From the Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, 33006-Oviedo, Spain
Received for publication, November 21, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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We have cloned a mouse brain cDNA encoding a
new protein of the ADAMTS family (a disintegrin
and metalloproteinase domain, with
thrombospondin type-1 repeats), which has been
called ADAMTS-20. This protein shows a domain organization similar to
that described for other ADAMTSs including signal sequence, propeptide,
metalloproteinase domain, disintegrin domain, central TS-1
motif, cysteine-rich region, and C-terminal TS module. However, this
last module is more complex than that of other ADAMTSs, being composed
of a total of 14 repeats. The structural complexity of ADAMTS-20 is
further increased by the presence of an additional domain 200 residues long and located immediately adjacent to the TS module. This domain has
been tentatively called GON domain and can also be recognized in some
ADAMTSs such as gon-1 from Caenorhabditis elegans and human
and mouse ADAMTS-9. The presence of this domain is a hallmark of a
novel subfamily of structurally and evolutionarily related ADAMTSs,
called GON-ADAMTSs. Expression analysis demonstrated that
ADAMTS-20 transcripts can be detected at low levels in
several human and mouse tissues, especially in testis. This gene is
also overexpressed in some human malignant tumors, including brain, colon, and breast carcinomas. Western blot analysis using polyclonal antibodies raised against recombinant ADAMTS-20 produced in
Escherichia coli showed the presence of a 70-kDa band in
mouse brain and testis extracts. This recombinant ADAMTS-20 hydrolyzed
a synthetic peptide used for assaying matrix metalloproteinases. These
data suggest that this novel enzyme may play a role in the tissue
remodeling process occurring in both normal and pathological conditions.
The ADAMTSs1
(a disintegrin and
metalloproteinase domain, with
thrombospondin type-1 modules) are a growing
family of zinc-dependent metalloproteinases that play
important roles in a variety of normal and pathological conditions (1,
2). These enzymes show a complex domain organization including signal
sequence, propeptide, metalloproteinase domain, disintegrin-like
domain, central TS-1 motif, cysteine-rich region, and a variable number
of TS-like repeats at the C-terminal region. To date, 18 genetically
different ADAMTSs have been identified in human tissues (3). Structural characterization of these enzymes has demonstrated that ADAMTSs are
distinct from ADAMs, a related family of metalloproteinases that
exhibit a similar domain organization. However, both proteinase families differ in some important aspects. Thus, ADAMs lack TS-1 repeats but contain a transmembrane domain that mediates their anchorage to the plasma membrane and a cytoplasmic tail that can participate in signal transduction events (4, 5). By contrast, ADAMTSs
display an organization of TS repeats of variable complexity and are
secreted proteins devoid of transmembrane and cytoplasmic domains
in their C-terminal region. The complexity of studies on
ADAMTSs has further increased after the finding of a family of proteins that resemble ADAMTSs and that have been called ADAMTSLs (ADAMTS-like) or punctins (6). These proteins lack the
metalloproteinase and disintegrin-like domains of ADAMTSs but
contain all the remaining ADAMTS domains, including several TS-1
repeats. ADAMTSLs have been proposed to participate in the endogenous
regulation of ADAMTS activity (6).
Functional analysis of ADAMTSs has demonstrated their participation in
a wide diversity of processes. Thus, ADAMTS-1 (or METH-1) and ADAMTS-8
(or METH-2) have angioinhibitory properties (7). Disruption of the
mouse adamts-1 gene results in decreased growth, renal
abnormalities, partial obstruction in the ureteropelvic junction, and
alterations in adipose tissue and adrenal medullary architecture (8).
Fertilization is also impaired in female mice deficient in ADAMTS-1,
indicating that this protease is necessary for proper function of the
female genital organs (8). ADAMTS-2, ADAMTS-3, and ADAMTS-14 are
procollagen N-proteinases (9-11), and deficiency in
ADAMTS-2 causes Ehlers-Danlos syndrome VIIC in humans (12). Mutant mice
lacking ADAMTS-2 develop fragile skin as well as male sterility due to
impaired spermatogenesis (13). ADAMTS-4 and ADAMTS-5/11 are
aggrecanases, and their implication in aggrecan degradation in
arthritic diseases has been reported (14-16). ADAMTS-1 has also been
found to cleave aggrecan at multiple sites and displays all features to
be classified as an aggrecanase (17, 18). ADAMTS-1 and ADAMTS-4
also have the ability to degrade versican in human aorta (19), whereas
ADAMTS-4 is responsible for brevican degradation in glioma cells (20,
21), a critical aspect in the invasive capacity of these tumors.
ADAMTS-13 is a von Willebrand factor-cleaving protease, and mutations
in the gene encoding this enzyme cause thrombotic thrombocytopenic
purpura, a life-threatening disease mainly characterized by hemolytic
anemia, microvascular thrombosis, low platelet count, renal failure,
and neurological dysfunctions (22-25). Other ADAMTSs, such as
ADAMTS-6, -7, -9, -10, -12, -15, -16, -17, -18, and -19, have only been structurally characterized, but their functional roles remain unknown
(3, 26-28).
