From the Graduate School of Biomedical Sciences,
University of Texas, Houston, Texas, 77030, the § Center for
Extracellular Matrix Biology, Texas A&M University System Health
Science Center, Institute of Biosciences and Technology, Houston, Texas
77030, the ¶ Department of Cell Biology, University of Alabama,
Birmingham, Alabama 35294, and the
Department of Medical
Genetics, University of Texas M.D. Anderson Cancer Center,
Houston, Texas 77030
Received for publication, December 14, 2000
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ABSTRACT |
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We have discovered a new member of the class I
small leucine-rich repeat proteoglycan (SLRP) family which is distinct
from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the
molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is
probably not a proteoglycan. The tissue expression of
asporin partially overlaps with the expression of
decorin and biglycan. During mouse embryonic
development, asporin mRNA expression was detected
primarily in the skeleton and other specialized connective tissues;
very little asporin message was detected in the major
parenchymal organs. The mouse asporin gene structure is
similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome
9q22-9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2,
osteoadherin, and osteoglycin. Further analysis
shows that, with the exception of biglycan, all known SLRP
genes reside in three gene clusters.
The small leucine-rich repeat
proteoglycans (or
SLRPs)1 are a group of
extracellular proteins (ECM) that belong to the leucine-rich repeat
(LRR) superfamily of proteins (1, 2). The LRR is a protein folding
motif composed of 20-30 amino acids with leucines in conserved
positions. LRR-containing proteins are present in a broad spectrum of
organisms and possess diverse cellular functions and localization (3).
The members of the SLRP subfamily have core proteins of similar size
(about 40 kilodaltons) that are dominated by a central domain composed
of 6-10 tandemly repeated LRRs. This domain is flanked by smaller,
less conserved N-terminal and C-terminal regions containing cysteines
in characteristic positions.
Most of the SLRP proteins are proteoglycans, and the SLRP gene family
has been subdivided into 3 classes based on similarities in overall
amino acid sequence, spacing of cysteine residues in the N terminus,
and gene structure. The previously identified class I members, decorin
(4) and biglycan (5), are the most closely related SLRPs based on amino
acid sequences; the human sequences are 57% identical. The core
proteins contain 10 LRRs, and the N-terminal regions of decorin and
biglycan are substituted with one and two chondroitin/dermatan sulfate
chains, respectively. The cysteine-rich cluster in the N terminus of
class I SLRPs has an amino acid spacing of
CX3CXCX6C. The
mouse decorin (6) and biglycan genes (7) contain
8 exons.
The class II members, fibromodulin (8), lumican (9), PRELP (10),
keratocan (11), and osteoadherin (12), have a pairwise amino acid
sequence identity between 37 and 55% and have a common gene structure
composed of three exons. The cysteine spacing in the N-terminal region
of class II SLRPs is identical (CX3CXCX9C)
but different from the other SLRP classes. The core proteins of class
II SLRPs contain 10 LRRs and (with the exception of PRELP) can be
substituted with N-linked keratan sulfate glycosaminoglycan chain(s).
The class III members, epiphycan/PG-Lb (13, 14), osteoglycin/mimecan
(15, 16), and opticin (17), have a pairwise amino acid sequence
identity between 35 and 42% and have a common gene structure composed
of either 7 or 8 exons. Class III SLRPs contain only 6 LRRs, and the
cysteine spacing in the N-terminal region is identical
(CX2CXCX6C)
for all members of this class. The recently identified opticin is
substituted with O-linked sialylated oligosaccharides, and
consequently is a glycoprotein rather than a proteoglycan. On the other
hand, osteoglycin/mimecan and epiphycan can be substituted with
N-linked keratan sulfate glycosaminoglycan chain(s) and
O-linked chondroitin/dermatan sulfate chain(s),
respectively. Many of the SLRP proteoglycans can be isolated from
tissues without attached glycosaminoglycans, suggesting that they are
"part-time" proteoglycans (11, 16, 18).
Several SLRP proteins display potent effects in vitro. For
example, recombinant decorin, biglycan, and fibromodulin bind to transforming growth factor- The SLRPs have been shown to interact with a variety of extracellular
matrix proteins, such as collagens (24), fibronectin (25), and
thrombospondin (26), as well as serum proteins, heparin cofactor II
(27) and C1q (28). Biochemical assays have demonstrated that decorin
(29), fibromodulin (30), and lumican (31) bind to collagens in
vitro and modulate collagen fibril formation. Morphological
analysis of mouse "knockouts" demonstrates that decorin (32),
fibromodulin (33), and lumican (34), respectively, are necessary for
normal collagen fibril formation in specialized connective tissues of
skin, tendon, and cornea. Therefore, a role for SLRPs in collagen fiber
formation is clearly established both in vivo and in
vitro. Biglycan-null mice exhibit a mild osteoporosis-like
phenotype (35). However, it is not known if this phenotype is the
consequence of a primary defect in collagen fiber formation. Recently,
patients with cornea plana 2 (MIM 217300) were shown to have mutations
in the keratocan gene, a class II SLRP family member
(36).
Nucleotide sequencing of a human bacterial artificial chromosome (BAC,
RPCI11-917O5), and contigs of overlapping BAC clones revealed that
four SLRPs genes (decorin, lumican, keratocan, and epiphycan/PG-Lb) are physically linked on human chromosome
12q (36). Previous genetic linkage studies in the mouse suggested that
decorin, lumican, and epiphycan map together in a
cluster in close proximity to the Mgf gene on mouse
chromosome 10, and these genes are deleted in mice that have large
deletion mutations at the Steel locus (37).
Chromosomal localization of three other SLRPs, fibromodulin
(38), PRELP (39), and opticin (40, 41) to human
chromosome 1q32 by fluorescent in situ hybridization
analysis and/or radiation hybrid mapping raised the possibility that
these SLRP genes may also be physically linked. A computer homology
search of the genome data bases was, therefore, initiated to look for
additional, unidentified SLRP family members that might be associated
with these clusters or a yet unidentified cluster.
In this study, we identify a novel SLRP family member that belongs to
the class I subfamily and is closely related to biglycan and decorin.
We have named this new SLRP asporin due to the unique aspartate stretch at the N terminus of the translated open
reading frame. We report the molecular cloning of the full-length mouse and partial human cDNA and investigate asporin mRNA
expression in mouse embryonic development. In addition, we have
determined the mouse and human asporin gene structure and
discovered that the human asporin gene is part of a SLRP
gene cluster on human chromosome 9q21.3-9q22 that also contains
osteoadherin, osteoglycin/mimecan, and a gene encoding
another LRR-containing protein, ECM2 (42).
