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INTRODUCTION |
Members of the family of large aggregating chondroitin sulfate
proteoglycans include aggrecan, versican, neurocan, and brevican. All
are composed of a signal peptide, a globular domain named G1, a large
fragment for chondroitin sulfate modification
(CS1 sequence), and a
globular domain G3 (1). The G1 domain, which is homologous to link
protein, contains one immunoglobulin (IgG)-like domain and two tandem
repeat motifs (2-4). Aggrecan is unique in containing an additional
globular domain G2 that is structurally similar to the tandem repeats
of the G1 domain. An inter-globular domain (IGD) is located between the
G1 and G2 domains, and a segment modified by keratan sulfate (KS
sequence) is situated between the G2 domain and the CS sequence. The G3
domain is composed of one or two alternatively spliced epidermal growth
factor (EGF)-like motif(s), one lectin (also called carbohydrate
recognition domain or CRD)-like motif, one complement binding protein
(CBP)-like motif, and a short C-terminal tail (2, 5, 6). The structures of these motifs, especially the tail, are highly conserved in aggrecan
across species boundaries.
In the early stages of cartilage development, versican is the
predominant proteoglycan and is believed to play an important role in
tissue development. After cartilage maturation, versican is replaced by
aggrecan, which facilitates the formation of a matrix network for
resilience and load-bearing. Aggrecan processing seems to be regulated,
in part, by the G3 domain. This was initially observed in the chicken
disorder nanomelia, which produces a lethal phenotype in homozygous
form (due to failure of chondrogenesis and osteogenesis) and dwarfism
in heterozygote form (7). Cartilage of the nanomelic mutant
lacks aggrecan in its matrix. It was discovered that the core protein
of this mutant aggrecan is truncated as a result of a premature stop
codon at the N-terminal side of G3 (2). In addition, its secretion is
hindered and no modification by glycosaminoglycan (GAG) chains occurs
(7). Recently, it was reported that the G3 and G1 domains play roles in
processing of recombinant aggrecan and versican (8-14).
Extensive aggrecan degradation occurs during normal cartilage
metabolism, aging, and joint diseases (15, 16). In mature cartilage, up
to half of the aggrecan population loses its G3 domain. The other
domains are also subjected to cleavage, resulting in the release of
degraded fragments in the matrix. The exception is the G1 domain, which
is trapped and retained by hyaluronan. Loss of aggrecan is a major
feature of cartilage degradation associated with arthritis (6). In the
early stages of rheumatoid arthritis, the chondroitin sulfate-bearing
sequence of aggrecan, the CS domain, is preferentially released. The
synovial fluid of patients with osteoarthritis and joint injury
contains fragments of degraded aggrecan (17). A large number of
proteases are known to cleave aggrecan, and their target sites are
located in the IGD, KS, and CS domains (15, 18-21). No protease has
yet been reported to cleave inside the globular domains, or in the
carboxyl tail.
Our preliminary studies suggested that the carboxyl tail of aggrecan is
removed during product secretion. After transfecting COS-7 cells with a
His-tagged (at the C terminus) G3 construct, we did not detect the
His-tagged G3 product in culture medium but detected this product in
cell lysate. An antibody against another epitope at the amino region
allowed detection of product in both culture medium and cell lysate.
Hence, we hypothesized that the G3 tail was removed after product
secretion. The present study was designed to determine the mechanism of
this process, to identify amino acid motifs that may be involved in
this degradation, and to determine whether this process is specific to
aggrecan or common to the proteoglycan family.
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EXPERIMENTAL PROCEDURES |
Materials--
PCR amplification kit, Taq DNA
polymerase, and restriction endonucleases were from Roche Molecular
Biochemicals and New England BioLabs. DNA marker was from Fermentas
MBI. Bacterial growth medium was from Difco. Prestained protein marker
was from New England BioLabs. Lipofectin, Dulbecco's modified Eagle's
medium (DMEM), fetal bovine serum (FBS), Hanks' balanced salt
solution, trypsin/EDTA, and T4 DNA ligase were from Invitrogen. ECL
Western blot detection kit was from Amersham Biosciences, Inc.
Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was from
Sigma Chemical Co. Anti-His-tag monoclonal antibody and DNA Midi-prep
kit were purchased from Qiagen Inc. Tissue culture plates (6-well,
12-well, and 100 mm) were from Nunc Inc. Protease inhibitors Amastatin
and E64 were from Calbiochem. A furin inhibitor
(decanoyl-RVKR-chloromethylketone) was from Bachem (King of
Prussia, PA). All other chemicals were from Sigma. COS-7 cells were
from American Type Culture Collection. The cells were cultured in DMEM
supplemented with 5% FBS at 37 °C in a humidified incubator
containing 5% CO2.
Strategy for Generation of Recombinant Constructs--
In this
study, a total of 43 recombinant constructs was used: G3His, HisG3, C2
(C representing the last cysteine residue of aggrecan), C7, C13, C16,
C20, C2tail, C7tail, C13tail, C16tail, C20tail, Muttail, CR8P, CR9P,
CK12A, CR13S, del8-12, delR13S, delR16P, G3KRTS, G3allmut, HuC8-13,
BoC8-13, HuC13-16, HuC16-19, G3KS, G3G3, C2G3, C7G3, C13G3, CR9PG3,
CK12AG3, CR13SG3, G3KRTSG3, G3allmutG3, del8-12G3, huC8-13G3,
cvG3aG3, hvG3aG3, cvRSPRaG3, hvRSPRaG3, and cvG3
EGFHis. G3His, G3KS,
and cvG3EGFHis have been described in our previous publications (10, 22-25; for structures see Figs. 1, 6, and 9 of this paper). The
G3His construct was generated by linking together the leading peptide
(LP) of link protein, aggrecan G3 domain and a His-tag. The leading
peptide contained link protein signal peptide and an epitope recognized by the monoclonal antibody 4B6 (26). The G3KS construct contained the
leading peptide, aggrecan G3 domain, aggrecan KS domain, and a
His-tag.