As part of our studies on the human and mouse degradomes (29), and
considering the growing relevance of ADAMTSs in normal and pathological
processes, we have examined the possibility that additional yet
uncharacterized family members could be present in the genome of these
organisms. In this work, we report the identification of a novel ADAMTS
that has been called ADAMTS-20. We also report the structural
characterization of both human and mouse enzymes with the finding of a
novel domain present in ADAMTS-20, as well as in a long isoform of
ADAMTS-9, and in ADAMTSs from other organisms including
Caenorhabditis elegans, Drosophila melanogaster, Anopheles gambiae, and Fugu rubripes. Finally, we
report the tissue distribution of ADAMTS-20 and perform a preliminary
analysis of its enzymatic activity.
Materials--
Human and mouse cDNA libraries and Northern
blots containing polyadenylated RNAs from different adult and fetal
human and mouse tissues were from Clontech.
RNAs from human tumors were obtained from patients who had
undergone surgery for diverse malignancies at the Hospital Central de
Asturias (Banco de Tumores, Instituto Universitario de
Oncología del Principado de Asturias), Oviedo, Spain.
Restriction endonucleases and other reagents used for molecular cloning
were from Roche Molecular Biochemicals. Synthetic peptides used for
enzymatic analysis were from Novabiochem.
Isolation of cDNA Clones--
A search using the BLAST
(Basic local alignment
search tool) program in the public
(www.ncbi.nlm.nih.gov) and private (www.celera.com) mouse genome
databases allowed the identification of a mouse clone RP23-24F24
(GenBankTM accession number AC084384) containing an
uncharacterized ADAMTS proteinase-like domain and regions encoding
thrombospondin-like repeats. We designed specific oligonucleotides to
PCR-amplify the region contained between these two putative ADAMTS
domains, using several different commercially available cDNA
libraries (Clontech) and the Expand High Fidelity
PCR system (Roche Molecular Biochemicals). The following
oligonucleotides derived from the RP23-24F24 DNA clone were
synthesized: TS20F, 5'-CCAAGATACGTGGAAGTTATGGT-3'; TS20R,
5'-CTGTGCTCTTGATTCCACCTC-3', whereas the following
oligonucleotides were used for the nested PCR: TS20Fnd,
5'-GTTATGGTTACAGCCGATGCT-3', and TS20Rnd,
5'-TGCTCTTGATTCCACCTCCGC-3'. The PCR reaction was performed in a
GeneAmp 2400 PCR system from PerkinElmer Life Sciences for 40 cycles of
denaturation (94 °C, 20 s), annealing (64 °C, 15 s),
and extension (68 °C, 60 s). The amplified PCR product, of
about 0.9 kb, was treated with T4 polynucleotide kinase and T4 DNA
polymerase and ligated in the SmaI site of pUC18. The
identity of the product was confirmed by sequencing using the kit DRho terminator Taq FS (Applied Biosystems) and the automatic DNA
sequencer ABI-PRISM 310 (PerkinElmer Life Sciences). To obtain human
probes for human ADAMTS-20, the following specific oligonucleotides
were used in a nested PCR amplification: hTS20F,
5'-GTGAAATTGCTGCCTCAAAGG-3'; hTS20F-nd,
5'-GCTGCCTCAAAGGACCATCA-3', hTS20R, 5'-GACATAGTAAGCAGAAAGTGG-3'; and
hTS20R-nd, 5'-GGATTTCCAATCTAAGATAGC-3'; nd indicates the primers used for the nested PCR amplification.
5'- and 3'-Extension of Isolated cDNAs--
The 5'- and
3'-ends of cloned cDNAs were extended by successive rounds of RACE
(rapid amplification of c-DNAs ends) using RNA from different mouse
tissues and the MarathonTM cDNA amplification kit
(Clontech), essentially as described by the
manufacturer. Each cycle of RACE allowed the extension of about 200 bp
toward each end. Following gel purification, the PCR products were
cloned and sequenced as described above.
Northern Blot Analysis--
Nylon filters containing 2 µg of
poly(A)+ RNA of a wide variety of both human and mouse
tissues were prehybridized at 42 °C for 3 h in 50% formamide,
5× SSPE (1× SSPE = 150 nM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4), 10×
Denhardt's solution, 2% SDS, and 100 µg/ml denatured herring sperm
DNA. Hybridization was performed with a radiolabeled ADAMTS-20-specific
probe (0.7 kb long) corresponding to the PCR product amplified by using
the oligonucleotides TS20F and TS20R3 (5'-GTAGGGACACACTTGTGATCCA-3') as
forward and reverse primers, respectively. After hybridization for
20 h under the same conditions used for prehybridization, filters
were washed with 0.1× SSC, 0.1% SDS for 2 h at 50 °C, and
exposed to autoradiography. RNA integrity and equal loading were
assessed by hybridization with an actin probe.
