From the Department of Biomedical Engineering, Lerner Research
Institute, and Orthopedic Research Center, Cleveland Clinic
Foundation, Cleveland, Ohio 44195, § Vascular Biology, The
Hope Heart Institute, Seattle, Washington 98104-2046, and the
Department of Pharmacology, Faculty of Medicine,
Universite de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada
Received for publication, October 28, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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We demonstrate that in humans, two
metalloproteases, ADAMTS-9 (1935 amino acids) and ADAMTS-20
(1911 amino acids) are orthologs of GON-1, an ADAMTS protease required
for gonadal morphogenesis in Caenorhabditis elegans.
ADAMTS-9 and ADAMTS-20 have an identical modular structure, are
distinct in possessing 15 TSRs and a unique C-terminal domain, and have
a similar gene structure, suggesting that they comprise a new subfamily
of human ADAMTS proteases. ADAMTS20 is very sparingly
expressed, although it is detectable in epithelial cells of the breast
and lung. However, ADAMTS9 is highly expressed in embryonic
and adult tissues, and therefore we characterized the ADAMTS-9 protein
further. Although the ADAMTS-9 zymogen has many proprotein convertase
processing sites, pulse-chase analysis, site-directed mutagenesis, and
amino acid sequencing demonstrated that maturation to the active form
occurs by selective proprotein convertase (e.g. furin)
cleavage of the Arg287-Phe288 bond. Although
lacking a transmembrane sequence, ADAMTS-9 is retained near the cell
surface as well as in the ECM of transiently transfected COS-1 and 293 cells. COS-1 cells transfected with ADAMTS9 (but not
vector-transfected cells) proteolytically cleaved bovine versican and
aggrecan core proteins at the Glu441-Ala442
bond of versican V1 and the Glu1771-Ala1772
bond of aggrecan, respectively. In contrast, the ADAMTS-9 catalytic domain alone was neither localized to the cell surface nor able to
confer these proteolytic activities on cells, demonstrating that
the ancillary domains of ADAMTS-9, including the TSRs, are required
both for specific extracellular localization and for its versicanase
and aggrecanase activities.
The ADAMTS (A disintegrin-like
and metalloprotease (reprolysin type) with
thrombospondin type I motif) family consists of secreted zinc metalloproteases with a precisely ordered modular organization that includes at least one thrombospondin type I repeat
(TSR)1 (1, 2). Important
functions have been established for several members of the family.
ADAMTS-4, ADAMTS-5, and (less efficiently) ADAMTS-1 degrade the
cartilage proteoglycan aggrecan and are referred to as aggrecanases
(3-5). They play a major role in aggrecan loss in arthritis (6, 7).
ADAMTS-1 and ADAMTS-4 participate in the turnover of the
aggrecan-related proteoglycans versican and brevican in blood vessels
(8) and the nervous system, respectively (9). ADAMTS2
mutations cause dermatosparaxis, a recessively inherited disorder
characterized by severe skin fragility that results from incomplete
proteolytic removal of the procollagen I amino propeptide
(N-propeptide) (10). ADAMTS-3 and ADAMTS-14 are procollagen
N-propeptidases with probable roles in procollagen II processing in
cartilage or procollagen I processing in tissues other than skin,
respectively (11, 12). ADAMTS13 mutations lead to inherited
thrombocytopenic purpura, a coagulation disorder caused by deficient
proteolytic processing of von Willebrand factor (13).
Adamts1-null mice have abnormal adipogenesis, defective angiogenesis in the adrenal gland, and a defect of ureteric ECM turnover, leading to hydronephrosis (14). Adamts2-null mice have fragile skin, and males are infertile (15). Many other ADAMTS
enzymes have been discovered through molecular cloning, and their
functions are presently unknown. Altogether, 19 human ADAMTS symbols
identifying 18 distinct genes and their products have been assigned
(note that ADAMTS5 (1) and ADAMTS11 (4) designate the same
gene).2
ADAMTS are also present in invertebrates, which contain fewer ADAMTS
genes than mammalian genomes. A Caenorhabditis elegans ADAMTS gene, gon-1, has an essential role in reproduction
(16). The protease (GON-1) encoded by gon-1 is required for
migration of distal tip cells during gonadal morphogenesis. It may have a role in degradation of basement membrane or for processing of extracellular cues required for cell migration (16). GON-1 is the
largest of all ADAMTS enzymes described to date and contains 18 TSRs
(16). In addition, it has a presumed globular domain at the C terminus
without similarity to known proteins.
Human ADAMTS-9, as previously described (17) contains four TSRs.
Despite being a much smaller enzyme than GON-1, it had greater sequence
similarity to it than to any other human ADAMTS (17). Here, we
characterize a considerably longer form of ADAMTS-9 (designated
ADAMTS-9B, but referred to subsequently in this paper as ADAMTS-9) that
we propose is the authentic full-length product of ADAMTS9.
In addition, we have discovered a novel enzyme, ADAMTS-20, and
determined its complete primary sequence. ADAMTS-9 and ADAMTS-20 have
an identical domain organization and exon structure and a very similar
primary sequence, showing that they comprise a distinct subfamily of
GON-1-related ADAMTS proteases in the mammalian genome. We have
characterized the zymogen maturation and cellular localization of the
more highly expressed of these two proteins, ADAMTS-9, and have
investigated its role in proteolysis of the large aggregating proteoglycans versican and aggrecan. Our data demonstrate the critical
requirement of the ancillary domains for the proteolytic function and
localization of ADAMTS-9.
cDNA Cloning and Sequence Analysis of ADAMTS9 and
ADAMTS20--
BLAST (Basic Local Alignment Search Tool) programs from
the National Center for Biotechnology Information were used to search the data base of expressed sequence tags (dBEST), using the protein sequences of ADAMTS proteases previously discovered by us (1, 18). To
extend the initially identified ADAMTS9 cDNA to the 5'-end, human chondrocyte, muscle, heart, or fetal brain mRNA (Marathon cDNA, Clontech, Palo Alto, CA) was
used as the template for rapid amplification of cDNA ends as
previously described (1). To confirm that the overlapping cDNA
clones obtained represented a contiguous mRNA, the complete ORF was
amplified by PCR. The oligonucleotide primers
5'-AAGCGGCCGCACCATGCAGTTTGTATCC-3'
(NotI site underlined and start codon italicized) and
5'-CTCGAGAATAAAACTCGCACCTCCAGGC-3' (XhoI site underlined and modified stop codon italicized)
were used for PCR with human fetal skeletal muscle cDNA as template and Advantage 2 polymerase (Clontech, Palo Alto,
CA). The 5.8-kb PCR product was cloned into pGEM-T Easy (Promega,
Madison, WI) and sequenced completely. cDNA cloning of
Adamts9 will be reported elsewhere.3
To ask whether there existed additional ADAMTS proteases with a domain
organization similar to GON-1 and ADAMTS-9, the human genome sequence
(Celera, Rockville, MD) was searched using the amino acid sequence of
the unique C-terminal domain of ADAMTS-9. GENSCAN (available on the
World Wide Web at genes.mit.edu/GENSCAN.html) analysis of genomic DNA
upstream and downstream of the initially identified ADAMTS20
sequence was used to identify putative ADAMTS20 exons.
Oligonucleotide primers based on the sequences of these putative
ADAMTS20 exons were used for PCR spanning adjacent exons using cDNA derived from the human K562 (erythroleukemia) and A549 (lung cancer) cell lines.
The exon-intron structures of ADAMTS9 and
ADAMTS20 were deduced by comparison of the respective
cDNAs with human genome sequences using BLAST searches of private
(Celera) and public (GenBankTM) databases.