The HisG3 construct was generated by ligating two fragments, the
leading peptide containing a His-tag and the G3 domain, into pcDNA3. The leading peptide containing a His-tag was produced using
two primers, LP40CHisXhoI and LPNKozakEcoRI (for
sequences see Table I), in a PCR using
link protein as a template. The PCR product was doubly digested with
EcoRI and XhoI, purified, and ligated with the G3
fragment, which was derived by digesting the G3His construct, into
pcDNA3.
The remaining 39 constructs were based on the G3His construct. They all
contain the leading peptide, the CRD and CBP motifs, a modified tail,
and the His-tag. Modification of the tail was performed through either
systematic incremental deletion, site-directed mutagenesis, or
engineering of a potential site for cleavage from other species or
proteoglycans into the tail.
Ten constructs were made to contain incremental deletion of the tail.
Two types of deletion were made. In the first type, the entire tail was
incrementally deleted producing 5 constructs (C2, C7, C13, C16, and
C20). This type was based on the plasmid Type I (see Fig.
2A). The end of the tail contains a restriction enzyme site
XbaI (encoding two amino acids SR) followed by a His-tag. The original XbaI site on the vector was inactivated by
ligation with the restriction enzyme site NheI. The primers
NheIHisXbaIXhoI and
CRDNXhoI were used in a PCR with aggrecan G3 domain as a
template. The product was purified, digested with XhoI and
NheI, and inserted into XhoI- and
XbaI-digested G3His construct to generate the Type I
construct. To generate incremental deletion mutations, two primers were
used in each PCR: one complementary to the 5' terminus of G3 domain
(CRDNXhoI) whereas the other was complementary to the desired 3' region of the tail. For example, primer C2CXbaI
and CRDNXhoI were used in a PCR using G3 as template. The
PCR product was purified, doubly digested with XhoI and
XbaI, and ligated with XhoI- and
XbaI-digested Type I plasmid to produce construct C2. Primer
C7CXbaI was combined with CRDNXhoI to produce the
construct C7. C13CXbaI was used for production of construct
C13, whereas C16CXbaI was used for construct C16 and
C20CXbaI was used for construct C20 (see Fig.
2B). To produce the second type of incremental deletion,
nine amino acids from the extreme 3'-end of the tail were linked to the
His-tag in a PCR, using the primers XbaItailHis and
pcDNA3XmaI (complementary to nucleotides 2091-2096 of
the vector). The PCR product was digested with XbaI and
XmaI and inserted into XbaI- and
XmaI-digested construct C2 resulting in the construct C2tail. Replacement of the XbaI-XmaI fragment
from the construct C7 with the PCR product produced construct C7tail.
Constructs C13tail, C16tail, and C20tail were generated in the same way.
Another ten constructs contained site-directed mutagenesis in the tail
that could be divided into three groups. In Group 1, the first
potential consensus sequence (amino acids RRLYKR, see Fig.
3A) was mutated: in each construct, one basic amino acid was
mutated to a non-basic amino acid. For example, two primers, CSDFBamHI and CR8PXbaI, were used in a PCR with
C13tail as a template. The primer CR8PXbaI generated a
single mutation at amino acid C8 (R 
P). The PCR product was
purified, doubly digested with XhoI and XbaI, and
ligated into XhoI- and XbaI-digested vector from
the construct C13tail. Using primers CR9PXbaI and
CSDFBamHI, we generated a single mutation at amino acid C9
(R P) and produced the construct CR9P. Likewise, combined with
CSDFBamHI, primers CK12AXbaI and
CR13SXbaI were used to generate constructs CK12A and CR13S,
respectively. In the second group, we linked the second consensus
sequence to construct C2tail (see Fig. 3A). This was performed by using two primers, CSDFBamHI and del8-12,
which deleted the first motif, amino acids 8-12, in a PCR with G3
domain as a template. The PCR product was purified, digested with
XhoI and XbaI, and ligated into XhoI-
and XbaI-digested C2tail. Combined with primer
CSDFBamHI, primer delR13SXbaI was used to
generate a mutation at amino acid 13 to produce the construct delR13S, and primer delR16PXbaI was used to generate a mutation at
amino acid 16 to produce the construct delR16P. In the third group, each construct contained multiple mutations. Construct Muttail was
generated in a PCR using two primers, CSDFBamHI and
muttailXbaI. The PCR product was inserted into
XhoI- and XbaI-digested construct C20. Construct
G3KRTS was generated using two primers CSDFBamHI and
G3KRTSXbaI, and, after similar treatment, the PCR product was inserted into XhoI- and XbaI-digested plasmid
C20tail. Using a similar method, the primers G3allmutXbaI
and CSDFBamHI were used to produce the construct G3allmut
(see Fig. 4A).
Four constructs contained consensus sequences obtained from bovine,
human, dog, mouse, or rat aggrecan (see Fig. 5). These constructs have
similar structure: the putative cleavage motif was engineered into the
construct C7tail. For example, the sequence RRLQKR from the first
potential cleavage site of rat, human, or dog was linked to 3' of the
construct C2 by using two primers, CSDFBamHI and
HuC8-13XbaI, in a PCR. After treatment similar that described above, the PCR product was inserted into XhoI- and
XbaI-digested plasmid C7tail to produce the chimeric
construct HuC8-13. The sequence RHLQKR (from bovine potential cleavage
site) was linked to the 3' of the construct C7 using the primers
BoC8-13XbaI and CSDFBamHI to generate the
chimeric construct BoC8-13. The second potential protease site, RSSR,
from rat, human, or dog was linked to 3' of the construct C7 using
primers HuC13-16XbaI and CSDFBamHI to produce
the chimeric construct HuC13-16. The third potential protease site in
human aggrecan, RHPR, was linked to 3' of the construct C7 using primer
HuC16-19XbaI and CSDFBamHI to generate the
chimeric construct HuC16-19.
