THE JOURNAL OF BIOLCOICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 269, No. 2. Issue of January 14, PP . 1262-1269, 1994 Printed in U S A .

Thrombospondin 3 Is a Developmentally Regulated Heparin Binding Protein*

(Received for publication, July 30, 1993, and in revised form, September 16, 1993)

Aziz N. Qabar80, Zhiwu LinSO, Frederick W. Wolf$, K. Sue O’Shean, Jack Lawlerll, and Vishva M. Dixit§** From the §Department of Pathology, the Wepartment of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor, Michigan 48019 and the lllivision of Vascular Research, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

The thrombospondins (TSPs) are a growing family of cell surface and extracellular matrix molecules com- posed of multiple repeating elements. Thrombospondin 3 is a recently described member that possesses the cal- cium binding Type 3 repeats, has 4 epidermal growth factor receptor-like Type 2 repeats, a complete absence of the complement-like Type 1 repeats, and a distinct N terminus that has no significant homology to the other TSPs. Metabolic labeling and immunoprecipitation analysis of cells transfected with a TSPS expression vec- tor revealed it to be an oligomeric heparin binding pro- tein present in both the cell layer and medium. Finally, a combination of in situ hybridization and immunocyto- chemistry demonstrated TSPS to be expressed in a tem- poral and spatial manner during murine embryogenesis, especially in the gut, cartilage, lung, and central nerv- ous system.

The thrombospondins are an emerging family of extracellu- lar matrix proteins (see Refs. 1, 2, and 3 for reviews). Throm- bospondin 1 (TSP1)l was first described as a platelet cu-granule protein which was released on activation and contributed to the secretion dependent or secondary phase of platelet aggregation (4). With the availability of anti-TSP antibodies, it was soon apparent that TSP was synthesized, secreted, and incorporated into the extracellular matrix by a variety of cells including fibroblasts, smooth muscle cells, keratinocytes, endothelial cells, and carcinoma cell lines (5,6). Additional studies demon- strated TSP to be an attachment factor for these cells and in keeping with this was the identification of a number of cell surface receptors capable of binding TSP (7,8). TSPl is a large (-450 kDa) trimeric, modular glycoprotein having a domain- like structure made up of multiple repeating elements. The N terminus has a high affinity heparin binding domain that is capable of binding to the integral membrane proteoglycan re- ceptor syndecan and to sulfated glycolipids (9, 10). Just distal to the heparin binding domain are 2 cysteine residues respon- sible for interchain disulfide linkage and the Type 1 repeats

CA58182 (to V. M. D.) and HL28749 (to J. L.). The costs of publication * This work was supported by National Institutes of Health Grant

of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in ac- cordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession numbeds) L24434.

$ The first two authors contributed equally to this work. ** Established Investigator of the American Heart Association. The abbreviations used are: TSP, thrombospondin; DTT, dithio-

threitol; PBS, phosphate-buffered saline; PCR, polymerase chain reac- tion; IPTG, isopropyl-l-thio-/%o-galactopyanoside; PAGE, polyacryl- amide gel electrophoresis.

which have homology to properdin, complement proteins, and malarial circumsporozoite proteins (11). Embedded within these repeats is the VTCG sequence which mediates attach- ment of melanoma cells to TSP, possibly through the cell sur- face receptor GPIV or CD36 (12). Also contained within the Type I repeats are copies of the WSXW sequence, a recently identified heparin binding motif that represents the second, albeit lower affinity, heparin binding sequence in TSP (13). The Type 2 repeat contains 6 invariant cysteine residues that are spaced in a manner similar to that found in mouse epidermal growth factor precursor (11). The adjacent Type 3 repeats are enriched in aspartic acid residues and constitute the calcium binding domain of TSP (11). In addition, the last Type 3 repeat contains an RGD sequence that is recognized by the integrin receptor avP3 and contributes to the attachment of endothelial cells to TSP (14).

Besides promoting cell attachment, TSP has also been impli- cated in mediating neurite outgrowth, chemotaxis, haptotaxis, smooth muscle cell proliferation, tumor cell invasiveness, an- chorage independent growth, and inhibition of endothelial cell proliferation (1-3). These diverse and at times contradictory functions of TSP were originally assigned to a single gene prod- uct. However, the discovery of four additional TSP gene prod- ucts, designated TSP2, TSP3, TSP4, and COMP for cartilage oligomeric matrix protein, suggests that this myriad of func- tions may be ascribed to a growing list of molecules having homology to TSPl (3, 15-19).

In addition, more distantly related proteins which have ho- mology exclusively to the TSP Type 1 repeats have recently been described and include rat F-spondin and the Caenorhab- ditis elegans protein UNC-5 (20, 21). This suggests that the Type 1 repeat is a motif that is widely dispersed, presumably by exon shuffling (22).

