-40, 1 µg of trypsin
inhibitor (from chicken egg white type II-0, Sigma), and 1 unit of
N-glycosidase F (Roche Molecular Biochemicals). An aliquot
of the mixtures before and after digestion was diluted with sample
buffer (2% SDS, 0.125 M Tris-HCl, pH 6.8, 0.002%
bromphenol blue, and 20% glycerol), boiled at 100 °C for 4 min and
electrophoresed on the gradient polyacrylamide gel. Proteins were
visualized by staining with Coomassie Brilliant Blue R-250 (Serva).
Sample Preparation for Mass Spectrometry--
Coomassie-stained
bands on SDS-PAGE gels were excised and washed extensively using 40%
acetonitrile in 25 mM NH4HCO3, pH
7.8. After washing, the gel pieces were dried in a SpeedVac and
subsequently reduced and alkylated using 10 mM
dithiothreitol and 55 mM iodoacetamide at 56 °C (30 min)
and at 20 °C (30 min), respectively. Samples were then washed and
dried before digestion overnight at 37 °C using 10-20 µl of
sequencing grade endoproteinases such as trypsin (Promega) or Glu-C
(Roche Molecular Biochemicals) at 25 ng/µl in 25 mM
NH4HCO3, pH 7.8. The digestion was terminated
by the addition of 10 µl of 2% trifluoroacetic acid, which also
extracted the peptides out of the gel. After a minimum 1-h extraction
at room temperature, peptides were purified from buffer using
miniaturized C-18 reversed phase tips (ZiptipsTM,
Millipore). Purified peptides were eluted directly onto the sample
target using acetonitrile, 0.1% trifluoroacetic acid (1:1). Various matrices were used to increase the sequence coverage. When
using water-soluble matrices such as 2,4,6-thihydroxyacetophenone and
2,5-dihydroxybenzoic acid, an AnchorchipTM target (Bruker
Daltonik GmbH, Bremen, Germany), that confines the sample to a smaller
area increasing the sensitivity was used (33). The intact mass was
obtained after elution of intact protein from the gel (34) followed by
direct application to the AnchorchipTM target using ferulic
acid as the matrix. Carbonic anhydrase was used for external calibration.
Mass Spectrometry--
Mass spectrometric studies were performed
using a Bruker Scout 384 Reflex III matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. The
instrument was used in the positive ion mode with delayed extraction
and an acceleration voltage of 26 kV. Peptide samples were mainly
analyzed using the reflector detector and 50-150 single-shot spectra
were accumulated for improved signal-to-noise ratio. Spectra were
internally calibrated using autolysis fragments of trypsin. For
analysis of intact protein the linear detector was used, with an
acceleration voltage of 20 kV. The software used to identify the
obtained peptide masses and N-linked oligosaccharide
composition and structure were ProFound (35) and GlycoMod (36), respectively.
Partial Purification of Asporin from Cartilage--
Extraction of
human articular cartilage with 4 M GdnHCl followed by
cesium chloride gradient centrifugation separated the matrix proteins
from the bulk of the large proteoglycans in the cartilage.
Fractionation of the extract by gel filtration on Superose 6 resulted
in two pools, one containing large proteins and the other with proteins
of lower molecular masses (
Further attempts to separate asporin and fibromodulin by Superose 12 gel filtration with 1% SDS in the buffer, heparin-Sepharose chromatography, anti-fibromodulin antibody affinity chromatography, and
C-18 reverse phase chromatography were unsuccessful. A final attempt to
use the collagen affinity of fibromodulin (24) to precipitate the
protein with collagen I, gave the interesting result that also asporin
was recovered with the collagen precipitate (data not shown).
Partial Purification of Asporin from Meniscus--
The analysis of
a GdnHCl extract of human meniscus by electrophoresis showed a low
content of large proteoglycans (data not shown). The extract was thus
directly applied on a Q-Sepharose anion exchange column in 7 M urea, 20 mM Tris-HCl, pH 8.0. Asporin was
recovered in a few fractions identified by SDS-PAGE. These fractions
were pooled and further fractionated by gel filtration on two tandemly
arranged columns of Superdex 200 and Superose 6 (Fig.
2). Asporin eluted in a few fractions
together with a minor proportion of the fibromodulin.
Peptide Sequencing of Asporin--
After Lys-C digestion of the
intact protein excised from an SDS-polyacrylamide gel, peptides were
separated by reversed phase HPLC. Peaks were collected and analyzed.