Materials--
Chemicals and supplies were purchased from Sigma,
Fisher, and Intermountain Scientific. Total RNA was extracted from
confluent mouse ATDC5 cells (43) by using the QIAshredder kit and
purified with the RNAeasy mini kit (Qiagen, Santa Clarita, CA). Total
human heart RNA was obtained from Ambion (Austin, TX). Reverse
transcriptase used was SuperScript II (Life Technologies, Inc.,
Rockville, MD), and first strand cDNA was synthesized by 5' and
3'-rapid amplification of cDNA ends (5' and 3' RACE) using
SMARTTM (Switch Mechanism
At the 5' end of RNA Templates)
technology (CLONTECH, Palo Alto, CA). The QIAPREP
spin miniprep kit and the QIAEX II gel extraction kit (Qiagen) were
used to purify DNA. The plasmid vector used in subcloning was
pBluescriptKS (Stratagene, La Jolla, CA). Nucleotide sequencing
reactions were performed at a University of Texas sequencing core
facility. Oligonucleotides were purchased from Sigma-Genosys
(Woodlands, TX). Polymerase chain reactions (PCR) were performed with
one of the following polymerases: Taq polymerase (Life
Technologies, Inc.), Advantage 2 Polymerase Mix
(CLONTECH), Pfu Polymerase (Stratagene),
or Takara LA TaqTM polymerase (Takara
Biomedicals, Japan). PCR products were ligated into a TA-cloning vector
(44) or the pGEMTM T-easy vector system (Promega). The
mouse poly(A)+ multiple tissue Northern blot was from
OriGene Technologies, Inc. The mouse BAC library was from
Research Genetics (Huntsville, AL). Radioisotopes
[ Cloning Full-length Mouse cDNA--
The full-length mouse
cDNA was obtained by aligning nucleotide sequences of overlapping
PCR products. RNA was extracted from mouse ATDC5 cells and first-strand
cDNA was synthesized by reverse transcriptase SuperScript II (Life
Technologies, Inc.) using the reagents provided in the
SMARTTM RACE cDNA amplification kit
(CLONTECH). The gene-specific primers for mouse
asporin were designed from nucleotide sequences contained in
expressed sequence tags (ESTs) that were publicly available in
GenBankTM data bases. For 5' RACE reactions, reverse
oligonucleotide primers were designed against mouse EST
GenBankTM accession number AI 006670 (MS ASP RV 406, 5'-AGGCTTCACTGGCTCTTTCGTAGGAAAAAG; and MS ASP RV 343, 5'-CGTCATCATCTGTGTCTTCCATATCCTTC). For 3' RACE reactions, forward
oligonucleotides were designed against mouse EST GenBankTM
accession number AA980962 (MS ASP FW 983, 5'-CTTGAAGATCTTAAACGGTACAGGGAACTGC; and MS ASP FW 1077, 5'-CCACGTGTGAGAGAGATACACTTGGAACAC). First round PCR conditions for 5'
and 3' RACE were as follows: the template-RACE ready cDNA; gene
specific oligonucleotides, MS ASP RV 406 (5' RACE) and MS ASP FW 983 (3' RACE), 25 cycles (5 s 94 °C, 10 s 60 °C, 2 min
72 °C). First round PCR products were diluted and used as template
in a second round "nested" PCR as recommended by the instructions
provided with the kit. Nested PCR products for 14 clones harboring 5'
RACE products were resolved electrophoretically on an ethidium
bromide-stained 1% agarose gel. Five of the plasmids containing 5'
RACE products of different sizes were sequenced in both directions
using the T3 and T7 primers. The largest fragment was called p329, and
was used in subsequent Northern, Southern, and in situ
hybridization experiments. Analysis of this sequence in
GenBankTM failed to reveal any homology to known cDNA
or genomic sequences. Also, two clones harboring 3' RACE products of
identical size were sequenced in both directions using the T3 and T7 primers.
Since the complete open reading frame (ORF) for mouse asporin could not
be determined by alignment of overlapping mouse ESTs, two
primers (Ms Start FW, 5'-CGCGGATCCAAACCCTTCTTTAGCCCTTCCCAC; Ms Stop RV,
5'-CGCGGATCCTTATTTTCCAACATTCCCAAGCTG) were designed to amplify by
PCR the mouse asporin ORF (template of mouse RACE-ready cDNA,
Pfu polymerase, 20 cycles (20 s 94 °C, 30 s
60 °C, 2 min 72 °C). The amplified mouse asporin ORF was digested
with BamHI restriction enzyme and ligated to
BamHI-cleaved pBluescript KS+. The resulting subcloned ORF
plasmid was sequenced with three primers: T3, T7, and MS FW 775 (5'-GGACACGTTCAAGGGAATGAATGC) to determine the open reading frame of
mouse asporin.
Human Partial cDNA--
Human heart RNA (Ambion) was reverse
transcribed to first strand cDNA by using the SMARTTM
RACE cDNA amplification kit (CLONTECH). A
partial human cDNA was obtained that contained the open reading
frame and the 5'-untranslated region. The gene-specific primers for
human asporin were designed from nucleotide sequences contained in
human ESTs, AK000136, FLJ20129, and AI539334. PCR conditions were as
follows: template-human RACE ready cDNA; gene-specific
oligonucleotides, HU ASP RV STOP (5'-CCGCTCGAGTTACATTCCAAAGTTCCCAAGCTGAAC) and HU ASP RV 1503 (5'-ACTGCAATAGATGCTTGTTTCTCTCAACCC), 30 cycles (5 s 94 °C, 10 s
60 °C, 2 min 72 °C). PCR-amplified products from first round 5'
RACE reactions were sequenced.
Northern Hybridization--
Three consecutive Northern
hybridizations were performed on a single mouse multi-tissue
poly(A)+ RNA blot (Origene). DNA fragments of mouse
asporin, biglycan, and decorin cDNAs were
random-labeled in separate Northern hybridizations. The asporin probe
(p329) is a 478-base pair (bp) PCR-amplified 5' RACE product that
encodes for the 5' end of the mouse asporin cDNA that includes the
5'-untranslated region (region of cDNA that is encoded by exon I)
and a portion of the open reading frame (a fragment of the cDNA
that is encoded by exon 2). The biglycan probe (p368) is a 731-bp
PCR-amplified fragment that encodes for a portion of the
3'-untranslated region of mouse biglycan (7). PCR parameters
are as follows: primers are MS BGN3, 5'-CCTGAGACCCTGAACGAACTTCACCTGG, and MS BGN4, 5' CGGTGGCAGTGTGCTCTATCCATCTTTCC; template is mouse RACE-ready cDNA as described previously, 30 cycles (20 s 94 °C, 20 s 60 °C, 1 min 72 °C). The decorin probe (p280) is a
399-bp XbaI/HindIII fragment from the 3' end of
the mouse decorin open reading frame (6).