As an initial step in the study of these TSP-related gene products, we have further characterized the recently described TSP3 gene product (17) by cloning and sequencing a full-length mouse TSP3 cDNA. The predicted open reading frame indi- cated marked homology to TSP1, TSP2, TSP4, and COMP at the C terminus including the Type 3 repeats, the existence of an extra Type 2 repeat, a complete absence of Type 1 repeats, and a distinct N terminus. Immunocytochemistry using an anti- TSP3 peptide antibody and in situ hybridization analysis re- vealed a distinct pattern of TSP3 expression during mouse development. In addition, despite having no significant se- quence homology to the TSPl and TSPS heparin binding do- mains, TSP3 was found to be a heparin binding protein.

MATERIALS AND METHODS Cloning and Sequencing-A genomic clone, GPEM-2, containing the

last exon of human TSP3 (23) (kindly provided by Dr. Sandra Gendler, Imperial Cancer Research Fund, London) was used to screen both a

1262

Characterization of TSP3 1263

random-primed mouse Schwann cell cDNA library (Invitrogen) in the plasmid vector pcDNAl and an oligo(dT)-primed human embryonic lung library in the same vector (16). Plasmid from hybridizing colonies was cesium chloride banded and sequenced by the dideoxynucleotide method using modified T7 DNA polymerase (Sequenase, U. S. Bio- chemicals). Custom synthetic oligonucleotide primers were used to se- quence both strands of overlapping cDNA clones, and sequence analysis was performed using MacVector (Kodak) and DNASTAR (Madison, WI) software packages.

Cell Culture and I).ansfections"293T cells, a human renal epithelial cell line, were grown in Dulbecco's modified Eagle's medium supple- mented with fetal calf serum, nonessential amino acids, L-glutamine, and antibiotics (100 unitslml penicillin, 50 pg/ml streptomycin). For Western blot analysis, cells were grown in medium in the absence of serum to remove any exogenous source of TSP, being supplemented instead with insulin, transferrin, selenium, and linoleic acid (ITS, Col- laborative Research, Inc.).

A HindIIIINotI restriction fragment encompassing the entire mTSP3 coding sequence was directionally cloned into pCEP4, a mammalian episomal expression vector (Invitrogen) driven by the cytomegalovirus promoter/enhancer and containing a selectable marker for hygromycin resistance. 293T cells were transfected with the above expression con- struct, designated pCEP4-TSP3, as described previously (24). Following selection, stable cell lines were established that expressed recombinant mTSP3 as confirmed by both Western blot analysis and metabolic la- beling and immunoprecipitation using the anti-TSP3 peptide immune serum. Transfection of 293T cells with mTSPl and TSP2 expression constructs and subsequent analysis by metabolic labeling and immu- noprecipitation has been described previously (24).

Preparation of Anti-TSP3 Peptide Antiserum-A synthetic peptide having the sequence PLQTDRDED, which is conserved in human and mouse TSP3 but not in the other TSPs, was synthesized with a car- boxyl-terminal cysteine (PLQTDRDEDC; Multiple Peptide Systems, San Diego, CA) to enable coupling via the free sulfhydryl group to carrier protein.

The heterobifunctional cross-linker n-maleimidobenzoyl-N-hydroxy- succinimide was used to couple 25 mg of the peptide to 20 mg of keyhole limpet hemocyanin as per the manufacturer's instructions (Multiple Peptide Systems). 2 mg of the coupled peptide was emulsified with complete Freund's adjuvant for the initial injection or incomplete adju- vant for subsequent injections into the gluteus of New Zealand White female rabbits. Animals were bled 2 weeks following the sixth injection. Antisera specificity was confirmed by immunoblot analysis using a bacterially expressed recombinant TSP3 fusion protein encompassing the synthetic peptide sequence.

Affinity Purification ofAnti-peptide Antibody-An anti-TSP3 peptide affinity column was constructed by covalently cross-linking 20 mg of purified peptide to 3 ml of a cross-linked agarose resin modified with an N-hydroxysuccinimide ester according to instructions supplied with the Proton kit (Multiple Peptide Systems). 5 ml of immune serum was diluted with an equal volume of phosphate-buffered saline, pH 7.4 (PBS), and applied to the anti-peptide column. Following extensive washing with PBS, the column was eluted with a sodium phosphate buffer at low pH (2.5), the eluate was neutralized immediately by the addition of 1.0 M Tris base, pH 10.0, followed by extensive dialysis against PBS. For preparation of blocked antibody, immune serum was incubated overnight at 4 "C with an excess of peptide-Sepharose which was then pelleted by centrifugation, and the supernatant was saved as blocked antibody.