Reverse Transcription and PCR Amplification--
To analyze the
expression of ADAMTS-20 in human tumor specimens, total RNA was
isolated from diverse malignant tumors by guanidium thiocyanate-phenol-chloroform extraction and used for cDNA
synthesis with the RNA PCR kit from PerkinElmer Life Sciences. After
reverse transcription (RT) using 1 µg of total RNA and random
hexamers as primer according to the manufacturer's instructions, the
whole mixture was used for PCR with the following ADAMTS-20-specific oligonucleotides (hTS20F2, 5'-CTTTTACTATAGCCCATGAGC-3'; hTS20F2-nd, TTACTATAGCCCATGAGCTTGG-3'; hTS20R2, 5'-CAGGAAGTTCTGAAGGCAGAT-3'; and
hTS20R2-nd, 5'-TTGTCAAGAAGACATTCGGGC-3'), as described above. The PCR
products were analyzed in 1.5% agarose gels. cDNA quality was
verified by performing control reactions with primers derived from the
sequence of actin. Negative controls were also performed in all
cases by omitting the template or reverse transcriptase.
Production and Purification of Recombinant Proteins--
A
cDNA construct containing the metalloproteinase domain of ADAMTS-20
was made by PCR amplification using the following two oligonucleotides
containing BamHI and EcoRI sites, respectively: TS20expF, 5'-ATCGGATCCGTTTTTTATCATAC-3', and TS20expR,
5'-GAAGAATTCGAGACAGGTCATATGTTCTCC-3' (where the
BamHI and EcoRI sites are underlined). The PCR
amplification was performed for 25 cycles of denaturation (95 °C,
15 s), annealing (58 °C, 10 s), and extension (68 °C,
50 s) using the ExpandTM High Fidelity PCR system. The
PCR product was digested with BamHI and EcoRI and
cloned between these two sites of the pGEX-3X expression vector
(Amersham Biosciences). The resulting vector, pGEX-3XTS20(M), was
transformed into BL21(DE3)pLysE-competent Escherichia
coli cells, and expression was induced by the addition of
isopropyl-1-thio- Enzymatic Assays--
Enzymatic activity of the purified
recombinant GST-TS20(M) protein was assayed using the synthetic
fluorescent substrates QF-24
(Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2), QF-35
(Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2), and QF-41
(Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2). Routine assays were
carried out at 37 °C at substrate concentrations of 1 µM in a buffer containing 50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl2, 0.05%
Brij-35, pH 7.5, with a final concentration of Me2SO of 1%. The fluorimetric measurements were made in an LS 50-B PerkinElmer Life Sciences spectrofluorimeter (
Kinetic studies were performed using different concentrations of
the fluorogenic peptide (0.5-4 µM) in 2 ml of assay
buffer containing 48 nM recombinant ADAMTS-20, and peptide
hydrolysis was measured as the increase in fluorescence at 37 °C for
5 min. Initial velocities were calculated using the analysis package FL
WinLab 2.01 (PerkinElmer Life Sciences), and the
kcat/Km ratio was calculated
as described previously (30). For inhibition experiments, the reaction
mixture was preincubated for 30 min at 20 °C with EDTA, BB-94,
E-64, or 4-(2-aminoethyl)-benzenesulfonyl fluoride, and the
hydrolyzing activity of ADAMTS-20 against QF-35 was
determined by fluorimetric measurements as above. Purified recombinant
GST was used in all the experiments as a negative control.
Antibody Production and Western blot Analysis--
Purified
ADAMTS-20 was injected into rabbits, and the animals were bled 6 weeks after the injection The serum was then dialyzed for 24 h at
4 °C against 20 mM phosphate buffer, pH 7.2. After dialysis, the material was chromatographed in a column of
DEAE-cellulose equilibrated and eluted in the same 20 mM
phosphate buffer. IgG-containing fractions were collected and stored at
Identification and Characterization of ADAMTS-20--
An extensive
search for DNA genomic sequences encoding uncharacterized ADAMTS-like
domains allowed us to identify a mouse clone (RP23-24F24,
GenBankTM accession number AC084384) that fulfilled the
premises to be considered as a region containing a new
adamts gene. These features included the presence of
putative exons encoding metalloproteinase and thrombospondin-like
domains similar to those found in previously described ADAMTSs. To
generate a cDNA for this region, we carried out PCR amplification
experiments using mouse brain cDNA libraries and specific
oligonucleotides derived from the identified metalloproteinase and
thrombospondin-like domains. After cloning a PCR product of about 0.9 kb, we confirmed by conceptual translation that this sequence was
distinct from the equivalent regions present in all previously
identified ADAMTS family members. To obtain the full-length cDNA
for this putative new metalloproteinase, we performed successive rounds
of 5'- and 3'-RACE experiments using specific oligonucleotides deduced
from the end of the previously obtained sequences. This strategy
allowed us to extend the original cDNA clone toward both ends, and
finally, to identify the corresponding start and stop in-frame codons.