Northern Blot and Quantitative RT-PCR of ADAMTS9 and ADAMTS20 and
ADAMTS20 RNA in Situ Hybridization Analysis--
Multiple tissue
northern blots containing 1 µg/lane poly(A+) RNA from
mouse embryos and individual adult mouse and human tissues (Clontech, Palo Alto, CA) were hybridized to
[
RNA in situ hybridization was performed essentially as
previously described (19), using 35S-labeled antisense and
sense cRNA probes transcribed from a 600-nt cDNA template encoding
the unique domain of ADAMTS-20. Normal human breast and lung samples as
well as samples of squamous cell carcinoma of breast and adenocarcinoma
of lung were obtained under a Cleveland Clinic Foundation Institutional
Review Board-approved protocol and fixed in formalin (tissue samples
were provided by the Cooperative Human Tissue Network). 5-µm-thick
paraffin sections were hybridized to the probes prior to dipping in
photographic emulsion (Eastman Kodak Co.) and followed by
autoradiographic exposure for 7 days. Nuclei were stained with
4',6-diamidino-2-phenylindole.
ADAMTS9, ADAMTS4, and ADAMTS5 Expression Plasmids--
The
ADAMTS9 cDNA was excised as a
NotI-XhoI fragment and cloned into the
NotI and SalI sites of pFLAG-CMV-5a (Sigma) to
introduce an in-frame C-terminal FLAG tag (ADAMTS-9FLAG).
For expression of ADAMTS-91-508 (the signal peptide,
prodomain, and catalytic domain), PCR amplification was done using the
same forward primer as for the full-length ADAMTS9 cDNA,
the reverse primer 5'-AACTCGAGTTAGGCAAAGGGTAGGGTCTG-3' (XhoI site underlined), and fetal heart cDNA
(Clontech) as template. The resulting amplicon was
cloned in pFLAG-CMV-5a (Sigma) and pcDNA3.1 MYC/HIS B+ (Invitrogen)
to generate proteins with in-frame C-terminal FLAG or
myc-His tags, respectively, ADAMTS-91-508FLAG, and ADAMTS-91-508MYC/HIS. Site-directed mutagenesis of the convertase (e.g. furin) sites (Arg33
The insert of the KIAA0688 gene (20) encoding ADAMTS-4 (3)
in pBluescript SK (Stratagene) was excised with EcoRI and XhoI and inserted into the corresponding sites of
pcDNA3.1MYC/HIS A- (Invitrogen) to generate a mammalian expression
vector producing untagged ADAMTS-4. The ADAMTS4 and
ADAMTS5 ORFs from the convertase-processing site to the stop
codon were PCR-amplified and cloned into p3XFLAG-CMV-9 (Sigma) for
expression in frame with a preprotrypsin leader sequence and three
tandem FLAG tags present just downstream of the signal peptidase
cleavage site. These proteins are therefore secreted with N-terminal
FLAG tag (3×FLAGADAMTS-4 or 3×FLAGADAMTS-5). All expression plasmids and site-directed mutations were verified by
DNA sequencing.
ADAMTS-9 Localization in Transfected Cells--
COS-1 and
293-HEK cells were maintained and transfected with
ADAMTS-9FLAG, ADAMTS-91-508FLAG,
3×FLAGADAMTS-4, or 3×FLAGADAMTS-5, as
described previously (21). Transfected cell lysates and culture medium
were harvested separately after 48 h and were separated by
reducing SDS-PAGE followed by Western blot analysis using the FLAG M2
monoclonal antibody (Sigma). For immunolocalization of extracellular
ADAMTS-9FLAG, ADAMTS-91-508FLAG, 3×FLAGADAMTS-4, and 3×FLAGADAMTS-5, cells
were stained with anti-FLAG M2 monoclonal antibody 48 h
post-transfection without permeabilization as previously described
(21). Alternatively, transfected cells were stained following fixation
in 4% paraformaldehyde (staining with permeabilization). Nuclei were
stained with 4',6-diamidino-2-phenylindole. As controls, COS-1 and 293 cells were transfected with the empty FLAG vector alone, followed by
the immunostaining procedure, or the primary antibody was omitted for
FLAG staining.
To release ADAMTS-9 from the cell surface, transfected 293 cells and
ECM were harvested by scraping and resuspended in phosphate-buffered saline (10 mM phosphate buffer, pH 7.4, 2.7 mM
KCl, 137 mM NaCl). Cells and ECM were gently agitated by
end-over end rotation in PBS alone or in PBS plus 100 mM or
200 mM NaCl at 4 °C for 30 min.
ADAMTS-91--
508MYC/HIS Purification and
Analysis
Deglycosylation of lysate and conditioned medium from
ADAMTS-91-508FLAG-transfected cells was done using 10 units of PNGase F (Roche Molecular Biosciences) for 3 h at
37 °C in 150 mM sodium phosphate, pH 7.4, 50 mM EDTA, 0.1% SDS, 1% 2-mercaptoethanol, 0.5% Triton
X-100, followed by immunoprecipitation with anti-FLAG M2 as described
below. Stably transfected cells were cultured with and without
tunicamycin A homolog (Sigma) as previously described (21), followed by
Western blotting of conditioned medium and cell lysates.
Biosynthesis of ADAMTS-9--
Unless specified, reagents were
purchased from Sigma. QBI 293A cells (Quantum Biotechnologies,
Montreal, Canada) were maintained in complete DMEM containing 10%
heat-inactivated fetal bovine serum, 2 mM
L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cells were transiently transfected with
ADAMTS-91-508FLAG or ADAMTS-91-508MYC/HIS
using Fugene 6 (Roche Molecular Biosciences). 24 h following
transfection, cells were washed twice with warm phosphate-buffered
saline and incubated in Met/Cys-free medium (MEM SelectAmine kit;
Invitrogen), supplemented with 10% dialyzed FCS, 1 mM
glutamine, and a [35S]methionine/cysteine mixture
(EXPRE35S35STM; PerkinElmer Life Sciences). A
15- or 30-min labeling (pulse) was followed by incubation in complete
nonradioactive medium (chase) for the indicated times. The cell layer
was washed with PBS, and cells were lysed with 1 ml of radioimmune
precipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, and 4 mM EDTA) containing protease inhibitors (1 µM
aprotinin, 10 µM pepstatin, 10 µM
leupeptin, and 1 µM phenylmethylsulfonyl fluoride).
Samples were centrifuged to remove insoluble material. FLAG M2 antibody
or penta-His antibody (Qiagen, Mississauga, Canada) was added to cell
lysate and medium followed by overnight incubation at 4 °C. Protein
A/G Plus-agarose beads were added and incubated with samples for 1 h at 4 °C. Beads were washed three times with 1 ml of radioimmune
precipitation buffer, and labeled proteins were resolved by reducing
SDS-PAGE. Gels were treated with ENTENSIFY reagent (PerkinElmer Life
Sciences), dried, and exposed for fluorography.
Analysis Of Proprotein Convertase Processing--
CHO.RPE
40 cells (22) were maintained as previously described (32). They were
transfected with ADAMTS-91-508MYC/HIS alone or in
combination with furin. QBI 293A cells were transiently transfected
either with ADAMTS- 91-508MYC/HIS, or its derivatives obtained by site-directed mutagenesis. Cells were metabolically labeled
as above for 3 h, and immunoprecipitation and fluorography were
done as above.
