From the Shriners Hospital for Children, Tampa,
Florida 33612, the
Department of Surgery, University of
Washington School of Medicine, Seattle, Washington 98195, the
** Department of Molecular, Cell and Developmental Biology, University
of California at Los Angeles, Los Angeles, California 90095, and the
Molecular Biology Laboratory, Department of
Pathology, University of Zurich, 8091 Zurich, Switzerland
Received for publication, October 25, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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Mature human aorta contains a 70-kDa
versican fragment, which reacts with a neoepitope antiserum to the
C-terminal peptide sequence DPEAAE. This protein therefore appears to
represent the G1 domain of versican V1
(G1-DPEAAE441), which has been generated in
vivo by proteolytic cleavage at the
Glu441-Ala442 bond, within the sequence
DPEAAE441-A442RRGQ. Because the equivalent
aggrecan product (G1-NITEGE341) and brevican product
(G1-EAVESE395) are generated by ADAMTS-mediated cleavage of
the respective proteoglycans, we tested the capacity of recombinant
ADAMTS-1 and ADAMTS-4 to cleave versican at
Glu441-Ala442. Both enzymes cleaved a
recombinant versican substrate and native human versican at the
Glu441-Ala442 bond and the mature form of
ADAMTS-4 was detected by Western analysis of extracts of aortic
intima. We conclude that versican V1 proteolysis in vivo
can be catalyzed by one or more members of the ADAMTS family of metalloproteinases.
Versican is a member of the family of large aggregating
proteoglycans, which also includes aggrecan, neurocan, and brevican. Although aggrecan is most abundant in cartilages (1) and both neurocan
and brevican are largely restricted to nervous tissues (2), versican
has a rather wide tissue distribution (3). It has been identified in
loose connective tissues and in fibrous, articular, and elastic
cartilages. It is also detectable in the central and peripheral nervous
system, in the epidermis, and in all three wall layers of veins and
elastic arteries. Furthermore, versican can exist in a number of
isoforms, namely V0, V1, V2, V3, and Vint (4, 5-7). The V1 isoform is
composed of a G1 domain, chondroitin sulfate
(CS)1 attachment domain
(GAG-beta) and the G3 domain. The V0 and V1 isoforms differ only
by the presence of the GAG-alpha domain in the V0 form, which adds 987 amino acids and about five putative CS-attachment sites, adjacent to
the hyaluronan binding domain (4, 6). The V2 isoform, which is a
predominant brain proteoglycan (8), is composed of the G1 domain,
GAG-alpha domain, and the G3 domain. Both V0 and V1 are detected at the
mRNA level in the human aorta, and versican is also detected in the
aorta by immunohistochemistry with antibodies recognizing both variants
(3, 9). Furthermore, recent data show that cultured smooth muscle cells
contain mRNA for V0, V1, and V3 isoforms (7).
In contrast to aggrecan, for which the degradative pathways have been
described in detail (10), very little is known regarding versican
turnover. A 66-kDa protein, which is immunologically related to
versican, has been described in fetal human skin (11). However, the
metabolic origin of this protein was not described. On the other hand,
the findings (2, 12) that both aggrecan and brevican are degraded
in vivo by glutamyl endopeptidases, which appear to belong
to the ADAMTS family of metalloproteinases (13-15), suggested to us
that versican might also be degraded in this manner. In this paper we
provide the first evidence that versican is indeed processed in
vivo by a glutamyl endopeptidase that appears to belong to the
ADAMTS family.
Preparation of Versican from Human Aorta and Smooth Muscle Cell
Cultures--
Abdominal aorta was anonymously obtained from an organ
donor with approval from the University of Washington human subjects review committee. During preparation of the organs for transplantation, excess aortic tissue was trimmed from around the entry point of the
celiac artery. This tissue was kept in University of Wisconsin solution
(16) at 4 °C until the tissue was either frozen intact in liquid
nitrogen or dissected on ice in Ca2+- and
Mg2+-free phosphate-buffered saline with proteinase
inhibitors (10 mM EDTA, 0.1 mM AEBSF, 1 µg/ml
pepstatin) into the intimal, medial, and adventitial layers. Tissue
samples (100-500 mg of wet weight) were frozen for storage and
immediately after thawing were extracted twice for 24 h in 1 ml of
4 M guanidine-HCl, 10 mM MES, 50 mM sodium acetate, 5 mM EDTA, 0.1 mM AEBSF, 5 mM iodoacetamide, 0.3 M aminohexanoic acid, 15 mM benzamidine, 1 µg/ml pepstatin, pH 6.8, at 4 °C.
The samples were then centrifuged for 10 min at 12,000 × g at 4 °C. The clear supernatants were combined (1.5-2.0 ml), and three volumes of ice-cold ethanol/5 mM sodium
acetate were added. After 16 h at Source and Preparation of Antibodies for Western
Analyses--
Antiserum DPEAAE (abbreviated to anti-DP in the text
below) was raised in rabbits against the synthetic peptide CGGDPEAAE conjugated to ovalbumin (by Research Genetics, Huntsville, AL), and the
antibodies were affinity-purified on a peptide-substituted Sulfolink column from Pierce and Co. Anti-CDAGWLADQTVRYPI (also called HAL) was obtained from Dr. Steve Carlson; this antiserum is
known to detect a 21-residue sequence, which begins with CDAG and is
present in the proteoglycan tandem repeat loops of the G1 domain of
aggrecan, versican, and also in link protein (18). Anti-G1 (aggrecan)
was raised in rabbits against bovine aggrecan G1 domain supplied by Dr.
Larry Rosenberg. LF-99 (raised to the N-terminal 13-residue peptide
sequence of human versican) was from Dr. Larry Fisher (19), and the
antiserum Vc (raised to recombinant human versican V1 expressed in
Chinese hamster ovary cells) was from Dr. Richard Le Baron (20).
Antibodies specific for the GAG-alpha domain and the GAG-beta domain of
human versican have been previously described (6, 21). The anti-His tag antibody was from RDI Research Diagnostics. The anti-human TS-4 antiserum was raised in rabbits by injection of the synthetic peptide
CYNHRTDLFKSFPGP, conjugated to ovalbumin (by Research Genetics); this
peptide represents residues 590-603 of human ADAMTS-4. Western
analysis was done on Novex Mini-gels under reducing conditions with
primary antibodies at between 1:1000 and 1:3000 dilution followed by
ECL detection as previously described (18, 22). For identification of
the V0 and V1 isoforms of versican, SDS-PAGE was run on 16- × 18-cm gels.