Computer analysis of the obtained sequence (Fig. 1 and GenBankTM accession
number AJ512753) revealed an open reading frame coding for a protein of
1906 amino acid residues with a predicted molecular mass of
212,040. The sequence also contains 10 potential sites of
N-glycosylation (Fig. 1) that can contribute to increase the
predicted molecular mass for this protein. This structural analysis
also showed that the overall domain organization of this protein was
very similar to that of previously characterized ADAMTSs, containing
signal sequence, propeptide, metalloproteinase domain, disintegrin-like
domain, central TS1 motif, cysteine-rich region, and a C-terminal
module with several TS1 submotifs (Fig.
2A).
Further analysis of the identified amino acid sequence showed a
significant similarity with other ADAMTSs, the maximum percentage of
identities (52%) being with human ADAMTS-9. Additional hallmarks of the ADAMTS family were also apparent from this analysis. Thus, the
putative prodomain contains three conserved Cys residues found in other
ADAMTSs (2, 3). The first two Cys are within the conserved
sequences CFYRGQV (positions 129-135, consensus sequence CXYXGXV), and CGGLMG (positions
148-153, consensus sequence CXGLXG). The third
Cys residue is located close to the prodomain end (position 202) and
lies within a sequence (KPCEVSE) that does not resemble the Cys switch
consensus sequence (PRCGVPD) present in matrix metalloproteinases
(MMPs) (31, 32) and in some ADAMTSs such as ADAMTS-12 and ADAMTS-15 (3,
28). The propeptide ends in a basic region RNKR (positions 246-249),
which could correspond to a proprotein-convertase recognition sequence
for generation of the mature enzyme (consensus sequence
RX(K/R)R). The catalytic domain includes the sequence
HELGHVFNVPHD (positions 399-410) closely related to that involved in
the coordination of the catalytic zinc atom at the active site of
metalloproteinases (consensus sequence
HEXGHXXGXXH). This motif ends in an
Asp residue, which distinguishes ADAMs and ADAMTSs from other
metalloproteinases, such as MMPs that contain a conserved Ser residue
at this position. A few residues C-terminal to this site, there is a
Met residue (position 427) that forms the "Met turn" characteristic
of ADAMs, ADAMTSs, and MMPs. The catalytic domain also contains the
eight conserved Cys residues present in the corresponding region of all ADAMTSs.
The metalloproteinase domain is followed by a disintegrin domain, which
is very similar in size (76 residues) to that of other ADAMTSs and
includes the 8 conserved Cys residues characteristic of this region.
Furthermore, a central TS-1 motif, a Cys-rich domain, and a spacer
region can be clearly recognized after the disintegrin domain. These
regions are also very similar in size and structural features to the
equivalent ones from other ADAMTSs. Following the spacer region, the
characteristic C-terminal TS-1 module of these enzymes can be found. It
is remarkable that the number of TS-1 repeats located at this region of
the identified protein is higher than that present in all human or
mouse ADAMTSs whose sequences have been reported to date. Thus, a total
of 14 TS-1 repeats can be distinguished in this module, whereas most vertebrate ADAMTSs have just three or four repeats, the maximum number corresponding to ADAMTS-12, which contains seven TS-1 repeats in
this C-terminal module. Interestingly, the identified sequence ends
with a region of about 200 amino acids, which is not present in any
other sequence described to date, with the exception of some ADAMTSs
such as gon-1 (33), an ADAMTS family member from C. elegans.
Computer analysis of the human and mouse genome sequences has also
allowed us to identify enlarged isoforms of human and mouse ADAMTS-9
containing this additional domain present in ADAMTS-20 and gon-1. These
enlarged ADAMTS-9 variants have not been described yet in the
literature and are distinct from those reported previously for this
enzyme (27). Putative proteins containing this domain can also be
deduced from the genome sequence of other organisms whose sequence is
available, including D. melanogaster (GenBankTM
accession number AY094716), A. gambiae
(GenBankTM accession number AGCP2529), and F. rubripes (wrongly annotated as being part of two independent
proteins with GenBankTM accession numbers SINFRUP
00000055939 and 00000062760). This conserved domain present in all
these proteins is rich in Cys residues (Fig. 2B) and
represents a particular structural hallmark for these ADAMTSs.
Because the first published sequence containing this motif is that of
gon-1, we propose the name GON domain for this newly identified domain
present in a subset of ADAMTSs.