Fifteen constructs are based on a G3-G3 duplex. The G3G3 construct was
generated by replacing the His-tag in the G3His construct with a G3His
fragment (see Fig. 6). Two separate aliquots of the G3His construct
were doubly digested with (i) EcoRI and SalI and (ii) XhoI and EcoRI to purify LP60G3 and
G3His+pcDNA3, respectively. The two fragments were then ligated
together. Ten constructs (C2G3, C7G3, C13G3, CR9PG3, CK12AG3, CR13SG3,
G3KRTSG3, G3allmutG3, del8-12G3, huC8-13G3) contained the mutated or
truncated G3 in addition to normal aggrecan G3 (see Fig. 8). This was
achieved by replacing the junction of the mutated or truncated G3 and
pcDNA3 vector (XbaI-ApaI) with
XbaI-ApaI-containing normal G3. The latter G3 was
obtained in a PCR using aG3NXbaI and aG3CApaI as
primers. The cvG3aG3 and hvG3aG3 were produced by linking the chicken
versican G3 domain or human versican G3 domain with the normal aggrecan G3 domain as above (see Fig. 9). Generation of chicken versican G3
domain has been described by us previously (27, 28). We used an reverse
transcriptase-PCR (with hvG3NXhoI and hvG3CXbaI as primers and glioma total RNA as template) to produce human versican
G3 domain. Finally, we engineered the consensus sequence RSPR in the
tail of chicken and human versican G3 domains (in constructs cvG3aG3
and hvG3aG3, respectively) using primers cvG3CRSPRXbaI and
hvG3CRSPRXbaI in PCR reactions.
DNA Manipulation and Clone Selection--
DNA was amplified in
PCRs using pairs of appropriate primers. The reaction mixture (total
volume, 100 µl) contained 200 µM dNTPs, 0. 2 µg of
each primer, 50 ng of template DNA, 5 units of Taq DNA
polymerase, and the Mg2+-containing buffer. The reactions
were carried out at 94 °C for 5 min for one cycle; 94 °C (60 s),
55 °C (60 s), and 72 °C (60 s) for 25 cycles; and a final
extension at 72 °C for 10 min. DNA products were purified then
doubly digested with two appropriate restriction endonucleases. The DNA
was ligated into the linearized plasmid pcDNA3. The ligation
reaction was carried out at 16 °C overnight. The ligation mixture
was used for transformation of competent Escherichia coli
strain DH5
. After clone selection, all new constructs were confirmed
by DNA sequencing, performed by the Core Molecular Biology Laboratory
at York University (Toronto, Ontario). The results were then compared
with the published sequence.
Construct Expression and Product Analysis--
To analyze gene
expression, COS-7 cells or bovine chondrocytes were transfected
transiently with recombinant constructs using Lipofectin according to
the manufacturer's instructions (Invitrogen). Briefly, the cultured
COS-7 cells or bovine chondrocytes were seeded onto 12-well tissue
culture plates (1.5 × 105 cells/well). The cells were
allowed to attach and grow overnight in DMEM containing 5% FBS. Cells
were transfected once they reached 70% confluence. Lipofectin (2 µl)
was incubated with plasmid DNA (~2 µg) for 15 min in 100 µl of
DMEM followed by the addition of 900 µl of DMEM. Concurrently, COS-7
cultures were washed with 2 ml of DMEM. The Lipofectin-DNA mixture was
added to the cultures followed by incubation at 37 °C for 5 h
in an incubator. The DNA-Lipofectin mixture was replaced with 1 ml of
DMEM containing 5% FBS. Three days later, culture medium was
harvested, and dead and floating cells were removed by centrifugation.
Cell lysate in each well was obtained by using 200 µl of lysis
buffer. Because the volume of medium in each well is 5-fold the volume
of lysis buffer used, the percentage of the portions of cell lysate
used in each loading well (see below) represented 5-fold more than that
of the medium.
Cell lysate and culture medium were subjected to SDS-PAGE in separating
gel containing 10-12% acrylamide. The buffer system was 1× TG
(Tris-glycine buffer according to Amresco) containing 1% SDS.
Separated proteins were transblotted onto a nitrocellulose membrane in
1× TG buffer containing 20% methanol at 60 V for 2 h in a cold
room. The membrane was blocked in TBST (10 mM Tris-Cl, pH
8. 0, 150 mM NaCl, 0.05% Tween 20) containing 10% nonfat
dry milk powder (TBSTM) for 30 min at room temperature, and then
incubated at 4 °C overnight with the monoclonal antibody 4B6 or the
anti-His-tag monoclonal antibody in TBSTM. The membranes were washed
with TBST (3 × 30-min washes) and then incubated for 2 h
with HRP-conjugated goat anti-mouse IgG antibody (1:50,000 dilution) in
TBSTM. After washing as above, the bound antibody were visualized with
chemiluminescence (ECL kit, Amersham Biosciences, Inc.).
To compare the G3 tail cleavage, it was necessary to load an equal
amount of secreted products to each well on Western blot. To do so, the
products of each construct, as well as one control (product of C2tail
construct) were pre-analyzed on Western blot probed with 4B6, which is
much more sensitive than the anti-His antibody. Equal amounts of gene
products, obtained by dilution, were then analyzed for G3 tail cleavage
probed with anti-His antibody. Because the affinity of anti-His
antibody is much lower than the 4B6 antibody, we had to use larger
amounts of products (~ 3-fold) for anti-His staining. A reduction in
the intensity of bands probed with anti-His antibody would suggest G3
tail cleavage.
Effect of Furin Inhibitor on Product Cleavage--
COS-7 cells
were transiently transfected with G3G3 construct. After overnight
incubation, culture medium was replaced with fresh medium containing
5% FBS and furin inhibitor decanoyl-RVKR-chloromethylketone. Protease
inhibitors Amastatin and E64 were used as controls. Cell lysate and
culture medium were harvested and analyzed on Western blot as above.
Densitometer Analysis--
Relative protein concentration was
estimated by using a densitometer (Molecular Dynamics) according to the
manufacturer's instructions for scanning the densities of the signals
on the films after Western blot. The relative intensity of each band after Western blot development is shown below the blot. In the case of
product cleavage in duplex constructs, both protein bands (undegraded
and degraded) were scanned and density was calculated using the
following formula: degradation (%) = OD of degraded band/(OD of
degraded band + OD of undegraded band).