DNA probes were random-labeled by using the Strip-EZTM kit
(Ambion). Following an overnight hybridization at 42 °C, the blot was washed under high stringency (1% SDS, 2 × SSC) at 65 °C.
The same blot was subjected to 3 separate Northern hybridizations in
this order: asporin hybridization, strip blot of probe, decorin hybridization, strip blot of probe, and biglycan hybridization. Radiolabeled probes were removed using Ambion's Strip-EZ technology between consecutive hybridizations. The wash conditions following each
hybrization are as follows: asporin, 2 washes of 5 min, film exposure
16 h; decorin, 2 washes of 30 min, film exposure 2 h; biglycan, 3 washes of 10 min, film exposure 7 h. Radioactive
Northern blots were exposed to Kodak film (X-Omat AR).
Mouse Gene Structure--
A PCR-amplified 5' RACE product (p329)
described earlier was used as a radiolabeled probe in a Southern
hybridization to screen a mouse genomic BAC library (Research
Genetics). After an overnight hybridization at 65 °C, the blots were
washed under high stringency (1% SDS, 2 × SSC) at 65 °C
(3 × 15 min) and exposed to x-ray film. Two BAC clones
corresponding to positive signals seen on the developed film were
purchased from Research Genetics.
After annotation of the genomic nucleotide sequence from BAC number
AL137848, the exon/intron boundaries of the human asporin gene were determined by aligning homologous regions of this sequence with sequence from available human ESTs. Assuming that the mouse gene
structure is similar to the human gene structure, the regions in the
mouse cDNA that encoded for exons in the mouse gene were predicted.
Forward and reverse primers were designed from regions in the mouse
cDNA that were predicted to encode for consecutive exons
(i.e. forward primer in exon 1, reverse primer in exon 2). With purified mouse BAC DNA as template, such primer pairs were used in
long distance PCR reactions to amplify the introns of the mouse
asporin gene. Amplified fragments were separated by electrophoresis on an ethidium bromide-stained 0.8% agarose gel to
judge intron size and were subcloned using the pGEMTM
T-easy vector system. Subcloned fragments were sequenced with the T7
and SP6 primers to determine the sequence of the mouse exon/intron
boundaries. The primer pairs used to amplify the introns of the mouse
asporin gene are as follows: intron 1: Asp Ex1 Fw38, 5'-GCACATAGAGGCTGTTAGGAGGGCTGG; Asp Ex2 Rv343,
5'-CGTCATCATCTGTGTCTTCCATATCCTTC; Intron 2: Asp Ex2 Start,
5'-CGCGGATCCAAACCCTTCTTTAGCCCTTCCCAC; Asp Ex3 Rv, 5- 5'-CGAGTATCAAATGGAATGTTGTTTGGAACCG; Intron 3: Asp Ex3 Fw,
5'-GCGTTCCAAACAACATTCCATTTGATACTCG, Asp Ex4 Rv,
5'-GTTGGTTGTGGGATAAATATAGCCTTCTC; Intron 4: Asp Ex4 Fw,
5'-GAGAAGGCTATATTTATCCCACAACCAAC, Asp Ex5 Rv,
5'-CCCTGGTTCTATCCCGTTGTTCTCAAGAGG; Intron 5: Asp Ex5 Fw,
5'-CCTCTTGAGAACAACGGGATAGAACCAGGG, Asp Ex6 Rv,
5'-CTTTGCAGTTCCCTGTACCGTTTAAGATC; Intron 6: Asp Ex6 Fw,
5'-CTTGAAGATCTTAAACGGTACAGGGAACTGC, Asp Ex7 Rv,
5'-GAGTTCCAAGTGTATCTCTCTCACACGTGG; Intron 7: Asp Ex7 Fw, 5'
CCACGTGTGAGAGAGATACACTTGGAACAC, Asp Ex8 Stop,
5'-CGCGGATCCTTATTTTCCAACATTCCCAAGCTG. Cycling parameters are as
follows: template, 25 ng of purified BAC DNA, primers at a final
concentration of 1 µM, Takara LA
TaqTM polymerase, 25 cycles (10 s, 98 °C, 6 min 66 °C).
Human Gene Structure of Asporin--
During annotation of the
nucleotide sequence from BAC number AL137848, the exon/intron
boundaries of the human asporin gene were established by
aligning homologous regions of the genomic sequence with available
human ESTs (i.e. AK000136, FLJ20129, and AI539334), and
determining the regions in the human cDNA that encoded for exons in
the human gene. The ENSEMBL web site on the Sanger Center server
confirmed the location of an open reading frame (ENST00000026531) in
BAC number AL137848 that we have named asporin.
RNA in Situ Hybridization--
In situ hybridizations
were performed on sections from different stages of mouse embryos.
Sections were hybridized with [35S]UTP-labeled antisense
or sense RNA probes generated from the plasmid p329 that contains the
extreme 5' end of the mouse asporin cDNA (1-478 bp).
Pregnant C57Bl mice were sacrificed on various days post-coitus (dpc),
embryos were harvested, rinsed in phosphate-buffered saline/diethyl
pyrocarbonate, and fixed in 10% (v/v) formalin in phosphate-buffered
saline for 2-25 h. The fixed tissues were dehydrated through a series
of increasing ethanol concentrations and then cleared in xylene before
being embedded in paraffin. Sections of 7-µm in thickness were
mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh,
PA). Dimineralization was performed by placing the tissue into a
solution of 0.1 M Na phosphate, pH 6.5, containing 0.26 M EDTA for 2-3 days at room temperature with several
changes in between. The tissue was rinsed in diethyl pyrocarbonate/H2O, then dehydrated through a graded series
of ethanol concentrations, and embedded and sectioned as described for
embryonic tissue.
In situ hybridization was performed essentially as described
previously (45). Hybridization was carried out at 50 °C for 16-17
h. Two high stringency washes were performed at 55 °C in 50%
formamide, 2 × SSC for 20 min each. Autoradiography was carried out using NTB-2 Kodak emulsion. The slides were exposed for 16 h
to 7 days at 4 °C. Photomicrographs were taken using both bright and
dark-field optics.