Taking together these structural comparisons, we can conclude that the
identified murine sequence corresponds to a bona fide member
of the ADAMTS family of metalloproteinases whose officially approved
name is ADAMTS-20. In this work, and by using the mouse ADAMTS-20
sequence as query, we have also deduced the complete sequence of its
human orthologue (Fig. 1, and GenBankTM accession number
AJ515153). The gene encoding human ADAMTS-20 is located in chromosome
12q12, syntenic to a region of mouse chromosome 15, where the mouse
adamts-20 gene is located. Detailed pairwise comparisons
revealed that human ADAMTS-20 is 69% identical to its mouse
counterpart and also contains all structural hallmarks characteristic
of this family of metalloproteinases. Human ADAMTS-20 contains 15 potential sites of N-glycosylation, 6 of them being conserved with those predicted for mouse ADAMTS-20 (Fig. 1).
Interestingly, bioinformatic analysis of the sequence of human
ADAMTS-20 gene led us to predict the presence of an
alternative exon that was not found in the mouse orthologue. To provide
experimental support to this prediction, we performed PCR
amplifications of this region using the specific oligonucleotides
hTS20F, hTS20F-nd, hTS20R, and hTS20R-nd and a human brain cDNA
library. These experiments allowed us to amplify a cDNA sequence
that confirmed the occurrence of an alternative splicing event in the
analyzed region. This event would take place after the exon encoding
the TS-1 repeat number 10 and would lead to the synthesis of a short
form of human ADAMTS-20 with a total of 11 TS-1 repeats, due to the
presence of an in-frame stop codon at the end of this region
(GenBankTM accession number AJ515154). These splicing
events are common in the 3'-end of ADAMTS genes and can be
responsible for the previously observed variations in the C-terminal
end of these metalloproteinases (3, 26-28).
Production of Recombinant ADAMTS-20 in E. coli and Analysis of Its
Enzymatic Properties--
We have expressed the metalloprotease domain
of mouse ADAMTS-20 in bacterial cells to analyze its activity. To do
this, a cDNA coding for this catalytic domain was subcloned into
the expression vector pGEX-3X, and the resulting plasmid, called
pGEX-3XTS20(M), was transformed into E. coli. After
induction with isopropyl-1-thio- Expression Analysis of Mouse and Human ADAMTS-20--
To study the
expression pattern of mouse and human ADAMTS-20, we have hybridized
Northern blots containing poly(A)+ prepared from a variety
of human and mouse tissues. As shown in Fig.
4A, two transcripts of about
7.5 and 2.5 kb were detected in mouse brain and testis. In the case of
human ADAMTS-20, a single transcript of about 7 kb was observed in
testis, prostate, ovary, heart, placenta, lung, and pancreas (Fig.
4A). RT-PCR amplification and nucleotide sequencing of the
amplified products confirmed the expression of ADAMTS-20 in all these
human tissues in which transcripts were detected at low levels by
Northern blot (data not shown). To examine the possibility that
ADAMTS-20 was produced by human tumors, we performed RT-PCR
amplification with RNAs obtained from a panel of paired primary tumors
and adjacent normal tissue. As illustrated in Fig. 4B, which
shows some representative cases, ADAMTS-20 was overexpressed in several
brain, colon, and breast carcinomas when compared with the low or
undetectable levels observed in the paired adjacent normal tissues.
Finally, we performed Western blot analysis of protein extracts from
different mouse tissues with polyclonal antibodies against the
purified recombinant ADAMTS-20. As can be seen in Fig.
4C, a major band of about 70 kDa was observed in testis and
brain but not in other tissues in which the expression of this gene had
not been detected. These findings demonstrate the presence of the
ADAMTS-20 protein in these tissues. Furthermore, the absence of
significant amounts of a putative immunoreactive band of about 212 kDa,
which could correspond to the intact ADAMTS-20, strongly suggests that
as demonstrated previously for other ADAMTSs (28), this novel enzyme is
subjected to several proteolytic processing-mediated events to generate
the final active molecule.