Construction and Expression of TSP3 Fusion Protein-A TSPB glu- tathione S-transferase fusion protein was expressed using the prokary- otic expression vector pGEX2T (Pharmacia LKB Biotechnology Inc.). Polymerase chain reaction (PCR) primers were designed to amplify a 519-base pair fragment corresponding to the C-terminal 172 amino acids encoded by the partial human cDNA and containing the peptide sequence used to raise the anti-TSP3 antibody. In addition, the primers introduced BamHI restriction sites at both ends of the double-stranded PCR products. PCR was carried out using the human TSP3 cDNA as a template. The PCR products were digested with BamHI and ligated into BamHI-cut and phosphatased pGEX2T expression vector. The result- ant plasmid was introduced into competent E. coli DH5 cells, and plas- mid DNA isolated from individual colonies was sequenced in entirety to ensure fidelity of the PCR amplification. Plasmid from selected clones was then transformed into E. coli strain BL21 (DE3) pLysS, and syn- thesis of the TSP3-GST fusion protein was induced by addition of iso- propyl-P-o-thiogalactopyranoside (IPTG) as described in previous stud- ies (24).

Western Blot Analysis-Bacterial lysates solubilized in SDS-sample

buffer were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose using a semidry electroblotting apparatus (LKB Mul- tiphor, Pharmacia) according to the manufacturer's instructions. Effi- ciency of transfer was verified by staining a parallel nitrocellulose mem- brane with 0.1% amido black in 45% methanol and 10% acetic acid. After blocking the nitrocellulose membrane in Tris-buffered saline, pH 7.4, containing 3% nonfat dry milk, the membrane was incubated with either preimmune or immune rabbit anti-TSP3 peptide serum at a 1:lOOO dilution. Bound antibody was detected by incubating with horse- radish peroxidase-conjugated goat anti-rabbit secondary antibody (Bio- Rad) diluted 1:10,000.

For immunoblot analysis of TSP3 in 293T cell layer or medium, proteins in these samples were first precipitated using 10% trichloro- acetic acid, and the precipitate was washed extensively with ether be- fore solubilization in SDS-sample buffer.

Heparin-Sepharose Affinity Chromatography-1.5 ml of conditioned medium from transiently transfected 293T cells was incubated over- night at 4 "C with 0.5 ml of a 50% heparin-Sepharose slurry (Pharma- cia). To establish that the heparin affinity column was functioning ap- propriately, 10 pg of human platelet TSP1, a bona fide heparin binding protein, was also added to the slurry. Following incubation, a column was poured in a 1.0-ml pipette tip, and unbound material (flow through) was collected. Following extensive washing with 0.15 M NaCI, 20 mM Tris-HC1, pH 7.6, the column was subjected to a salt step gradient elution of 0.2, 0.25, 0.3, 0.35, 0.4, 0.5, and 1.0 M NaCI. 1-ml fractions were collected, the proteins were precipitated with trichloroacetic acid, dissolved in SDS-sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting using a monoclonal antibody specific to human platelet TSPl (A2.5; Ref. 25) or the affinity- purified anti-TSP3 peptide antibody.

Mouse Embryonic Tissue-Post-implantation staged embryos were obtained from matings of adult CD-1 strain (Charles River Laborato- ries) mice. The following morning was considered the first day of a 19-day gestation period. At sequential stages of development, embryos were removed from the uterus and decidua, frozen in OCT in hexane cooled over an acetone-dry ice slurry, and tissue-sectioned at 8 pm. Sections were processed without fixation for immunohistochemical lo- calization of TSP3. Sections were stored at -80 "C prior to additional processing.

In Situ Hybridization-In situ hybridization was carried out as de- scribed previously with modifications (26). Sections were fixed in fresh 4% paraformaldehyde in phosphate-buffered saline, pH 7.4 (PBS), rinsed in PBS, digested in 10 pg/ml proteinase K for 5 min, and refixed in 4% paraformaldehyde in PBS. Sections were placed in 0.25% acetic anhydride containing 0.1 M triethanolamine for 10 min, rinsed in PBS followed by 0.15 M NaC1, and dehydrated in ethanol.

Template for T7 and SP6 RNA Polymerases was generated by the polymerase chain reaction (PCR). The 5' primer (5'-gtaatacgactcac- tatagCCTGAGTGAATGTCCATTCC-3', T7 promoter in lower case) and 3' primer (5'ggatttaggtgacactataTACACTTCCATACAGTCCAC-3', SP6 promoter in lower case) encompass base pairs 634-912 of the mTSP3 cDNA. Single-stranded RNA transcripts uniformly labeled with PSIUTP (Dupont NEN) were transcribed according to the manufactur- er's instructions (Promega). Probes purified by ethanol precipitation were used for hybridization the same day.