Immunohistochemistry--
Pieces of human abdominal aorta from
organ donors were fixed in 10% neutral buffered formalin overnight at
4 °C. After being embedded in paraffin, transverse sections 8 µm
thick were cut and used for immunohistochemical staining by the
streptavidin-biotin/horseradish peroxidase method (Vectastain
Elite ABC, Vector Laboratories) with 3,3'-diaminobenzidine plus nickel
chloride as a chromogen. The affinity-purified anti-DP was used at 10 ng of IgG/ml, the versican (Vc) antiserum was used at 1:400, and the
biotinylated goat anti-rabbit conjugate (Vector Laboratories) was used
at 1:400. Sections were counterstained with hematoxylin.
Sequence Alignments--
The alignment and consensus sequences
for the enzyme cleavage sites were generated using Multalin version
5.3.2 (23) with a gap weight of 12, gap length weight of 2, and
consensus levels of high = 90% and low = 50%. The proposed
cleavage sites were identified using PattInProt version 5.4 with
searches having a minimum similarity level of 70%.
Enzyme and Substrate Preparation and Digestion
Conditions--
Recombinant human ADAMTS-1 was expressed and purified
as previously described (24). Purified rhADAMTS-4 was a generous gift from Genetics Institute, Boston, MA. Preparation of the substrate recombinant fragment A by expression of the
Gly357-Asp567 portion of the GAG-beta domain of
versican V1 in Escherichia coli has been described (21). The
230-residue substrate (shown below) includes a 21-residue leader
sequence, including an His-Tag and protease X cleavage site spliced to
the Gly357-Asp567 versican peptide. The ADAMTS
clip site at Glu441-Ala442 is shown as
follows. MRGSHHHHHHGSKALLAIEGR
G357HPIDSESKEDEPCSEETDPVHDLMAEILPEFPDIIEIDLYHSEENEEEEEECANATDVTTTPSVQYINGKHLVTTVPKDPEAAE441(CLIP);
A442RRGQFESVAPSQNFSDSSESDTHPFVIAKTELSTAVQPNESTETTESLEVTWKPETYPETSEHFSGGEPDVFPTVPFHEEFESGTAKKGAESVTERDTEVGHQAHEHTEPVSLFPEESSGEIAID567.
For ADAMTS digestion, the GAG-beta substrate was first dialyzed
against water for 4 h to remove phosphate-buffered saline. Then
GAG-beta was incubated with ADAMTS (1 µg of proteinase per 20 µg of
substrate) in 100 µl of 50 mM Tris, 100 mM
NaCl, 10 mM CaCl2, pH 7.5, at 37 °C for
16 h and digests (10 µg for Western analysis or 20 µg for
Coomassie Blue) were analyzed on 4-20% SDS gels (Invitrogen).
For digestion of native substrates, 10 µg of versican-rich protein
purified as above from human aortic intima (estimated from
Coomassie-stained gel at about 3 µg of versican core protein) or 40 µg of rat chondrosarcoma aA1D1 (a gift from Dr. Jim Kimura) was
incubated with ADAMTS-1 or ADAMTS-4 in 100 µl 50 mM Tris,
100 mM NaCl, 10 mM CaCl2, pH 7.5, at 37 °C for 16 h. The samples were then
chondroitinase-digested as above, dried, and run on 4-12% SDS gels
(Invitrogen) for Western analysis.
Generation of Neoepitope Antiserum--
Computer-generated
alignment of the five known aggrecanase (ADAMTS)-mediated cleavage
sites of human aggrecan (10, 14, 15, 25, 26) with the single known
cleavage site of rat brevican by ADAMTS-4 (2, 27) is shown in the
top alignment in Table I. The
substrate length for the alignment was based on the cleavage sequences
located within the "gap" regions between the clusters of
chondroitin sulfate chains in the CS-2 domain of aggrecan and thus
support the view that these "nodal" regions may represent proteolytically sensitive sites (28). Most importantly, this alignment
suggests that the activity of the ADAMTS proteinases toward aggregating
proteoglycans is promoted by an extended motif found in the 23 residues
on the upstream side of the scissile bond along with a short 3-residue
stretch on the downstream side of the bond. A rather loose consensus
sequence for cleavage is apparent as
pt(V/I)XX(V/I)(t/d)XXlvEXvtpXXXXeXE*Xrg,
where the asterisk represents the scissile bond,
uppercase residues are 100% conserved, lowercase
residues are 50% or more conserved, and X represents
nonconserved residues. Using a search string of
[P]-X-[VI]-X-X-[VI]-X-X-X-X-X-[E]-X-[PVQ]-X-[PQAE]-X
(5,6)-[E]-[ASGL] based on this consensus, the sequences of
the known isoforms of human versican were inspected for evidence of
potential "aggrecanase-like" cleavage sites.
This approach suggested the presence of a very likely cleavage site in
the GAG-beta region at the Glu441-Ala442 bond
in V1 versican and at the equivalent
Glu1428-Ala1429 site in V0 versican (see Table
I, lower alignment). The likelihood of such a cleavage site
at Glu441 in V1 versican was strengthened by the fact that
the position of this site relative to the N terminus of the protein is
similar to the location of the known cleavage sites in aggrecan at
Glu373 and brevican at Glu395. The same search
strategy also suggested a likely cleavage site at
Glu405-Gln406 in the GAG-alpha region of both
the V0 and V2 isoforms of versican (see Table I), and studies on the
in vivo localization of the product of this cleavage will be
described elsewhere. Because the anti-NITEGE neoepitope antiserum has
been widely used previously (18, 29, 30) to specifically detect the
aggrecanase-generated species G1-NITEGE373, it seemed
likely that an equivalent antiserum to the putative versican neoepitope
sequence DPEAAE would be capable of detecting the equivalent
G1-DPEAAE441 versican product. We therefore generated a
rabbit polyclonal antiserum to the ovalbumin-conjugated peptide CGGDPEAAE.