Human and Mouse Asporin cDNA--
A novel member of the class
I SLRP gene family has been identified and named asporin. The cDNA
sequence of human decorin (4) was used as a query to search
the human dbEST data base of GenBankTM using the BLAST-N
algorithm. At the time, the nucleotide sequence of several human
expressed sequence tags (ESTs AK000136, FLJ20129, and AI539334)
exhibited strong homology to the nucleotide sequence of the class I
SLRPs, but individual ESTs did not encode a full-length ORF.
Furthermore, alignment of overlapping human ESTs did not establish a
complete, "in-frame" ORF, so human genomic sequence from BAC
AL137848 was used to correct the sequencing errors present in the human
ESTs and to fill any gaps that were missing from the alignment of
overlapping ESTs. These results revealed an open reading frame of 380 amino acids and were later confirmed experimentally by sequencing PCR
products generated from 5' RACE reactions that used reverse transcribed
human heart RNA (first-strand cDNA) as template. The transcription
start site of human asporin, as well as the open reading
frame and 5'-untranslated region, were determined by nucleotide
sequencing of 5' RACE products obtained with the SMART cDNA
amplification kit. Several 5' RACE products were resolved
electrophoretically on an ethidium bromide-stained 1% agarose gel
(data not shown). No attempts were made to clone the 3'-untranslated
region of human asporin.
An open reading frame of mouse asporin could not be obtained from
overlapping mouse ESTs thus leaving a central gap in the computer-derived sequence. Furthermore, the genomic sequence of mouse
asporin was not available in the public data bases.
Therefore, oligonucleotide primers were designed from nucleotide
sequences present in available 5' and 3' mouse ESTs, and a conventional PCR strategy was used to amplify a PCR fragment that bridged the gap in
the open reading frame. The 5' and 3' cDNA ends of mouse asporin cDNA were determined by sequencing 5' and 3'
RACE PCR products, and the transcriptional start site of the mouse
asporin message was determined by sequencing the largest PCR-amplified 5' RACE product. This RACE product was subcloned and used as a probe
for Northern hybridization, Southern hybridization, and RNA in
situ hybridization experiments.
A full-length mouse cDNA of 2357 nucleotides was generated by
aligning the nucleotide sequences of overlapping PCR reactions and is
shown in Fig. 1. The mouse asporin ORF
(including the stop codon) is 1122 bp. The 5'-untranslated and
3'-untranslated regions of the mouse cDNA are 305 and 930 bp,
respectively. A noncanonical polyadenylation signal sequence of AATAA
(cDNA 2326-2331) is present very close to the end of the
3'-untranslated region of the mouse cDNA. The translated open
reading frame encodes a protein of 373 amino acids that contains a
putative signal peptide sequence of 15 amino acids assigned using the
Signal P V1.1 program (46). The central domain is composed of an array
of 10 LRRs, and each LRR contains 24 amino acids with the central
consensus sequence X-L-X-X-L-X-(L/I)-X-X-N-X-(L/I).
The LRR domain is flanked by smaller cysteine containing N- and
C-terminal regions. A cluster of four cysteines (C) in the N-terminal
region conform to the amino acid spacing of
CX3CXCX6C that
is also found in the other class I SLRP members. The C-terminal region
contains two cysteines with 32 intervening amino acids, and this exact
spacing is also found in decorin and biglycan.
In contrast to decorin and biglycan, a serine/glycine dipeptide
consensus sequence for O-linked glycosaminoglycan
substitution is not present in the translated open reading frame of
asporin. One putative N-linked oligosaccharide attachment
site, located between LRRs 8 and 9, can be found in the asporin ORF. A
stretch of 14 amino acids N-terminal to the first cysteine cluster
contains 10 aspartic acid residues. A similar stretch of
acidic residues is not present in the other class I SLRPs, and hence
the new member was named asporin.
Comparison of Human and Mouse Asporin ORFs with Decorin and
Biglycan--
The translated human asporin ORF was aligned with mouse
asporin, mouse biglycan (7), and mouse decorin (6) ORFs using the
CLUSTAL program (Identity) contained in the MacIntosh MacVector software package (version 6.0.1) and is shown in Fig.
2. The mouse and human asporin ORFs are
91% identical. The major difference is located in an acidic stretch in
the N-terminal region of the translated human ORF (380 aa) that is 7 amino acids longer than the corresponding acidic stretch in the mouse
ORF (373 aa), and hence accounts for the size difference between the
two ORFs. The human ORF, like the mouse, also lacks a dipeptide
serine/glycine consensus sequence for glycosaminoglycan substitution,
but contains the same potential N-linked glycosylation
substitution site located between the eighth and ninth LRR.
The three mouse class I SLRPs have remarkably similar amino acid
sequences. The translated ORF of mouse asporin (373 aa) is most
homologous to that of mouse biglycan (369 aa) with 52% identical and
an additional 17% similar residues. The amino acid identity between
mouse asporin (373 aa) and mouse decorin (354 aa) is slightly less at 49% with an additional 19% similar residues. The
amino acid identity between mouse biglycan (369 aa) and mouse
decorin (354 aa) is 54% with an additional 14% similar residues. The
region of lowest homology among the three mouse translated ORFs is
N-terminal to the first cysteine cluster. The aspartate-rich stretch of
asporin is contained in this region, as is the serine residue(s)
involved in O-linked glycosaminoglycan substitution of
decorin and biglycan.
Tissue Distribution of Class I SLRPS--
The mRNA expression
for the class I SLRPs is broadly distributed in mammalian tissues (see
Fig. 3). Three separate Northern hybridizations of a single mouse multitissue Northern blot using radiolabeled cDNA fragments of decorin (Fig. 3,
top panel), biglycan (Fig. 3, center
panel), and asporin (Fig. 3, bottom panel)
were performed. The Northern results for mouse biglycan and
mouse decorin confirm previously published work (6, 7). The
asporin probe recognized a single mRNA of 2.4 kb in the tissues
tested. The asporin mRNA is comparable in size to the
biglycan message of 2.4 kb and slightly larger than the
decorin message of 1.8 kb.
For the 12 organs that were represented in the mouse adult
multiple-tissue Northern blot, asporin message was
most prominent in the heart. Asporin message was also
detected in kidney, stomach, testes, and skin but only weakly in lung,
skeletal muscle, small intestine, and thymus. However,
asporin message in brain, liver, and spleen was virtually
undetectable at the longest exposure tested.
