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Originally published In Press as doi:10.1074/jbc.M501903200 on August 8, 2005

J. Biol. Chem., Vol. 280, Issue 41, 34441-34446, October 14, 2005
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Plasmin- and Thrombin-accelerated Shedding of Syndecan-4 Ectodomain Generates Cleavage Sites at Lys114–Arg115 and Lys129–Val130 Bonds*

Annette Schmidt{ddagger}1, Frank Echtermeyer§, Anthony Alozie{ddagger}, Kerstin Brands§, and Eckhart Buddecke{ddagger}§

From the {ddagger}Leibniz-I Institute of Arteriosclerosis Research, §Department of Physiological Chemistry and Pathobiochemistry, University of Muenster, D-48149 Muenster, Germany

Received for publication, February 18, 2005 , and in revised form, June 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Syndecans are transmembranous heparan sulfate proteoglycans abundant in the surface of all adherent mammalian cells and involved in vital cellular functions. In this study, we found syndecan-1, -2, -3, and -4 to be constitutively expressed by human umbilical vein endothelial cells. The exposure of the ectodomains of syndecan-1 and -4 to the cell surface and their constitutive shedding into the extracellular compartment was measured by immunoassays. In the presence of plasmin and thrombin, shedding was accelerated and monitored by detection and identification of 35S-labeled proteoglycans. To elucidate the cleavage site of the syndecan ectodomains, we used a cell-free in vitro system with enzyme and substrate as the only reactants. For this purpose, we constructed recombinant fusion proteins of the syndecan-1 and -4 ectodomain together with maltose-binding protein and enhanced yellow fluorescent protein as reporter proteins attached to the N and C termini via oligopeptide linkers. After protease treatment of the fusion proteins, the electrophoretically resolved split products were sequenced and cleavage sites of the ectodomain were identified. Plasmin generated cleavage sites at Lys114{downarrow}Arg115 and Lys129{downarrow}Val130 in the ectodomain of syndecan-4. In thrombin proteolysates of the syndecan-4 ectodomain, the cleavage site Lys114{downarrow}Arg115 was also identified. The cleavage sites for plasmin and thrombin within the syndecan-4 ectodomain were not present in the syndecan-1 ectodomain. Cleavage of the syndecan-1 fusion protein by thrombin occurred only at a control cleavage site (Arg{downarrow}Gly) introduced into the linker region connecting the ectodomain with the enhanced yellow fluorescent protein. Because both plasmin and thrombin are involved in thrombogenic and thrombolytic processes in the course of the pathogenesis of arteriosclerosis, the detachment of heparan sulfate-bearing ectodomains could be relevant for the development of arteriosclerotic plaques and recruitment of mononuclear blood cells to the plaque.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The family of syndecans forms a group of transmembrane heparan sulfate proteoglycans that are composed of a core protein with covalently attached glycosaminoglycan chains. The four mammalian syndecan genes (syndecan-1, -2, -3, -4) have been cloned and sequenced and are expressed in most human cells and tissues (for review, see Refs. 1 and 2), including human umbilical vein endothelial cells (3, 4). The syndecans contain an N-terminal extracellular domain or ectodomain, a hydrophobic transmembrane domain, and a short C-terminal cytoplasmic domain. The ectodomain bears, near the N terminus, three consecutive consensus Ser-Gly sequences for heparan sulfate chain attachment and may also contain Ser-Gly sequences near the plasma membrane that serve as attachment sites for chondroitin sulfate side chains. The length of the ectodomains varies markedly among family members, whereas the length of the transmembrane and cytoplasmic domain is highly conserved (5).

The function of syndecans includes anchorage of cells to extracellular matrix components with associated heparan sulfate binding domains (6), maintenance of epithelial and endothelial morphology (1), binding to and modulation of the activity of heparan sulfate binding growth factors (7), and modulation of the activity of several proteases and their inhibitors (for review, see Refs. 2 and 8), but they may also serve as signaling molecules (9, 10) and as arterial counterparts for monocyte L-selectin in the vascular endothel (10).