Proteolytic Processing of the Versican and Aggrecan Core
Proteins--
Versican monomer (a mixture of the V1 and V0 forms) was
isolated from bovine aorta, as previously described (8). COS-1 cells
were transfected with ADAMTS-9FLAG,
ADAMTS-91-508FLAG, or ADAMTS-4 expression plasmids or with
empty vector pFLAG-CMV-5a (as negative control) in six-well plates in
DMEM plus 10% fetal bovine serum. Cells from a single well were used
for each experiment. 48 h after transfection, cells were scraped
off and suspended in serum-free DMEM followed by six washes in fresh
serum-free DMEM. Cells were resuspended in a final volume of 75 µl of
serum-free DMEM. Versican (5 µg in a volume of 25 µl) was added,
and the reaction was incubated at 37 °C for 18 h. The reaction
was centrifuged briefly, and the cell pellets were retained for Western
blotting of ADAMTS-9FLAG using the anti-FLAG M2 monoclonal
antibody (Sigma). An equal volume of 2× glycosaminoglycan digestion
buffer (200 mM Tris, pH 6.5, 100 mM sodium
acetate) containing 0.5 units of chondroitinase ABC (Seikagaku) was
added to the supernatant, followed by incubation for 16-18 h at
37 °C. Protein was precipitated with 5 volumes of acetone at
Aggrecan monomer was isolated from bovine articular cartilage as
previously described (23). Aggrecan (20 µg) was incubated with
transfected cells as described above. Neoepitope Western blot analysis
was as performed for versican (above), except that the proteolytic
cleavage at the Glu1771-Ala1772 bond of
aggrecan was detected using anti-Ala1772-Gly-Glu-Gly (AGEG)
antiserum (24) (provided by Micky Tortorella).
Cloning of ADAMTS9 and ADAMTS20 cDNAs--
Our search for
novel ADAMTS proteases identified a human expressed sequence tag
(GenBankTM accession number AA205581 encoded by IMAGE clone
646675) from neuroepithelium-derived NT2 cells treated with retinoic
acid. The ORF of this expressed sequence tag was homologous to ADAMTS proteases and encoded four TSRs followed by a C-terminal domain containing 10 cysteines that was similar to the C terminus of a
polypeptide predicted by the C. elegans F25H8.3 cosmid
(C. elegans protein data base Wormpep,
www.sanger.ac.uk/Projects/C_elegans/wormpep) and subsequently
identified as GON-1. The novel human ORF was designated ADAMTS-9.
Completion of the full-length protein coding sequence to the putative
start codon required several rounds of rapid amplification of cDNA
ends. Together, the cloned cDNA sequences represent an mRNA of
8 kb (Fig. 1a). The
3'-untranslated region in IMAGE clone 646675 contained a consensus
polyadenylation signal (AATAAA) 15 nucleotides upstream of the poly(A)
tail. The most 5' clone obtained (TS9-B10) contained 32 bp of the
5'-untranslated region. The putative signal peptide coding sequence was
preceded by a methionine codon within a satisfactory Kozak consensus
sequence (A at
The search for ADAMTS-9-related proteins led to identification of a
polypeptide (Celera hCP1629711) predicted by exons on human chromosome
12. The complete 5733-nt-long ADAMTS-20 ORF was assembled from
overlapping cDNA clones (Fig. 1a). The
ADAMTS20 mRNA was found in low quantities, routinely
requiring 35 cycles of PCR or nested PCR for visualization of the PCR
products on a gel. Because of the rarity of ADAMTS20
transcripts as well as the presence of numerous regions that are
difficult to PCR-amplify, we have been so far unable to obtain the
complete ORF in a single PCR reaction.
Identical Domain Organization and Similar Primary Structure of
ADAMTS-9 and ADAMTS-20--
ADAMTS-9 and ADAMTS-20 are similar in
length, containing 1935 and 1911 amino acids, respectively (Figs.
1b and 2). Each contains a
C-terminal array of 14 TSRs (15 TSRs/enzyme) that is interrupted by
short "linker" peptides located between TSR-6 and -7 and TSR-8 and
-9 that do not have similar sequences. ADAMTS-9 and ADAMTS-20 are very
similar to each other, with 48% identity and 64% similarity. The cysteine signatures of individual modules in ADAMTS-9 and ADAMTS-20
are identical to those of most other ADAMTS enzymes, with the exception
of the procollagen aminopropeptidases (ADAMTS-2, ADAMTS-3, ADAMTS-14)
and ADAMTS-13, which have distinctive prodomains and catalytic domains
(12). Each module in ADAMTS-9 and ADAMTS-20 (with one exception,
described below) contains an even number of cysteines, suggesting
participation in internal disulfide bonds. There are 126 cysteines in
mature ADAMTS-9, predicting 63 intrachain disulfide bonds. ADAMTS-20
has a Cys to Tyr substitution in TSR-13 (Fig. 2). Since the substituted
Cys is the fourth of six conserved cysteines in TSRs, TSR-13 in
ADAMTS-20 may contain two intrachain disulfide bonds instead of three
and have an unattached cysteine.
The predicted molecular mass of the full-length enzymes is 216 kDa
(ADAMTS-9) and 214 kDa (ADAMTS-20). The mass will decrease by ~2-3
kDa following signal peptide processing. In addition, both enzymes have
a prodomain that is likely to be proteolytically processed prior to or
during secretion. ADAMTS-9 contains five consensus furin cleavage sites
in its prodomain, whereas ADAMTS-20 contains three (Figs. 1b
and 2). Two sites, those corresponding to Arg74 and
Arg287 in ADAMTS-9, are conserved with ADAMTS-20. Following
processing at the furin recognition sequence closest to the C terminus,
mature ADAMTS-9 is predicted to have a molecular mass of 184,000 and mature ADAMTS-20 a molecular mass of 185,000. Both enzymes contain consensus sites for N-linked glycosylation
(Asn-X-Ser/Thr, where X is any amino acid except
Pro), 9 in ADAMTS-9 and 15 in ADAMTS-20 (Fig. 1b). Five such
sites, including three in the unique C-terminal domain, are conserved
in ADAMTS-9 and ADAMTS-20. Because of the high likelihood of
utilization of these sites, the molecular mass of ADAMTS-9 and
ADAMTS-20 will probably be in excess of that predicted (i.e.
>185,000). Although there are a number of Ser-Gly or Gly-Ser motifs in
both ADAMTS-9 and ADAMTS-20, most are within presumed disulfide-bonded
domains and lack the expected sequence context for xylosyltransferase
recognition (25, 26). However, one motif in the middle of the ADAMTS-9
spacer domain with the sequence Glu-Tyr-Ser830-Gly-Ser832-Glu-Thr-Ala-Val-Glu
lies within a sequence context that is compatible with GAG attachment
to Ser830 or Ser832. A similar sequence is
present at this location in ADAMTS-20 (Fig. 2). ADAMTS-9 and ADAMTS-20,
respectively, contain three and two Cys-Ser-Val-Thr-Cys-Gly (CSVTCG)
motifs that are believed to mediate binding to the cell surface
molecule CD36 (27, 28) (Fig. 2). In addition, each enzyme contains two
BBXB motifs (where B represents basic amino acid and
X represents any amino acid) that have been shown to mediate
heparin and sulfatide binding (27, 29) (Fig. 2). Neither enzyme
contains an Arg-Gly-Asp motif.
ADAMTS-9 and ADAMTS-20 Do Not Have Identical Zinc-binding Catalytic
Site Motifs--
The ADAMTS-9 catalytic site is identical to that of
ADAMTS-1 and ADAMTS-15 and very similar to that of ADAMTS-4 (Fig.
3a). The unique feature of
ADAMTS-9, ADAMTS-1, and ADAMTS-15 is the presence of a proline residue
preceding the third zinc-coordinating histidine (Fig. 3a).
The corresponding amino acid is leucine in ADAMTS-4, the next most
closely related enzyme. The ADAMTS-20 zinc-binding site is not
identical to that of any other ADAMTS but is most closely related to
that of ADAMTS-7 and ADAMTS-12 with 4/12 variant amino acids (Fig.
3a). Moreover, all of the substitutions in the ADAMTS-20
active site relative to ADAMTS-7 and ADAMTS-12 are conservative ones.
Alignment and clustering of the published ADAMTS proteases confirm the
unique place of ADAMTS-9 and ADAMTS-20 in the ADAMTS family (Fig.
3b) and indicate that they constitute a distinct subfamily
of proteases.