Similarities and differences in the relative RNA expression pattern of
the three genes were found. A message for all three genes was detected
in heart, kidney, skin, testes, and small intestine, but the message in
brain was extremely weak for all the genes. Biglycan message
in spleen and lung was fairly robust, yet asporin and
decorin message were very weak in these organs.
Asporin message was virtually undetectable in the liver, yet
expression of biglycan and decorin mRNA was observed in this organ.
Expression of Asporin in Mouse Development--
To obtain a more
complete picture of asporin expression, particularly during
mammalian embryonic development, we used RNA in situ
hybridization analysis of sagittally sectioned mouse embryos at
different stages of development. No asporin mRNA was
detected at the two earliest time points tested, 9.5 and 10.5 days dpc of mouse embryonic (ME) development (data not shown).
Asporin mRNA was detected at 12.5 dpc in the maxilla and
mandible (Fig. 4). At ME 12.5 dpc, a
groove forms between the lower surface of the anterior tip of the
tongue and the mandibular component of the first brachial arch. At this
stage, asporin message is absent from the tongue, but is present in the
mandibular (shown as an arrow in the 12.5 dpc panel) as well
as maxillary components of the first branchial arch. A signal is also
detected in the thoracic body wall adjacent to the heart. At ME 13.5 dpc, Meckel's cartilage is recognizable, and asporin
mRNA expression is detected in the mesenchyme lateral to Meckel's
cartilage, but not in Meckel's cartilage. Pronounced expression of
asporin is observed in the perichondrium of the humerus, ribs, and
scapula. At ME 14.5 dpc, the mesenchymal condensations lateral to
Meckel's cartilage are clearly positive, and the asporin expression
pattern appears as a "cusp" surrounding Meckel's cartilage. This
"cusp-like" area will eventually ossify and give rise to
intramembranous alveolar bone of the mandible. Asporin
expression is also found in the perichondrium surrounding the central
cartilaginous elements of the vertebrae. Weak asporin
expression is detected in dermal mesenchyme.
At ME 15.5 dpc (Fig. 5), sagittal
sections reveal a robust expression of asporin in the
perichondrium/periosteum of the long bones (i.e femur, tibia, and
fibula), some of the flat bones at the base of the skull (i.e sphenoid
bone), ribs, clavicle, and vertebrae. The intramembranous bones of the
maxilla and mandible (alveolar bone) are also positive for asporin. A
strong signal was observed in sagittal sections of the subcutaneous
muscles or panniculus carnosus of the thorax, trunk, and head/neck
(platysma muscle) region and are shown as arrows in Fig. 5.
Very little asporin message was detected in the major
parenchymal organs (with the exception of the large bronchi of the
lung). The strong expression of asporin in the perichondrium is
underscored in a sagittal section of the digits of a 15.5 dpc forelimb
(Fig. 5, panel C). Comparisons of dark and bright field
micrographs of the distal end of the third digit (magnification of
×20, panel D) suggests that asporin signal is prominent
throughout the perichondrium but not in differentiated cartilage.
Asporin RNA expression is prominent in the developing mouse
skeleton, particularly in the perichondrium/periosteum of
cartilage/bone, and is also found in other specialized connective
tissues such as tendon, sclera, the connective tissue sheath
surrounding muscle and dermis. Tendon expression of asporin at 15.5 dpc
is shown in Fig. 6 (panel A).
Expression of asporin in the sclera of the eye was first
detected at 15.5 dpc and stronger expression was detected at 17.5 dpc
(see Fig. 6, panel B). The section of the eye shown in Fig.
6, panel B, is from an albino BALB/c mouse (the sagittal
section of the 15.5 dpc embryo shown in Fig. 5 is from a C57Bl/6J
mouse). Parasagittal sections of the tongue at ME 18.5 dpc reveals that
the connective tissue layer of the lamina propria and the lingual
fascia ensheathing the skeletal muscle bundles of the tongue are
positive for asporin RNA expression (Fig. 6, panel
C). The positive signal for the fascia surrounding the skeletal muscle bundles appears as parallel striations in the center of the
section, and the myofibers are negative. Prior to embedding the 18.5 dpc embryos during the in situ hybridization protocol, the
skin is peeled away from the embryo to allow for adequate penetration
of fixatives. Fortuitously, some skin shavings remained on the slide
during the procedure, and positive signal for asporin was observed in
the dermis but not in the epidermis (Fig. 6, panel D).
Gene Structure of Asporin--
A mouse BAC was screened with an
asporin probe and two unique BAC clones were obtained. Purified BAC DNA
was used as template to PCR amplify the introns of the mouse
asporin gene in a long distance PCR strategy. The amplified
introns were resolved electrophoretically on an ethidium
bromide-stained 0.8% agarose gel (data not shown), and their sizes
were estimated (see Table I). The seven
introns were subcloned and the exon/intron junctions were sequenced
(see Table I). The mouse asporin gene spans about 23 kilobases and contains 8 exons.
The gene structure of human asporin (see Table I) was
determined by alignment of annotated nucleotide sequence from genomic BAC clone AL137848 with overlapping human ESTs. The human gene spans at
least 25 kb and also contains 8 exons. The size of the first exon was
determined by nucleotide sequencing the largest PCR-amplified product
from 5' RACE reactions. The size of the last exon was determined by
comparing all the overlapping asporin 3' ESTs available on the public
data bases and choosing the ones that were the longest in the 3' direction.
The gene size and structure of asporin in both species
examined are very similar. The largest intron for both genes is the first, whereas the smallest intron is the sixth. The codon phasing at
the exon/intron boundaries of the mouse and human genes is identical,
and the intron sizes are similar (Table I).
Asporin Is Part of a SLRP Gene Cluster on Human Chromosome
9--
The nucleotide sequence of three overlapping human BAC clones
(AL157827, AL137848, and AL354924) localized to chromosome 9q22-9q21.3
was subsequently annotated. A diagram depicting a 188-kb region of
three overlapping BAC clones is shown in Fig. 7 and reveals a cluster of 4 genes that
code for LRR-containing proteins: ECM2, asporin,
osteoadherin, and osteoglycin. It was necessary to
determine the location and orientation of a 16-kb contig located in the
center of BAC AL137848, between asporin and
osteoglycin, by performing bridging PCR reactions to
neighboring contigs. Three gaps within intronic sequences could not be
annotated and are shown as vertical dotted lines in Fig. 7.