The syndecan ectodomains are released from the cell surface in a process commonly known as shedding (10–12). Shedding of the ectodomain occurs as part of the normal turnover and involves the activity of a not-identified cell surface zinc metalloproteinase that is specifically inhibited by tissue inhibitor of matrix metalloproteinase-3 (8). So far the site of cleavage has not been identified. For syndecan-1, a cleavage site has been localized within nine amino acids adjacent to the extracellular face of the plasma membrane (13). Shedding of syndecan-1 and -4 can be regulated by various external stimuli and intracellular signaling pathways such as growth factors (14), cell stress (13), or soluble microbial pathogens (15, 16). Shedding of syndecan-1 and -4 can be accelerated via receptor activation, such as the thrombin receptor and epidermal growth factor (EGF)2 receptor family or by the direct action of proteases. Subramanian et al. (14) describes an accelerated shedding by plasmin and thrombin and the presence of the soluble ectodomains in fluids accumulating following injury and inflammation, but no information about the cleavage site of the ectodomains are available.

In the present study, we investigated the plasmin- and thrombin-mediated shedding of syndecan-1 and -4 in a cell-free in vitro system and identified, in the ectodomain of syndecan-4, two cleavage sites for plasmin and one for thrombin. For this purpose, recombinant fusion proteins were constructed that contained the ectodomain of syndecan-1 or -4 with reporter proteins attached to the N and C terminus via linker oligopeptides.

The pathophysiological relevance of our findings for the pathogenesis of arteriosclerosis lays in the fact that syndecans are constitutively expressed by endothelial cells of the vascular system and that plasmin and thrombin are involved in thrombogenic and thrombolytic processes at the site of arteriosclerotic lesions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Sodium [35S]sulfate (carrier free, 0.8–1.5 TBq mg–1 sulfur) was obtained from ICN Biomedicals GmbH (Eschwege, Germany). Heparan sulfate lyase (heparitinase, EC 4.2.2.8 [EC] ) and chondroitin sulfate lyase (chondroitinase ABC, EC 4.2.2.4 [EC] ) were from Medac (Hamburg, Germany). Plasmin (human plasma fibrinolysin, EC 3.4.21.7 [EC] ) 5.7 units/mg protein and thrombin (human plasma, factor IIa, EC 3.4.21.5 [EC] ) 2800 NIH units/mg protein were from Sigma. Monoclonal mouse anti-syndecan-1 (DL101), anti-syndecan-4 (5G9), polyclonal rabbit anti-syndecan-1 (H174), and anti-syndecan-4 (H140) were from Santa Cruz Biotechnology, polyclonal goat anti-mouse HRP-conjugated IgG (P-0447) from DakoCytomation, and polyclonal goat anti-rabbit HRP-conjugated IgG from Vector Laboratories. All other chemicals were of analytical grade or the best grade available.

Cell Culture—Human umbilical vein endothelial cells (HUVEC) were harvested from fresh human umbilical cords as previously described (15). Primary isolated cells were cultured in gelatin-precoated tissue flasks at 37 °C under 5% CO2/95% air. Culture medium consisted of RPMI 1640 (Invitrogen) and supplements, as previously described (17), including 10 µg/ml ciprofloxacin (Bayer, Wuppertal, Germany). Cultures of the second to the fifth passage were used for the experiments.

Immunoassays—Cell surface-exposed syndecan ectodomains were identified according to a protocol described previously (17) for cell adhesion molecules. Native cells were exposed to monoclonal anti-syndecan-1 or -4 antibodies (1:200), which recognize the glycosylated form of syndecans. Plates were finally incubated with HRP-conjugated secondary antibodies (1:10,000). Sandwich immunoassays were designed for detection of the shed syndecan-1 and -4 ectodomains according to Rioux et al. (18), with the exception that mouse monoclonal antibodies were employed as capture, polyclonal rabbit anti-syndecans as secondary antibodies, and HRP-conjugated goat anti-rabbit IgG (1:50,000) reserved for color development. The serum-free cell supernatant containing the shed ectodomains of syndecan-1 and -4 was freeze-dried, redissolved in a small volume, dialyzed, and deglycosylated by heparitinase and chondroitin sulfate lyase for an enhanced binding of antibodies. For Western blot analysis, syndecan-1 and -4 were collected from the cell supernatant by protein A-Sepharose precoated with both polyclonal anti-syndecan-1 and anti-syndecan-4 antibodies that recognize the glycosylated form of syndecans. Thereafter, the protein A-Sepharose suspension was degraded by heparitinase and chondroitin sulfate lyase, boiled in SDS buffer, and submitted to PAGE. The blotted deglycosylated syndecans were detected according to a standard protocol using monoclonal anti-syndecan-1 or anti-syndecan-4 antibodies.

