Versican V1 Proteolysis in Human Aorta in Vivo Occurs at the Glu441-Ala442 Bond, a Site That Is Cleaved by Recombinant ADAMTS-1 and ADAMTS-4*

John D. SandyDagger §, Jennifer WestlingDagger §, Richard D. Kenagy||, M. Luisa Iruela-Arispe**, Christie VerscharenDagger , Juan Carlos Rodriguez-Mazaneque**, Dieter R. ZimmermannDagger Dagger , Joan M. Lemire||, Jens W. Fischer||, Thomas N. Wight||, and Alexander W. Clowes||

From the Dagger  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 Dagger Dagger  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



    ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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.

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EXPERIMENTAL PROCEDURES
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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 -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).

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.

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INTRODUCTION
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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.

                              
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Table I
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)
Upper case, residue conserved 100%; lower case, residue conserved for 50% to less than 100%.

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.


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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.

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.


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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.

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).


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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.

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.


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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.

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 alpha -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.

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. 


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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.

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.


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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).

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).


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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

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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