Analysis of Versican Isoforms in Conditioned Medium from Human
Smooth Muscle Cell Cultures--
Cultures were labeled for 24 h
with [35S]methionine, and the proteoglycans were isolated
from the culture medium by DEAE cellulose chromatography (17) and
digested with chondroitinase ABC for analysis on a 16- × 18-cm,
4-12% gradient gel (Fig.
1A). Autoradiography (lane 1) showed the presence of two major very high
molecular mass products (bands 1 and 2),
which migrated in the 350- to 400-kDa range. On Western analysis both
of these products were reactive with anti-Vc (lane 2), LF99
(lane 3), and the anti-GAG-beta antiserum (lane
5), whereas only band 1 reacted with the GAG-alpha
antiserum (lane 4). When taken together with the established
specificity of these antisera (6, 21), these results identify
band 1 as the V0 isoform and band 2 as the V1
isoform of versican.
Portions of a similar proteoglycan preparation from human smooth
muscle cells (9) were separated on a 4-12% Mini-gel system for
Western analysis with four different antisera, namely Vc, HAL, LF99,
and DP (Fig. 1B). A single major high molecular mass species
(labeled as band 2) was present, and this was highly
reactive with Vc (lane 1), reactive with HAL (lane
2), and very weakly reactive with LF99 (lane 3). With
Vc and HAL there was also evidence for the presence of a low abundance
but discrete immunoreactive product (labeled as band 1),
which migrated more slowly than band 2. It seems likely that
band 1 and band 2 represent versican isoforms V0
and V1, respectively (as shown in A); however, it is also
possible that V0 and V1 did not separate effectively on this Mini-gel
system. In addition to full-length versican, the Vc antiserum (Fig.
1B, lane 1) also detected abundant products in
the 180- to 300-kDa range, and the LF-99 antiserum (Fig. 1B,
lane 3) detected products at about 70 and 120 kDa, but none
of these were further studied. The DP antiserum (Fig. 1B,
lane 4) showed no reactivity with any species in the smooth
muscle cell versican preparation, demonstrating that the antibody does
not react with the DPEAAE sequence when it is present in intact
versican (band 2). Furthermore, the DP antiserum did not
react with any of the Vc-reactive fragments that migrated between 180 and 300 kDa, suggesting that they are generated by proteolysis of
versican at sites other than the Glu441-Ala442 site.
Detection of Versican Cleavage Products in Extracts of Human
Aorta--
To examine the structure of versican in human aorta we next
analyzed extracts of whole aorta and of the intima, media, and adventitia on the Mini-gel system. The general pattern of
immunoreactive products with each of the four antisera (Vc, HAL, LF99,
and DP) was similar in each zone of aorta, so only the results with
intima are shown (Fig. 1C). Intact versican (labeled as
band 2, probably the V1 isoform) was the only
detectable very high molecular mass product. This was highly reactive
with Vc (lane 1) and also weakly reactive with HAL
(lane 2). The absence of detectable reactivity of band 2 with LF99 in this sample (lane 3) is probably due to the
poor reactivity of this antiserum relative to Vc and HAL (see Fig.
1B). The Vc antiserum (lane 1) also detected
abundant species in the 160- to 300-kDa range, which appeared similar
to those in the smooth muscle cell-conditioned medium (Fig.
1B, lane 1), however, these have not been
identified. Of particular interest was the finding that a 70-kDa band
(labeled as band 4 on Fig. 1C) was detected with
Vc (very weakly), HAL, and LF-99, and this species exhibited very
strong reactivity with the neoepitope antiserum DP (lane 4).
The migration behavior and immunoreactivity profile of this product
suggested that it represents the G1-DPEAAE441 cleavage
product of human versican V1 predicted from the consensus cleavage site
search strategy (Table I). In addition, a low abundance but discrete
DP-reactive product at about 220 kDa (labeled as band 3) was
also seen in these extracts, and its properties are consistent with the
G1-DPEAAE1428 species generated by cleavage of the versican
V0 isoform at the same site in the GAG-beta domain. The relatively low
abundance of band 3 relative to band 4 is
consistent with the very low apparent abundance of V0 relative to V1
versican in these samples. The specificity of immunoreactivity of the
band 3 and band 4 products was confirmed by
showing that the reactivity of both species was eliminated by
preadsorption of the antiserum with the immunizing peptide CGGDPEAAE at
10 µM concentration for 2 h (Fig. 1C,
lane 5). The band at about 60 kDa in lanes 2-5
appears to be nonspecific.
Because of the possibility of false positives with affinity-purified
C-terminal neoepitope antisera (18), we tested antiserum DP against a
wide range of aggrecan preparations. ADAMTS-generated fragments
with the C-terminal sequences TASELE, TFKEEE, APTAQE, PTVSQE, and
NITEGE (10, 18, 22) did not react with the affinity-purified DP under
Western blot conditions in which they reacted strongly with their
cognate antisera (results not shown).
Versican Immunohistocytochemistry in Human
Aorta--
Immunostaining with Vc and DP (Fig.
2) of sections prepared from the same
human aorta samples used for the Western analyses (Fig. 1) showed a
similar distribution for the two epitopes. In this sample, both the
total versican (Vc) and the truncated product(s) (DPEAAE) appeared to
be most abundant in the intima, detectable in patches in the media, and
difficult to detect in the adventitia. The same immunohistochemical
analysis of four additional human aorta samples (which ranged from
normal with diffuse intimal thickening to a vessel with atherosclerotic
lesions), showed that the vast majority of both the Vc-reactive and
DP-reactive species were present in the intima and media.
ADAMTS-1 and ADAMTS-4 Cleave Versican at the
Glu441-Ala442 Bond--
Because the human
aorta extracts contained versican fragments with strong and specific
immunoreactivity to the DP antiserum, we examined the possibility that
the C-terminal at DPEAAE441 can be generated by digestion
of versican with ADAMTS proteinases. For this purpose we incubated
purified recombinant human ADAMTS-1 and ADAMTS-4 with a recombinant
human versican GAG-beta domain substrate and examined the products by
Coomassie staining on SDS-PAGE (Fig. 3).