Construction of Syndecan-1 and Syndecan-4 Ectodomain Fusion Proteins—The pMAL-c2 vector (New England Biolabs GmbH, Frankfurt/Main, Germany) was used to express and purify fusion proteins produced from the cloned ectodomain of syndecan-1 and -4 with the 5'linker to the maltose-binding protein (MBP) DNA and 3'linker to the enhanced yellow fluorescent protein (EYFP) DNA. The coding sequences of the syndecan-1 and -4 ectodomains were amplified by PCR from normal human fibroblast cDNA using primers containing a 5'EcoRI and a 3'XbaI site (syndecan-1) or a 5'EcoRI and 3'KpnI site (syndecan-4). The sequence coding for EYFP was amplified from the plasmid pIRES-EYFP (BD Biosciences/Clontech) using primers containing a 5'XbaI and 3'HindIII site. The syndecan-1 and EYFP PCR products were digested with XbaI, ligated, and digested again with EcoRI and HindIII. The purified Syn-1-EYFP product was cloned into the EcoRI and HindIII sites of the pMal-c2 expression vector downstream of the MalE gene coding for MBP. For construction of the syndecan-4 fusion protein, a different cloning protocol was used. First, the EYFP PCR product was digested with XbaI and HindIII and ligated into the respective sites of pUC19 vector (Invitrogen), which was designated as pUC19-EYFP. Second, the syndecan-4 PCR fragment was cloned via the EcoRI and KpnI sites upstream of EYFP into the pUC19-EYFP vector. Third, after digestion with EcoRI and HindIII, the syndecan-4-EYFP construct was cloned downstream of MPB into the respective sites of the pMAL-c2 expression vector. After sequence verification, the plasmids for MBP-Syn-1-EYFP and MBP-Syn-4-EYFP fusion products were transfected into Escherichia coli BL21 plysS cells. Overexpression and purification of the MBP fusion proteins were performed following the manufacturer's protocol. Briefly, E. coli BL21 plysS cells were grown until A600 reached a value of 0.7. Thereafter, expression was induced with isopropyl 1-thio-{beta}-D-galactopyranoside (0.3 mM) for 3 h. The cells were harvested by centrifugation, resuspended in column buffer (20 mM Tris/HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA), and sonicated. Cell extracts were centrifuged, and the supernatant was bound to a 5-ml amylose resin column. After extensive washing with column buffer, the fusion proteins were eluted from the column with column buffer containing 10 mM maltose. The elution fractions were examined on SDS-PAGE to check for their purity. Fractions containing recombinant MBP-Syn-1-EYFP or MBP-Syn-4-EYFP protein were pooled and used for further digestions with plasmin or thrombin.

