©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tenascin-C Binds Heparin by Its Fibronectin Type III Domain Five (*)

(Received for publication, October 20, 1994; and in revised form, December 22, 1994)

Peter Weber Dieter R. Zimmermann (1) Kaspar H. Winterhalter Lloyd Vaughan (§)

From the Laboratorium für Biochemie I, Universitätstr. 16, ETH Zentrum, 8092 Zürich and the Institut für klinische Pathologie, Department für Pathologie, Universität Zürich, 8091 Zürich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two sites on tenascin mediate interactions with glycosaminoglycan chains of proteoglycans. One is situated on the fibrinogen-like domain, whereas the other lies within the fibronectin type III homology region (Aukhil, I., Joshi, P., Yan, Y. Z., and Erickson, H. P.(1993) J. Biol. Chem. 268, 2542-2553.). We now characterize the latter binding site more closely by means of recombinant protein fragments derived from the type III homology region of tenascin. Using a heparin-Sepharose column, we localize the second heparin binding site to the fifth fibronectin type III domain. This is confirmed in solid phase assays by incubation of fusion proteins with biotin-labeled heparin. In addition, we demonstrate the binding of heparan sulfate and dermatan sulfate to domain five. Molecular modelling of this domain reveals a conserved heparin-binding motif that we propose as the putative binding site. The fact, that different glycosaminoglycans may bind to this domain, implies that different classes of proteoglycans may in vivo compete for the same site.


INTRODUCTION

Tenascin-C is a large extracellular matrix glycoprotein implicated in pattern formation during development and in malignant progression. It commonly occurs as a homohexamer, each polypeptide chain disulfide linked at its amino terminus to give the characteristic hexabrachions observed in electron microgaphs(1, 2, 3) . Each tenascin polypeptide is composed of colinear domains, similar to epidermal growth factor, fibronectin type III domains (TNfn) (^1)or fibrinogen (TNfbg)(4, 5) . A prominent structural feature is the variability of a section in the TNfn region arising from alternative splicing. In chick, for example, three major tenascin variants exist with either one (TN200) or three (TN220) additional TNfn domains inserted between TNfn5 and TNfn6 of the TN190 variant (Fig. 1).


Figure 1: Schematic localization of fusion proteins on the tenascin-C isoforms. The three isoforms of chick tenascin-C differ in their number of TNfn domains. The TNfn domains of the TN190 isoform are numbered from 1 to 8. The alternatively spliced domains are designated with letters, based on the sequence of the human protein with seven additional type III domains (14) . The fusion proteins TNfn56, TNfn5D, TNfnD6, and TNfn5ABD6 were constructed as outlined under ``Experimental Procedures.'' They contain at their amino-terminal end 6 histidines and a factor Xa cleavage site (Ile-Glu-Gly-Arg). The bordering domains of the fusion proteins do not start or end with their theoretical first or last amino acid but extend 4 or 5 amino acids into the adjacent TNfn domain. For more details consult sequences listed in Table 1. EGF, epidermal growth factor.





The alternatively spliced region has been linked to ligand binding of tenascin. Contactin/F11, a neuronal cell receptor anchored in the membrane by glycosyl phosphatidylinositol, is a ligand for tenascin-C and binds specifically to the TN190 isoform(6) . This contrasts with the recent isolation and identification of a 35-kDa cell surface tenascin-C receptor as annexin II, which binds specifically to the alternatively spliced region itself(7) . As annexin II binds within the alternatively spliced region and contactin/F11 is specific for the TN190 variant, missing these domains, alternative signaling may be mediated by different tenascin-C isoforms, underlining the functional importance of this region. Consistent with a functional role, the expression of these variants is closely regulated during development. For example, embryonic cartilage contains only the TN190 isoform(2, 8) , whereas the TN220 isoform expression is timed to pave the migration of neural crest cells in the cornea(3) .