The substrate preparation (lane 1) ran as a single major Coomassie-stained band of about 50 kDa, which represents the
full-length substrate (band 1). Digestion with ADAMTS-1
(lane 2) eliminated much of the substrate and generated a
major stained product of about 28 kDa (band 2), which
appeared as a doublet, and a minor stained product at about 18 kDa
(band 3). Under the same conditions the ADAMTS-4 (lane
3) eliminated more of the substrate than the ADAMTS-1 digestion
but generated the same two product bands (bands 2 and
3).
To further characterize these digestion products another set of
incubations were used for Western analysis with the general GAG-beta
antiserum and the specific neoepitope DP antiserum (Fig. 4). The 50-kDa substrate (band
1) reacted strongly with the GAG-beta antiserum (lane
1) and also, unexpectedly, reacted weakly with the DP antiserum
(lane 2). After digestion with either ADAMTS-1 (lane
3) or ADAMTS-4 (lane 6) the GAG-beta-reactive substrate was completely eliminated, and two GAG-beta-reactive products were
obtained at about 28 kDa (band 2) and 18 kDa (band
3), which corresponded in migration behavior to the
Coomassie-stained products labeled as band 2 and band
3 (Fig. 3). When these digests were analyzed with the DP antiserum
(lanes 4 and 7) only the 28-kDa band (band
2) was reactive, identifying this as the N-terminal fragment with
a C-terminal at Glu441. In addition, the DP reactivity of
the 28-kDa fragment generated by ADAMTS-1 was eliminated by
preadsorption of the antiserum with the immunizing peptide (lane
5). Another TS-4 digest was also analyzed with the GAG-beta
antiserum, anti-DP, and an anti-His-tag antibody. On image overlay, the
results (not shown) confirmed that the slower of the GAG-beta-positive
products (band 2, Fig. 4) reacted with both anti-DP and the
anti-His tag antibodies, whereas the faster of the GAG-beta-positive
products (band 3, Fig. 4) reacted with neither of these
antibodies. Taken together, these data clearly show that both ADAMTS-1
and ADAMTS-4 are capable of cleaving the 50-kDa versican substrate
at the Glu441-Ala442 bond to generate an
N-terminal fragment of 105 residues (band 2, which migrates
at about 28 kDa) and a C-terminal fragment of 125 residues (band
3, which migrates at about 18 kDa). The reason for the anomalous
migration behavior of the band 2 and band 3 products, with respect to their predicted molecular masses, is unknown.
To confirm the identity of the products of ADAMTS digestion of the
recombinant GAG-beta substrate, another set of digests with both
ADAMTS-1 and ADAMTS-4 were analyzed by matrix-assisted laser desorption
time-of-flight mass spectrometry and m/z values generated from two separate digestions, calibrated both externally and
internally using egg ovalbumin and ADAMTS Proteinases Cleave Native Versican at the
Glu441-Ala442 Site, and ADAMTS-4 Is Present in
Human Aortic Intima--
To determine whether the ADAMTS proteinases
can also cleave native versican at the
Glu441-Ala442 bond, we next digested versican
from human aortic intima with TS-1 and TS-4. The products were analyzed
by Western blot with anti-Vc and anti-DP (Fig.
5). The substrate-only controls
(lanes 1 and 4) contained some full-length
versican (band 2), abundant versican fragments in the 120- to 250-kDa range, and, as expected, some of the 70-kDa
G1-DPEAAE441 (lane 4, band 4).
The apparent low abundance of the G1-DPEAAE in this material, relative
to that shown in Fig. 1C, is due to the very short film
exposure time required with the anti-DP antiserum in the digestion
experiment. Digestion with ADAMTS-1 (lane 2) did not
markedly alter the pattern of Vc-reactive species but did generate a
marked increase in the amount of 70-kDa G1-DPEAAE441
(lane 5, band 4). Digestion with ADAMTS-4
(lane 3) eliminated all of the full-length versican and also
most of the intermediate-sized Vc-reactive versican and generated a
Vc-reactive band at 70 kDa, which was highly reactive with anti-DP
(lane 6, band 4). Taken together these results
show that the naturally occurring 70-kDa G1-DPEAAE441 can
be generated by incubation of aortic versican with ADAMTS-1 and -4.
To determine whether human aortic intima contains ADAMTS proteinase
we analyzed extracts by Western blot with an anti-peptide antiserum
(anti-YNHRTD) to human ADAMTS-4 (Fig.
6). The extract (lane 1)
contained a major band of immunoreactive protein at about 70 kDa, which
comigrated with the mature form of recombinant TS-4 (lane
3). Confirmation of the presence of TS-4 in these extracts was
obtained by showing that immunoreactivity was eliminated (lanes 2 and 4) by preadsorption of the antiserum with the
immunizing peptide at 10 µM for 1 h at room
temperature.
The Relative Activity of ADAMTS-1 and -4 against Versican and
Aggrecan--
Digestion of the recombinant versican substrate or
native versican with purified recombinant enzymes (see Figs. 3 and 5)
consistently showed that the ADAMTS-4 was more active than the ADAMTS-1
per µg of enzyme protein. To examine this further, native versican was digested with 0, 0.1, 1.0, and 5.0 µg of each enzyme for 16 h, and the abundance of G1-DPEAAE product was determined by Western analysis. This confirmed (Fig. 7,
upper panel) that the ADAMTS-4 was about 5- to 10-fold more
active than an equivalent microgram amount of the ADAMTS-1 over this
concentration range. ADAMTS-4 was also found to be more active when
rat aggrecan was used as substrate (Fig. 7, lower panel),
showing that the high activity of ADAMTS-4 was not confined to versican
as a substrate. These digestions also showed that both enzymes degraded
aggrecan more efficiently than versican under the conditions used here.
Thus, complete aggrecan digestion, as indicated by elimination of
substrate bands (not shown), was obtained with 0.1 µg of TS-4 or 1.0 µg of TS-1, whereas complete versican digestion required about 1-5 µg of TS-4 and could not be obtained even with 5 µg of TS-1, the highest amount tested. (also see Fig. 5).