ADAMTS-9 and ADAMTS-20 Are Related to GON-1--
The domain
organization and primary sequence of ADAMTS-9 and ADAMTS-20 have a
greater similarity to GON-1 than any other mammalian ADAMTS enzyme
(Figs. 1b and 2). ADAMTS-9 and ADAMTS-20 are equally related
to GON-1 in paired BLAST comparisons. The percentage identity of
ADAMTS-9 protein to GON-1 is 33% (that of ADAMTS-20 is 32%), and the
percentage similarity (including conservative substitutions) is 46%
for both ADAMTS-9 and ADAMTS-20 relative to GON-1. The zinc-binding
active site sequence of GON-1 resembles ADAMTS-9 more closely than
ADAMTS-20, with just 2 of 14 variant amino acids (Fig. 3a).
The conserved C-terminal-most convertase-processing site is at an
identical location in ADAMTS-9, ADAMTS-20, and GON-1. The unique
C-terminal domain varies slightly in length but nevertheless is highly
similar in the three enzymes, including an identical cysteine signature
(Fig. 2). TSR-1 is well conserved in these ADAMTS enzymes, but there is
less similarity between TSRs 2-15 of ADAMTS-9 and ADAMTS-20 and TSR-2
to -18 of GON-1.
ADAMTS9 and ADAMTS20 Are Located on Different Chromosomes but Have
a Highly Conserved Gene Structure--
Exons corresponding to the
overlapping ADAMTS9 and ADAMTS20 cDNA clones
were found arranged sequentially on human chromosomes 3p14 (as
previously mapped (18)) and 12q11, respectively (GenBankTM
and Celera Genomics, Rockville, MD). ADAMTS9 and
ADAMTS20 are large, being 137 and 200 kb in size,
respectively. The ADAMTS9 and ADAMTS20 ORFs are
each encoded by 39 exons (Fig. 2). Notably, all of the splice
boundaries between exons are identical in ADAMTS9 and
ADAMTS20 mRNAs (Fig. 2). The exons vary in size, with
the largest, coding exon 2, encoding half the prodomain and the
smallest, exon 7, encoding just 14 amino acids within the catalytic
domain. Like other ADAMTS proteases, the active site sequence is split by an intron. The majority of the TSRs are encoded by single exons whose 5' end is just upstream of the first cysteine codon, but there
are three exceptions to this rule: TSR-1, TSR-2, and TSR-3. TSR-1 and
TSR-2 are each split by an exon junction and are thus encoded by two
separate exons, although the exon junctions are not at the same
location in each TSR. The 5' exon junction in TSR-3 is 8 amino acids
upstream of the first cysteine residue. Linker 2 is encoded by the exon
encoding TSR-8, whereas a separate exon encodes linker 1. Interestingly, the unique C-terminal domain is encoded by six exons, so
that with the exception of 13 of 15 TSRs, none of the presumed
intrachain disulfide-bonded domains are encoded in their entirety by
single exons.
ADAMTS9 and ADAMTS20 Are Differently Regulated--
In contrast to
ADAMTS9, expression of ADAMTS20 was not
detectable in Northern blots of human adult mRNA. In mouse embryos, a single ADAMTS9 mRNA of ~8.5 kb was detected (Fig.
4a). Expression was highest in
7- and 17-day-old embryos and lower in 11- and 15-day-old embryos. A
number of adult human tissues expressed ADAMTS9, with
highest expression in heart, placenta, and skeletal muscle (Fig.
4b). All these tissues contained an 8.0-kb mRNA, but
kidney and ovary contained additional mRNAs of 4.5 kb, and kidney
contained a hybridizing mRNA of 3.0 kb. Spleen, thymus, prostate,
testis, small intestine, and peripheral blood leukocytes had low to
undetectable levels of ADAMTS9 on Northern blots.
Quantitative RT-PCR was undertaken to determine which tissues, if any,
expressed ADAMTS20 and to measure relative levels of ADAMTS9 and ADAMTS20 mRNAs. In all human
fetal and adult tissues examined (other than peripheral blood
leukocytes), ADAMTS20 mRNA levels were 1-3 orders of
magnitude lower than ADAMTS9 (Fig. 4, c and
d). ADAMTS9 was expressed at higher levels than
ADAMTS20 in ovary and testis, the two tissues relevant to
GON-1 function (Fig. 4d). By Northern blot analysis,
expression of ADAMTS9 was substantially higher in ovary than
in testis (Fig. 4b).
Since ADAMTS20 expression levels were so low, we asked
whether it was detectable at the single cell level using the highly sensitive RNA in situ hybridization approach. The data
demonstrate that in normal breast (Fig. 4f) and lung (Fig.
4g), as well as in breast cancer (Fig. 4i) and
lung cancer (Fig. 4l), ADAMTS20 mRNA was
detectable at low levels in epithelial cells but was not expressed in
stromal cells (Fig. 4, f, g, i, and
l). Sense probe showed no hybridization (Fig. 4,
e, h, and k).
ADAMTS-9 Is Located Near the Cell Surface but Not in Conditioned
Medium--
ADAMTS-9FLAG was detected in lysates of
transiently transfected COS-1 and 293 cells as two major anti-FLAG
reactive bands migrating at ~180 and >250 kDa under reducing
conditions (Fig. 5a), although
the 250-kDa band was inconsistently seen. In addition, a number of
smaller FLAG-tagged bands, presumably derived from the full-length
ADAMTS-9 were also seen (Fig. 5a, upper
panel). Treatment of ADAMTS-9-expressing cells with an
increasing concentration of NaCl demonstrated a
concentration-dependent release of ADAMTS-9 from the cells
(Fig. 5a, lower panel). Due to the
unfavorable effects of supraphysiological salt concentrations on cell
viability, concentrations higher than 340 mM were not
tested.
To identify the cellular or extracellular location of ADAMTS-9 and
contrast it with ADAMTS-4, ADAMTS-5, and the ADAMTS-like protein, punctin (21), transiently transfected COS-1 and 293 cells were
immunostained with anti-FLAG M2 antibody with or without permeabilization. In permeabilized COS-1 cells, there was cytoplasmic staining characteristic of localization to endoplasmic reticulum and
Golgi apparatus as well as cell surface staining (data not shown). In
nonpermeabilized cells, ADAMTS-9 was localized to the cell surface of
transiently transfected cells and/or to their substratum (Fig. 5,
b and c). Negative controls did not show
immunostaining with the FLAG M2 antibody. ADAMTS-4 immunolocalization
resembled ADAMTS-9, with granular staining present in the cell
substratum and on the cell surface (Fig. 5d), whereas
ADAMTS-5 was exclusively present in the substratum (Fig.
5e). The substratum staining of ADAMTS-4 or ADAMTS-9 and
ADAMTS-5 were qualitatively different; ADAMTS-5 staining was of fine
granularity and was spread under and around the transfected cell in a
nebulous appearance (Fig. 5e), whereas that of ADAMTS-9 and
ADAMTS-4 was coarsely granular and limited to cell boundaries.
Processing of Versican and Aggrecan Core Proteins by
ADAMTS-9-transfected Cells--
Under the incubation conditions used,
versican and aggrecan underwent proteolytic cleavage mediated by cells
transfected with ADAMTS-9 or ADAMTS-4 but not with the vector control
(Fig. 5f). The proteolyzed peptide bond was identified using
neoepitope antibodies that demonstrated cleavage at previously
characterized "versicanase" and "aggrecanase" sites but not in
intact versican or aggrecan core protein (8, 24). The
anti-DPEAAE-immunoreactive 70-kDa band seen in ADAMTS-9- and
ADAMTS-4-digested versican represents the neoepitope derived from the
V1 form (Fig. 5f, left panel). The
anti-AGEG immunoreactive band following ADAMTS-9 digestion corresponds
in size to that obtained following proteolytic digestion of aggrecan by
ADAMTS-4 (24) (Fig. 5f, right panel).