Shedding of Cell Membrane-integrated/associated Sulfated Proteoglycans—50,000 human umbilical vein endothelial cells were seeded in 35 mm diameter plastic dishes, cultured to confluence, and labeled with 370 kBq (10 µCi) of [35S]sulfate/ml medium for 48 h. Thereafter, the cultures were washed three times with phosphate-buffered saline, and the cells were incubated in 1 ml of chase medium (serum-free medium, Invitrogen) containing 0.5 mg of bovine serum albumin, 10 µg of ciprofloxacin/ml in HEPES at 20 mM in the absence or presence of 15 milliunits of plasmin or 15 units of thrombin for 3–6 h. In pilot experiments, the concentration of plasmin and thrombin was adjusted to maximum activity in our system. At the end of the experiment, the cell-free medium and 1 ml of washing solution (phosphate-buffered saline) were pooled (chase medium) and stored at 4 °C until use. The pericellular compartment was obtained by trypsinization of the cells and centrifugation at 800 x g for 3 min (trypsin pool). The chase medium and trypsin pool were used for further analysis. Reference values for the cell number/dish were obtained from parallel cultures. The chase medium and trypsin pool were used in direct route or after digestion with chondroitinase ABC for 4 h at 37 °C, lyophylized, and submitted to a size exclusion chromatography on calibrated 25-ml Sephadex G-50 medium columns equilibrated in 1 M NaCl. The 35S-labeled radioactivity of the V0 fraction represents the total amount of total 35S-labeled proteoglycans or after chondroitinase ABC degradation the [35S]heparan sulfate-containing proteoglycans. The chondroitin sulfate/dermatan sulfate-containing proteoglycans are calculated as the difference of the total and heparan sulfate-containing proteoglycans.

Electrophoresis and Microsequence Analysis—SDS-PAGE was performed under reducing conditions using a 12% polyacrylamide separating gel and a 3.5% stacking gel. The ectodomains were treated (4 h, 37 °C) with plasmin or thrombin at an enzyme/substrate ratio of 1:5 (w/w) in a total volume of 50 µl. The digest was diluted with concentrated sample buffer to the standard SDS concentration used for electrophoresis, boiled for 5 min, separated by electrophoresis (MiniProtean II, Bio-Rad), and transferred to polyvinylidene difluoride membrane, and the resolved fusion protein fragments were stained with Ponceau red. Bands indicated in Fig. 4, B and C, were cut and subjected to N-terminal sequencing (10 cycles) on an Applied Biosystems 492 gas-phase protein sequencer.



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  		FIGURE 1.
Evidence of syndecans on transcriptional and translational level. A, total RNA was isolated from confluent cultures (HUVEC). 2 µg of RNA were reverse-transcribed into cDNA and submitted to PCR using syndecan-specific forward and reverse oligonucleotide primers. PCR products were visualized by agarose gel electrophoresis. The bands are representative for five experiments. B, exposure of syndecan-1 and -4 to the cell surface. Confluent HUVEC cultures were incubated with monoclonal mouse anti-syndecan-1 or -4 antibodies and with HRP-conjugated goat anti-mouse IgG. Syndecans shed into the cell supernatant were detected by sandwich immunoassays designed for syndecan-1 and -4 (see"Experimental Procedures"). Because quantified standards were not available, values are expressed in arbitrary units normalized for cell number. C, Western blot analysis of deglycosylated shed syndecans. Syndecans of the cell supernatant were collected by binding to protein A-Sepharose precoated with a combination of polyclonal rabbit anti-syndecan-1 and -4 antibodies, degraded with heparitinase, and chondroitin lyase and submitted to PAGE. The blotted syndecans were made visible by monoclonal anti-syndecan-1 or -4 antibodies and HRP-conjugated anti-mouse IgG. OD, optical density; SYN, syndecan.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Syndecan-1 and -4 on Transcriptional and Translational Levels—Human umbilical vein endothelial cells express syndecan-1, -2, -3 and -4 in cell culture at the cell surface. Fig. 1A shows the cDNA transcripts of the corresponding mRNA. The exposure of syndecan to the cell surface was evidenced by immunoassays (see "Experimental Procedures") using monoclonal antibodies directed against the ectodomains of syndecan-1 and -4. The values given in Fig. 1B depended on the different affinities of the antibodies and did not reflect differences of concentration. For detection of the shed ectodomain, sandwich immunoassays for syndecan-1 and -4 were designed. Because quantified standards were not available, the values are given in arbitrary units normalized for cell number. Western blot analysis demonstrates the deglycosylated protein core of syndecan-1 and -4. Fig. 1C shows bands in the order of 25–35 kDa, but a correct molecular size could not be predicted, because the cleaving sites of the ectodomain generated under cell culture conditions were not known.