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RESULTS |
Cleavage of G3 Tail--
To assess the possibility of cleavage of
the C-terminal tail of the chicken aggrecan G3 domain, we initially
generated a G3 construct that contains the leading peptide of link
protein at the N terminus. This leading peptide harbors the signal
peptide and an epitope recognized by the monoclonal antibody 4B6 (26). At the C-terminal end of G3, a His-tag recognized by anti-His monoclonal antibody was added (G3His, Fig.
1A). After transfection of
COS-7 cells with this G3 construct, the 4B6 antibody detected products
(~48 kDa) in both culture medium and cell lysate on Western blot
(Fig. 1B). However, the anti-His antibody failed to detect the product in culture medium, but could detect the product (~48 kDa)
in cell lysate (Fig. 1C). This suggested that the G3 tail has been removed after product secretion. To test this, we generated another construct (HisG3) containing both the 4B6 epitope and the
His-tag at the N-terminal region of the construct. Analysis of cell
lysate and culture media from COS-7 cells transfected with HisG3 on
Western blot indicated that products of HisG3 (~48 kDa) were detected
by 4B6 (Fig. 1D) and anti-His antibody (Fig. 1E)
in cell lysate and culture medium.
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Fig. 1.
Detection of G3 products in cell lysate and
media using different epitopes. COS-7 cells were transiently
transfected with the recombinant constructs G3His and HisG3
(A). Cell lysate and culture media were subjected to
SDS-PAGE in 12% gels in the amounts indicated, which represent the
percentage of total products. The separated proteins were then
transblotted onto a nitrocellulose membrane and probed with the
monoclonal antibody 4B6 or anti-His-tag antibody. Vector-transfected
cells were used as controls. Products of G3His (~48 kDa) were easily
detected in cell lysate with 4B6 (B) or anti-His antibody
(C). However, secreted G3His could be detected by 4B6 but
only negligible amounts were detected by anti-His antibody. Products of
the N-terminally tagged construct HisG3 (~48 kDa) were detected by
4B6 (D) and anti-His antibody (E) in both the
cell lysate and culture medium.
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Identification of the Amino Acids Involved in the Cleavage of G3
Tail--
To investigate how the G3 tail was cleaved, ten incremental
deletion constructs (C2, C7, C13, C16, C20, C2tail, C7tail, C13tail, C16tail, and C20tail) were made based on two types of parental constructs, Type I and Type II (Fig. 2,
A and B). In addition, the last nine amino acids
were completely mutated resulting in the construct muttail. Because all
the mutations and deletions were made between two restriction sites,
EcoRV on G3 domain and ApaI on the extreme 3'
cloning site, the EcoRV-ApaI fragment of the
above 12 cDNA constructs (including G3His as a control) was examined using polyacrylamide gel electrophoresis to ensure that deletion had been made (data not shown). Culture medium and cell lysate
from COS-7 cells transiently transfected with these 12 constructs were
analyzed on Western blot probed with 4B6. All constructs were equally
well expressed in both the lysate (L) and medium (M) (Fig.
2C). Probed with anti-His antibody, no significant difference in expression levels could be seen in cell lysate. However,
little product was detected in the medium from G3His-, C13-,
C16-, C20-, C13tail-, C16tail-, C20tail-, and muttail-transfected COS-7
cells probed with the anti-His antibody (Fig. 2D) suggesting that cleavage had occurred in all of these constructs. These results indicated that the sequence RRLYKR (C13) was the minimum for sufficient cleavage and that this sequence was also recognized in the extended sequences RRLYKRSPR (C16) and RRLYKRSPRSRLR (C20). On the other hand,
the sequence SRPGVVHRPTH (C2tail) was not sufficient for cleavage,
although its presence did not interfere with cleavage within the
adjacent active motif RRLYKR (see C13 tail).
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Fig. 2.
Identification of potential sites for
cleavage by systematic deletion. A, two types of
constructs were generated (Type I and Type II) to produce incremental
deletions in the G3 tail. B, ten deletion constructs (C2,
C7, C13, C16, C20, C2tail, C7tail, C13tail, C16tail, and C20tail) were
made based on the G3His construct. In the construct muttail, the last 9 amino acids were completely mutated. C, culture medium
(M, 1% of total product) and cell lysate (L,
2.5% of total product) from transfected COS-7 cells were analyzed on
Western blot probed with 4B6. All constructs were equally well
expressed. D, products (3% of total medium and 7.5% of
total lysate) of the 12 constructs were also analyzed on Western blot
probed with anti-His antibody. Equivalent levels of 4B6 and anti-His
labeling were observed in cell lysate. However, the anti-His antibody
detected reduced amounts of secreted product from G3His-, C13-, C16-,
C20-, C13tail-, C16tail-, C20tail-, and muttail-transfected cells. The
relative densities of bands after Western blot development are shown
below the figure.
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It has long been known that the amino acid motif RXXR, a
pair of arginine residues separated by 2 amino acid residues, is a
potential cleavage site for proprotein convertase. The experiments above had indicated that the arginine-rich sequence RRLYKRSPRSRLR promotes tail cleavage, although the short motif RRLYKR alone is
sufficient. Because RRLYKRSPRSRLR contains two potential sites for
furin-like activity (RRLYK and RSPR), we next investigated the
contribution of individual basic residues in cleavage at these sites.
Using site-directed mutagenesis technique, we generated four mutants
from the construct C13tail: CR8P, CR9P, CK12A, and CR13S (Fig.
3A). In these constructs, the
basic amino acids arginine and lysine are individually mutated to
non-basic residues. Expression of these constructs in COS-7 cells and
analysis of the product on Western blot probed by 4B6 indicated that
all constructs were equally well expressed (Fig. 3B).
Anti-His antibody staining indicated that the uncleaved product of all
these mutated constructs was present in the medium at 30-60% of the
levels of the C2tail construct (Fig. 3C), which totally
lacked the cleavable sequence. This suggested that each individual
basic residue in the minimum necessary sequence (RRLYKR) is important
for promoting substrate cleavage.
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Fig. 3.
Site-directed mutagenesis of basic amino
acids in the potential sites for cleavage. A,
site-directed mutagenesis (boldface residues) produced seven
constructs: CR8P, CR9P, CK12A, CR13S, del8-12, delR13S, and delR16P.