Probes (500,000 cpndsection) were hybridized to sections in 75 pl of a solution containing 50% formamide (Fluka, Buchs, Switzerland), 0.3 M NaCI, 20 mM Tris, pH 8.0, 5 m~ EDTA, 10 m~ NaPO,, pH 8.0, 10% dextran sulfate, 5 x Denhardt's (50 x is 1% Ficoll, 1% polyvinylpyrrol- idone, and 1% bovine serum albumin), 0.5 mg/ml yeast tRNA, and 0.1 M DTT. Following incubation for 1G18 h at 55 "C, the slides were washed in 5 X SSC (1 x is 0.15 M NaCl and 0.15 M sodium citrate, pH 7.0) containing 10 m~ DTT at the same temperature. Slides were then stringently washed in buffer (HS) containing 50% formamide, 2 x SSC, and 0.1 M DTT for 30 min at 65 "C. After four washes in 0.5 M NaCl containing 10 mM Tris, pH 7.6, and 5 m~ EDTA (RN buffer), the slides were incubated with 1 mg/ml RNase Ain RN buffer for 30 min at 37 "C, again rinsed in RN buffer, placed a second time into HS buffer for 30 min at 65 "C, washed in 2 x SSC and 0.1 x SSC, and dehydrated in ethanol containing 0.3 M ammonium acetate.

The slides were dipped in NTB-2 emulsion (IBI, New Haven, CT) diluted 1:l with 2% glycerol and exposed for 2-4 weeks at 4 "C. The emulsion was developed in Dl9 developer, fixed, and then counter- stained with hematoxylin and eosin. The slides were photographed on a Wild M420 darkfield microscope.

Immunohistochemistry-Selected sections of developing embryos were processed for indirect immunofluorescence localization of TSPB essentially as described previously (27-29). Briefly, OCT was removed

1264 Churucterizution of TSP3

1 ~ M E K P E L W G V L A L L L L C S Y T C G ~ ~ D L ~ V I O L L T V G E S R mTSP3

38 Q M V A V A E K I R T A L L T A G D I V L L S T F R L P P K ~ G G V L F G L Y S R ~ O N T R W L E A S V V G K I N K V L V R Y ~ R E O G K V H A V N L ~ ~ A G L mTSP3

118 A O G R T H T A L L R L R G P S R P S P G L ~ L Y V O C K L G O ~ H A G L P A L A P I P P A E V S G L E I R T G ~ K A Y L R M ~ G F V E S M K I l L G C S M A R mTSP3

198 V C A L S E C P F ~ C D D S l H N A V T S A L ~ S l L G E ~ T K A L V T ~ L T L F N ~ l L V E L R O O l R O ~ V K E M S L l R N T l M E C ~ V C G F H E ~ R S H mTSP3

551 mTSPI

278 mTSP3 553 mTSP2

e -"----~ a a m ............................................. ............................................. - - - - - - - "-

803 mTSPl

7 hTSP3 589 rTSP4 375 COMP

805 mTSP2 568 mTSP3

903 mTSPI 905 mTSP2 668 mTSP3 107 hTSP3 669 rTSP4 475 COMP

e 1099 mTSP1 1101 mTSP2 868 G R V mTSP3 307 G R V hTSP3 869 S rTSP4 671 COMP

A FIG. 1. Comparison of derived amino acid sequences of mTSP1, mTSP2, mTSP3, hTSP3 (partial), xTSP4. and rat COMP. Coneerved

sequences are shaded, and conserved cysteines are indicated by a dot above thr nlignmrnts. The cysteine that serves as a free sulfhydryl is denoted by an arrowhead ahove the alignments, and the additional cysteine by an arroujhead hdouj the alignments. Sequence reprats arr hrarkptd and lightly shadrd. The s i e a l sequence and RGD motif are hoxed. Amino acid numbering is indicated to the lrf l .

from the sections with PBS. nonspecific staining was blocked with nor- mal goat serum (1:20), and primary antibody a t a 150 dilution applied for 2 h a t room temperature in a moist chamber. Sections were then exposed to the secondary antibody a t a 1:20 dilution (goat anti-rabbit I&-FITC, Organon Teknika Corp.. Durham, NC). rinsed. viewed, and photographed using a Leitz Orthoplan photomicroscope. Controls in- cluded the use of preimmune serum and anti-TSP3 antibody that had been preincubated with an excess of TSP3 peptide-Sepharose.

Purification and Iodination of Human Platelet TSPI-TSP1 was pu- rified from the supernatant of thrombin-activated platelets, and iodin- ations were performed using NalZ5I (ICN) and Iodo-Beads (Pierce Chemical Co.) as detailed in past publications (30. 31).