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  		FIGURE 2.
Shedding kinetics of cell membrane-integrated/associated 35S-labeled proteoglycans. Confluent endothelial cells prelabeled with inorganic [35S]sulfate were washed exhaustively and incubated in serum-free medium at 37 °C in the presence or absence of plasmin or thrombin. A, at the specified time intervals, the amount of 35S-labeled proteoglycans shed into the medium was determined. B, at the specified time intervals, the amount of 35S-labeled cell membrane-integrated/associated material obtained by trypsinization was determined. cpm, counts/min.

 
Kinetics of Constitutive and Accelerated Shedding of 35S-Labeled Proteoglycans—The shedding of sulfated proteoglycans was monitored after pulse labeling of the cells with [35S]sulfate for 48 h. The kinetics of the constitutive and protease-accelerated shedding is shown in Fig. 2A. After a hyperbolic increase of 35S-labeled material, a nearly constant level was reached after 3–6 h. The constitutive shedding is accelerated in the presence of plasmin or thrombin 1.5-fold over the control values (p < 0.05). The 35S-labeled material that was shed into the medium included the 35S-labeled heparan sulfate and chondroitin sulfate bearing ectodomain of syndecans but also chondroitin sulfate/dermatan sulfate-containing proteoglycans exported by HUVEC under physiological conditions. The shedding process could be followed also by examination of the cell membrane-integrated/associated 35S-labeled material after releasing it from the cell surface by trypsin treatment (Fig. 2B). In the control cells (constitutive shedding), the zero time value 5490 cpm/105 cells (100%) decreased within 6 h to 3304 cpm/105 cells (60.1%). In the presence of thrombin, a decrease to 40%, and of plasmin, to 33.8% was found (p < 0.05). No significant difference between the effect of plasmin and thrombin could be observed.

The ectodomains of all syndecans have attachment sites for heparan sulfate at the N terminus. In addition, syndecan-1 and -3 possess two and syndecan-4, one, membrane-proximal attachment sites for chondroitin sulfate. To evaluate the heparan sulfate-specific shedding in further experiments, the 35S-labeled chondroitin sulfate chains of syndecan and other chondroitin and dermatan sulfates containing proteoglycans were eliminated by exhaustive degradation of the shed 35S-labeled material with chondroitin sulfate lyase (EC 4.2.2.4 [EC] ). Fig. 3A shows that >50% of the total proteoglycans that were shed into the medium accounts for heparan sulfate in plasmin- or thrombin-treated cells, suggesting that the protease-accelerated shedding involves the membrane-integrated syndecans. The cell-associated 35S-labeled radioactivity obtained by and quantified after trypsin treatment of the cells is shown in Fig. 3B. The shedding activity of the plasmin and thrombin can be completely inhibited by 4-amidinophenyl-methanesulfonyl fluoride (not shown).



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  		FIGURE 3.
Accelerated shedding of 35S-labeled heparan sulfate proteoglycans. Confluent HUVEC prelabeled with inorganic [35S]sulfate were washed exhaustively and incubated in serum-free medium at 37 °C in the presence or absence of plasmin or thrombin. A, after 6 h, the 35S radioactivity incorporated into the total and HS-containing proteoglycans of the medium was determined. B, after 6 h, the 35S radioactivity incorporated into the total and HS-containing cell membrane-integrated/associated proteoglycans was determined. HSPG, heparan sulfate proteoglycans; DSPG, dermatan sulfate proteoglycans; CS, chondroitin sulfate.

 
Construction of Ectodomain Fusion Proteins—To elucidate the protein cleavage site of syndecans generated in response to plasmin and thrombin treatment, cell-free in vitro experiments with recombinant ectodomains of syndecan-1 and -4 were performed. This system contained substrate (fusion protein) and enzyme (plasmin, thrombin) as the only reactants excluding the cellular shedding activities. As the cleavage site was suggested to be proximal to the C terminus of the ectodomains, split products with free N termini were expected to be in the order of oligopeptides containing a few amino acids only. For better detection of split products, the syndecan ectodomains were linked with EYFP at the C terminus by a decapeptide linker. The N terminus of the ectodomain was tagged via an octapeptide linker with MBP to facilitate purification of the fusion protein.