B, COS-7 cells were transiently transfected with these
constructs, and the products from cell lysate (L) and
culture medium (M) were analyzed on Western blot probed with
4B6. The control construct was C2tail. All constructs were well
expressed. C, these products were also analyzed on Western
blot probed with anti-His antibody. No significant difference was
observed in cell lysate. However, the amount of CR8P, CR9P, CK12A,
CR12S, del8-12, delR13S, and delR16P products in the medium was
reduced compared with the control C2tail product.
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To investigate the effect of the consensus sequence RSPR on product
degradation, an RSPR sequence was inserted into the construct C7tail
(which was not subject to cleavage in the preceding experiments). This
created the construct del8-12, in which the arginines were individually replaced to create delR13S and delR16P (Fig.
3A). PAGE analysis of the EcoRV-ApaI
fragments of these three constructs showed that the constructs had the
expected sizes (data not shown). Cell lysate and culture medium
from COS-7 cells transiently transfected with these three constructs
were analyzed on Western blot. Staining with 4B6 detected no difference
in expression levels (Fig. 3B). However, when probed with
anti-His antibody, the band intensities of del8-12, delR13S, and
delR16P in culture medium decreased somewhat indicating that there was
some limited cleavage at these RSPR sites (Fig. 3C).
We next investigated the effects of mutating both consensus sequences
simultaneously. Mutating one basic amino acid in each consensus
sequence generated the construct G3KRTS, which also converted LR to SR
as in other constructs. Mutating all the important basic amino acids
that may be involved in the tail cleavage resulted in the construct
G3allmut (Fig. 4A). The
EcoRV-ApaI fragments of these two constructs were
slightly larger than that from C2tail, as expected (data not shown).
Transfected COS-7 cells expressed these constructs well, as indicated
by Western blot probed with 4B6 (Fig. 4A). Staining with
anti-His antibody demonstrated that medium from G3KRTS-transfected
cells contained only 10% detectable product as compared with medium
from C2tail-transfected cells (Fig. 4B). Therefore, it
appears that the double basic KR in the sequence RRLYKRSPRSRLR, which
contributes basic residues to both motifs (RRLYKR and RSPR) is not
absolutely required for activity, because the KR to TS mutation did not
completely eliminate cleavage activity. Mutations of all basic amino
acid residue completely inhibited tail cleavage.
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Fig. 4.
Mutation of all basic amino acid residues
completely inhibits tail cleavage. A, site-directed
mutagenesis was carried out at multiple sites to remove important basic
amino acids in the G3 tail. The first construct (G3KRTS) contained
mutations in C12 (K T) and C13 (R S). The second construct
(G3allmut) contained mutations of 6 amino acids (boldface).
B, cell lysate (L) and culture medium
(M) from cultures transfected with these two constructs and
C2tail (as control) were analyzed on Western blot. 4B6 probing showed
that all constructs were well expressed. Probing with anti-His antibody
revealed consistent levels of products in cell lysate. However, the
amount of secreted products from G3KRTS-transfected was only 10% of
that from C2tail-transfected cells.
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Cleavage of the Consensus Sequence Obtained from Other Aggrecan
Species--
Thus far, our studies had focused on chicken aggrecan.
However, the aggrecan G3 tail sequence is conserved across rat, human, dog, mouse, cow, and chicken species (Fig.
5A). All of them contain the
consensus sequences, (R/H)(R/H)XXKR and
RXXR, for cleavage (Fig. 5B). Only in mouse is
histidine substituted for the initial arginine, and only in the bovine
sequence is the second arginine replaced by histidine. To investigate
if the RRLQKR (in human and dog) and RHLQKR (in cow) in the former
consensus sequence, and RSSR (rat, human, and dog) and RHPR (in human)
in the latter consensus sequence, were involved in tail degradation,
these sequences were inserted into the construct C7tail producing
HuC8-13, BoC8-13, HuC13-16, and HuC16-19 (Fig. 5C). The
EcoRV-ApaI fragments from these four constructs
were examined in PAGE analysis (data not shown). Products from COS-7
cells transiently transfected with these constructs were analyzed
on Western blot probed with 4B6 (Fig. 5D) and anti-His
antibody (Fig. 5E). All constructs were equally well
expressed as detected with 4B6, and the amount of secreted (uncleaved)
product from these four constructs was greatly reduced compared with
the uncleavable construct C2tail. This suggests that all individual
sequences (RRLQKR, RHLQKR, RSSR, and RHPR) were quite efficiently
cleaved by furin-like activity in these constructs.
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Fig. 5.
Cleavage of consensus sequences in aggrecan
of other species. A, comparison of aggrecan tail
sequences from rat, human, dog, mouse, cow, and chicken. B,
consensus sequence for potential degradation sites. C, four
constructs containing consensus sequences for tail cleavage
(boldface) from these species were generated by inserting
these sequences into construct C7tail. D, products of these
four constructs and C2tail were analyzed on Western blot probed with
4B6. E, products of these five constructs were also probed
with anti-His antibody. No difference in detection levels could be seen
in cell lysate. However, secreted products of HuC8-13, BoC8-13,
HuC13-16, and HuC16-19, detected with the anti-His antibody, greatly
decreased compared with the product of C2tail.
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Cleavage of G3-G3 Duplex and Effects of GAG Chains, Protease
Inhibitors, and Tail Mutations on the Cleavage--
To study the
degradation process at the domain level, two constructs, G3G3 and G3KS,
were generated, and transiently expressed in COS-7 cells along with
G3His (Fig. 6). G3G3 was composed of two
G3 domains joined C-terminal to N-terminal. G3KS consisted of a G3
domain and appended KS sequence. Cell lysate and culture medium were
analyzed on Western blot probed with 4B6. G3G3 products in lysate
fraction exhibited the size of 72 kDa. However, the majority of
secreted product was 48 kDa, a size similar to G3His, suggesting
degradation of G3G3 (Fig. 6). It should be pointed out that the size of
G3G3 in cell lysate did not double the size of secreted G3. This may be
due to incomplete glycosylation of the second G3. Another possibility
is that secretion of G3G3 product is a fast step following G3G3
glycosylation. The G3KS product had expected size (62 kDa), and the
observed smear suggested GAG chain attachment to the KS sequence.