Within the 188-kb region, the 4 genes are arranged in a head-to-tail
fashion with the same transcriptional orientation. The three SLRP genes
are physically linked and include one member from each SLRP class: asporin (class I)-osteoadherin (class
II)-osteoglycin/mimecan (class III). The 5'- and
3'-untranslated region of each gene was estimated by comparison with
nucleotide sequences contained in human ESTs that exhibited homology
specifically to the extreme 5' and 3' ends of the genes. The four genes
are also physically linked in the mouse.
We have discovered a new member of the class I subfamily of SLRPs
that we have named asporin. The size and amino acid sequence of the
asporin protein are remarkably similar to those of the core proteins of
the other members of the class I subfamily, decorin and biglycan.
Almost 70% of the residues in these proteins are identical or
conserved. Furthermore, they all contain 10 highly conserved LRRs in
the central region, and the number and amino acid spacing of the
cysteine residues in the N- and C-terminal domains are conserved. The
region of the class I proteins that is least similar lies N-terminal to
the first cysteine cluster. For the proteoglycans decorin and biglycan,
the serine/glycine dipeptide sequence(s) required for xylosyl transfer
and glycosoaminoglycan assembly are located in this region. Asporin
does not contain this dipeptide, thus asporin is probably not a
proteoglycan. Instead, asporin contains a stretch of aspartic acid
residues in this region. This acidic motif in human asporin is composed
of 18 residues, and in mouse asporin the acidic motif is 7 residues
shorter. Two other identified SLRPs, osteoadherin and epiphycan have
acidic regions. In epiphycan, this stretch is composed of glutamic acid residues and interestingly the acidic motif in human and bovine sequences (14, 47) is longer than the corresponding motif in the mouse
sequence (48). The C-terminal region of osteoadherin is rich in both
aspartic and glutamic acid residues (49). The importance of these
acidic motifs is unclear.
A dendrogram of the SLRP gene family is shown in Fig.
8. With the introduction of asporin, 11 members are contained in the three SLRP gene family classes. Class I
members include asporin, biglycan, and decorin; class II includes
osteoadherin, lumican, fibromodulin, PRELP, and keratocan; class III
includes osteoglycin/mimecan, opticin, and epiphycan/PG-Lb. Although
chondroadherin (50, 51) and the recently identified nyctalopin (52, 53)
have been granted membership to the SLRP gene family, they may have
diverged from the other three SLRP classes early in evolution because
their structures are significantly different from the conventional
SLRPs. Human ECM2 (42) also has a LRR domain that is 34% identical to
the corresponding domain in human decorin, but ECM2 is much larger and
structurally different from the conventional SLRPs. However, ECM2 is
physically linked to asporin on human chromosome 9 so it has been
included in the dendrogram.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in vitro (19), and
decorin can interfere with transforming growth
factor-
-dependent proliferation of Chinese hamster ovary
cells (20). Furthermore, injection of decorin into rats with
experimental glomerulonephritis curtailed the abnormal deposition of
matrix suggesting that decorin may affect transforming growth
factor-
activity also in vivo (21, 22). Recently, it has
been shown that decorin can down-regulate epidermal growth factor
receptor leading to growth suppression, and decorin may act as a
natural inhibitor of the epidermal growth factor receptor signaling
pathway (23).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP and [
-32P]dCTP were
purchased from PerkinElmer Life Sciences. Random labeling kits, T7
QuickPrime kit (Amersham Pharmacia Biotech), and DNA Strip-EZ (Ambion,
Austin, TX) kit were used. Hybridization fluids used were either
Rapid-Hyb (Amersham Pharmacia Biotech) or UltraHyb (Ambion). Imaging
film and emulsion were purchased from Kodak (X-Omat AR film and NTB-2 emulsion).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Complete cDNA of mouse asporin
(2357 bp) was determined by nucleotide sequence of overlapping 5'
RACE and 3' RACE reactions. The transcription start site was
determined by sequencing the largest amplified PCR fragment from 5'
RACE reactions. The numbering of the cDNA and the deduced amino
acid sequence (boldface and underlined) of the
open reading frame (373 aa) are given at the right margin.
The putative signal peptide of 15 amino acids was predicted using the
Signal P V1.1 program (46), and the cleavage site is shown as an
arrow. A stretch of 14 amino acids at the N terminus of the
ORF contains 11 acidic residues and is labeled as acidic stretch.
Cysteines in the N terminus with the amino acid spacing of
CX3CXCX6C are
boxed. Ten homologous LRRs with the central consensus
sequence
X-L-X-X-L-X-(L/I)-X-X-N-X-(L/I)
are underlined. The only putative N-linked
oligosaccharide attachment site is circled and is located
between LRR 8 and LRR 9 as predicted by the NetOGlyc 2.0 program.
Unlike decorin and biglycan, a serine/glycine dipeptide consensus
sequences for O-linked oligosaccharide substitution is not
present in the asporin ORF. Two cysteines in the C terminus are
boxed and are conserved among the SLRPs. A noncanonical
polyadenylation signal sequence of AATAA in the 3'-untranslated region
of the mouse asporin cDNA is depicted in boldface, underlined
letters.
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Fig. 2.
Multiple alignment of human asporin, mouse
asporin, mouse biglycan, and mouse decorin ORFs with the CLUSTAL
program (Identity) using the MacIntosh MacVector version 6.0.1 software. The human asporin ORF of 380 amino acids is 91%
identical to the mouse asporin ORF of 373 amino acids. The acidic
stretch at the N terminus of human asporin (amino acids 33-53 of ORF)
is 7 amino acids longer than the corresponding stretch in mouse
asporin, and hence, accounts for the size difference between the two
open reading frames. Neither the human nor the mouse asporin ORF
contains a potential O-linked glycosaminoglycan substation
site. However, both the human (asn number 282) and mouse (asn number
275) contain one potential N-linked glycosylation site. The
amino acid identity between mouse asporin (373 aa) and mouse biglycan
(369 aa) is 52% with an additional 17% similar residues. The amino
acid identity between mouse asporin (373 aa) and mouse decorin (354 aa)
is 49% with an additional 19% similar residues. The amino acid
identity between mouse biglycan (369 aa) and mouse decorin (354 aa) is
54% with an additional 14% similar residues. Using the Clustal W
(1.4) multiple alignment program, the number of identical amino acids
shared by the three mouse aligned sequences is 141 amino acids.
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Fig. 3.