ADAMTS-4-transfected cells produced a stronger immunoreactive band than
ADAMTS-9-transfected cells. The ADAMTS-9 catalytic domain without the
ancillary domains (encoded by ADAMTS-91-508) did not
process aggrecan or versican at these sites (data not shown).
Intracellular Maturation of ADAMTS-9 Involves N-Glycosylation of
the Prodomain and Furin Processing at the
Arg287-Phe288 Bond--
The predicted
molecular masses of signal peptide processed and
ADAMTS-91-508 that is processed at the consensus
proprotein convertase sites are shown in Fig.
6a. Transient expression of ADAMTS-91-508FLAG in 293 cells followed by pulse-chase
analysis, immunoprecipitation using anti-FLAG M2 antibody, and
fluorography identified three major immunoreactive bands in cell
lysates with molecular masses of ~66, 56, and 54 kDa, respectively.
The relative intensity of these bands varied with the duration of pulse
and chase. After a 15-min pulse and 60-min chase, the amount of the 66-kDa protein seen was significantly greater than that seen after a
15-min chase (Fig. 6b). Conversely, the 54-56-kDa doublet
was more prominent after a 15-min chase (Fig. 6b). The
66-kDa band intensified substantially after a 135-min chase with very
little of the 54-56-kDa doublet being detectable. When cell lysate and culture medium were immunoprecipitated and immunoblotted with anti-FLAG
M2 antibody 48 h following transfection of QBI 293A cells, the
cells contained the 66-kDa band and essentially no 54-56-kDa doublet
(Fig. 6c). When these cell lysates were treated with PNGase
F, this 66-kDa band was reduced to a doublet of ~54-56 kDa (Fig.
6c). Collectively, these observations suggest that the 66-kDa band is derived from a 54-56-kDa precursor by
N-linked glycosylation. N-Glycosylation of
ADAMTS-91-508HIS was confirmed by culture of stably
transfected cells in the presence of the tunicamycin A homolog (data
not shown). Under the pulse-chase conditions used, no labeled protein
could be immunoprecipitated from the conditioned medium (Fig.
6b), and protein corresponding in size to the active form
(28 kDa) was not seen in cell lysate. However, in stably transfected
cells (not shown) or immunoprecipitation 48 h after transfection,
the mature, tagged protein could be detected in culture medium (Fig.
6c). Deglycosylation did not alter the migration of the
secreted mature enzyme (Fig. 6c). N-terminal sequencing of
the secreted mature ADAMTS-91-508HIS gave the sequence
Phe-Ser-Leu-Tyr-Pro-Arg-Phe.
Furin-deficient CHO.RPE 40 cells did not process ADAMTS-9 (Fig.
7a). Processing was rescued by
transfection with furin (Fig. 7a). In QBI 293A cells, the
Arg33 Identification of the Full-length Product Of ADAMTS9--
Although
a report of the ADAMTS9 mRNA (GenBankTM
accession number AF 261918) and ADAMTS9 chromosomal
localization was published (17) while our work was in progress, the
novel sequence data we report here extend the predicted C terminus of
that protein further to include an additional 10 TSRs and the unique
C-terminal domain. Our data suggest that the ADAMTS9
transcript presented here encodes the full-length, authentic product of
this gene for several reasons. First, the previously described
ADAMTS9A cDNA diverges from our ADAMTS9
sequence at an unspliced intron (deduced by comparison of the
ADAMTS9A and mRNA sequence with the ADAMTS9 genomic sequence). Another ADAMTS9 product predicted by the
sequence of the KIAA1233 gene (GenBankTM
accession number AB037733) is incomplete at both the amino and carboxyl
termini. Comparison of this sequence with the cDNA sequence
reported here and the ADAMTS9 genomic sequence suggests the
inclusion of an unspliced intron leading to a premature stop codon.
Intron inclusion suggests cloning of partially processed pre-RNA, not
authentic mRNA. Second, the ADAMTS-9A transcript does not contain a
consensus polyadenylation sequence upstream of the poly(A) tail, in
contrast to the ADAMTS9 transcript reported here.
Third, Northern analysis demonstrated that probes from the novel
sequences we describe here, as well as a probe from the region shared
by all transcripts (data not shown), hybridized to the same major 8-kb
band on Northern blots, suggesting that the dominant transcript in most
tissues encodes the longer form, ADAMTS-9B.
Previous studies of ADAMTS9 had shown widespread expression
in fetal and adult tissues by RT-PCR (18). Our studies using Northern
blots and quantitative RT-PCR are in agreement with this and provide
additional information about the mRNA size. The 8-kb ADAMTS9 mRNA is compatible with the long ORF we have
cloned. The identity of the smaller mRNAs found in kidney and ovary
is presently unclear. These could represent alternative splice forms or
unrelated transcripts that cross-hybridize with the probe. Since
ADAMTS20 mRNA is undetectable on Northern blots, both
its size and the existence of alternative forms is unknown. In fact,
ADAMTS20 transcripts are extremely rare in all of the
tissues we have examined, and there are only two human
ADAMTS20 expressed sequence tags (AU132053 and BG212007)
reported in GenBankTM. Nevertheless, a sensitive RNA
in situ hybridization approach did demonstrate low levels of
expression in epithelial cells of breast and lung origin. The
prevalence and biological significance of this low level expression is
unknown. Therefore, at the protein level, detailed characterization of
the more abundantly expressed enzyme, ADAMTS-9, was subsequently undertaken.
ADAMTS9 and ADAMTS20 Constitute a Distinct Subfamily of ADAMTS
Proteases--
ADAMTS proteases can be clustered into subfamilies of
closely related enzymes on the basis of their domain organization and primary sequences. The procollagen aminopropeptidase subfamily (ADAMTS-2, -3, and -14) represents the most striking example, and other
enzymes such as ADAMTS-7 and -12 and ADAMTS-6 and -10 occur in closely
related pairs. The ADAMTS-9 and ADAMTS-20 subfamily is
particularly interesting, because it is the first such ADAMTS subfamily
with a closely related ortholog in invertebrates, indicating, perhaps,
a highly conserved physiological role. However, unlike the other ADAMTS
subfamilies, ADAMTS-9 and ADAMTS-20 do not have identical
zinc-binding active site sequences. Furthermore, their expression
patterns are quite different, suggesting they may have nonredundant
biological roles.
The genomic organization of ADAMTS9 and ADAMTS20
bears little resemblance to other genes in the family. ADAMTS-1 is
encoded by nine exons, and the prodomain, disintegrin-like domain, and central TSR are each encoded by single exons, whose boundaries coincide
with the domain boundaries (30). In ADAMTS-1, a single terminal exon
encodes the spacer and two C-terminal TSRs (30). This is clearly not
the case with ADAMTS9 and ADAMTS20, where few
domains other than the TSRs are encoded by single exons.
ADAMTS13 (13) has 29 protein-coding exons whose boundaries
are different from ADAMTS-1, -9, and -20. The procollagen
aminopropeptidases share a different genomic organization (12).
Therefore, gene structure may be conserved in ADAMTS subfamilies, but
there is not a characteristic gene structure that is shared by the
entire family.
The Cys to Tyr substitution in TSR-13 is not an artifact of cloning,
because we found it both in the genomic DNA (in Celera and
GenBankTM databases), in the cloned cDNA, and in a
small number of normal human alleles in which the corresponding exon
was subjected to PCR-direct sequencing (data not shown). It may
represent a non-synonymous single nucleotide (4715A Intracellular Maturation Of ADAMTS-9 Involves Glycosylation of the
Prodomain and Processing at a Single Proprotein Convertase-processing
Site--
Following removal of the signal peptide and entrance into
the secretory pathway, ADAMTS proteases, like ADAMs and some MMPs, are
processed further by one or more proprotein convertases to remove the
prodomain and undergo additional post-translation modification such as
glycosylation. Proprotein convertases (e.g. furin) are serine proteases present in the Golgi apparatus or at the cell surface
that typically cleave immediately following a consensus recognition
sequence rich in basic residues (31). Our studies showed that
processing did not occur in the absence of furin but could be rescued
by transfection of furin, demonstrating that proprotein convertases
were essential for pro-ADAMTS-9 maturation.