Another prominent group of ligands are proteoglycans, some of which interact with tenascin-C through their glycosaminoglycan moieties. This includes the chondroitin sulfate proteoglycans cytotactin-binding proteoglycan(9) , and receptor tyrosine phosphatase beta (10) as well as the heparan sulfate proteoglycans syndecan (11) and glypican(12) . For the latter two, binding occurs in part through the heparan sulfate chains. In contrast to contactin/F11 and annexin II, the binding sites of these proteoglycans have yet to be established. As a first step in this direction, the binding of tenascin to heparin has been investigated. Tenascin-C can be isolated over heparin-Sepharose(13) , an interaction that may be due to one of two putative heparin-binding regions(14) . Given that one of these sites lies within the TNfn region and may be adjacent to the functionally important alternatively spliced domains, we prepared recombinant protein fragments covering this region of chick tenascin-C. Here we demonstrate that this heparin-binding site is located on TNfn5. With molecular modelling, we show that the Tnfn5 domain contains a cluster of basic amino acids that forms a conserved heparin binding motif.


EXPERIMENTAL PROCEDURES

Materials

Chondroitin sulfates A and C (chondroitin sulfate A from bovine trachea and chondroitin sulfate C from shark cartilage), dermatan sulfate (DS from bovine mucosa), heparan sulfate (HS from bovine kidney) and heparin (from porcine intestinal mucosa), avidin, and 2-(4`-hydroxyazobenzene)benzoic acid (HABA) reagent from Pierce, heparin-Sepharose CL-6B was from Pharmacia Biotech Inc. and protamine sulfate was from Eli Lilly & Co. (Indianapolis, IN). Alkaline phosphatase-conjugated streptavidin, antibiotics, and isopropyl-beta-D-thiogalactopyranoside were obtained from Boeringer Mannheim. The SDS-polyacrylamide gel electrophoresis broad range molecular weight standards are from Bio-Rad. Ni-NTA resin was from Qiagen (Germany), and factor Xa was from New England Biolabs, Inc. Rabbit anti-tenascin antibodies have been described previously (3) . Alkaline phosphatase-coupled goat anti-rabbit polyclonal antibody was from Caltag Laboratories. TNfbg (14) was a kind gift of Harold P. Erickson. The chicken tenascin cDNA clone pCTN230 was generously provided by Ruth Chiquet-Ehrismann. All other chemicals were from Fluka (Switzerland).

Fusion Proteins

The alternatively spliced fibronectin type III domains TNfn56, TNfn5D, TNfnD6, and TNfn5ABD6 were expressed as fusion proteins in a bacterial expression system(15) . Expression constructs were prepared essentially according to (16) . cDNA fragments encoding the TNfn domains were amplified by polymerase chain reaction from the full-length construct pCTN230 (donated by Dr. R. Chiquet-Ehrismann). The resulting polymerase chain reaction products were ligated into the multiple cloning site of pDS9 (16) yielding in the constructs pDS9/56, pDS9/5D, and pDS9/D6. The multiple cloning site is located at the 3` end of the translation initiation codon and a sequence coding for 6 histidines. To obtain the construct pDS9/5ABD6, we digested the construct pDS9/56 with BstXI and ligated into the remaining plasmid the appropriate fragment of a BstXI digestion of pCTN230. All constructs were controlled by DNA sequencing. The amino and carboxyl termini of the resultant fusion proteins are presented in Table 1.

Recombinant proteins containing a stretch of 6 histidines amino-terminal of the factor Xa cleavage site were prepared from M15 (pREP4), transformed with the constructs pDS9/56, pDS9/5D, pDS9/D6, or pDS9/5ABD6, respectively. Transformed bacteria were grown at 37 °C overnight in a rotary shaker (250 rpm) in 2 times YT medium containing ampicillin and kanamycin. The production of the fusion proteins was initiated with isopropyl-1-thio-beta-D-galactopyranoside as described(15) .