Northern hybridization of a multiple tissue
mouse poly(A)+ blot (Origene) of 12 tissues with 3 different random-labeled DNA probes (top panel,
decorin; center panel,
biglycan; bottom panel,
asporin). The blot was commercially prepared so
that it contains about 2 µg of poly(A)+ RNA per lane, and
the tissues were taken from 9-10-month-old Swiss Webster mice (thymus,
8-12 weeks old). The RNA was loaded in 12 lanes (left to right) from
the following tissues: brain (1), heart (2),
kidney (3), liver (4), lung (5),
muscle (6), skin (7), small intestine
(8), spleen (9), stomach (10), testis
(11), and thymus (12). Markers on the
left side of the blot (dots representing Ambion
RNA Millenium marker) from bottom to top are 0.5, 1, 1.5, 2.0, 2.5, 3.0, 4, 5, 6, and 9 kb. The RNA message size of 1.8 kb for mouse
decorin (6) and 2.4 kb for mouse
biglycan (7) confirms previous reports. The mouse
asporin message is about 2.4 kb. Hybridization and wash
conditions are given under "Experimental Procedures."
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Fig. 4.
Sagittal sections of mouse embryos at three
different stages of development probed with an asporin antisense
riboprobe. Dark field micrographs are shown to the
right of the bright field images (magnification ×1).
Top panels (A), at 12.5 dpc, asporin
RNA is detected in the maxillary (Mx) and mandibular
(Mn) components (arrow) of the first branchial
arch and the thoracic body wall (Bw) adjacent to the heart.
Middle panels (B), at 13.5 dpc,
asporin is detected in the perichondrium of the scapula
(Sp), ribs (Ri), and humerus (Hu).
Asporin mRNA is not detected in Meckel's cartilage, but
instead the mesenchymal cells lateral to Meckel's cartilage (shown
with arrow). Bottom panels (C), at
14.5 dpc, asporin expression is detected in the perichondrium of the
vertebrae (Ve). Condensing mesenchymal cells in the mandible
surrounding Meckel's cartilage are positive for asporin RNA, and this
cusp-like expression pattern is highlighted by the arrow.
Strong expression of asporin is maintained in the mandible and maxilla,
at future sites of intramembranous bone formation. Weak expression is
also detected in the dermal mesenchyme (Dm) at 14.5 dpc.
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Fig. 5.
Sagittal sections of a mouse embryo at 15.5 dpc probed with asporin sense and antisense riboprobes (magnification
×1). The section hybridized with the antisense riboprobe is shown
at the top left (Panel A), and a serial section
that was hybridized with a "control" sense probe is shown at the
top right (Panel B). Asporin RNA is
detected in the perichondrium/periosteum of the long bones such as the
tibia (Ti), fibula (Fi), femur (Fe),
iliac bone (Il), the flat bones at the base of the skull
such as the sphenoid bone (Sh), ribs (Ri),
clavicle (Cl), and vertebrae (Ve). Some of the
intramembranous bones of the maxilla (Mx) and mandible
(Mn) are also positive for asporin. A positive
signal for asporin is detected in the region of the subcutaneous
muscles of the thorax, trunk, and head (platysmal muscle), and these
muscles are delineated with arrows. Very little
asporin mRNA is detected in the major parenchymal
organs, with the exception of the lung bronchi (arrow). A
nonspecific signal is evident in the major parenchymal organs of heart
(He), lung (Lu), and liver (Li)
perhaps due to the nonspecific binding of the probe to the
erythrocytes. A sagittal section of the digits from a forelimb at 15.5 dpc is shown in panel C (bright field to left;
dark field to right; magnification of ×4). The tip of the
third digit from panel C is shown in panel D
(bright field to left, dark field to right;
magnification of ×20). Asporin has a prominent expression
in the fibroblast (Fb) layer of the perichondrium.
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Fig. 6.
Sagittal sections of specialized connective
tissues at different stages of mouse embryonic development probed with
an asporin antisense riboprobe. Bright field
images are shown to the right of dark field images.
Panel A, asporin expression in tendon
(Te) and the perichondrium/periosteum of scapula
(Sp) and humerus (Hu) at 15.5 dpc. Panel
B, asporin expression in the eye is restricted to the
sclera (Sc) at 17.5 dpc (albino mouse). Panel C,
asporin expression in connective tissues of tongue at 18.5 dpc: 1) the lamina propria (Lp) underlying the tongue
epithelia; 2) lingual fascia (Lf) or connective tissue
sheath surrounding the muscle bundles of the tongue. The positive
asporin signal for the fascia appears as parallel striations in the
section and the muscle fibers do not give a positive signal. The lower
surface of the tongue faces the mandible (Mn). Panel
D, asporin expression in dissected skin from an 18.5 dpc embryo is detected in the dermis (Dm) but not in the
epidermis (Ep).
All exon sequences are represented by boldface uppercase letters and
intron sequence is represented by lowercase letters. The dinucleotide
consensus sequence at the splice sites is depicted in italic lowercase
letters. All nucleotide sequences are shown in the 5' to 3' direction.
Superscripts following the amino acids at the splice junction denote
codon phasing of the open reading frame.
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Fig. 7.
Annotation of the nucleic acid sequences of
three BAC clones to give the genomic organization of ECM2,
asporin, osteoadherin, and osteoglycin
on human chromosome 9q21.3-9q22. Wherever possible,
comparison was made between overlapping clones. However, it was
necessary to determine the location and direction of 16 kb of one
contig in the center of AL137848, between asporin and osteoglycin, by
performing bridging PCR reactions to neighboring contigs. Three gaps
within intronic sequences could not be annotated.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
A dendrogram showing predicted relationships
between SLRP family members and other LRR proteins of the ECM.
Horizontal distances of bars are proportional to
evolutionary distance and are based on human protein sequences. The
SLRP family is subdivided into 3 classes: class I contains 3 members;
class II contains 5 members; and class III contains 3 members. Asporin
is a class I member, and biglycan and decorin are more related.
Although chondroadherin (50, 51) and the recently identified nyctalopin
(52, 53) have been granted membership to the SLRP gene family, they may
have diverged from the other three SLRP classes early in evolution
because their structures are significantly different from the
conventional SLRPs. Likewise, ECM2 is structurally different from
the conventional SLRPs, but has a LRR domain that shows some homology
with the SLRPs. Since ECM2 is physically linked to asporin on human
chromosome 9, it has been included in the dendrogram. This analysis was
done with public software using ClustalW version 1.81 and the
output was generated with TreeViewer.