RESULTS AND DISCUSSION

Comparison of TSPI, TSPZ, TSP3, TSP4, a n d COMP Se- quences-Fig. 1 compares the derived amino acid sequences of

mouse TSPl (mTSPI), mouse TSF'2 (rnTSP2). mouse TSPS (mTSP3). human TSP3 (hTSP3). Xenopus TSP4 (xTSP4). and rat COMP. The human TSP3 (hTSP3) sequence is partial, miss- ing the N-terminal half. The full-length mTSP.7 sequence re- ported here is in agreement with the sequence reported hy Bornstein and colleagues (17, 32). At first approximation, it is evident that the molecules are very similar at their C termini. In contrast, the N terminus of TSP3 is distinct, and significant homology to the other TSPs only becomes evident a t t h e start of the first Type 2 repeat. Thus, TSP3 appears to possess a n N terminus with no homology to the N-terminal heparin binding domains of TSPl and TSP2 or with the N terminus of TSP4 or COMP and is completely missing the Type 1 repeats. The same also holds true for xTSP4 and COMP in that they, too. have

Characterization of TSP3 1265

distinct N termini, lack the Type 1 repeats, and have 4 Type 2 repeats instead of the 3 seen in mTSPl and mTSP2 (18, 19). This dichotomy suggests that TSP family members fall into two categories (3). The first category, as exemplified by TSPl and TSP2, possess Type 1 repeats but have only 3 Type 2 repeats. The second category would include TSP3, TSP4, and COMP that display a complete absence of Type 1 repeats but have 4 Type 2 repeats (3). In addition, TSP3, TSP4, and COMP have an additional cysteine residue at the very C terminus (Fig. 1, arrow) that is absent in TSPl and TSP2 where it is replaced by a valine. Similarly, the last Type 3 repeat contains an RGD motif in both TSPl and TSP2. This motif is responsible for recognition of TSPl by the a& integrin receptor (14) and is conspicuously absent in mTSP3, xTSP4, and rat COMP, imply- ing that a,Ps may not bind to these TSPs. However, an excep- tion to this is bovine COMP that contains an RGD sequence in the 3rd Type 3 repeat (19).

A feature unique to TSPl is the presence of a presumed free thiol a t position 992 that participates in disulfide exchange reactions resulting in covalent association of TSPl to itself and to other proteins including protease nexin-1 (33,34). The other TSPs have a serine at this position.

The N terminus of TSP3 is distinct and does not contain the heparin binding sequences defined for human platelet TSPl (ARKGSGRR and MKKTRG) (35) nor the general heparin binding sequences XBBXBX and XBBBXXBX where B is a basic residue and X a nonbasic residue (36). The sequence of the very N terminus of TSP3 is consistent with that of a signal peptide, and application of the von Heijne (37) algorithm pre- dicts that the mature polypeptide should begin with the serine following the boxed signal sequence (see Fig. 1).

Characterization of the Anti-TSP3 Peptide Antibody-The anti-TSP3 peptide antibody was characterized by immunoblot- ting lysates of E. coli transformed with the prokaryotic expres- sion vector (pGEX2T) encoding a fusion protein between glu- tathione S-transferase and a 172-amino acid C-terminal portion of TSPB encompassing the sequence of the injected peptide (PLQTDRDED). The pGEX2T-TSP3 recombinant con- struct was transcriptionally driven by the IPTG-inducible tac promoter and as such the chimeric GST-TSP3 protein was syn- thesized only in the presence of the inducer IPTG. This was verified as shown in Fig. 2 where the Coomassie blue- and Amido black-stained transfers show the induction of a protein of the predicted molecular mass (48 kDa) occurring only in the presence of IPTG. Furthermore, only this chimeric protein was recognized by the affinity-purified anti-peptide antibody on im- munoblotting. No reactivity was seen in the absence of induc- tion. In addition, preblocking the antipeptide antibody with a molar excess of free peptide totally abolished binding of the antibody to the GST-TSP3 chimera. Preimmune serum dis- played no activity. Taken together, these data indicated that the anti-TSP3 antibody was monospecific.

Labeling and Immunoprecipitation-To confirm antibody specificity and to study TSP3 biosynthesis in mammalian cells, we undertook immunoprecipitation studies using the antipep- tide antibody and cells transfected with a TSP3 expression vector.

The human renal epithelial cell line 29313 which is an ad- enovirus Ela transformed cell, was chosen for the transfection studies since it does not express any of the known TSPs (24). Furthermore, this cell line is highly transfectable and supports replication of Epstein Barr virus-based vectors which are main- tained extrachromosomally in high copy number and produce significant levels of recombinant protein. Stable 293T cell lines were obtained following transfection with the Epstein Barr virus-based TSP3 expression vector designated pCEP4-TSP3 and selection with hygromycin. Metabolic labeling and immu-

e c U

29-

18-

14-

FIG. 2. Specificity of anti-TSPS peptide antibody. The C-termi- nal 172 amino acids of TSPB whlch encompass thr ptptide sequence used for immunization was expressed as a bacterial recombinant glu- tathione S-transferase fusion protein using the pGEX expression sys- tem. In the presence of IPTG. expression of the recombinant protein was