Some peaks gave two sequences, but by analysis of the relative yields
of the amino acids at each cycle, it was possible to determine both
sequences with a high degree of confidence. As the protein was not
reduced and carboxymethylated, no peptides were isolated that
contained cysteine.
Determination of the Asporin Nucleotide and Amino Acid
Sequence--
TBLASTN searches with the 9 peptide sequences obtained
(Table I) showed that six of these were
contained within an EST clone (GenBankTM accession number
AK000136). One peptide was derived from fibromodulin. Two peptides were
too short to produce BLAST hits, but the sequences of these are present
in the AK000136 sequence (Fig. 3).
AK000136 is an EST sequence deposited in GenBankTM as a
putative extracellular matrix protein. The deduced AK00136 sequence contains several leucine-rich repeats and the two COOH-terminal cysteine residues typical of the extracellular matrix LRR-repeat protein family. When AK000136 was used as the query in further BLAST
searches, a number of other EST sequences were identified. Assembly of
these sequences produced a longer open reading frame that included a
signal peptide and the amino-terminal 4-cysteine motif of the
extracellular matrix LRR proteins. The cDNA of the novel LRR
protein was cloned through reverse transcriptase PCR from human femoral
head osteoarthritic cartilage, using primers corresponding to the 5'-
and 3'-untranslated regions of the assembled consensus sequence. The
mouse homologue was similarly identified through BLAST searches of the
mouse EST data base with the human sequence and cloned from mouse aorta
cDNA through reverse transcriptase PCR using primers based on the
EST sequences.
The human and mouse asporin sequences are shown in Fig. 3. The
predicted amino acid sequences of the two proteins are 90% identical.
The four amino-terminal cysteines show the
C-X3-C-X-C-X6-C pattern typical of decorin and biglycan (37), which clearly identifies
asporin as a member of the class I branch of the LRR proteins. Indeed,
like decorin and biglycan, asporin contains a highly conserved putative
propeptide sequence (amino acid residues 15-32). The putative
propeptide cleavage site conforms to the bone morphogenetic protein-1
cleavage site in biglycan (38). Alignment of the LRRs of asporin to
decorin and biglycan reveal a striking conservation in amino acid
sequence as well as repeat length (Fig.
4). Construction of a phylogenetic tree
of the extracellular matrix LRR proteins using Clustal W confirmed that
asporin belongs to the type I group, i.e. the decorin and
biglycan branch (Fig. 5). Unlike decorin
and biglycan, asporin contains no consensus glycosaminoglycan
attachment sites (Ser-Gly) in its amino terminus. There is, however,
one conserved consensus site for N-linked glycosylation (Asn281 and Asn275 in the human and mouse
asporin sequences, respectively). In contrast to all previously
identified extracellular matrix LRR proteins, asporin has a stretch of
13 aspartic acid residues in its amino-terminal region. Interestingly,
we found that the number of consecutive aspartic acid residues is
variable. When performing direct sequencing of the human asporin PCR
product, the sequence trace ended abruptly after the first 13 Asp
residues in the human sequence (Asp30). Sequencing
subcloned cDNA revealed that some clones contained an additional
Asp codon at this position. Indeed, several clones with varying numbers
of Asp residues (11-15) were identified in the human EST data base
(not shown). In addition, the genomic sequence of human asporin (see
below) coded for 15 contiguous Asp residues. Since the first-strand
cDNA used in cloning the human asporin cDNA was prepared from
tissue pooled from several individuals, we believe that this represents
a polymorphism. We found no corresponding variation in the Asp stretch
of the mouse protein, which comprises 7 Asp and 1 Asn residues.
A UniGene search of GenBankTM with AK000136 yielded
clusters Hs.10760, Rn.43324, and Mm.132637 for the human, rat, and
mouse homologues, respectively. The human asporin gene is located on chromosome 9q31.1-32, within the interval D9S1842-D9S196. This interval also contains the genes for the LRR proteins
osteoadherin/osteomodulin (OMD) and mimecan/osteoglycin
(OGN). A full-length asporin cDNA was assembled from our
sequence and a number of overlapping EST sequences to obtain the 5'-
and 3'-untranslated regions. BLAST searches of the high
throughput genomic sequence division of GenBankTM
identified a contig from chromosome 9 that contained the full asporin
sequence (GenBankTM accession number AL137848). The first
exon is also present in the overlapping contig AL157827. As shown in
Fig. 6, the asporin gene spans over 26 kilobases and consists of 8 exons. All the intron boundaries follow the
gt-ag rule and the introns show the same codon phases as the
corresponding introns in decorin and biglycan (Table
II). Indeed, the introns are positioned
in the exact corresponding locations as in decorin and biglycan (Fig. 4). It is presently unknown whether any additional alternatively spliced untranslated exons are present in the 5'-end of the gene, as is
the case in decorin. Like biglycan no consensus TATA box is found 5' of
the first exon of the asporin gene. A number of transcription factor
binding sites (including AP-1) were, however, identified immediately
upstream of the asporin exon 1 (not shown).