Interestingly, we did not observe secreted product with a size of 48 kDa (G3 fragment), implying that the secreted product of G3KS was not
cleaved and suggesting that the presence of the KS domain inhibited
proteolysis of an otherwise cleavable tail sequence.
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Fig. 6.
Cleavage of G3 tail inhibited by
glycosaminoglycan chains. Cell lysate and culture medium from G3G3
and G3KS were analyzed on Western blot probed with 4B6. The G3G3
products in the lysate had the expected size. However, most of the
secreted G3G3 product had a size similar to that of G3His. The faint
band at 70 kDa probably represents intact G3G3. The G3KS product also
exhibited the expected size, and the smear (the larger sizes) suggested
GAG chain attachment to the KS sequence. No cleavage was observed for
G3KS product.
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To investigate how the tail was cleaved, furin inhibitor
decanoyl-RVKR-chloromethylketone was added to the culture medium. Cells
transfected with G3G3 construct were incubated with this medium
to test the effect of the inhibitor on the cleavage of G3G3 product. At
both concentrations (25 and 50 µM) used, the inhibitor
reduced cleavage of G3G3 product (Fig.
7).
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Fig. 7.
The effect of furin inhibitor on tail
cleavage. COS-7 cells were transiently transfected with G3G3
construct, and the cultures were maintained in the presence or absence
(control) of furin inhibitor decanoyl-RVKR-chloromethylketone (25 and
50 µM), or protease inhibitors Amastatin (50 µM) and E64 (50 µM) for 3 days. Cell lysate
and culture media were analyzed on Western blot probed with 4B6. The
furin inhibitor reduced cleavage of the G3G3 product.
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Based on the cleavage of G3G3 product, we generated 10 constructs
(C2G3, C7G3, C13G3, CR9PG3, CK12AG3, CR13SG3, G3KRTSG3, G3allmutG3,
del8-12G3, and HuC8-13G3) containing a mutated or truncated G3
followed by a normal G3, to examine the tail degradation in a more
specific way. These constructs, shown in Fig.
8A, were expressed in COS-7
cells, and the products were analyzed on Western blot as for the other
products. The experiment indicated that all of these 10 constructs were
equally well expressed, and the products were secreted to culture
medium (Fig. 8B). However, their cleavage patterns differed.
The product of G3G3 received extensive cleavage (95.8%), whereas C2G3
received little degradation (5.4%). C13G3 and huC16-19G3, each of
which contains RRLYKR and RHPR, respectively, appeared to have
undergone extensive cleavage (at round 50%). Interestingly, CR9PG3,
CK12AG3, CR13SG3, G3KRTSG3, and G3allmutG3, which contains point
mutations in the RRLYKR sequence or both RRLYKR and RSPR, still
received decent amount of cleavage. These results suggested that some
clusters of basic amino acids are involved in the degradation. It
should be pointed out that C2G3 and C7G3, which contain no basic amino
acids in the tail region, still received some degradation, indicating
some nonspecific activity. In particular, degradation of C7G3 is
unexpectedly severe, a level even higher than some constructs
containing basic amino acids in this furin cleavage motif.
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Fig. 8.
Cleavage of aggrecan G3 tail in constructs
lacking the consensus sequence RXXR.
A, ten constructs were generated by linking the native G3
cDNA with each of the mutated or truncated G3 constructs, resulting
in C2G3, C7G3, C13G3, CR9PG3, CK12AG3, CR13SG3, G3KRTSG3, G3allmutG3,
del8-12G3, and HuC16-19G3. B, products of these 10 constructs and G3G3 were analyzed on Western blot probed with 4B6. All
constructs were well expressed, and the amounts of products in cell
lysate were similar in sizes and expression levels as expected
(top). Detection of two bands in culture medium suggested
that all products were cleaved after secretion (bottom).
Notably, however, the G3G3 control underwent 95.8% cleavage while
mutants exhibited lower cleavage levels. The densitometry of both bands
and percentage of cleavage were given below the gel.
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RXXR-mediated Cleavage Patterns Are Specific to Aggrecan and Not
Observed in Versican--
Versican, another member of the large
aggregating chondroitin sulfate proteoglycan family, has also been
extensively studied. Versican G1 and G3 domains are highly conserved
(2, 29, 30). The versican tail also contains an RXXR
consensus sequence. To investigate if the tail of versican is also
involved in degradation, human (hvG3) and chicken (cvG3) versican G3
domains were joined to aggrecan G3 clones (aG3) to generate hvG3aG3 and
cvG3aG3 constructs (Fig. 9A).
These constructs were transiently expressed in COS-7 cells, and the
products from the cell lysate and culture medium were analyzed on
Western blot probed with 4B6. All constructs were equally well
expressed and were secreted to culture medium as shown by 4B6 probing
(Fig. 9B). However, little degradation of the products was
detected in the culture medium, while the control construct aggrecan
G3G3 received severe cleavage. The tail sequences of human versican G3
domain and chicken versican G3 domain were modified to contain RSPR
sequence (constructs hvRSPRaG3 and cvRSPRaG3), which is identical
to that detected in the chicken aggrecan G3 domain. Surprisingly, no
tail degradation was detected even with this modification (Fig.
9B), whereas the aggrecan G3 construct containing this
sequence (del8-12G3) received notable cleavage (Fig.
8B).
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Fig. 9.
Human and chicken versican contain the
consensus sequence RXXR in their G3 tail but are not
cleaved. A, linking native human or chicken versican G3
cDNA with the aggrecan G3 domain produced hvG3aG3 and cvG3aG3
constructs. Two additional constructs (hvRSPRaG3 and cvRSPRaG3) were
engineered to contain the consensus sequence RSPR in the tails of human
and chicken versican G3 domains. B, cell lysate
(L) and culture medium (M) of hvG3aG3, cvG3aG3,
cvRSPRaG3, hvRSPRaG3, and G3G3 were analyzed on Western blot probed
with 4B6. Little product cleavage was detected in the culture medium
from hvG3aG3-, cvG3aG3-, hvRSPRaG3-, and cvRSPRaG3-transfected cells,
but the majority (~90%) of aggrecan G3G3 product was cleaved.