After 5 h, the cells were collected by centrifugation and then resuspended in one-thirtieth of the original volume in ice-cold sonification buffer (25 mM Tris/HCl; 150 mM NaCl; 0.2 mM phenylmethylsulfonyl fluoride; 2 mM iodacetamide; pH 8.0) and lysed in aliquots of 15 ml with a sonifier (cell disrupter, SKAN; setting 4, 6 min, 75%). During the sonification, the suspension was cooled with ice-water. The extract was cleared by centrifugation at 12,000 times g for 15 min at 4 °C (Sorvall, SS34). The fusion protein containing supernatant was diluted with 1.5 volumes of loading buffer (67 mM sodium phosphate; 300 mM NaCl; 0.02% NaN(3); pH 8.0) and loaded onto a Ni-NTA column. Under these conditions, fusion proteins containing the 6-His tag bound tightly to the column, whereas other bacterial proteins were eluted with washing buffer (67 mM sodium phosphate; 300 mM NaCl; 0.02% NaN(3); pH 6.0). The fusion proteins were subsequently eluted with 100 mM sodium acetate; 300 mM NaCl; 0.02% NaN(3); pH 3.8. The pH of the collected fractions was adjusted to 7.5 with 1 M Tris/HCl, pH10, and subjected to analysis on 10-15% SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining.

Affinity Chromatography

The affinity chromatography with the fusion proteins on heparin-Sepharose was carried out as follows. The fusion protein concentration was adjusted to 0.5 mg/ml in Tris buffer (10 mM Tris/HCl; 20 mM NaCl; 0.02% NaN(3); pH 7.5), and 1 ml was applied to the heparin-Sepharose column. The column was washed with the Tris buffer and subsequently eluted with a linear salt gradient from 20 mM to 1.5 M NaCl. The fractions were analyzed by enzyme-linked immunosorbent assay using polyclonal rabbit anti-tenascin antibodies and alkaline phosphatase-conjugated goat anti-rabbit antibodies.

Labeling of Glycosaminoglycans with Biotin

The glycosaminoglycans were labeled with biotin as described(17) . The amount of biotin/unit of glycosaminoglycan was estimated using the HABA-avidin reagent. To avoid precipitation during the color reaction, the biotin-labeled glycosaminoglycans were first digested at 37 °C overnight by the addition of chondroitinase ABC (EC 4.2.2.4) or heparinase III (EC 4.2.2.8). The concentrations of biotin in these solutions were then determined as described by the supplier.

Solid Phase Assays

The labeled glycosaminoglycans were used in solid phase assays for which 96-well plates were coated at room temperature overnight with 100 µl/well of fusion protein diluted to final concentration of 1 µM in TBS. Protamine sulfate coated at a concentration of 100 µg/ml was used as a positive control; 1% bovine serum albumin in TBS was used as negative control. Where bacterial proteins were used as negative control, dilutions with an OD (1 cm; 280 nm) of 0.1 in TBS were applied.

The wells were blocked for 1 h at room temperature with 200 µl of 1% heat denatured bovine serum albumin in TBS (5 min, 85 °C). The binding of glycosaminoglycans was performed for 2 h at room temperature. Bound biotin labeled glycosaminoglycans were detected with alkaline phosphatase conjugated to streptavidin (1:2500) in TBS and p-nitrophenyl phosphate-Na(2) as substrate. Between each step, the wells were washed 3 times with 200 µl of TBS.

Modelling of the Three-dimensional Structure of TNfn5

Chicken TNfn5 was modelled based on the known structure of human TNfn3(18) . The structure of TNfn3 was obtained as file ``pdb1ten.ent'' from the Protein Data Bank at the Brookhaven National Laboratory. The human TNfn3 contains two additional amino acids (Ile-838 and Glu-852 in TNfn3) compared with chicken TNfn5. To model the chicken TNfn5, we deleted first these 2 amino acids with the program RIMINI (molecular mechanics program for rings; M. Dobler, ETH Zürich, 1989). In a first step, we deleted the Ile at the end of the beta-strand C. The beta-strands C and C` were held in place, and 2 amino acids amino-terminal and 6 amino acids carboxyl-terminal of the deleted Ile were moved by changing their torsion angles to close the nick. With the same procedure, the Glu between the beta-strands C` and E was deleted. These two beta-strands were held in place, and 3 amino acids on either side of the deleted Glu were moved to close the gap. The calculations with RIMINI were performed on a VAX 9000-420. In a second step, the amino acids were mutated with the program QUANTA version 3.3 from Molecular Simulations running on a personal IRIS 4D25G. After mutating the amino acids, their side chains were orientated using the rotamer tool. A CHARMm minimization of this structure resulted only in small changes, suggesting that the model obtained of chicken TNfn5 is reasonable.