Plasmin Cleaves the Syndecan-4 Fusion Protein at the Lys114–Arg115 and at the Lys129–Val130 Bonds—SDS-PAGE of the affinity-purified fusion protein revealed, besides the expected MBP-Syn-4 ectodomain-EYFP (predicted molecular mass 92.5 kDa), fragments with lower molecular mass obviously because of a truncated synthesis. A control blot of the native syndecan-4 fusion protein is shown in Fig. 4A and gave a similar band pattern for syndecan-1 fusion protein (not shown). The fusion protein of the syndecan-4 ectodomain was used as the substrate for plasmin. The proteolytic split products obtained after plasmin treatment were resolved by SDS-PAGE, and bands I–VI were excised and analyzed by gas-phase protein sequencing. Fig. 4B shows the band pattern of the syndecan-4 fusion protein obtained after incubation with plasmin. Bands I–III showed the sequence MKTEEGKLVI corresponding to the N-terminal sequence of MBP; band IV gave ambiguous values. Bands V and VI revealed the N-terminal sequences RISPYEESE (band V) and VSMSSTVQG (band VI). These sequences indicate cleavage sites between Lys114 and Arg115 and between Lys129 and Val130 of the ectodomain of syndecan-4 localized at a distance from the cell membrane of 33 and 17 amino acids.



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  		FIGURE 4.
SDS-PAGE of syndecan-4 ectodomain proteolysates. A, 10 µg of syndecan-4 fusion protein was submitted to PAGE, transferred to polyvinylidene difluoride membrane, and stained by Ponceau red. B, 60 µg of syndecan-4 fusion protein was incubated with plasmin in an enzyme/substrate ratio of 1:5 in a total volume of 50 µl. The electrophoretically resolved lysate was transferred to polyvinylidene difluoride membranes, and the Ponceau red-stained bands (I–VI) were excised for gas-phase protein sequencing. C, same conditions as in B but incubation with thrombin.

 
Thrombin cleaves the syndecan-4 fusion protein at the Lys114–Arg115 bond and the bond between the C-terminal linker oligopeptide and EYFP. The degradation pattern of the syndecan-4 ectodomain fusion protein after degradation with thrombin is shown in Fig. 4C. Bands I–III had the same N-terminal sequence (MKTEEGKLVI) as the corresponding bands obtained by plasmin treatment. Sequence analysis of band V gave an identical sequence as the corresponding plasmin-generated band (RISPVEESE). Thus, plasmin and thrombin recognize the same sequence (Lys-Arg) and deliver identical split products. The linker oligopeptide connecting the ectodomain and EYFP that had the C-terminal sequence RGAG served as a control for the proteolytic activity and specificity of thrombin. Thrombin is known to cleave the Arg–Gly bond of fibrinogen, thereby converting fibrinogen to fibrin monomers. Thus, the second split product after thrombin treatment (band VI) gave the expected sequence GAGMVSKGEE, containing the start sequence of EYFP (MVSKGEE) with the last three amino acids of the decapeptide linker at the N terminus. Fig. 5 shows the amino acid sequence of the complete fusion protein of syndecan-4 and the position of the identified cleavage sites. The sequences cleaved in the syndecan-4 ectodomain are not present in syndecan-1, and therefore neither plasminnor thrombin-catalyzed cleavage of the ectodomain could be observed. However, plasmin-specific cleavage sites were found to exist in MBP (K{downarrow}V, K{downarrow}R) and EYFP (K{downarrow}R) of the fusion proteins. The corresponding split products of MBP were not found within the analyzed bands I–VI, and the molecular weight of the split products of EYFP were too low for detection in the electrophoretic system used.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Fundamental data on enzymatic shedding of syndecans have been reported (1, 2, 5, 8, 9, 13), but cleavage sites for plasmin and thrombin (two enzymes involved in thrombogenic and thrombolytic processes within the circulation) have not been described. The present studies demonstrated by system that plasmin and thrombin can hydrolyze the Lys114–Arg115 and Lys129–Val130 peptide bonds of the ectodomain of syndecan-4 independently of the cellular shedding activities. The cleavage sites found are located within the juxtamembrane domain of the syndecan-4 molecule in a distance of 33 and 17 amino acids to the cell membrane. These data identify and localize, for the first time, two cleavage sites of the syndecan-4 ectodomain. No cleavage of the syndecan-1 ectodomain was observed under the action of thrombin and plasmin. This is in apparent contrast to a report about an accelerated shedding of syndecan-1 by thrombin (14). However, the data of this report (14) are based on experiments with cultured endothelial cells where a direct proteolytic action of thrombin cannot be shown. However, these data (14) indicate a receptor-mediated proteolysis of syndecan-1 and -4 under cell culture conditions. Thus, it appears that the thrombin-accelerated shedding requires the interaction of thrombin with the cellular thrombin receptor and that the thrombin effect can also be triggered by the thrombin receptor agonist peptide L-1-tosylamido-2-phenylethyl-chlormethyl ketone that is proteolytically inactive by itself. Moreover, accelerated shedding of syndecan-1 and -4 could also be achieved by phorbol 12-myristate 13-acetate, EGF, heparin binding EGF, and tumor necrosis factor-{alpha}. Thus, the authors conclude that at least the activation of two receptor classes, namely the G-protein-linked thrombin receptor and the EGF-specific tyrosine kinase receptor, regulates syndecan shedding by activation of a probably unknown cell membrane-associated proteolytic system. As other growth factors, such as vascular EGF, FGF-2 (basic fibroblast growth factor), platelet-derived growth factor, and transforming growth factor-{beta}, failed to accelerate syndecan shedding (14), the growth factor-induced shedding is considered to be selective. These findings suggest that distinct signaling pathways converge to activate a cell surface-associated metalloproteinase that cleaves the syndecans by a common but unknown mechanism.