Northern blot analysis suggests that the members of the class I SLRP subfamily appear to have a relatively broad tissue distribution in the adult mouse, but the distribution for asporin seems to be the most restricted. An earlier study of the RNA expression pattern of biglycan and decorin during human fetal development showed that biglycan and decorin expression patterns were "substantially divergent and sometimes mutually exclusive" (54). In mouse embryonic development at 14.5 dpc, biglycan is expressed in the perichondrium of the vertebrae, ribs, and large bones of the hind limbs (55), but decorin is not expressed in cartilage and bone (6, 55). At this stage of mouse embryonic development, the RNA expression of asporin in the skeleton is similar to that of biglycan and is specifically localized to the perichondrium. Asporin mRNA was observed in the periosteum of the long bones at ME 18.5 dpc (data not shown) and biglycan is clearly expressed in the periosteum at 2 days of postnatal development (55). The strong RNA expression of asporin observed in the fascia surrounding the muscle bundles of the tongue, and presumably the fascia surrounding subcutaneous muscles as well, coincides with a similar connective tissue expression pattern observed for mouse and human decorin. During human fetal development, decorin was localized to the connective tissue sheathes surrounding skeletal myofibers or fascia, whereas biglycan was localized within the connective tissue sheathes (endomysium) of the skeletal myofibers (54). Likewise, in the mouse, decorin was localized to the fascia (perimysium) of the subcutaneous muscle (32). We now report that asporin RNA expression, during mouse embryogenesis, partially overlaps with biglycan expression in the skeleton and with decorin expression in the fascia of skeletal muscle.
The close structural similarity and overlapping tissue distribution suggest that the SLRPs could represent a family of molecules with redundant functions. This hypothesis is supported by the observation that despite the potent in vitro effects of individual SLRPs, analyses of mice with inactivated or deleted genes reveal suprisingly mild phenotypes. The RNA expression pattern of decorin and biglycan appears to be divergent and in some cases completely nonoverlapping. Thus, it appears unlikely that these two molecules can have completely redundant functions. Our identification of a novel class I subfamily member may impact on our understanding of redundancy among class I members. Asporin has a partially overlapping RNA expression pattern with decorin and biglycan in mouse embryonic development, and consequently asporin must be considered as a candidate for functional redundancy with the class I SLRPs.
A certain degree of compensation has already been observed in targeted mutations of the SLRP class II genes in mice. Recently, morphological analysis of early tendon development in mice for the double "knockout" of lumican and fibromodulin revealed an additive phenotypic effect for the double mutant as compared with the single mutants (56). An increased deposition of lumican protein was observed in whole protein extracts of tails from fibromodulin-null mice suggesting that lumican and fibromodulin may share a binding site on the collagen fibril (33) which was subsequently demonstrated (57).
Some functional redundancy may be present between SLRPs of different subfamily classes. Abnormal collagen fibril formation was observed in the targeted mutations of both class I SLRPs and class II SLRPs, and in some cases, similar phenotypes were seen in the same tissue. For example, the decorin-null (32) and fibromodulin-null mice (33) exhibit a collagen fibril defect in tendon, and abnormal collagen fibril formation in the skin of decorin-null (32) and lumican-null mice (34) leads to skin fragility. In this study, asporin RNA was detected in the dermal mesenchyme, and this expression pattern has also been observed for certain members of the class I, class II, and class III (osteoglycin)2 SLRPs. It is unclear whether it is possible for SLRP proteins of different classes to have interchangeable functions in certain tissues. The importance and extent of compensation may become more apparent as more SLRP double (and multiple) knockouts are generated and subsequently analyzed.
In this report, we describe a novel SLRP cluster in mammals and a
schematic depiction of three SLRP clusters is shown in Fig. 9. In this figure, the horizontal
distance between genes in a cluster is not to scale. SLRP class
I gene members always lie 5' to class II members in a cluster.
Likewise, class III members always lie 3' to class II members in a
cluster. The transcriptional orientation of the genes located on
chromosome 12 follows published reports (36). The relationship among
human fibromodulin, PRELP, and opticin on human chromosome 1q32 was
derived from the annotation of sequences present in
GenBankTM (NT_004523). Biglycan resides on human chromosome
Xq28 (58).
|
The class members can be aligned vertically among paralogous genes in the three clusters (see Fig. 9). Upon examination of additional overlapping BACs from human chromosome 1q32, we have failed to discover a SLRP class I member upstream or downstream of the fibromodulin gene. Additionally, the biglycan gene on the X-chromosome does not appear to be physically linked to other genes that encode for leucine-rich repeating proteins. With the identification of asporin as a class I member on human chromosome 9, we propose that biglycan may have been previously linked with the SLRP cluster on chromosome 1, but early in evolution the gene migrated from this cluster and came to reside on the X-chromosome. If the three clusters are aligned based upon the evolutionary distances depicted in the dendrogram tree (see Fig. 8), it appears that the paralogous genes on chromosomes 1 and 12 are the most similar. Therefore, the SLRP genes clustered on human chromosome 9 may have arisen independently from the clusters on chromosome 1 and 12. Perhaps, the clusters on chromosome 1 and 12 arose from a second duplication of a common cluster.
The significance of the SLRP gene clusters is unclear. Since several of
the SLRP genes have been "retained" in the clusters during
evolution, it is tempting to speculate that a degree of functional
redundancy has also been retained. We speculate that asporin,
like the other class I molecules, plays a structural and/or signaling
function in the extracellular matrix of skeletal tissues and other
specialized connective tissues.
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ACKNOWLEDGEMENTS |
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We thank Yi Xu and Richard B. Jones for assistance with computer submission of the manuscript, and James F. Martin for consultation. We thank Alice Woodsworth for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by Grants P01 AR42919-05 (to M. H.) and R37 AR30481 (to R. M.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Dept. of Cell Biology, Box 302 Volker Hall, University of Alabama, Birmingham, AL 35294-0019. Tel.: 205-934-2053; Fax: 205-934-7029; E-mail: rmayne@ cellbio.bhs.uab.edu.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M011290200
2 W. Zhou, J. Johnson, T. Shinomura, H. Eberspaecher, T. Cook, R. Mayne, B. deCrombrugghe, and M. Hook, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: SLRP, small leucine-rich repeat proteoglycan; LRR, leucine-rich repeat; BAC, bacterial artificial chromosome; ORF, open reading frame; RACE, rapid amplification of cDNA ends; dpc, days postcoitum; ECM, extracellular matrix; PCR, polymerase chain reaction; EST, expressed sequence tag; bp, base pair(s); aa, amino acid; kb, kilobase pair(s); ME, mouse embryonic.
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