(CBB )-stained gel and Amido black-stained transfer of total bacterial induced. This can be seen (orrow) in the Coomassie Brilliant Blue

lysates made from uninduced ( U I and induced I I I E. roli. Following transfer to nitrocellulose, the bacterial lysates were probd with either anti-TSP3 peptide antibody (aTSP). hlocked antibody (prepared by pre- incubating anti-TSP3 peptide antibody with an excess of TSPB peptide- Sepharose), or preimmune serum. Only the anti-TSP.7 peptide antibody displayed reactivity against the TSP3-GST fusion protein. Molrcular mass markers in kilodaltons are shown to the lrft.

noprecipitation of a representative line revealed TSP3 to be present in both cell layer and medium in keeping with the presence of a signal peptide sequence and with the observation that the other characterized TSPs. namely TSPl and TSP2, are both secreted and cell surface-associated molecules (24; see Fig. 3 A ) . The apparent molecular mass of -140-150 kDa in the presence of reductant is appreciably larger than the predicted mass of -105 kDa. This discrepancy is likely due to post-trans- lational modifications, especially glycosylation, given that there are four potential N-linked glycosylation sites in TSPB and that TSPl is known to be modified by both 0- and N-linked glycosylation (1. 6). In addition, both TSPl and TSP2 run anomalously on SDS-PAGE, being larger than their predicted molecular masses (24). Besides post-translational modifica- tions, another factor contributing to the anomalous migration of the TSPs may be their low isoelectric point (-44.7). in that being negatively charged they do not effectively complex with SDS (6). TSPl has a predicted molecular mass of -140 kDa, but it migrates as a 160-180-kDa species on SDS-polyacryl- amide gels (see Fig. 3A ). TSP2, which has a similar predicted molecular mass migrates even slower, in the 180-190-kDa range. In the absence of reductant, TSPl is a trimeric molecule (1, 24) and migrates correspondingly slower f -440 kDa) on SDS-polyacrylamide gels (Fig. 3R). Surprisingly, TSP3 mi- grates even slower in the absence of reductant which suggest9 that it is comprised of more than 3 subunits (Fig. 3 8 ). In this regard, it is of note that COMP is a pentameric protein (19). However, given the poor resolution of SDS-acrylamide gels a t such high molecular masses, i t is impossible to determine with certainty the oligomeric nature of TSP3 by this method. It is certainly possible that TSP3. like COMP, exists as a pentamer. Once purified, recombinant TSP3 is available in significant quantities, it should be possible to use analytical ultracentri- fugation or rotary shadowing electron microscopy to accurately determine its molecular mass and subunit number.

1266 Characterizar

""

C M C M C M C M

200-

97 - ""

FIG. 3. mTSP3, like mTSP1 and mTSP2, is expreseed in both the cell layer and medium of transfected 293T cells. A. 293T cells were transfected with the indicated expression constructs and meta- bolically labeled with [""Slcysteine and -methionine, and the harvested cell layer ( C ) and medium ( M ) were immunoprecipitated with the cor- responding antibodies (vector control was immunoprecipitated with anti-TSP3 antibody). Cells transfected with vector alone did not syn- thesize TSP3, whereas cells transfected with the expression constructs synthesized the corresponding protein which was detectable in both cell layer and medium. The apparent order of mobility of the molecules on SDS-PAGE is mTSP2 being the slowest followed by mTSPl and mTSP3. Minor proteolytic forms can he seen in some of the immunoprecipitates. Molecular mass markers in kilodaltons are shown to the left. All samples were resolved in the presence of reductant (p-mercaptoetha- nol). B, in the absence of reductant, immunoprecipitated mTSP3 mi- grates slower on SDS-PAGE than does iodinated human platelet TSPl (hTSP1) which is a trimer with an apparent molecular mass of -440 kDa.

Heparin-Sepharose Afinity Chromatography-Given the lack of significant sequence homology between TSP3 and the heparin binding domains of TSPl and TSP2, it was important to establish whether or not TSP3 was a bona fide heparin binding protein.

To address this, serum-free conditioned medium from 293T cells transfected with the TSP3 expression construct (pCEP4- TSP3) was applied to a heparin-Sepharose affinity column fol- lowed by stepwise salt elution. Collected fractions were ana- lyzed by immunoblotting for the presence of TSP3. As shown in the upper panel of Fig. 4, recombinant TSP3 in the conditioned medium bound to the affinity column, and peak elution oc- curred a t a salt strength of between 0.3 and 0.35 M. This result is in keeping with TSP3 being a heparin binding protein.