C, bovine chondrocytes were transiently transfected with
G3G3, G3allmutG3, cvG3aG3, hvG3aG3, and a control vector pcDNA3.
Cell lysate (L) and culture medium (M) were
analyzed on Western blot probed with 4B6. All constructs were well
expressed and secreted. The G3G3 product in the culture medium
was completely cleaved, and a small amount of the cell-associated G3G3
product was cleaved. D, the constructs G3His and
cvG3EGFHis (from chicken versican G3 domain) were expressed in COS-7
cells. Cell lysate (L) and culture medium (M)
were analyzed on Western blot probed with anti-His antibody. Only the
His epitope in the secreted product of cvG3EGFHis (but not of G3His)
was detected.
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We then examined whether the tail cleavage could be obtained by using
chondrocytes. Bovine chondrocytes isolated from caudal discs were grown
on 6-well tissue culture plates as monolayer cultures, followed by
transfection with the recombinant constructs G3G3, G3allmutG3, cvG3aG3,
and hvG3aG3 and the control vector pcDNA3. Product analysis on
Western blot probed with 4B6 indicated that similar results were
obtained (Fig. 9C). The little discrimination was that the
G3G3 product in the culture medium was completely cleaved and that a
small amount of the cell-associated G3G3 product was cleaved as
compared with those expressed by COS-7 cells.
To ensure that the native tail of versican G3 was not cleaved, we
expressed aggrecan G3 construct (G3His) and versican G3 lacking two
EGF-like motifs (cvG3EGFHis, structurally similar to G3His) in COS-7
cells. Product analysis indicated that only the secreted product of
cvG3His could be detected with anti-His antibody (Fig. 9D).
These results indicate that, despite the structural similarities of
aggrecan and versican G3 domains, only aggrecan tail is vulnerable to
protease-mediated cleavage processes.
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DISCUSSION |
The large aggregating chondroitin sulfate proteoglycan aggrecan is
highly expressed in cartilage, and its structure, especially the G1,
G2, and G3 domains, is highly conserved (2, 5). Aggrecan serves a
structural function in the formation of cartilage. In aging and in
disease states, aggrecan is severely degraded. This process has been
extensively investigated, and many proteases have been implicated in
aggrecan degradation (15, 19-21, 31). For example, degradation of
aggrecan, along with disruption of the collagenous network, is a major
feature of the cartilage deterioration associated with arthritis (18,
20). As a result, the synovial fluid and afflicted joints of arthritis
patients contain fragments of aggrecan (17). It has been reported that
aggrecan is degraded by aggrecanase, cathepsin B, cathepsin L,
leukocyte elastase, and MMPs (including MMP-1, MMP-2, MMP-3, MMP-7,
MMP-8, MMP-9, and MMP-13) (6, 32-35). However, our study was the first
to show that the C-terminal tail of aggrecan is cleaved. This study was
initiated by our observation that the product of a G3 construct was not
detected in culture medium using antibody against a C-terminal His-tag
epitope but could be detected in cell lysate. An antibody against an
epitope (4B6) at the N-terminal region detected product in both culture
medium and cell lysate. Hence, we hypothesized that the G3 tail
cleavage is associated with product secretion. The present study was
designed to test this hypothesis.
Because the tail consists of only 20 amino acids, the cleaved tail is
too small to be detected. As well, removal of the tail from the G3
domain reduces the size of the domain only slightly. This makes it
difficult to observe the phenomenon. Glycosylation of the G3 domain
obscures the effect even more. To overcome these problems, we used
incremental deletion and site-directed mutagenesis techniques to
generate a large number of constructs. We also engineered a His-tag at
the end of the G3 domain and added a leading peptide of link protein at
the N-terminal. This leading peptide serves as a signal sequence for
product secretion and contains an epitope recognized by the monoclonal
antibody 4B6. With this method, we were able to demonstrate that two
tandem boxes, RRXXK and RXXR, were the consensus
sequences and involved in tail cleavage. Inclusion of the last 9 amino
acids of chicken aggrecan in the constructs did not affect the results,
indicating these 9 amino acids are not involved. Further confirmation
was obtained by replacing these 9 amino acids with 9 unrelated amino
acids. It should be pointed out that the results were reproducible, but
they were not clear-cut, because the extent of cleavage was variable.
This might be affected by culture and transfection conditions. It might
also be affected by the sensitivity of the Western blot detection
method, although we standardized our methods. Because we isolated and
used at least two clones for transfection, repeated transfection
assays, and included positive controls, these varying factors do not
affect the interpretation of our results.
The structure of the aggrecan tail is highly conserved. Among rat,
human, dog, mouse, and cow, there is 80% homology. The homology of the
consensus sequences, (R/H)(R/H)XXK and RXXR, is even greater. Although the sequence of chicken aggrecan tail is relatively different from those discussed above, the chicken aggrecan tail does contain the consensus sequences, RRXXK and
RXXR. Using this knowledge, we were able to pinpoint these
sequences as essential for cleavage. We confirmed this by testing the
effects of these motifs in aggrecan from other species. Because the
tail structure of aggrecan is highly conserved, our results do not
exclude the possibility that other amino acids may also play a role in
tail cleavage. As discussed below, the tail cleavage is very complex.
Proprotein convertases are a group of proteases that cleave sequences
composed of pairs of basic amino acids. It has been reported that the
minimum required site for convertase cleavage is RXXR
(36-38). Proprotein convertases often act in the trans-Golgi and on
cell surfaces. Cleavage is often the step that converts a protein or
peptide to its active form. Because the aggrecan C-terminal tail
contains many basic amino acids and RXXR sties, we thus
designed experiments to examine whether these sites are cleaved during
product processing. We used the chicken aggrecan as it contains only
one RXXR.