Asporin mRNA Expression--
Northern blot analysis
demonstrated that the asporin gene codes for a single message of 2.56 kilobases (Fig. 7). Using a commercial human tissue RNA filter (Multiple Tissue Northern) we found that the
highest amount of message was present in the heart tissue, followed by
the liver whereas the message was almost undetectable in the other
tissues. As articular cartilage is not included on the commercial
membrane it was not possible to directly compare asporin expression in
cartilage with that in other tissues. However, the Northern blot
analysis of human osteoarthritic cartilage total RNA showed a strong
hybridization signal. Considering that 10 µg of total cartilage RNA
(less than 1 µg of mRNA) was loaded, as compared with 2 µg of
poly(A)+ RNA from the other tissues, and that the
autoradiograms were exposed equally long, the expression of asporin may
well be higher in articular cartilage than in the other tissues
investigated.
A broader screening for the presence and relative abundance of asporin
was done by hybridization of a normalized mRNA dot blot (Human RNA
Master Blot) which covers adult and fetal tissues (Fig.
8). The asporin cDNA probe hybridized
with a wide range of human tissues with the highest signal levels in
aorta and uterus. Moderate expression levels were found in small
intestine, heart, liver, bladder, ovary, stomach, and in the adrenal,
thyroid, and mammary glands. Low asporin expression was observed in
trachea, bone marrow, and lung. There was a notable lack of signal in
the central nervous system as well as in spleen and thymus. A similar asporin expression pattern was observed in fetal tissues.
Characterization of the Asporin Protein--
Peptide mapping using
MALDI-TOF mass spectrometry was used to verify the previously obtained
protein sequence. After digestion with endoproteinases the peptide
masses obtained were compared with the peptides expected from the novel
protein. The identified peptides of asporin are listed in Table
III. The identified peptides cover 84%
of the mature protein sequence. The only major peptide missing in the
sequence is the absolute amino-terminal peptide (amino acids 33-62)
containing the consecutive Asp residues. This is probably due to the
extreme acidity of this peptide, which makes ionization and thus mass
spectrometry very difficult.
The protein has one potential N-glycosylation site at
Asn281. Treatment of asporin with N-glycosidase
F confirmed that the protein contains N-linked
oligosaccharides as shown by the change in its mobility on SDS-PAGE
after reduction (Fig. 9). This enzymatic deglycosylation resulted in the identification of the peptide containing Asn281 by MALDI-TOF mass spectrometry (see Table
III). Furthermore, this nonglycosylated peptide (amino acids 276-289)
was not observed in untreated protein digests. A deviation of 1 mass
unit was observed which can be explained by deaminidation of Asn to
Asp, a possible modification when running SDS-PAGE (39). The
N-linked glycopeptides could be detected both in reflector
as well as in linear mode. A mass accuracy of <100 ppm was obtained,
allowing identification of the oligosaccharide composition and putative
structures of the glycans linked to Asn281 in asporin
obtained from cartilage and meniscus (Table
IV). Apparently, there are no major
differences in the glycosylation pattern of the investigated tissues.
The composition of N-linked oligosaccharides at
Asn281 was confirmed using endoproteinase Glu-C. This
enzyme has a different cleavage pattern resulting in an easily detected
mass shift. This mass shift fits perfectly with the expected
glycopeptide masses.