In contrast, when human platelet TSPl was applied to the same affinity column, its peak elution occurred between 0.4 and 0.5 M salt, confirming its greater affinity for heparin (1,24) (Fig. 4B). Even though TSPS was not examined in this particu- lar study, previous studies from our laboratory indicate a hep- arin affinity less than that of TSPl with elution from a heparin column occurring a t between 0.35 and 0.4 M NaCl (24). Taken together, these studies indicate that TSPl has a higher heparin affinity while TSP2 and TSP3 have lower but comparable af- finities. Since both heparan sulfate proteoglycans and sulfated glycolipids bind to TSPl in a heparin-inhibitable manner (9, 10) and given that TSP2 (24) and TSP3 possess heparin binding activity, the present results raise the possibility that the TSPs bind to a common set of receptors in a hierarchical manner based on local concentration and affinity.

In Situ Hybridization-The pattern of expression of mTSP3 was analyzed by in situ hybridization using a mTSP3 cRNA probe and sagittal sections of murine embryos from days 15-19 of development. The mTSP3 probe was designed from the 5' region of the open reading frame, which is unique among mem- bers of the thrombospondin gene family and shares no similar-

!ion of TSP3

97 I I

B NaCI (M)

"""" "-7

2001 - 97 - !

ho. 4. mTSpS bind. hepuin-8eplmrwe with m lower r i l l n i t y than in hTSP1. A, conditioned medium from 2931 cells transfected with a TSP3 rxpression construct was applied to a heparin-Sepharose column. Following extensivr washing. the column was eluted uslng the indicated salt step gradient, and the flow through ( F I T ) , wash, and collected fractions were trichloroacetic acid-precipitated, dissolved in SDS-sample buffer, resolved by SDS-PAGE. transferred to nitrocellu- lose, and probed with a anti-TSP.? peptide antibody. Peak elution of mTSP3 is seen a t 0.3-0.35 M NaCI. R. condltions identical with A except that 10 pg of human platelet TSPl was added to the conditioned me- dium and the Western blot was carried out using a monoclonal antibody specific to hTSP1. Peak elution of hTSPl occurs a t 0 . 4 4 . 5 H NaCI.

ity with other known sequences (3). Sense strand "%-labeled riboprobe hybridized to serial sections as a control for nonspe- cific hybridization produced an unpatterned scatter of silver grains (Fig. 5, lower panel ).

Intense TSP3 hybridization was apparent in the forming musculature of the anterior body wall and along the dia- phragm, most prominently a t day 19 (Fig. 5, upper panel ). At this stage of development, mesenchyme is condensing to form the musculature associated with the skeleton and body wall. The intestine, from its herniated position on day 15, through its relocation into the peritoneal cavity of days 17-19 of develop- ment, also expressed TSP3. The outer muscular wall of the intestine is derived from mesenchyme and provides structural support for the endodermal lining. TSP3 expression was also high in the sternum and in vertebral bodies, especially on day 19, when it was conspicuous in the perichondrium and in hy- pertrophic chondrocytes. I t was also expressed a t high levels during membranous bone formation in the head, particularly in Meckel's cartilage (Fig. 5, upper panel ).

In addition to TSP3, COMP is expressed (almost exclusively) by chondrocytes of postnatal bovine tissues ( 19). TSPl is pre- sent in chondrocytes during development (29). while TSP2 is restricted to the perichondrium and regions of hypertrophied cartilage (38).* These data taken together suggest that TSPS may participate in specifying or maintaining mesenchyme dif- ferentiation into muscle, bone, and cartilage.

Both the spinal cord and the brain expressed moderate levels of TSPS late in gestation. Higher levels of TSP3 were present in the olfactory bulb on days 1-5-17, the telencephalon on day 17, and in the thalamic region and cortex just prior to birth. It was also present in peripheral nerve during these stages.

Expression of TSP3 in the lung was first apparent on day 15, _ _ ~ _ _ ~ ~ ~

K. S . OShea and V. M. Dixit, unpublished observations

Chamcterization of TSP3 1267

FIG. 5. Localization of mTSP3 during murine development. Midllne sapttal sectlnns of e rnhyos a t 1.5 I /‘*/I I. 17 I rvntrr I , and 13 1 ri#ht) dayn gestation hyhridizrd with antisense 1 n p p w pnnrl I or control sense ( louvr p o n d I prohcs for rnTSI’:1. Ahhre\iatmnci arr \’h. vrr t rhrar ; I,r. liver.

decreasing by day 19. By RNase protection, TSP3 is expressed at high levels in the adult lung (17). suggesting that it may have separate development and homeostatic pulmonary func- tions. Unlike TSPl(29) and TSP22 which are expressed at high levels in the developing liver, very little TSP3 was present in this region at any stage of development examined.

Immunocytochemistry-Immunocytochemical localization of mTSP3 indicated that it was highly restricted during early embryonic development (days 9-13), significantly increasing during organogenesis, and peaking on days 17-19 of gestation. Control embryos in which the primary antibody was preab- sorbed with an excess of TSP3 peptide-Sepharose (Fig. 6C) were uniformly negative, confirming antibody specificity.