This paper shows that the mature human aorta contains a fragment
of V1 versican (G1-DPEAAE441), which can be generated by
ADAMTS-1 or ADAMTS-4 digestion of intact human versican. This product
has been characterized by Western blot with three general versican
antisera (Vc, HAL, and LF-99) and a new antiserum raised to an
alignment-predicted C-terminal neoepitope (DPEAAE). The novel fragment
appears to arise in vivo, because it is present in extracts
made in the presence of proteinase inhibitors, and the immunoreactivity
can be detected by immunohistochemistry of formalin-fixed
paraffin-embedded sections. Moreover, because the extracts that contain
the versican fragment also contain the mature form of human ADAMTS-4
(Fig. 6), it is possible that this member of the ADAMTS family
is responsible for its formation in vivo.
The abundance of the 70-kDa fragment relative to other versican species
in the aorta cannot be determined from these analyses, although
Coomassie stains of SDS-PAGE gels (not shown) suggest that it
represents about 10% of the total protein in intimal extracts. Interestingly, the same product appears to be present in human skin
extracts, because the 66-kDa versican fragment described by Sorrell and
colleagues (11) reacts strongly with the anti-DPEAAE antiserum
characterized in this
report.2 This 66-kDa protein
is more abundant in fetal skin compared with adult skin, supporting an
important biological role for this protein in the extracellular
matrix of versican-rich tissues. Aortic extracts also contained a
220-kDa fragment (Fig. 1C, lane 4, band
3) that appears to represent the product generated by ADAMTS
cleavage at DPEAAE1428 of the V0 isoform of versican, and
this product is also present in human skin extracts.2
The present data also support the existence of a consensus motif for
ADAMTS cleavage of specific Glu-X bonds in aggregating proteoglycans
(Table I). The inability of matrix metalloproteinase 2, matrix
metalloproteinase 7, and plasmin to cleave versican at
Glu441-Ala442, despite a clear ability to
degrade versican (31),3 also
supports the specificity of this ADAMTS motif. Moreover, it is possible
to predict from this 26-residue consensus sequence another likely
ADAMTS cleavage site in versican at
Glu405-Gln406 that is present in versican
isoforms V0 and V2 (Table I, lower panel). In this regard,
because versican V2 is the most abundant isoform in brain (8), we have
examined human brain extracts for the presence of the predicted
G1-NIVSFE405 (Table I). This versican fragment is indeed
abundant in this tissue.4 In
the same way, the developmentally regulated and proteolytically generated major forms of neurocan (32) may result from cleavage at an
ADAMTS site, which is predicted from similar homology searching to be
present in human neurocan at Glu653-Ala654.
If the recognition sequence suggested here (Table I) plays a
pivotal role in the susceptibility of these different sites to
proteolysis, it would suggest that recognition of various proteoglycan substrates by the ADAMTS family of proteinases may involve an exosite
binding domain (33) adjacent to, but distinct from, the active site
cleft. Such multiple binding sites for the interaction of the aggrecan
interglobular domain with ADAMTS-4 has also been recently indicated
by the use of mutated substrates in activity studies (34, 35).
Delineation of such interactions might offer therapeutic potential in
situations, such as osteoarthritis, where ADAMTS activity appears to be
uncontrolled. In addition, the ADAMTS subgroup of proteinases has one
or more thrombospondin motifs in addition to the metalloproteinase and
disintegrin domain of all ADAM family members. It has been suggested
that the thrombospondin motif confers heparan sulfate binding
properties on this subgroup, which unlike other ADAMs is not anchored
to cells by a transmembrane segment (36). In addition, recent data (37,
38) has suggested that the interaction between aggrecan and
ADAMTS-4 is mediated through the keratan sulfate chains of the
substrate so that control of ADAMTS-mediated proteolysis by sulfated
glycosaminoglycans appears to warrant further investigation. In this
regard, it is possible that disruption of the activation or
localization of an ADAMTS proteinase by heparin (36) might provide an
additional explanation for how heparin inhibits migration and
proliferation of smooth muscle cells (39, 40). Thus an inhibition of
ADAMTS-dependent versican degradation might lower the local
concentration of versican G1 and G3 domain fragments, which have been
reported to stimulate cell proliferation and migration (41, 42).
The data presented here on ADAMTS-1 and ADAMTS-4 cleavage of versican,
along with the observations on ADAMTS-1, -4, and
-5-dependent degradation of aggrecan (15, 43, 44) and
ADAMTS-4 cleavage of brevican (27) suggests that these three
ADAMTS family members do not target individual substrates. Instead they
are all particularly suited to glutamyl-endopeptidase cleavage of one
or more aggregating proteoglycans. In this regard, the concept of
linking particular ADAMTS family members to individual substrates, for
example as in the description of ADAMTS-4 as aggrecanase-1 (14), may
now require revision. It will be interesting to determine the
structural features of ADAMTS-1, -4, and 5, which are responsible for
this apparent preference for cleavage of specific Glu-X
bonds in the core proteins of the proteoglycans, and also perhaps to
define which family members are primarily responsible for the
degradation of each proteoglycan substrate in vivo. In this
regard, it may be significant that we found (Fig. 7) that both
ADAMTS-1 and -4 appear to degrade aggrecan more effectively than
versican and that for both substrates ADAMTS-4 was the most active
proteinase. Comparisons of this kind, however, may be misleading in
that the high relative activity against aggrecan might be due to the
higher substrate concentration for aggrecan (500 nM)
relative to versican (about 50 nM) or the type of
glycosaminoglycan substitution (38) present on the proteoglycan
substrates used in these incubations. In addition, the apparent high
activity of the TS-4 relative to the TS-1 might be related to a
different stability of the recombinant enzymes to purification or
storage. Freshly prepared and active-site titrated enzymes with more
fully characterized natural or artificial substrates will be needed to
adequately address this issue in the future.
Finally, with respect to the physiologic role of TS-1, it is
interesting that the ADAMTS-1-null mouse (45) exhibits a broad pathology, including growth retardation with adipose tissue
malformation, fibrotic changes in the ureter, and a lack of capillary
formation in the adrenal medulla. Whether this profile results from an
inability to degrade proteoglycans such as aggrecan and/or versican or
other extracellular matrix proteins during organogenesis remains to be determined.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C, the precipitate was
collected by centrifugation at 13,000 × g for 20 min
at 4 °C. The pellet was dried, dissolved in 50 mM Tris,
50 mM sodium acetate, 10 mM EDTA, pH 7.6, and
deglycosylated by digestion for 1.5 h at 37 °C with
chondroitinase ABC (25 milliunits/100 µg of GAG, protease-free, Seikagaku). Versican was prepared from human smooth muscle
cell-conditioned medium after labeling with
[35S]methionine for 24 h as described previously
(17).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Alignment and consensus sequence of the known ADAMTS cleavage sites for
aggrecan and brevican (top) and the versican cleavage sites described
in this report (bottom)
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Fig. 1.