RESULTS

Constructs of Recombinant DNA

Comparing the sequences of our constructs with the published sequence ((5) , M23121) revealed a point mutation in the domain five (nucleotide 3200: CA), changing the amino acid 989 in the published sequence from a Thr to an Asn. The latter corresponding to the human (M55618, X56160) and mouse (D90343, X56304) sequence. Sequencing showed the mutation was already present in the pCTN230 construct.

A second mutation was observed in domain D (nucleotide 3915: AC) of the construct 5D but not D6. The nucleotide substitution changes the amino acid 1227 from a Glu to an Asp. This mutation close to the priming site of DL2 was introduced by polymerase chain reaction and could not be avoided by repeating the polymerase chain 1 reaction with the same or other primers.

Production and Purification of Fusion Proteins

After sonification and centrifugation of the bacteria, about 95% of fusion protein can be recovered from the supernatant, whereas 5% remains in the pellet. The fusion proteins are soluble in isotonic buffer and do not require additional agents for solubilization. The highest yield of fusion protein is obtained with the bacteria expressing TNfnD6, where from 1 liter of bacteria culture, 160 mg of TNfnD6 is isolated. The recoveries of the fusion proteins TNfn56, TNfn5D, and TNfn5ABD6 are approximately 3-5 mg of fusion protein/liter of bacterial culture. The one-step purification over a Ni-NTA column yields fusion proteins free of contaminants and soluble in isotonic solution (Fig. 2). The amino-terminal fusion sequence is readily removed by factor Xa. The resulting polypeptide includes the correct amino-terminal sequence and covers only sequences specific for tenascin.


Figure 2: Purity of the isolated fusion proteins. The fusion proteins TNfn56 (lane1), TNfn5D (lane2), TNfnD6 (lane3), and TNfn5ABD6 (lane4) were analyzed on 10-15% SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue.



Binding of Fusion Proteins to Heparin-Sepharose

TNfn5ABD6, the fusion protein containing all domains of the variably spliced region of chicken tenascin, displays moderate affinity to heparin-Sepharose, being eluted with a linear NaCl gradient in a major peak at about 0.6 M NaCl (Fig. 3). To examine whether binding is due to a site within the alternatively spliced region itself or on the neighboring domains TNfn5 or TNfn6, fusion proteins TNfn56 and TNfnD6 were applied to the column. TNfnD6 elutes in the break-through without binding. In contrast, TNfn56 is retained by the heparin-Sepharose and elutes with 0.6 M NaCl, indicating that TNfn5 carries the heparin-binding site. Nonspecific interactions resulting from the net charge of the fusion proteins (Table 2) is unlikely, as TNfn5ABD6 with pI 5.01 binds just as well to the heparin column as TNfn56 with a pI of 10.08. Although TNfnD6 has pI 6.1 and is therefore similarly charged at pH 7.5 as TNfn5ABD6, it is not retained by the heparin column.


Figure 3: Binding of fusion proteins to heparin-Sepharose. Fusion proteins TNfn5ABD6 (left), TNfn56 (middle), and TNfnD6 (right), were each applied to heparin-Sepharose in 20 mM NaCl and eluted with a linear gradient of NaCl. Detection was by enzyme-linked immunosorbent assay using tenascin-specific polyclonal antibodies and phosphatase-coupled secondary antibodies, with absorbance measured at 405 nm.





Solid Phase Assays with Biotin-labeled Heparin

To further investigate the heparin binding of different fusion proteins directly, a solid phase assay was established using biotin-labeled heparin and alkaline phosphatase-conjugated streptavidin. On average, 25 biotins (range 16-36) were introduced per 1000 disaccharide units of glycosaminoglycan, as estimated using the HABA-avidin reagent. This permitted ready detection, without unduly influencing the properties of the glycosaminoglycans themselves.