TSP3 was first detected in the amnion of the day 9 embryo, where it was present in the outer layer formed from extraem- bryonic mesoderm, but was absent from the underlying endo- dermal layer (Fig. 6 A ) . There was little expression of TSP3 until day 13 of development when chondrocytes began to ex- press slight quantities of TSP3; Meckel’s cartilage was more strongly stained. At this stage, hepatocytes and Schwann cells associated with forming peripheral nerve also began to express TSP3. Expression continued at low levels until day 17, when skeletal muscle cells also began to express small amounts of TSPB (Fig. 6, B and F ) .

Unlike TSPl (27, 29) and TSP22 which are present at very early developmental stages (prior to neurulation), TSPB was

not expressed in the developing central nervous system in quantity until later stages of gestation. TSPB was particularly densely deposited in the peripheral nervous system where i t was associated with forming dorsal root ganglion and develop- ing peripheral nerve (Fig. 6E ). Interestingly, TSP3 immunore- activity was typically associated with cell surfaces. with con- siderably less immunoreactivity in the surrounding extracellular matrix than TSPl (29) or TSF?L2

The relative abundance and pattern of expression of TSP3 was also very different from TSPl and TSW. In the forming cerebellar cortex, where TSPl(28) and TSP22 are most densely deposited at the interface between the external granule cell layer and forming molecular layer where cells prepare to mi- grate, TSP3 was found predominantly in the Purkinje cell layer and in the forming white matter region (Fig. RG), although i t was also present on the granule cell surface. In the eye (Fig. 6D), TSP3 was present in the inner plexiform layer and in Bruch’s membrane and was particularly highly expressed in the forming lens. Other TSPs are present in the developing lens,2 but are enriched in the region of delamination of lens fibers from the capsule at the equator, while TSP3 is absent from the capsule and is expressed throughout the lens fibers.

Many of these differences, e.g. cell surface rather than extra- cellular matrix localization, may result from the lack in the TSP3 molecule of identified sequences within the Type 1 re- peats for cell-matrix interaction (3) . The expression of TSPs by

1268 Characterization of TSP3

FIG. 6. Immunocytochemistry. A. cross-scctlon through the arnnlon of B day 9 r rnhcc~ Illustrating tht. t i v n w d ~ p ~ ~ ~ ~ t l o n of TSP3 in the outer layer of extraembryonic rncsodrrm (arrowhends~. Thcrr was littlr TSP3 associated with the 1ntPrrndodrrmal 1 r . n I u*I l layer. R . sagittal section through a day 19 embryo illustrating the intense immunoreactivity for TSP3 In developing cartilagr ( C I and in thr rrgion of calcified cartilage/ forming bone (arrowheads). Developing skeletal muscle cells ( r n ) also express some TSP3. C, similar sagittal section through the developing femur of a day 19 embryo in which the primary antibody was preabsorbed with an excess of TSP3 peptide-Sepharose, illustratln~ the lack of immuno- reactivity in this preparation. D. cross-section through developing eye of day 19 embryo illustrating the dense deposition of TSP3 in the body of the lens ( L ) , with little TSP3 at the equator or in the lens capsule. TSP3 is also present at the region of the forming iris (double arrowheads), in the inner plexiform layer (arrowhead) of the retina ( R ) , and in association with Rruch's membrane. E , parasagittal section through developing dorsal root ganglia ( C ) illustrating the considerable TSP3 associated with both ganglia and forming peripheral nerve bundles rdouhlp arrowhrads I. F , sagittal section through a day 15 embryo illustrating the high level of expression of TSP3 in forming peripheral nerve rn ). Chondrocytes ( c ) as well as smooth muscle cells also express TSP3. but not at the very high levels seen in Schwann cells. C , sagittal section through the developing cerebellar cortex in a day 19 embryo. TSP3 was present on the surface of external granule cells (g) and was particularly densely drposited in deeper regions of the cortex, the Purkinje cell layer ( p ), and future white matter. The pial surface is located toward the fop of the micromaph. The wrftr-nl while bar indicates the depth of the (external) granule cell layer. Scale bars = 50 pm.

most mammalian tissues indicates that the conserved regions of the molecules may serve a function that is common to most cell types. The unique distribution patterns of the thrombo- spondins, in combination with the recent observation that TSPl and TSPL are able to form both homo- and heterotrimers (24), suggest a degree of functional complexity and redundancy which likely plays a crucial role in both the early development of the embryo and in its later response to injury.

Acknowle&rnents-We thank Karen ORourke for assistance in all aspects of the project, Dr. Josephine C. Adams for helpful discussion and for help in obtaining the human TSP3 genomic clones, Dr. Sandra Gendler for providing the genomic clone that contains the TSP3 gene, and to Mark Duquette and Katherine McHenry for technical assistance.

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