A, identification of versican V0 and V1
in human smooth muscle cell cultures.
[35S]Methionine-labeled and purified proteoglycans were
treated with chondroitin ABC lyase and analyzed on 4-12% SDS-PAGE
(16- × 18-cm gels). The gel was either autoradiographed (lane
1) or probed with antisera Vc (lane 2), LF99
(lane 3), GAG-alpha domain (lane 4), and GAG-beta
domain (lane 5). The migration position of the 250-kDa
marker is shown. B, Western analysis of versican. Versican
from human smooth muscle cell-conditioned medium was analyzed on
4-12% SDS-PAGE Mini gels with four different antisera. The antibodies
used were in lane 1, Vc, an antiserum to recombinant
versican V1; lane 2, HAL, an antiserum to a sequence
starting at CDAG of the hyaluronate binding site in aggrecan, versican,
and link protein; lane 3, LF99 an antiserum to the
N-terminal 16 amino acids of versican; lane 4, anti-DP,
which recognizes a neoepitope at Glu441 (V1 versican) and
Glu1428 (V0 versican). Bands 1 and 2 refer to the apparent migration positions of versican V0 and V1,
respectively. C, Western analysis of versican in human
aortic intimal extracts. Five portions of an extract of mature human
aortic intima were separated on a 4-12% Mini-gel and probed with the
antiserum Vc (lane 1), HAL (lane 2), LF-99
(lane 3), DP (lane 4), and anti-DP preadsorbed
with the immunizing peptide (DP ( ), lane 5).
The identification of bands 2, 3, and
4 as versican V1, G1-DPEAAE1428 derived from
versican V0, and G1-DPEAAE441 derived from versican V1,
respectively, is described in the text.
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Fig. 2.
Distribution of versican and versican
neoepitope in human aorta. Immunohistochemical staining of mature
human aorta (400× magnification) with anti-Vc (top
panel) or anti-DPEAAE (bottom panel) antibodies. The
vessel layers are labeled as adventitia (A), media
(M), and intima (Int). Nuclei were counterstained
with methyl green.
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Fig. 3.
Digestion of recombinant versican with
ADAMTS-1 and ADAMTS-4. Recombinant versican GAG-beta substrate (20 µg) was incubated alone (lane 1), with 1 µg of ADAMTS-1
(lane 2), or 1 µg of ADAMTS-4 (lane 3) for
16 h, and the samples were run on 4-12% SDS-PAGE and stained
with Coomassie Blue. Identification of bands 1,
2, and 3 as the substrate, the N-terminal
fragment, and the C-terminal fragment, respectively, is given in the
text.
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Fig. 4.
Western analysis of digestion products of
recombinant versican with ADAMTS-1 and ADAMTS-4. Recombinant
versican GAG-beta substrate (10 µg) was incubated alone (lanes
1 and 2), with 0.5 µg of ADAMTS-1 (lanes
3, 4, and 5) or 0.5 µg of ADAMTS-4
(lanes 6 and 7) for 16 h, and the samples
were run on 4-12% SDS-PAGE for Western analysis. The blots were
probed with the anti-GAG-beta antiserum (lanes 1,
3, and 6), anti-DP (lanes 2,
4, and 7) and anti-DP preadsorbed with the
immunizing peptide (DP ( ), lane 5).
Identification of bands 1, 2, and 3 as
the substrate, the N-terminal fragment, and the C-terminal fragment,
respectively, is given in the text.
-lactalbumin. The predicted molecular weight for the full-length GAG-beta substrate is 25651.5, and
the two products expected from cleavage at the
Glu441-Ala442 bond alone are 11844.8 (N-terminal product), and 13824.7 (C-terminal product). The digest with
ADAMTS-1 contained a peak at m/z 25697 ± 108, which corresponds to undigested full-length GAG-beta substrate. This peak was not seen in the ADAMTS-4 digest consistent with our
general observation of more complete digestion with TS-4. The digests
with both enzymes contained products that were very close to the
theoretical size for the C-terminal fragment; the ADAMTS-1 product was
at m/z 13841 ± 17 and the ADAMTS-4 product at m/z 13848 ± 21. There were several peaks
in the region of both spectra near the position of the theoretical
N-terminal product; however, none corresponded exactly to the expected
size. This appears to be due to further very limited proteolysis of the
N-terminal product from the N terminus, because this product reacts
with anti-DP, which detects the C-terminal (Fig. 4), and also the
anti-His-tag, which detects residues 5 through 10 at the N terminus.
View larger version (66K):
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Fig. 5.
Digestion of human aortic versican with
ADAMTS-1 and ADAMTS-4. Versican-rich material (10 µg of protein)
prepared from human aortic intima was incubated alone (lanes
1 and 4), with 5 µg of ADAMTS-1 (lanes 2 and 5) or 1 µg of ADAMTS-4 (lanes 3 and
6) for 16 h, and the samples were run on a 4-12%
Mini-gel for Western analysis. The blots were probed with anti-Vc
(lanes 1, 2, and 3) and anti-DP
(lanes 4, 5, and 6). Identification of
bands 2 and 4 as versican V1 and
G1-DPEAAE441 derived from versican V1, respectively, is
described in the text.
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Fig. 6.
Western analysis of human aortic intima
extracts for ADAMTS-4 protein. Versican-rich material (10 µg of
protein) prepared from human aortic intima (lanes 1 and
2) and 0.3 µg of recombinant human ADAMTS-4 (lanes
3 and 4) were probed with antiADAMTS-4
(anti-YNHRTD) either directly ((+), lanes 1 and
3) or after preadsorption of the antiserum with the
immunizing peptide (( ), lanes 2 and 4).
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Fig. 7.