In this work, we describe the identification and characterization
of a novel member of the ADAMTS family of secreted metalloproteinases with disintegrin and thrombospondin domains. The approach to identify ADAMTS-20 involved a search of human and mouse genome databases followed by a combination of RT-PCR amplifications of cDNA
libraries and successive 5'- and 3'-RACE experiments to extend the
originally amplified cDNA fragments. This strategy allowed us to
isolate a full-length cDNA for mouse ADAMTS-20 and to deduce the
complete sequence of its human orthologue. Both proteins exhibit an
identical domain organization that is similar to that of previously
described ADAMTSs. Thus, they harbor signal sequence, propeptide,
metalloproteinase-, disintegrin-, central TS-, and cysteine
rich-domains and a complex C-terminal TS-like module. Likewise, mouse
and human ADAMTS-20 show several conserved residues and motifs
characteristic of each of these domains, including a proprotein
convertase activation sequence at the end of the propeptide, a
zinc-binding site with the reprolysin signature in the catalytic
domain, and conserved patterns of cysteine arrangements in the
disintegrin and cysteine-rich regions. However, ADAMTS-20 also exhibits
some characteristic features that allow us to distinguish this enzyme
from other family members as well as to define a novel subfamily of
ADAMTSs. Thus, it contains an unusually complex organization of TS
repeats at the C-terminal module, being composed of a total of 14 repeats, the highest number among all equivalent modules present in
vertebrate ADAMTSs identified to date. Interestingly, we have
previously reported that although ADAMTS-9 has been described to
possess three TS repeats at the C-terminal module (27), information retrieved from databases reveals the occurrence of an alternative transcript of ADAMTS-9, which encodes a protein isoform also containing 14 TS-1 repeats (Fig. 1 and data not shown) (3). The second distinctive
feature of ADAMTS-20 is the presence of an additional domain in its
C-terminal region, immediately adjacent to the TS-terminal module. This
structural motif, which we have designated as GON domain, is
characterized by the presence of several conserved cysteine residues
and can be recognized in gon-1 from C. elegans, in the large
isoforms of human and mouse ADAMTS-9, and in proteins predicted from
sequence analysis of the genomes of D. melanogaster, A. gambiae, and F. rubripes. On the basis of
these structural features, we propose that all these proteins define a
novel subset of ADAMTSs that could be known as GON-ADAMTSs.
The dendrogram shown in Fig. 5 confirms
the structural relationships among these ADAMTS family members and
allows the classification of the family of human ADAMTSs into seven
subfamilies of closely related members. The first of these subfamilies
should be that of hyalectanases (36), comprising ADAMTS-1, -4, -5/11,
-8, and -15 and characterized by structural and enzymatic similarities including proteoglycanase activities, and in some cases,
angioinhibitory properties (7, 14-21). The second subfamily should
be that of procollagen N-propeptidases and includes
ADAMTS-2, -3, and -14 (9-12). ADAMTS-9 and ADAMTS-20 should conform to
the subfamily of GON-ADAMTSs. ADAMTS-13, with unique properties among
all described ADAMTSs, should be the only representative of von
Willebrand factor-cleaving proteases (22-25). Finally, ADAMTS-6,
-7, -10, and -12; ADAMTS-16 and -18; and ADAMTS-17 and -19 form
groups of structurally related family members that might also be
indicative of putative common enzymatic and functional properties.
Further studies aimed at identifying the substrates targeted by the
proteases belonging to these three last ADAMTS subfamilies will be
necessary to confirm that the structural similarities here defined are
also supported by functional relationships between them. In this
regard, the structural similarities between vertebrate and invertebrate
members of the GON-ADAMTS subfamily of ADAMTSs may also allow us to
speculate about putative functional roles for ADAMTS-20. To date, no
physiological role has been ascribed to ADAMTS-9 nor to those related
proteins identified in Drosophila, Anopheles, or
Fugu; however, gon-1 is an active metalloproteinase
essential for controlling morphogenesis in C. elegans (33).
Thus, mutagenesis studies have suggested that this ADAMTS permits and
directs expansion of the gonad by remodeling the extracellular matrix
and basement membrane. Interestingly, the region containing TS-1
repeats is critical for gon-1 activity because some mutations
inactivating this gene are located in regions encoding these repeats
(33). Since it has been suggested that similar activities may control
organ morphogenesis throughout the animal kingdom, it is tempting to
speculate that ADAMTS-20 may play similar roles in vertebrates to those
played by gon-1 in nematodes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (final
concentration 0.5 mM). The cells were collected by
centrifugation, washed, and resuspended in 0.05 volumes of
phosphate-buffered saline. Then, the cells were lysed by sonication and
centrifuged at 20,000 × g for 20 min at 4 °C. The
soluble extract was purified using a glutathione-Sepharose 4B column
(Amersham Biosciences), and the glutathione S-transferase
GST-TS20(M) fusion protein was eluted with 20 mM reduced
glutathione, following manufacturer's instructions. The GST-TS20(M)
purified protein was used for enzymatic assays.
ex = 328 nm, and
em = 390 nm). The pH optimum analysis was carried out in
assay buffer as above but using 50 mM Bis-Tris and Tris (pH
6-9), or 50 mM glycine (pH 10) as buffer for the indicated
pH range and containing 2 µM fluorogenic QF-35 substrate
and 29 nM enzyme. The resulting data were fit to Equation 1, describing two pKa values (pKa1 and
pKa2) and one limiting
kcat/Km value.