First, we examined the effect of increasing NaCl concentrations on the binding of heparin to fusion proteins. Protamine sulfate, TNfn56, TNfn5D, TNfnD6, and TNfn5ABD6 were coated in TBS, and, after incubating heparin at 150 mM NaCl with these substrates, washing was performed under increasing NaCl concentrations (Fig. 4). Heparin binds to all fusion proteins carrying the TNfn5 domain (TNfn56, TNfn5D, or TNfn5ABD6) but not to TNfnD6. The binding to TNfn5 decreases with increasing NaCl concentrations and is fully abolished between 450 and 600 mM NaCl, similar to the concentration required to elute TNfn56 from heparin-Sepharose. The binding of heparin to protamine sulfate is not influenced by the NaCl concentrations tested. In a separate set of experiments (not shown), the binding of heparin to TNfn56 was found to be saturable. The binding to TNfn56 was also similar to that between heparin and the fibrinogen-like domain of tenascin, consistent with previously published data(14) .


Figure 4: Heparin binding to tenascin fusion proteins. The substrates were coated to 96-well plates as outlined under ``Experimental Procedures.'' After a 2-h incubation with 1 µM biotin-labeled heparin, the wells were washed with different concentrations of NaCl, and the remaining heparin was detected. The strong binding of heparin to the positive control protamine sulfate was not influenced by the NaCl concentrations used.



Solid Phase Assay with Biotin-labeled Glycosaminoglycans

As heparin itself is unlikely to be the physiological ligand for tenascin-C, we incubated various biotin-labeled glycosaminoglycans in the solid phase assay with TNfn56 as substrate. Both heparan sulfate and dermatan sulfate bind to TNfn5 with a similar half-maximal concentration of around 50 µg/ml (Fig. 5). Half-maximal binding of heparin occurs at 0.8 µg/ml, indicating a 50-100 fold greater affinity. Neither chondroitin sulfate A nor chondroitin sulfate C bind to TNfn56 under these conditions.


Figure 5: Binding of other glycosaminoglycans to TNfn56. Biotin-labeled glycosaminoglycans were incubated at different concentrations with coated TNfn56. Heparin shows the strongest binding. Chondroitin sulfates A and C (CSA and CSC) do not bind to TNfn56. Dermatan sulfate (DS) and heparan sulfate (HS) bind with a similar half-maximal concentration of 50 µg/ml




DISCUSSION

Here we present evidence for a conserved heparin binding site in tenascin-C on the TNfn5 domain. This conclusion is based on the binding of heparin at physiological NaCl concentrations to fusion proteins containing TNfn5. This is true for the fusion proteins derived from chicken tenascin, shown here, and for fusion proteins derived from human tenascin(14) . Heparin binds most strongly of the glycosaminoglycans tested, likely due to its higher level of modification. Heparin and heparan sulfate differ in their degree of sulfation and extent of epimerization of D-glucuronic to L-iduronic acid(19) . The lower affinity of heparan sulfate and dermatan sulfate may not only be explained by their lower degree of sulfation, but also by their reduced content of L-iduronic acid. The latter may be essential for the binding to TNfn5, as glycosaminoglycans lacking L-iduronic acid (chondroitin sulfates A and C) did not bind to TNfn5. Involvement of L-iduronic acid has also been observed for the binding of heparin or heparan sulfate to basic fibroblast growth factor(20, 21) .

The functional conservation of heparin binding between chicken and human TNfn5 implies that the binding site itself must also be conserved. To identify this binding site within domain five, we modeled chicken TNfn5 (Fig. 6), based on the known structure of human TNfn3(18) . The fibronectin type III repeats are formed from two beta-sheets, giving rise to a minor face of three beta-strands (designated A, B, and E) and a major face of four beta-strands (C, C`, F, and G). The beta-strands F and G, shown facing the viewer in Fig. 6, contain a consensus heparin-binding motif (BXBXBXXXXB)(22) , which is highly conserved in tenascin-C between different species ranging from chick, mouse, human, and pig(23) . The spacing between residues Lys-1032 and Lys-1039 (Table 3), which are separated by 21 Å in the model (Fig. 6), is likely to be critical here. In both apolipoprotein E and antithrombin III, a heparin-binding motif is formed from similarly conserved basic amino acids, which are spaced about 23 Å apart along beta-strands (Table 3). This is consistent with that predicted for efficient binding to a pentasaccharide sequence(22) , thought to be the minimal functional unit of heparin(24) . The conservation of the consensus heparin-binding motif stands in contrast to the poor conservation of domain five as a whole(25) , providing further evidence for a likely functional importance. However, other basic residues may also be involved in heparin binding. Inspection of the TNfn5 model reveals that other pairs of conserved basic amino acids lie within a radius of 20-23 Å on the major face of TNfn5. In particular Arg-985 and Arg-987 on the C-strand are possible candidates in combination with Lys-1034 and Lys-1039, respectively. Which combination of amino acids are involved in binding to heparin can now be studied using site-directed mutagenesis or by x-ray diffraction methods should crystallization of the fusion protein-glycosaminoglycan complex prove to be possible.