Relative activity of ADAMTS-1 and ADAMTS-4
for versican and aggrecan. Human versican (at about 50 nM) or rat chondrosarcoma aA1D1 (at about 500 nM) was incubated with 0, 0.01, 0.1, 1.0, or 5 µg of
ADAMTS-1 or ADAMTS-4 in 100 µl of 50 mM Tris, 100 mM NaCl, 10 mM CaCl2, pH 7.5 at
37 °C for 16 h. The samples were then chondroitinase-digested
and dried, and portions were run on 4-12% SDS gels (Invitrogen) for
Western analysis. The blot of versican digests were probed with
anti-DP, and the blot of aggrecan digests were probed with anti-G1. The
product bands shown for comparison are the G1-DPEAAE product for
versican and the G1-NITEGE product for aggrecan.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. James Perkins and Beverly Nass for the human aortic specimens. The expert technical assistance of Vivian Thompson and the Protein Core Facility at the University of Florida, Gainesville, is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Services Grants HL30946, RR00166, and HL07828, by an award from the American Heart Association (to J. W.), by National Institutes of Health Grant R01CA 77420 (to M. L. I. A.), and by a grant from the Swiss Science Foundation (31-55718.98, to D. Z.).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.
§ Both authors contributed equally to this work.
¶ Supported by the Shriners of North America and the Arthritis Foundation. To whom correspondence should be addressed: Shriners Hospital for Children, 12502 North Pine Dr., Tampa, FL 33612-9499. Tel.: 813-972-2250; Fax: 813-975-7127; E-mail: jsandy@shctampa.usf.edu.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M009737200
2 D. A. Carrino and A. I. Caplan, personal communication.
3 R. Kenagy, unpublished data.
4 J. D. Sandy and P. Gottschall, unpublished.
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ABBREVIATIONS |
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The abbreviations used are: CS, chondroitin sulfate; ADAMTS, a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif; AEBSF, aminoethyl butane sulfonyl fluoride; GAG, glycosaminoglycan; MES, 4-morpholineethanesulfonic acid; anti-DP, antiserum DPEAAE; PAGE, polyacrylamide gel electrophoresis; Vc, antiserum to human versican V1.
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REFERENCES |
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---|
1. | Hascall, V. C., Calabro, A., Midura, R. J., and Yanagishita, M. (1994) Methods Enzymol. 230, 390-417[Medline] [Order article via Infotrieve] |
2. |
Yamada, H.,
Watanabe, K.,
Shimonaka, M.,
and Yamaguchi, Y.
(1994)
J. Biol. Chem.
269,
10119-10126 |
3. |
Bode-Lesniewska, B.,
Dours-Zimmermann, M. T.,
Odermatt, B. F.,
Briner, J.,
Heitz, P. U.,
and Zimmermann, D. R.
(1996)
J. Histochem. Cytochem.
44,
303-312 |
4. |
Ito, K.,
Shinomura, T.,
Zako, M.,
Ujita, M.,
and Kimata, K.
(1995)
J. Biol. Chem.
270,
958-965 |
5. |
Zako, M.,
Shinomura, T.,
Ujita, M.,
Ito, K.,
and Kimata, K.
(1995)
J. Biol. Chem.
270,
3914-3918 |
6. |
Dours-Zimmermann, M. T.,
and Zimmermann, D. R.
(1994)
J. Biol. Chem.
269,
32992-32998 |
7. |
Lemire, J. M.,
Braun, K. R.,
Maurel, P.,
Kaplan, E. D.,
Schwartz, S. M.,
and Wight, T. N.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1630-1639 |
8. |
Schmalfeldt, M.,
Dours-Zimmermann, M. T.,
Winterhalter, K. H.,
and Zimmermann, D. R.
(1998)
J. Biol. Chem.
273,
15758-15764 |
9. | Yao, L. Y., Moody, C., Schonherr, E., Wight, T. N., and Sandell, L. J. (1994) Matrix Biol 14, 213-225[CrossRef][Medline] [Order article via Infotrieve] |
10. | Sandy, J. D., Thompson, V., Doege, K., and Verscharen, C. (2000) Biochem. J. 351, 161-166[CrossRef][Medline] [Order article via Infotrieve] |
11. | Sorrell, J. M., Carrino, D. A., Baber, M. A., and Caplan, A. I. (1999) Anat. Embryol. 199, 45-56[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Sandy, J. D.,
Boynton, R. E.,
and Flannery, C. R.
(1991)
J. Biol. Chem.
266,
8198-8205 |
13. |
Kuno, K.,
Kanada, N.,
Nakashima, E.,
Fujiki, F.,
Ichimura, F.,
and Matsushima, K.
(1997)
J. Biol. Chem.
272,
556-562 |
14. |
Tortorella, M. D.,
Burn, T. C.,
Pratta, M. A.,
Abbaszade, I.,
Hollis, J. M.,
Liu, R.,
Rosenfeld, S. A.,
Copeland, R. A.,
Decicco, C. P.,
Wynn, R.,
Rockwell, A.,
Yang, F.,
Duke, J. L.,
Solomon, K.,
George, H.,
Bruckner, R.,
Nagase, H.,
Itoh, Y.,
Ellis, D. M.,
Ross, H.,
Wiswall, B. H.,
Murphy, K.,
Hillman, M. C., Jr.,
Hollis, G. F.,
Arner, E. C.,
et al..
(1999)
Science
284,
1664-1666 |
15. |
Abbaszade, I.,
Liu, R. Q.,
Yang, F.,
Rosenfeld, S. A.,
Ross, O. H.,
Link, J. R.,
Ellis, D. M.,
Tortorella, M. D.,
Pratta, M. A.,
Hollis, J. M.,
Wynn, R.,
Duke, J. L.,
George, H. J.,
Hillman, M. C., Jr.,
Murphy, K.,
Wiswall, B. H.,
Copeland, R. A.,
Decicco, C. P.,
Bruckner, R.,
Nagase, H.,
Itoh, Y.,
Newton, R. C.,
Magolda, R. L.,
Trzaskos, J. M.,
Hollis, G. F.,
Arner, E. C.,
and Burn, T. C.
(1999)
J. Biol. Chem.
274,
23443-23450 |
16. | Wahlberg, J. A., Southard, J. H., and Belzer, F. O. (1986) Cryobiology 23, 477-482[Medline] [Order article via Infotrieve] |
17. |
Olin, K. L.,
Potter-Perigo, S.,
Barrett, P. H.,
Wight, T. N.,
and Chait, A.