The amino-terminal part of the protein contains a potential
O-glycosylation site at Ser54, which may be
substituted with an oligosaccharide since we do not find the core
peptide, neither using MALDI-TOF, nor electrospray ionization
quadropole time-of-flight mass spectrometers. However, two peptide ions
with m/z 4494.84 and 4203.76 that could
correspond to the amino-terminal part carrying an
O-glycosidically linked oligosaccharide were found. The mass
difference between the two peptides equals one
N-acetylneuraminic acid, which could be part of an
O-glycosidically linked oligosaccharide structure. This notion is further supported by the detection of an oxidized
methionine in these peptides, matching the presence of a
methionine residue in the amino-terminal peptide sequence (amino
acids 33-59). Assuming that we have 13 consecutive Asp residues in the
sequence, a corresponding glycomass of 1329 and 1038 is obtained for
the peptide with and without sialic acid, respectively.
Another approach to verify that the protein contains the proposed
sequence is to measure its intact mass. Since this could not be
obtained directly from the partially purified asporin sample, we
investigated the possibilities of extracting the protein directly from
the gel avoiding the problems of contamination, e.g. by
fibromodulin. An intact mass of 43,200 ± 500 Da was derived for
the nontreated protein after extraction from the gel. The large mass
deviation was due to difficulties in assigning the peak maximum caused
by the heterogeneity of glycosylation together with the fact that formylation reactions usually occur upon extraction of the gel with
formic acid. This results in peak broadening with a shift toward higher
masses. The theoretical mass of the mature protein with 13 consecutive
Asp residues in the amino-terminal is 39,609 Da. However, by adding a
mass for an N-linked oligosaccharide of ~2,000 Da and for
an O-linked oligosaccharide of ~1000 Da to the theoretical
mass, the observed mass range indicates that the suggested
amino-terminal is present in the tissue.
Asporin is a new member of the LRR protein family most closely
related to decorin and biglycan. The four amino-terminal cysteines show
the
C-X3-C-X-C-X6-C
pattern typical of the class I LRR proteins. Furthermore, like decorin
and biglycan asporin contains a putative propeptide with a conserved
cleavage site corresponding to the recognition sequence for bone
morphogenic protein-1, i.e. the enzyme shown to cleave
probiglycan (38). Moreover, the sequence and length of the LRR
repeats of asporin are more similar to those of decorin and biglycan
than to other members of the ECM LRR-repeat proteins. This is also
evident from the evolutionary tree of the LRR proteins. Finally, like
the decorin (40) and biglycan (41) genes, the human asporin gene is
divided into 8 exons. The introns are inserted in the coding sequence
at exactly the corresponding positions to those of decorin and biglycan.
The eight exons of the human asporin gene span 26 kilobases on
chromosome 9q31.1-32. It is not yet clear if the asporin gene also
contains an additional alternatively spliced exon 1, as does the
decorin gene (40). Like in the biglycan gene (41), no TATA box was
found in the 5'-flanking region of exon 1 of asporin. We did, however,
locate a number of recognition sites for transcription factors in the
400 base pairs upstream of the deduced transcription start.
The extracellular matrix LRR protein genes appear to be organized in
clusters of four. Decorin, lumican, keratocan, and epiphycan (class I,
II, II, and III, respectively) map to chromosome 12q23. Asporin,
osteoadherin, and mimecan (class I, II, and III, respectively) are
found on chromosome 9q32. Interestingly, ECM2, a gene
encoding a LRR protein containing an amino-terminal von Willebrand
factor repeat, has been located between asporin and osteoadherin (42). Fibromodulin, PRELP, and opticin (class II, II, and III, respectively) locate to chromosome 1q32. Biglycan (class I) is unique in not being
part of such a cluster but rather found in isolation on chromosome X. It appears that several duplications have occurred during evolution,
resulting in the clustered organization of the LRR genes. The biglycan
gene may then have relocated to chromosome X. Alternatively, four more
LRR protein genes remain to be identified, one on chromosome 1 and
three on chromosome X.
The asporin amino-terminal extension is unusual in containing an
extended stretch of aspartate residues. Messenger RNAs very similar to
asporin were recently identified in the zebrafish and the cichlid
Oreochromis (43). These proteins (referred to as biglycan-3
by the authors) belong to the class I LRR proteins based on the
amino-terminal cysteine spacing and the amino acid sequence of the
LRRs. Stretches of aspartic acid residues in the amino-terminal
extensions clearly identify these proteins as fish homologues of
asporin. Unlike in human asporin, the mouse and the two fish
polyaspartate sequences are interrupted by other amino acid residues.
Nevertheless, the conservation of a number of aspartates and glutamates
in this region in the amino terminus suggests an important function for
this negatively charged amino acid cluster.