(Eq. 1)
20 °C until used. Western blots were blocked in 5% milk in PBT
(phosphate-buffered saline containing 0.1% Tween 20) and then
incubated for 1 h with 1 µg/ml rabbit antiserum in PBT. After
three washes in PBT, blots were incubated for 1 h with horseradish
peroxidase-conjugated goat anti-rabbit IgG at 1:20,000 and developed
with the Renaissance chemiluminescence kit (PerkinElmer Life Sciences).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequences of
mouse and human ADAMTS-20. The different domains are
overlined, and the potential sites of N-linked
glycosylation indicated by asterisks. The zinc-binding site
characteristic of metalloproteinases is boxed.
(H_ADAMTS-20 indicates human ADAMTS-20;
M_ADAMTS-20 indicates mouse ADAMTS-20;
TS indicates thrombospondin domains).
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Fig. 2.
Domain organization of ADAMTS-20 and related
proteins. A, the domain structure of the indicated
members of the ADAMTS family. B, sequence of the GON domains
found in identified or predicted ADAMTS proteins of different
organisms. Mm, Mouse musculus; Hs,
Homo sapiens; Fr, F. rubripes;
Ce, C. elegans; Dm, D. melanogaster; Ag, A. gambiae.
-D-galactopyranoside, a
protein band of the expected size (51 kDa) was detected by SDS-PAGE analysis of the protein extract (Fig.
3A). The recombinant fusion protein was purified using glutathione-Sepharose chromatography as
described previously (34) (Fig. 3A). The soluble GST-TS20(M) fusion protein eluted from the column was directly used for enzymatic analysis, employing as putative substrates a series of synthetic quenched fluorescent peptides commonly used for assaying other metalloproteinases, including MMPs. The recombinant catalytic domain of
ADAMTS-20 exhibited significant proteolytic activity against the
fluorogenic peptide QF-35 (38 pM product/min). Other peptides, including QF-24 and QF-41, were not significantly hydrolyzed by this recombinant protein. The proteolytic activity of ADAMTS-20 against QF-35 was substantially abolished by EDTA and BB-94 but not by
inhibitors of other classes of proteolytic enzymes distinct of
metalloproteinases (data not shown). To further examine the catalytic
activity of ADAMTS-20, we performed an analysis of the pH profile of
activity of this enzyme. As shown in Fig. 3B, recombinant ADAMTS-20 exhibited a pH optimum of 8.1. Finally, we also carried out a
kinetic study using QF-35 as substrate. To this purpose, the
recombinant protease was incubated with different concentrations of
fluorogenic substrate, and the
kcat/Km was deduced as
described previously (30). The observed
kcat/Km of ADAMTS-20 for
substrate QF-35 at pH 8.1 was 46 M
1
s
1, similar to that calculated for some MMPs such as
Mcol-A with the ability to hydrolyze the same substrate
(kcat/Km = 58 M
1 s
1) but substantially lower
than values determined for most members of this family of
metalloproteinases (35)). Finally, it is remarkable that preliminary
experiments aimed at evaluating the ability of the recombinant
catalytic domain of ADAMTS-20 to hydrolyze diverse endogenous
substrates, including several proteoglycans targeted by other ADAMTSs,
have not revealed any significant degrading activity against them.
These results suggest that the presence of ADAMTS-20 ancillary domains
may be necessary for the in vivo function of this enzyme,
although the possibility that it could target a novel substrate cannot
be ruled out.
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Fig. 3.
Production and enzymatic characterization of
the metalloprotease domain of ADAMTS-20. As shown in A,
5 µl of bacterial extracts transformed with pGEX-3X (lane
2) or pGEX-3XTS20(M) (lane 3) and purified
catalytic domain of ADAMTS-20 (TS20(M))
(lane 4) were analyzed by SDS-PAGE. The sizes of the
molecular size markers (MWM, lane 1) are
indicated on the left. As shown in B, the
fluorogenic peptide QF-35
(Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2) was incubated with 10 nM purified catalytic domain of mouse ADAMTS-20 using 50 mM Bis-Tris, Tris, or glycine as buffer (see
"Experimental Procedures"). The fluorimetric measurements were made
at ex = 328 nm, and
em = 393 nm.
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Fig. 4.
Analysis of the expression
of ADAMTS-20 in normal and tumor tissues. A, Northern
blot analysis of ADAMTS-20 expression in mouse and human tissues. About
2 µg of polyadenylated RNA of the indicated mouse and human tissues
were hybridized with specific probes isolated from the mouse and human
ADAMTS-20 cDNAs. The position of the size markers is shown. The
filters were subsequently hybridized with a mouse and a human actin
probe, respectively, to ascertain the differences in RNA loading among
the different samples. B, RT-PCR analysis of ADAMTS-20
expression in paired normal and tumor tissues. A 199-bp fragment
corresponding to a segment of human ADAMTS-20 was amplified with
primers indicated under "Experimental Procedures" in a volume of 50 µl, and 10 µl of the reaction were separated on a 1.5% agarose gel
run in Tris borate-EDTA buffer. Amplification of -actin was used to
ascertain RNA integrity and equal loading. C
indicates
negative control. C, Western blot analysis of protein
extracts from the indicated mouse tissues with 1 µg/ml polyclonal
antibody against ADAMTS-20 in PBT. As a positive control (C+) we used
purified recombinant ADAMTS-20. The sizes of the molecular size markers
are shown to the left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
The human ADAMTS subfamilies. A
phylogenetic tree of the human ADAMTS family was generated using the
amino acid sequences of their metalloprotease domains and the program
supplied by the Human Genome Mapping Project
(www.hgmp.mrc.ac.uk).