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  		FIGURE 5.
Position of protease cleavage sites within the syndecan-4 ectodomain. The maltose-binding protein (N-terminal sequence underlined) and EYFP were linked to the syndecan-4 ectodomain via an oligopeptide linker. The plasmin and thrombin cleavage sites are indicated by arrowheads. The sequences registered by the gas-phase protein sequencer are underlined, and the oligopeptide linker is in small bold letters.

 
Taking together our data and the findings of Ref. 14, we conclude that thrombin/plasmin-accelerated shedding may result, at least partly, from a direct proteolytic cleavage of the syndecan-4 ectodomain, whereas the thrombin-mediated cleavage of syndecan-1 is restricted to an indirect receptor-triggered shedding. This is in accordance with differences of the primary structures of syndecan-4 and -1. The amino acid sequences we found susceptible for direct thrombin and plasmin cleavage of syndecan-4 are not present in syndecan-1. Furthermore, shedding by plasmin and thrombin releases the ectodomain of syndecan-4 that still contains the complete set of attachment sites for three heparan sulfate chains (SG consensus sequences 39/40, 61/62 and 62/63) and for two chondroitin sulfate chains (95/96). These covalently linked glycosaminoglycan chains are absent in our fusion proteins, and therefore it remains to be clarified whether the native ectodomain bearing the complete set of heparan- and chondroitin sulfate chains would also be split off by plasmin or thrombin.

Matrix metalloproteinases (MMPs) and membrane-type MMPs (MT1-MMPs) may also be involved in the shedding and catabolic processes of syndecans. Thus, the ectodomain of the basic fibroblast growth factor receptor (FGFR-1) could be split off by MMP-1 (15). Matrilysin (MMP-7) was shown to mediate shedding of a syndecan-1-CXC chemokine (KC) complex (16). In addition, syndecan-1 has been identified as a substrate of MT1-MMP that cleaves the Gly245–Leu246 bond of a recombinant syndecan-1 fusion protein (19). However, it has to be clarified whether MMPs and MT-MMPs are involved in constitutive shedding by themselves or whether they merely induce it.