Our studies suggest that there is rapid glycosylation of the ADAMTS-9
prodomain following synthesis that is essentially complete in about
2 h. There is no N-glycosylation of the catalytic
domain, consistent with the observation that the prodomain contains
three consensus N-glycosylation sites, whereas the catalytic domain has
none. Our data indicated processing of the
Arg287-Phe288 peptide bond, whereas none of
the other furin sites appear to be used for enzyme maturation. We
should emphasize that the Arg280 mutation would abrogate
two furin sites, since this residue serves as the P1 Arg for the
Arg-Glu-Lys-Arg287 site as well as the P4 residue for the
Arg-Thr-His-Arg283 site. We could detect the 28-kDa mature
form intracellularly in the wild-type and Arg33
Western blotting of full-length ADAMTS-9 suggested that it undergoes
substantial post-translational modification. In keeping with the number
of consensus sites for N-linked glycosylation and the large
number of serine and threonine residues, glycosylation of full-length
ADAMTS-9 has also been noted (data not shown), as is shown in the
prodomain. Expression of full-length ADAMTS-9 demonstrated the
existence of a number of smaller FLAG-tagged fragments that were
presumably derived from it by proteolysis. Regulated processing has
been noted in ADAMTS-1 (32), ADAMTS-4 (33), and ADAMTS-12 (34) and is a
potentially intriguing phenomenon because the released ancillary
domains could have interesting biological functions or modify the
function of ADAMTS-9 (33). Proteolytic fragments of the native enzyme
will be sought in tissues and cells once specific high affinity
antibodies are available.
ADAMTS-9 Is Located near the Cell Surface and Is Involved in
Versican and Aggrecan Degradation--
Neither ADAMTS-9 or ADAMTS-20
nor any of the other known ADAMTS proteases has a potential
transmembrane sequence or a glycophosphatidylinositol signal anchor
sequence. Therefore, these are not predicted to be membrane-anchored
enzymes. Accordingly, studies with various ADAMTS proteases have shown
that they are soluble or associated with the ECM (3, 4, 35). ADAMTS-9
and ADAMTS-4 are therefore the first ADAMTS proteases shown to localize
near the cell surface, as demonstrated by immunofluorescence
microscopy, although their precise location relative to the cell
membrane or the binding mechanism is presently unknown. In contrast,
both the localization and appearance of ADAMTS-5 distribution are
different. Furthermore, although restricted to the ECM, ADAMTS-5
presents a different distribution than punctin, an ADAMTS-like protein
comprising only ancillary domains (21). Punctin localization to the
cell substratum (21) and the failure of ADAMTS-91-508 or
C-terminally truncated ADAMTS-1 (35) to be located in either the ECM or
cell surface strongly validates the role of the ancillary domains in anchoring these enzymes near the cell. ADAMTS-9 has consensus sites for
binding to heparin (and therefore to heparan sulfate proteoglycans) and
CD36, and these may be candidate cell surface and pericellular ECM
ligands. In support of this possibility, ADAMTS-9 was released from
cells and ECM by gentle washes with low concentrations of salt.
To identify potential substrates for ADAMTS-9, we relied upon
comparison of the ADAMTS active site sequences, the phylogenetic profile of the ADAMTS family, and the previous descriptions of their
enzymatic activities. The ADAMTS enzymes (ADAMTS-1, ADAMTS-4, and
ADAMTS-5) that process the large aggregating proteoglycans versican,
aggrecan, and brevican have very similar (although not identical)
active site sequences, but they have different domain structures.
Because ADAMTS-9 has an active site sequence identical to that of
ADAMTS-1 and similar to that of ADAMTS-4, we considered that it might
be a proteoglycan core protein-degrading enzyme. Since ADAMTS-9 was not
secreted into the culture medium of cells, we used a cell-based ADAMTS
assay. Serum-free culture medium has the appropriate pH and salt
concentration for ADAMTS activity and, when supplemented with calcium,
provided the reaction conditions necessary for the versicanase and
aggrecanase assays.
By analogy with aggrecanase-susceptible sites in aggrecan, Sandy
et al. (8) had previously predicted two putative ADAMTS cleavage sites in human versican and had prepared polyclonal antisera recognizing one such predicted neoepitope generated by proteolysis of
the V1 Glu441-Ala442 bond (8). Versican V0 and
V1 forms differ in the inclusion of the GAG-
A refined comparison of ADAMTS-4 and ADAMTS-9 cannot be done in the
cell-based assay, since transfection efficiency, expression levels,
secretion, and zymogen processing may be different. Purified ADAMTS-9
is not yet available, and given its complex domain structure and cell
surface localization, it may be difficult to obtain. We have purified
ADAMTS-91-508, but it does not process versican or
aggrecan, demonstrating the essential role of the ancillary domains in
substrate recognition and/or binding. Therefore, this form cannot be
used in kinetic studies to compare with ADAMTS-4. With these
limitations, however, it appears from our studies that ADAMTS-9 may be
an efficient versicanase, comparable with ADAMTS-4, but a less
efficient aggrecanase. Versican is widely distributed during
development and in adult tissues. Like aggrecan, it may have a
mechanical role, and since it interacts with fibrillin, fibulin-1, and
fibulin-2 through its lectin-like domain, it may have a specific role
in matrices enriched in these molecules (36-38). In addition, versican
is believed to provide guidance cues for migrating neural crest cells
(39). The presence of ADAMTS-9 near the cell surface and its homology
to an enzyme required for cell migration certainly make it an
appropriate enzyme for involvement in the migratory process. In
addition, the ability to process aggrecan warrants further
investigation of its involvement in skeletal development and cartilage
destruction in arthritis.
Comparison of Proteoglycan-degrading ADAMTS Enzymes and the
Procollagen Amino Propeptidases Suggests Differences in Stringency of
Enzyme-Substrate Interaction--
The procollagen N-propeptidases have
identical domain organizations and identical active site sequences (in
fact, ~70 amino acids around the zinc-binding site are identical in
these enzymes) (12). This identity suggests that the structural
requirements for procollagen processing are very stringent, and indeed,
the catalytic sites of the procollagen N-propeptidases have a
distinctive cysteine signature not found in other ADAMTS. Similarly,
ADAMTS-13, the von Willebrand factor protease, is unlike any other
ADAMTS in its domain organization and is clearly the major, if not
only, von Willebrand factor-processing enzyme (13), suggesting that the
structural requirements for this function are stringent as well. In
contrast, the four ADAMTS enzymes that degrade proteoglycan core
proteins have neither an identical domain organization nor identical
active site sequences. This dissimilarity suggests a relatively relaxed
structural requirement for proteoglycan processing and supports the
likelihood that additional ADAMTS enzymes may have activity against
proteoglycans. In the future, it will be important to define the
relative prevalence of each of the proteoglycan-degrading ADAMTS
enzymes in different tissues as well as in diseases such as arthritis
and to determine their tissue-specific role by targeted inactivation of
the corresponding mouse genes. In future studies, it will also be
important to ask whether ADAMTS-20 can process versican and aggrecan
and to ask whether ADAMTS-9 and ADAMTS-20 have biological roles similar
to GON-1.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-32P]dCTP-labeled ADAMTS9,
ADAMTS20, or Adamts9 probes, followed by
autoradiographic exposure for 3-7 days. cDNA panels derived from
human adult and fetal organs normalized with respect to
GAPDH mRNA levels were purchased from
Clontech. Real time PCR of these cDNA templates
was performed in an ABI Prism 7700 sequence detector using SYBR Green
PCR Core Reagents (Applied Biosystems, Foster City, CA), as previously
described (12). PCR amplifications were performed in triplicate for all
templates, along with parallel measurements of GAPDH
cDNA for normalization. The GAPDH-normalized quantitative data for
ADAMTS9 and ADAMTS20 were used to determine the
ADAMTS9/ADAMTS20 transcript ratio in all
templates examined. The following primers were used for amplification
at a concentration of 300 nM each: ADAMTS9
forward, 5'-GGACAAGCGAAGGACATCC-3'; ADAMTS9 reverse,
5'-ATCCATCCATAATGGCTTCC-3'; ADAMTS20 forward,
5-GGTGGCATGTTATTGGCAAAA-3'; ADAMTS20 reverse,
5'-CACAGTTACCATGGCATAGTTCTTG-3'; GAPDH primers were
described previously (12). RT-PCR performed in the absence of template
was negative with all primer pairs.