Although the site-directed mutagenesis technique allowed us to confirm
that the consensus sequences were important in tail cleavage, it led to
a puzzling situation. For example, in the seven constructs listed in
Fig. 3A (CR8P, CR9P, CK12A, CR13S, del8-12, delR13S, and
delR16P), we initially expected to detect tail cleavage in del8-12
construct, because it does contain RXXR. However, we did not
observe a convincing difference between del8-12 and the rest. Given
that a construct completely lacking a consensus sequence was degraded
somewhat, it seems likely that other amino acids in the tail,
especially other basic amino acids, play a role in nonspecific
degradation. Despite this, our studies do indicate that the consensus
sequences were important: This interpretation was supported by the
results obtained from two other constructs (G3KRTS and G3allmut).
G3KRTS contains mutations in both consensus sequences in the tail.
Product analysis indicated that G3KRTS was still severely degraded. On
the other hand, when all basic amino acids in both consensus
sequences were altered, as in the construct G3allmut, the resulting
product received no degradation.
To clarify this issue, we sought to develop a better method for
studying tail cleavage. A construct containing two G3 domains was then
produced. This G3G3 product was cleaved in the middle producing a
smaller fragment equal to G3 that could be detected by 4B6. This G3G3
construct thus served as an ideal model to identify the exact site of
tail cleavage. It should be pointed out that in the G3G3 construct, the
tail cleavage is not complete in COS-7 cells: ~96% of the products
were cleaved. The intact product was detected in culture medium using
the 4B6 antibody but not with the anti-His antibody. The former
antibody is more sensitive. Intact G3 with His-tag was also
successfully purified from culture medium with nickel-nitrilotriacetic
acid beads (11, 13). It may be that a small proportion of G3 is kept
intact to participate in specific interactions. However, in the case of
chondrocyte transfection, the G3G3 product in culture medium was
completely cleaved. Perhaps, the proteolytic activity in chondrocyte
culture was more active. Furthermore, the cell-associated fraction
contained a small portion of cleaved G3 product. It is possible that
the cleaved G3 fragment interacted with cell surface proteins. The CRD
and CBP motifs of aggrecan G3 domain are structurally similar to the
CRD and CBP motifs of versican G3 domain, which have been shown by us
to bind to cell surface and 
1 integrin is
involved in this interaction (39, 40). Our experiments indicate that, although G3 tail cleavage occurred in both COS-7 cells and
chondrocytes, there were some differences between these two types of
cells in the extent of cleavage. Because our study was performed in a
non-chondrocyte model system, to what extent this tail cleavage occurs
during the normal processing of aggrecan in cartilage is unclear at present.
Using the G3G3 duplex as a model, we were able to demonstrate that tail
cleavage does not occur prior to product secretion, because the G3G3
product from cell lysate migrated almost exclusively as a single band.
We occasionally observed a very faint band in the G3G3-transfected cell
lysate that migrated at a position equal to G3. This could be a result
of the interaction of secreted G3 with the cell surface during
lysate or post secretory G3 bound to the cell surface, although the
cells were rinsed with PBS before harvest. The possibility that the CRD
motif binds to the GAG chains on the cell surface is not excluded.
In addition to confirming our initial observations, the G3G3 model also
revealed the motifs necessary for tail cleavage. For example,
HuC16-19G3 (containing RHPR) received more cleavage than del8-12G3
(containing RSPR) did. Using the G3G3 strategy, we observed that the
products of C2G3, C7G3, and G3allmutG3 received trace amounts of
degradation, which could not be previously detected by using the
constructs C2, C7, and G3allmut. The C2G3 contains no tail sequence but
still received trace amounts of degradation producing a faint band with
of G3 size after overexposure. It appears that the one or more enzymes
responsible for the tail cleavage not only recognize the tail sequence
but may also recognize the C terminus of the CBP motif. Because no
known proprotein convertase has such a function, it is likely that
there may be a new protease involved in aggrecan tail cleavage.
Although we have demonstrated that furin-like protease inhibitors were
able to reduce the cleavage, the nature of the protease(s) involved in
this action is still not known.
The idea that basic amino acids are important in tail cleavage was
further supported by experiments involving G3KS product. G3KS contains
the G3 domain joined to a fragment of keratan sulfate modifying
sequence (KS domain). The product of G3KS was not cleaved. Although
there was no direct evidence that keratan sulfate chains were added to
the KS-modifying sequence in these cells under these conditions, the
increase in size of product in culture medium and diffusion patterns of
bands suggested modification by GAG chains. Because the GAG chains
contain a high density of negative charge, our results may imply that
the GAG chains protect the cleavage site either sterically or
electrostatically (or by a combination of effects).
Our study has shown that the cleavage mechanism examined here is
specific to aggrecan. Versican, another member of the large aggregating
chondroitin sulfate proteoglycan family, also contains a G1 domain, a
G3 domain, and a CS sequence for GAG modification. The structures of
the G1 and G3 domains are very conserved between aggrecan and versican.
Analysis of the products from chicken and human versican G3 domains
indicated that the tail in versican was not cleaved. Because both
versican and aggrecan G3 domains are processed in a similar fashion,
the difference in tail cleavage suggests additional roles for the tail.
Sequence alignment indicated that the tails are different between
aggrecan and versican. There is a major difference in the sequence
upstream of the "furin-cleavage area" in the C-terminal tail of
aggrecan and versican. Versican has an "insert"
(KYFKNSSSAKDNSINTSKHDH) between the PSSYQR motif and the furin-cleavage
motif RXXR, which is absent from aggrecan. This
insert, although eliminating the RRLYKR motif present in aggrecan, instead puts TSKHDH upstream of the RXXR motif,
which may be a "pseudo" furin site. Although RXXR is the
minimal requirement for furin recognition, additional Arg or Lys around
this recognition motif is important for furin cleavage (41, 42).
In conclusion, the studies reported here have revealed for the first
time that the carboxyl tail of the aggrecan G3 domain is subject to
cleavage. Two consensus sequences RRXXK and RXXR are involved in the cleavage, which appears to be specific to aggrecan.
These findings may have implications for the future development of
therapies for the loss of aggrecan function associated with aging and arthritis.
§¶,
**,
, and
TOP
ABSTRACT
INTRODUCTION
Deceased February 22, 2001; was on sabbatical from the University
of Calgary.