(1999)
J. Biol. Chem.
274,
34629-34636 |
18. | Sandy, J. D., Plaas, A. H., and Koob, T. J. (1995) Acta Orthop. Scand. Suppl. 266, 26-32[Medline] [Order article via Infotrieve] |
19. | Fisher, L. W., Stubbs, J. T., 3rd, and Young, M. F. (1995) Acta Orthop. Scand. Suppl. 266, 61-65[Medline] [Order article via Infotrieve] |
20. | du Cros, D. L., LeBaron, R. G., and Couchman, J. R. (1995) J. Investig. Dermatol. 105, 426-431[Abstract] |
21. | Zimmermann, D. R., Dours-Zimmermann, M. T., Schubert, M., and Bruckner-Tuderman, L. (1994) J. Cell Biol. 124, 817-825[Abstract] |
22. | Sandy, J. D., Gamett, D., Thompson, V., and Verscharen, C. (1998) Biochem. J. 335, 59-66[Medline] [Order article via Infotrieve] |
23. | Corpet, F. (1988) Nucleic Acids Res. 16, 10881-10890[Abstract] |
24. |
Rodriguez-Manzaneque, J. C.,
Milchanowski, A. B.,
Dufour, E. K.,
Leduc, R.,
and Iruela-Arispe, M. L.
(2000)
J. Biol. Chem.
275,
33471-33479 |
25. | Sandy, J. D., Flannery, C. R., Neame, P. J., and Lohmander, L. S. (1992) J. Clin. Invest. 89, 1512-1516[Medline] [Order article via Infotrieve] |
26. | Lohmander, L. S., Neame, P. J., and Sandy, J. D. (1993) Arthritis Rheum. 36, 1214-1222[Medline] [Order article via Infotrieve] |
27. |
Matthews, R. T.,
Gary, S. C.,
Zerillo, C.,
Pratta, M.,
Solomon, K.,
Arner, E. C.,
and Hockfield, S.
(2000)
J. Biol. Chem.
275,
22695-22703 |
28. | Hering, T. M., Kollar, J., and Huynh, T. D. (1997) Arch. Biochem. Biophys. 345, 259-270[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Lark, M. W.,
Gordy, J. T.,
Weidner, J. R.,
Ayala, J.,
Kimura, J. H.,
Williams, H. R.,
Mumford, R. A.,
Flannery, C. R.,
Carlson, S. S.,
Iwata, M.,
and Sandy, J. D.
(1995)
J. Biol. Chem.
270,
2550-2556 |
30. |
Fosang, A. J.,
Last, K.,
and Maciewicz, R. A.
(1996)
J. Clin. Invest.
98,
2292-2299 |
31. |
Halpert, I.,
Sires, U. I.,
Roby, J. D.,
Potter-Perigo, S.,
Wight, T. N.,
Shapiro, S. D.,
Welgus, H. G.,
Wickline, S. A.,
and Parks, W. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9748-9753 |
32. | Matsui, F., Nishizuka, M., Yasuda, Y., Aono, S., Watanabe, E., and Oohira, A. (1998) Brain Res. 790, 45-51[CrossRef][Medline] [Order article via Infotrieve] |
33. | Baykal, D., Schmedtje, J. F., Jr., and Runge, M. S. (1995) Am. J. Cardiol. 75, 82B-87B[Medline] [Order article via Infotrieve] |
34. |
Mercuri, F. A.,
Maciewicz, R. A.,
Tart, J.,
Last, K.,
and Fosang, A. J.
(2000)
J. Biol. Chem.
275,
33038-33045 |
35. | Horber, C., Buttner, F. H., Kern, C., Schmiedeknecht, G., and Bartnik, E. (2000) Matrix Biol. 19, 533-543[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Kuno, K.,
and Matsushima, K.
(1998)
J. Biol. Chem.
273,
13912-13917 |
37. |
Tortorella, M.,
Pratta, M.,
Liu, R. Q.,
Abbaszade, I.,
Ross, H.,
Burn, T.,
and Arner, E.
(2000)
J. Biol. Chem.
275,
25791-25797 |
38. |
Pratta, M. A.,
Tortorella, M. D.,
and Arner, E. C.
(2000)
J. Biol. Chem.
275,
39096-39102 |
39. | Majack, R. A., and Clowes, A. W. (1984) J. Cell. Physiol. 118, 253-256[Medline] [Order article via Infotrieve] |
40. | Castellot, J. J., Jr., Wright, T. C., and Karnovsky, M. J. (1987) Semin. Thromb. Hemost. 13, 489-503[Medline] [Order article via Infotrieve] |
41. | Ang, L. C., Zhang, Y., Cao, L., Yang, B. L., Young, B., Kiani, C., Lee, V., Allan, K., and Yang, B. B. (1999) J. Neuropathol. Exp. Neurol. 58, 597-605[Medline] [Order article via Infotrieve] |
42. |
Yang, B. L.,
Cao, L.,
Kiani, C.,
Lee, V.,
Zhang, Y.,
Adams, M. E.,
and Yang, B. B.
(2000)
J. Biol. Chem.
275,
21255-21261 |
43. | Kuno, K., Okadab, Y., Kawashimac, H., Nakamurab, H., Miyasakac, M., Ohnoa, H., and Matsushimad, K. (2000) FEBS Lett. 478, 241-245[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Tortorella, M. D.,
Pratta, M.,
Liu, R. Q.,
Austin, J.,
Ross, O. H.,
Abbaszade, I.,
Burn, T.,
and Arner, E.
(2000)
J. Biol. Chem.
275,
18566-18573 |
45. |
Shindo, T.,
Kurihara, H.,
Kuno, K.,
Yokoyama, H.,
Wada, T.,
Kurihara, Y.,
Imai, T.,
Wang, Y.,
Ogata, M.,
Nishimatsu, H.,
Moriyama, N.,
Oh-hashi, Y.,
Morita, H.,
Ishikawa, T.,
Nagai, R.,
Yazaki, Y.,
and Matsushima, K.
(2000)
J. Clin. Invest.
105,
1345-1352 |