In contrast to decorin and biglycan, asporin is not a proteoglycan. It
contains no consensus sequences for glycosaminoglycan attachment
between the propeptide and the amino-terminal cysteine motif, whereas
decorin (1) and biglycan (2) have one and two such Ser-Gly motifs,
respectively. There is, however, a conserved consensus sequence for
asparagine-linked glycosylation (Asn281 in the human
sequence). This was confirmed by N-glycosidase F treatment,
which resulted in a mobility shift on SDS-PAGE and allowed
identification of the Asn281-containing peptide by mass
spectrometry. The single N-glycosidically linked
oligosaccharide shows variability in structure, although all variants
represent typical N-linked structures. Whether the variability results from different tissue compartments or whether all
variants are present at a given location in the tissue is not known.
The fact that the protein analyzed was extracted from a pool of tissue
from several donors can of course also contribute to the observed
variation. Additional data on structure-function relationships in
relation to oligosaccharide variability may provide important
information on the role of such substituents.
The protein contains an additional putative glycosylation site
(Ser54 in the human sequence) that appears to be
substituted with an O-glycosidically linked
oligosaccharide. O-Linked glycosylation has previously been
described in the amino-terminal extension peptides of epiphycan and
opticin. It remains to be elucidated whether oligosaccharide
substituents in this region may modify the properties of this structure.
The role of the propeptides of class I LRR proteins is unclear. It has
been implied that this sequence affects the glycosaminoglycan structure of decorin (44) and biglycan (45). The presence of a
conserved propeptide in asporin, which does not contain any glycosaminoglycan attachment consensus sequence, suggests that the
propeptides may have other primary functions.
Asporin mRNA is expressed in a number of different tissues,
including articular cartilage. Beside cartilage, the highest expression levels were found in aorta and uterus, suggesting expression by smooth
muscle cells. Indeed, intermediate expression levels were detected in
other tissues with high content (large abundance) of smooth muscle cells.
The functional implications of the protein are not clear. However, in
attempts to separate the protein from fibromodulin in a collagen
co-precipitation assay, both proteins appeared to bind to collagen.
This would be in analogy with properties of other LRR proteins of the
type containing 10-11 repeats where most members have been shown to
bind tightly to collagen with equilibrium dissociation constants in the
nanomolar range.
Extended stretches of aspartic acid residues like in asporin are
unusual. Osteopontin, however, a prominent component of the mineralized
extracellular matrix of bone and teeth, has a polyaspartic acid
sequence in the center of the core protein (46). This protein binds to
hydroxyapatite and may have a role in bone mineralization (47). Whether
asporin also has a role in mineral deposition is not clear. In this
context the increased synthesis of the protein in early osteoarthritis
is of interest, particularly in view of the frequently altered
deposition of calcium phosphate in this disorder (reviewed in Ref. 48).
A similar analogy may be drawn between the polyglutamate sequences
found in epiphycan (9) and in bone sialoprotein (49). The polyglutamate
stretch of the latter protein has been shown to nucleate hydroxyapatite
crystal formation (50, 51). Another putative role for asporin would be
to involve the polyanionic stretch in interactions with other matrix
constituents in analogy with fibromodulin and lumican and also
potentially reminiscent of the glycosaminoglycan chains of decorin and
biglycan, the closest family members. This opens up possibilities for
interactions stabilizing the collagen network in the tissue.
The other two members of the family decorin and biglycan have been
shown to bind growth factors, particularly transforming growth
factor-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP
by using the Random Primed DNA labeling kit (Roche Molecular Biochemicals). Hybridization and washing of the membranes were according to the manufacturer's instructions. The membranes were allowed to expose x-ray film (Biomax MS, Kodak) or analyzed by the
Bas2000 phosphoimaging system (Fuji).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
67 kDa). The proteins in this
latter pool were fractionated by DEAE ion exchange chromatography, where asporin was observed in the fractions also containing
fibromodulin. These fractions were then chromatographed on a
Q-Sepharose column at low pH. Although asporin and fibromodulin still
coeluted, they were separated from other proteins in the pool. We then
tried to separate asporin from fibromodulin on a Mono Q column using elution at low pH with a linear NaCl gradient. Again the two proteins eluted together with fibromodulin as the predominant component (Fig.
1).
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Fig. 1.