Figure 6: Chicken TNfn5 modelled on human TNfn3. The blue and red highlighted stretches represent the basic amino acids Lys and Arg of chicken TNfn5. The basic amino acids, which are conserved in human, pig, mouse, and chicken are represented by red, and nonconserved residues are represented by blue. The conserved basic amino acids are concentrated on the major face of the domain. The heparin binding motif (BXBXBXXXXB) is located on the beta-strand G, with Lys-1032 (K`) and Lys-1039 (K) separated by 21 Å.





Presumably the correct conformation of the binding region is critical for the specificity of interaction with glycosaminoglycan chains(22) . An advantage of fusion proteins compared with peptides is that a correct folding of the domains should lead to the binding site being presented to the glycosaminoglycan in a close to native form. With this in mind, the strategy for preparation of the fusion proteins was chosen to maximize the likelihood of producing native proteins that are correctly folded and soluble under nondenaturing conditions. To this end, the constructs begin 4-5 amino acids amino- and carboxyl-terminal outside the theoretical domains. The length of the extraneous sequence at the amino termini (polyhistidine tail for purification and factor Xa cleavage site) was kept to a minimum to reduce the influence on folding of the type III homology domains. This approach appears to be successful, as the fusion proteins were released in soluble form from the Escherichia coli merely by sonification in isotonic buffer. A further indication of the native structure comes from the ready cleavage by factor Xa. Incorrect folding of fusion proteins would lead to the factor Xa recognition sequence being buried, which then only becomes exposed to the proteinase after denaturation/renaturation cycles. Both our fusion proteins (^2)and those produced by a similar protocol (14, 18) can be crystallized for structural analysis. This further strengthens the case for native folding. The proposed heparin-binding site is likely to be available in the intact tenascin-C as fusion protein TNfn1-5 from human (14) and TNfn56 (shown here), both encompassing TNfn5, are active.

A number of candidate proteoglycan ligands have been reported for tenascin-C(9, 10, 11, 12) . Among them, evidence for binding via the glycosaminoglycans has been presented for syndecan (11) and glypican (12) . The degree of modification of the heparan sulfate chains of syndecan and glypican is likely to be tissue-dependent. Whether a particular sequence of charged residues is necessary for optimal binding to TNfn5, such as the heparin pentasaccharide specific for antithrombin or the dermatan sulfate heptasaccharide binding to heparin cofactor(19) , is the subject of further investigations. The assignment of glycosaminoglycan binding activity to TNfn5 and the availability of the recombinant fragments should stimulate the search for further in vivo proteoglycan ligands and lead to a better understanding of the role of tenascin-C in development and disease.


FOOTNOTES

*
This work was supported by grants from the Swiss National Science Foundation and the ETH-Zürich (to L. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 41-632-25-72; Fax: 41-632-11-21; l_vaughan{at}ezinfo.vmsmail.ethz.ch.

(^1)
The abbreviations used are: TNfn, fibronectin type III homology domain of tenascin; ECM, extracellular matrix; TNfbg, fibrinogen like domain of tenascin; HS, heparan sulfate; GAG, glycosaminoglycan; TBS, Tris-buffered saline.

(^2)
P. Weber and L. Vaughan, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. H. P. Erickson for the donation of fusion protein TNfbg corresponding to the fibrinogen-like domain derived from human tenascin and Dr. R. Chiquet-Ehrismann for providing pCTN230. The calculations to estimate the structure of cTNfn5 was possible through the friendly help and encouragement of Prof. M. Dobler, ETH Zürich.


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