Ala,
Arg74
Ala, Arg280
Ala, and
Arg287
Ala) in ADAMTS-91-508MYC/HIS
was done using the QuikChange site-directed mutagenesis kit (Stratagene).
To obtain stably transfected 293 cells expressing
ADAMTS-91-508MYC/HIS, selection with G418 (750 µg/ml)
was applied after transfection, and selected clones were maintained in
culture medium containing 5% serum and 250 µg/ml G418. Conditioned
medium was dialyzed into binding buffer (20 mM sodium
phosphate, 500 mM NaCl, pH 7.8, containing 0.03% Brij-35
(Sigma)) prior to binding on a 5-ml Ni2+-Sepharose column
(ProBondTM; Invitrogen). The column was washed with 3 column volumes
of binding buffer. A gradient of 0-42.5 mM imidazole in
binding buffer was used to remove nonspecifically bound molecules from
the column. Stepwise elution was done using one-column volume batches
of 0-250 mM imidazole in binding buffer. Elution was
monitored by Western blotting using antibody 9E10. The majority of
protein was determined to elute at 50 mM imidazole. ADAMTS-91-508MYC/HIS was electrophoresed on 10% SDS-PAGE, electrotransferred to polyvinylidene difluoride membrane, and lightly
stained with modified Coomassie Blue (Simply Blue Safe Stain;
Invitrogen). The 28-kDa band was excised and subjected to Edman
degradation on an Applied Biosystems Procise 492 sequencer in the
Molecular Biotechnology Core Facility of the Lerner Research Institute.
20 °C for 15 min, dissolved in 50 µl of Laemmli sample buffer,
and electrophoresed on 7% SDS-PAGE prior to blotting to
nitrocellulose. A rabbit polyclonal antiserum to the versican
Asp-Pro-Glu-Ala-Ala-Glu (DPEAAE) neoepitope (8) (provided by Dr. John
Sandy) was used at 1:1000 dilution for Western blotting, followed by
enhanced chemiluminescence detection of antibody binding. Anti-DPEAAE
recognizes the new C terminus resulting from cleavage of versican at
the Glu441-Ala442 bond (this enumeration
describes the site in the V1 isoform); the corresponding peptide bond
is Glu1428-Ala1429 in the V0 isoform, since
this form contains an additional GAG-bearing region, GAG-
, as a
result of alternative splicing (8).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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3 relative to ATG), but there was no upstream,
in-frame stop codon.
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Fig. 1.
a, cloning of ADAMTS9 and ADAMTS20
cDNA. DNA ruler (in kb) is shown at the top. Each
box shows the overlapping cDNA clones from which the
complete ORF encoded by the respective mRNA was deduced, in
alignment with the encoded domains. Clone names are shown
above each clone. AATAAA denotes polyadenylation signal at
the 3' end of IMAGE clone 646675. b, domain organization of
ADAMTS-9B and ADAMTS-20 shown relative to GON-1. The key to
the domain structure is shown at the bottom. The amino acid
scale bar is at the upper
right. Since ADAMTS-9 and ADAMTS-20 have an identical domain
organization, only that of ADAMTS-9 is shown. The convertase-processing
sites and N-linked glycosylation sites shown are for
ADAMTS-9. The precise location of these sites in each enzyme is
indicated in Fig. 2.
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Fig. 2.
Alignment of primary sequences of ADAMTS-9,
ADAMTS-20, and the carboxyl domain of GON-1 (amino acids
1943-2165). The entire sequence of GON-1 (GenBankTM
accession number NP501792) is not aligned, because it has more TSRs
than ADAMTS-9 and ADAMTS-20, and these cannot be accommodated in the
alignment. Single-letter amino acid codes are used. The
vertical arrowheads indicate potential proprotein
convertase cleavage sites. The open arrow shows
the N terminus of mature ADAMTS-9. Consensus sequences for
N-linked glycosylation (Asn-X-Ser/Thr, where
X is any amino acid except proline) are
overlined. The metalloprotease active site sequence is
enclosed in a box, as is the "Met-turn" (VMA). The
estimated start of the disintegrin-like and spacer domains is
indicated. The disintegrin-like domain extends from this point until
TSR-1. The spacer domain extends to TSR-2. TSRs are
underlined and numbered consecutively.
Two linker peptides between TSR-6 and -7 and between TSR-8 and -9 are
indicated. Cysteines in the carboxyl-terminal domain (but not
elsewhere) are indicated by filled asterisks. A
Cys-to-Tyr change in TSR-13 of ADAMTS-20 is indicated by the filled
circle. Prospective GAG attachment sites are indicated by
open asterisks. Exon junctions are indicated by
open circles. An open
circle over an amino acid indicates that the exon
junction splits the codon, whereas an open circle
between amino acids indicates that the exon junction spares
the codon. The ADAMTS-9A ORF diverges from our sequence at the exon
junction in TSR 5.
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Fig. 3.
a, comparison of the active site
sequences of mammalian ADAMTS and GON-1 proteases. These are arranged
in descending similarity to ADAMTS-9. The overlined residues
are conserved in all ADAMTS proteases. b, phylogenetic
relationship between human ADAMTS protein sequences. These were
obtained using the ClustalW algorithm (Megalign software; DNAStar Inc.,
Madison, WI). In both a and b, ADAMTS-9 and
ADAMTS-20 are in boldface type, the previously
identified aggrecanases are italicized, and the procollagen
amino propeptidases are enclosed in a box.
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Fig. 4.
ADAMTS9 and ADAMTS20
mRNA expression. a, Northern blot of mRNA
from mouse embryos hybridized to an Adamts9 cDNA probe.
b, Northern blots from human tissues hybridized to an
ADAMTS9 cDNA probe. RNA kilobase markers are shown at
the left of the autoradiogram, and tissue origin is
indicated above each lane. c and
d, comparative quantitative RT-PCR analysis of
ADAMTS9 and ADAMTS20 mRNA in fetal human
tissues (c) and in adult human tissues (d).
Values for each tissue were normalized to GAPDH levels and
are indicated as -fold increase of ADAMTS9 over
ADAMTS20, except in leukocytes, where there was a relative
excess of ADAMTS20 over ADAMTS9 (indicated
by hatched bar and vertical
scale to the right). Tissue source is indicated
under each bar. e-m, RNA in
situ hybridization of ADAMTS20 mRNA in normal
breast, normal lung, and squamous cell carcinoma of breast and lung
adenocarcinoma. e, h, and k
show a lack of hybridization of normal breast, breast cancer, and lung
cancer, respectively, to sense probe. f and g,
hybridization of antisense probe to epithelial cells in the normal
breast duct epithelium (arrow) and bronchial lining
epithelium (arrow) respectively. i and
l, hybridization of antisense probe to carcinoma cells in
breast cancer (arrow in i) and lung cancer
(arrow in l). Note that tumor stroma
(asterisk) does not express ADAMTS20, and no
expression is seen in necrotic lung tumor (N). e
and f, h-j, and k-m, serial
sections. j and m, hematoxylin and eosin-stained
sections corresponding to i and j,
respectively.