Our present knowledge of syndecan points to different functions of individual syndecan types. Thus, syndecan-4 is capable of binding phosphatidylinositol 4,5 bisphosphate and activating protein kinase C and also conferring signaling functions on syndecan-4 as a bFGF coreceptor (20, 21), whereas such a property is not known for syndecan-1. Furthermore, syndecan-4-specific functions concern the link to anti-thrombin III (22), the up-regulation of interleukin-1{beta} in response to lipopolysaccharide (20), and the predominant expression of syndecan-4 in chronic venous ulcers where syndecan-1 is expressed in smaller quantities. These different functions are accentuated by a different sensitivity to the proteolytic activity of plasmin and thrombin that is able to cleave the ectodomain of syndecan-4 directly but not that of syndecan-1.

The susceptibility of syndecan shedding to multiple effectors (13, 14, 18, 23, 24) that act directly or via receptors implies that the ectodomains of syndecans have physiological roles as soluble proteoglycans. Shed ectodomains retain their heparan sulfate chains and hence the ligand binding activity of their cell surface counterparts. The release of the ectodomain of syndecan-4 leads to a down-regulation of its membrane-anchored co-receptor function but confers the ectodomain new functions as a soluble effector, which can compete for ligands with the membrane-integrated counterparts or other encounters, as extensively reviewed (24–29).

It is tempting to speculate, based on our data, that cleavage of the ectodomains of syndecans expressed by vascular endothelial and smooth muscle cells could determine the development and fate of arteriosclerotic plaques. Syndecan-4 has been shown to modulate events relevant to arteriosclerosis, such as acute tissue repair (30), cell motility (31), focal adhesion formation (32), and matrix remodeling (33). Shedding of syndecans could lead to an accumulation of soluble HS-containing ectodomains that may interfere with the membrane-bound syndecan-4 functioning as bFGF co-receptor (34). This would suppress the mitogenic activity of bFGF (35) and abolish the lipoprotein lipase binding (36, 37) and anticoagulative properties of membrane-integrated syndecans. Furthermore, the endothelial syndecans could interact via their heparan sulfate chains with L- and P-selectin expressed by mononuclear blood cells (10, 38) and thereby modulate leukocyte rolling as well as binding and recruitment of leukocytes into arteriosclerotic plaques. Moreover, the soluble ectodomain can modify the activity of leukocyte-derived elastase and cathepsin G (39) or bind these proteases and reduce their interaction with the subendothelial fibrous cap of arteriosclerotic plaques. Chemokine binding of syndecans has high impact for the regulation of inflammatory events. Syndecan-1 and/or -4 can generate a transendothelial interleukin-8 gradient by binding interleukin-8 via their HS chains (40), thereby increasing leukocyte/monocyte transmigration into arteriosclerotic lesions. Destroying the gradient by activated plasmin, which sheds the syndecan-interleukin-8 complex (40), results in a reduced recruitment of mononuclear cells to arteriosclerotic plaques. The syndecan-4 turnover of rat aortic smooth muscle cells is regulated by oxidized linoleic and linolic acid, the major oxidizable fatty acids in low density lipoprotein (41). Both oxidized fatty acids induce a dose-dependent up-regulation of syndecan-4 mRNA expression, but simultaneously the oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid) induces an accelerated shedding of syndecan-4 that would increase the amount of mobile HS-bearing ectodomains. As a net effect of syndecan overexpression and the associated accelerated shedding, a variety of pro- and anti-atherogenic events could be created.


   FOOTNOTES
 
* This work was financially supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 492, Project B12) (to F. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Institute of Arteriosclerosis Research, Dept. of Molecular Cardiology, Domagkstr. 3, D-48149 Muenster, Germany. Tel.: 49-251-835-8626; Fax: 49-251-835-8628; E-mail: annschm{at}uni-muenster.de.

2 The abbreviations used are: EGF, epidermal growth factor; EYFP, enhanced yellow fluorescent protein; FGF, fibroblast growth factor; HRP, horseradish peroxidase; HS, heparan sulfate; HUVEC, human umbilical vein endothelial cells; MBP, mannose-binding protein; MMP, matrix metalloproteinase. Back


   ACKNOWLEDGMENTS
 
We thank Michaela Tirre for skillful technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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