Partial purification of asporin from
articular cartilage. A, elution profile after Mono Q
anion exchange chromatography. B, SDS-PAGE analysis of some
of the fractions after reduction on a 4-16% gradient gel. The gel was
stained for proteins with Coomassie Brilliant Blue. Molecular mass
markers are indicated on the left and the position of
asporin by an arrow.
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Fig. 2.
Partial purification of asporin from
meniscus. A, elution profile after gel filtration on
tandemly arranged columns of Superdex 200 and Superose 6. B,
SDS-PAGE analysis of some of the fractions after reduction on a 4-16%
gradient gel. The gel was stained for proteins with Coomassie Brilliant
Blue. Molecular mass markers are indicated on the left and
the position of asporin by an arrow.
Identification of peptide sequences
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Fig. 3.
Human and mouse asporin sequences. The
cDNA and deduced amino acid sequences of human and mouse asporin
are shown. Peptide sequences obtained by Edman degradation are
underlined. The arrow and arrowhead
indicate the putative start of the propeptide and the mature protein,
respectively. Nucleotides in boldface flank the positions
where introns are inserted in the genomic human asporin sequence.
Asterisks highlight cysteine residues, and the
glycan-substituted asparagine residue is indicated by
N.
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Fig. 4.
Alignment of asporin, decorin, and
biglycan. Human asporin, decorin, and biglycan amino acid
sequences are aligned. Cysteines are marked with C, and the
glycosaminoglycan attachment sites on decorin and biglycan are
underlined. The conserved residues of the LRRs are shown in
boldface. The residues where introns are positioned within
the codons in the genomic sequences are marked with 0, 1, or 2 indicating the codon phase.
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Fig. 5.
Phylogenetic tree of extracellular matrix LRR
proteins. The tree was constructed with Clustal W. Roman
numerals indicate the branches of the extracellular matrix LRR
protein family.
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Fig. 6.
The human asporin
gene. The map shows the human asporin gene (ASPN)
drawn to scale. Filled boxes indicate the coding region of
the mRNA. The translation start and stop codons are indicated by
ATG and TAA, respectively. ASPN has been approved by the
Hugo Gene Nomenclature Committee.
Exon-intron boundaries in the human asporin gene
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Fig. 7.
Northern blot analysis of asporin
expression. Total RNA (10 µg) isolated from human osteoarthritic
articular cartilage and 2 µg of poly(A) mRNA from different human
tissues were hybridized with a 32P-labeled asporin cDNA
probe and detected by autoradiography. The positions of the RNA size
standards are indicated on the left. kbp,
kilobase pair(s).
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Fig. 8.
Expression of asporin mRNA in different
human tissues. A Human RNA Master Blot was hybridized with the
32P-labeled asporin cDNA probe. A,
autoradiography of the hybridization signal. B, diagram
showing the type and position of poly(A)+ RNAs.
C, PhosphorImager quantification of the hybridization
signal.
Peptides from asporin identified by MALDI-TOF mass spectrometry
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Fig. 9.
Asporin carries N-linked
glycans. Partially purified asporin from cartilage and meniscus
was subjected to N-glycosidase F digestion (+) or taken
through the same procedure without enzyme addition ( ). Products of
the digestions were resolved after reduction on a 4-16%
polyacrylamide gradient gel. The gel was stained for proteins with
Coomassie Brilliant Blue. Molecular mass markers are indicated on the
left and arrows point to asporin.
Composition and putative structures of N-linked glycans on human
asporin
, GlcNAc;
, HexNAc;
, mannose;
, hexose;
, fucose.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(52). Whether also asporin has this capacity remains to be demonstrated.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Thomas Larsson and Carol Nilsson at the Department of Medical Chemistry, Göteborg University, Sweden, for help with electrospray ionization quadropole time-of-flight mass spectrometry.
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FOOTNOTES |
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* This work was supported by the Medical Faculty of Lund University, the Swedish Medical Research Council, IngaBritt and Arne Lundbergs Research Foundation, Greta and Johan Kocks Stiftelser, Konung Gustaf V:s 80-årsfond, Reumatikerförbundet and Alfred Österlunds Stiftelse.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) AF316824 and AF316825.
§ Contributed equally to the results of this work.
** To whom correspondence should be addressed. E-mail: dick.heinegard@medkem.lu.se; Tel.: 46-46-222-8571; Fax: 46-46- 211-3417.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M010932200
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ABBREVIATIONS |
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The abbreviations used are: LRR, leucine-rich repeat; GdnHCl, guanidinium hydrochloride; HPLC, high-pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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