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Fig. 5.
a, Western analysis of ADAMTS-9
expressed in COS-1 cells using anti-FLAG M2 antibody. Top
panel, lane 1, cell plus ECM lysate from cells
transfected with empty vector; lane 2, cell plus ECM lysate
from ADAMTS-9FLAG-transfected cells. The arrow
shows the expected size of ADAMTS-9, whereas the arrowhead
shows substantially modified ADAMTS-9. Lower panel, salt
elution of ADAMTS-9 from COS-1 cells. Salt concentrations are indicated
above each lane. b-e,
confocal microscopic localization of ADAMTS-9 (b and
c), ADAMTS-4 (d), and ADAMTS-5 (e) in
transfected COS-1 cells. ADAMTS localization is detected by the green
fluorescence. 4',6-Diamidino-2-phenylindole-stained nuclei are
blue. Sequential images were collected and reconstructed
into a z axis image shown below the corresponding
x-y axis image. f, proteolysis of versican
(left) and aggrecan (right) by 293 cells
transfected with ADAMTS-9 or ADAMTS-4. Cells were transfected with
ADAMTS-9 or ADAMTS-4 expression plasmids as indicated and incubated
overnight with versican or aggrecan. Versican proteolysis was detected
with anti-DPEAAE serum, and aggrecan proteolysis was detected
with anti-AGEG. As a control, versican and aggrecan were incubated
under identical conditions with cells transfected with the expression
plasmid lacking an insert. The 70-kDa versican fragment and the 100-kDa
aggrecan fragments containing the neoepitopes are indicated by
arrows.
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Fig. 6.
a, scheme of the protein encoded by
ADAMTS91-508FLAG. The domains included in the expressed
proteins and the locations of N-linked sugar attachment
(lollipops) and FLAG tag are shown. Below this
are the protein species predicted following signal peptidase cleavage
or cleavage at each of five consensus furin cleavage sites. The
expected molecular mass of each unmodified protein species
is shown at the right. b, pulse-chase analysis of
ADAMTS91-508FLAG-transfected QBI 293A cells. Cells were
pulsed with radiolabeled amino acids and chased for varying times as
indicated. Control cells were transfected with empty expression vector.
Cell extracts and media were immunoprecipitated with anti-FLAG M2
monoclonal antibody and detected by fluorography. The
arrowhead indicates a doublet at 54-56 kDa, and the
arrow indicates a major N-glycosylated band at 66 kDa. C, cell lysates; M, medium. Molecular mass
markers are shown at left. c, deglycosylation of
ADAMTS91-508FLAG by PNGase F. Transiently transfected QBI
293A cell lysates and culture medium were immunoprecipitated with
anti-FLAG M2 48 h after transfection. Western blot analysis was
done using anti-FLAG-M2. Ig, the immunoglobulin heavy chain.
The arrow indicates the mature form in culture medium, and
the arrowheads indicate the intracellular zymogen
form.
Ala, Arg74
Ala, or
Arg280
Ala mutants did not affect the appearance of the
mature protein in the medium, but abrogation of the most
C-terminal processing site (Arg287
Ala) resulted
in failure of processing to the mature form (Fig. 7b).
Expression of the Arg74
Ala mutant resulted in
anomalous bands of ~40 and ~45 kDa in conditioned medium in
addition to the mature protein (Fig. 7b). Instead of the
mature 28-kDa form, expression of the Arg287
Ala mutant
resulted in the appearance of ~37- and ~42-kDa proteins in culture
medium whose identity is not known (Fig. 7b).
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Fig. 7.
a, requirement of furin for proenzyme
maturation. CHO.RPE 40 cells do not secrete the mature
ADAMTS-91-508MYC/HIS into culture medium
(center panel) unless co-transfected with furin
(right panel). The control panel is derived from
untransfected cells. C, cell lysate; M, culture
medium. b, analysis of the site-directed mutants of furin
processing sites. QBI 293A cells were transfected with
ADAMTS-91-508MYC/HIS or site-directed mutants as indicated
and metabolically labeled, and cell lysates (C) and media
(M) were immunoprecipitated followed by reducing SDS-PAGE
and fluorography. Molecular mass markers are on the left. In
both a and b, an arrow indicates the
mature secreted enzyme, and an arrowhead indicates the
proenzyme.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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G)
polymorphism, since TSR-13 in mouse ADAMTS-20 4 has the
typical six-cysteine signature. The prevalence and significance of this
amino acid change in humans is not presently known and will be
investigated further.
Ala
mutant. This then accumulates in the medium following secretion through
the constitutive secretory pathway. On the other hand, in the
Arg287
Ala mutant, the precursor is not processed
intracellularly and accumulates in the medium along with other
unidentified bands. The N terminus of mature ADAMTS-9 determined by
amino acid sequencing was in agreement with the location of the N
terminus of mature ADAMTS-1, ADAMTS-4, and ADAMTS-13, suggesting that
although more than one processing site may be present, the C-terminal
furin-processing site is generally used for production of the mature
ADAMTS enzymes.
region that is present
in the V0 form but missing in the V1 form. Accordingly, the peptide
bond cleaved has a different location in the two forms (8). Consistent
with the mixed population of versican made by smooth muscle cell
cultures, two bands (70 and ~180 kDa corresponding to
G1 versican fragments DPEAAE (V0 form) and DPEAAE
(V1 form)) were seen in previous studies of ADAMTS-4 processing of
versican (8). Of these, the 70-kDa band was considerably stronger,
consistent with there being more of the V1 form in the versican
preparation (8). In contrast, neither ADAMTS-4 nor ADAMTS-9 proteolysis
gave an anti-DPEAAE reactive band at 180 kDa in our experiments.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Satya Yadav for amino acid sequence analysis; Dr. Takahiro Nagase for providing the KIAA0688 (ADAMTS4) cDNA; Dr. John Sandy and Micky Tortorella for providing antisera to the versican and aggrecan neoepitopes, respectively; Christine Kassuba and members of the Apte laboratory for comments on the manuscript; and Christina Tsoi, Patrick Smits, and Veronique Lefebvre for technical advice and assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Northeast Ohio Chapter of the Arthritis Foundation, a Yamanouchi USA Foundation Award, and National Institutes of Health (NIH) Grant AR47074 (to S. A.), by NIH Grant HL18645 (to T. W.), and by Canadian Institutes of Health Research Grant MOP-13755 (to R. L.). Tissue samples were provided by the Cooperative Human Tissue Network, which is funded by NCI, NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF488803 (ADAMTS9) and AF488804 (ADAMTS20).
¶ A Fonds de la Recherche en Sant du Quebec Senior Scholar.
** To whom correspondence should be addressed: Dept. of Biomedical Engineering (ND20), Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-3278; Fax: 216-445-4383; E-mail: aptes@bme.ri.ccf.org.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M211009200
2 Gene nomenclature (ADAMTS9 and ADAMTS20) was assigned after consultation with the Human Gene Nomenclature Committee. Adamts9 and Adamts20 are the respective mouse orthologs. The protein products of these genes are designated as ADAMTS-9 and ADAMTS-20. Similar nomenclature is used for other ADAMTS genes and their products. GON-1 refers to the product of the C. elegans gon-1 gene.
3 K. A. Jungers and S. S. Apte, unpublished data.
4 Rao, C., Foernzler, D., Loftus, S. K., Liu, S., McPherson, J., Apte, S. S., Pavan, W. J., and Beier, D. R., submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: TSR, thrombospondin type I repeat; DMEM, Dulbecco's modified Eagle's medium; GAG, glycosaminoglycan; ORF, open reading frame; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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