As a previous step to evaluate this hypothetical function of ADAMTS-20 as an extracellular matrix remodeling enzyme, we have performed a preliminary analysis of the catalytic properties of a recombinant form of this protease produced in bacterial cells. Interestingly, ADAMTS-20 is able to hydrolyze a synthetic peptide used for analysis of vertebrate MMPs, and this hydrolyzing activity is abolished by inhibitors of metalloproteinases, demonstrating that the identified protein is an active member of this class of proteolytic enzymes. To our knowledge, this is the first report showing that a member of the ADAMTS family is able to degrade peptides such as QF-35 commonly used to assay the activity of MMPs, thus confirming the connections between both proteolytic systems (reviewed in Ref. 32). Nevertheless, it is remarkable that kinetic analysis has revealed that the catalytic efficiency of ADAMTS-20 against QF-35 is much lower than that of most MMPs with the ability to hydrolyze this peptide, suggesting the occurrence of important differences in the active site of both types of metalloproteinases. Consequently, studies of substrate specificity and resolution of the three-dimensional structure of the ADAMTS-20 catalytic domain will be required to clarify the similarities and differences of this novel enzyme with members of the MMP family.
Previous studies have shown that ADAMTSs may be of relevance in tumor processes (20, 28). Therefore, in this work, we have also explored the potential significance of ADAMTS-20 in human cancer through analysis of its expression pattern in a panel of malignant tumors. These studies have shown that ADAMTS-20 is overexpressed in several brain, colon, and breast carcinomas when compared with the paired adjacent normal tissues, suggesting that this protease could play some role in the progression of these tumors. Also, in this regard, it is interesting that the region containing the human ADAMTS-20 gene (12q12) has been found to be a recurrent site of translocations and other alterations in human malignancies (37-40). Genetic lesions in this region have also been linked to several diseases, including a new locus for Parkinson's disease (41), whose responsible gene remains to be characterized. It will be interesting to examine the possibility that ADAMTS-20 could be a target of some of these genetic abnormalities, as already demonstrated for other ADAMTS family members linked to relevant genetic diseases (12, 22). To this purpose, as well as to clarify the role of ADAMTS-20 in physiological processes, it will be very helpful to create a mouse deficient in this protease. This work is currently in progress in our laboratory and has been facilitated by the availability of cDNA and genomic clones for mouse ADAMTS-20 generated in the present study.
In conclusion, we have cloned and characterized ADAMTS-20, a protease
that, according to our exhaustive analysis of both mouse and human
genomes, represents the only member of the ADAMTS family that remained
to be identified in these organisms. ADAMTS-20 is an active protease
with a profile of activity and sensitivity to inhibitors characteristic
of metalloproteinases. However, it also exhibits a series of structural
peculiarities including the presence of the newly identified GON
domain, which has allowed us to define the occurrence of a novel
subfamily of ADAMTSs: the GON-ADAMTSs. This structural analysis,
together with that performed with other family members, has also
prompted us to propose that ADAMTSs can be organized into seven
different subfamilies. Hopefully, this classification may facilitate
future studies aimed at exploring the multiple roles that this large
and complex family of proteases may play in processes involving cell
migration, tissue remodeling, and changes in cell adhesion.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. J. P. Freije, X. S. Puente, and G. Velasco for helpful comments and support and C. Garabaya for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by grants from Comisión Interministerial de Ciencia y Tecnología-Spain (SAF00-0217); and European Union (QLG1-CT-2000-01131). The Instituto Universitario de Oncología is supported by Obra Social Cajastur-Asturias.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ512753, AJ515153, and AJ515154
Recipients of research fellowships or contracts from Ministerio de
Ciencia y Tecnología, Spain.
§ To whom correspondence should be addressed. Tel.: 34-985-104201; Fax: 34-985-103564; E-mail: CLO@correo.uniovi.es.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M211900200
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ABBREVIATIONS |
---|
The abbreviations used are: ADAMTS, a disintegrin and metalloproteinase domain, with thrombospondin type-1 modules; ADAMTSL, ADAMTS-like; GST, glutathione S-transferase; MMP, matrix metalloproteinase; RT, reverse transcription; Mca, 7-methoxycoumarin-4-acetyl; Dpa, L-dinitrophenyl-diamino propionic acid; Cha, cyclohexylalanine; Nva, norvaline; RACE, rapid amplification of cDNA ends.
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