©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Human Thrombospondin-4 (*)

(Received for publication, October 25, 1994)

Jack Lawler (1) Katherine McHenry (1) Mark Duquette (1) Laura Derick (2)

From the  (1)Division of Vascular Research, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the (2)Department of Biomedical Research, St. Elizabeth's Hospital, Boston, Massachusetts 02135

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The thrombospondins are a family of extracellular calcium binding proteins that are involved in cell proliferation, adhesion, and migration. We have sequenced full-length human thrombospondin-4 and characterized the recombinant protein. In contrast to Xenopus laevis thrombospondin-4, the human protein contains an RGD cell binding sequence in the third type 3 repeat. Transfection of mouse NIH3T3 fibroblasts or C2C12 myoblasts with a full-length human thrombospondin-4 cDNA results in the expression of a polypeptide with a reduced molecular weight of 140,000. In the absence of reducing agent, the expressed protein has an apparent molecular weight of 550,000. Recombinant thrombospondin-4 has been purified from the culture supernatant by heparin-Sepharose and anti-thrombospondin-4 antibody-Affi-gel affinity chromatography. Electron microscopy indicates that thrombospondin-4 is composed of five subunits with globular domains at each end. The observation of a calcium-dependent change in the electron microscopic appearance of thrombospondin-4 is consistent with limited tryptic digestion data that indicate that thrombospondin-4 is resistant to digestion in the presence of calcium. These data indicate that thrombospondin-4 is a pentameric protein that binds to heparin and calcium.


INTRODUCTION

The thrombospondins are a family of extracellular calcium binding proteins (for a review, see Adams and Lawler (1993a) and Bornstein (1992)). Thrombospondin-1 has been shown to modulate cell attachment, spreading, migration, and proliferation of various cell types in vitro. Thrombospondin-1 is expressed in many embryonic and adult tissues. By contrast, thrombospondin-4 appears to be specific to cardiac and skeletal muscle (Lawler et al., 1993a). Thrombospondin-4 was identified as cDNA clones from a Xenopus laevis stage 45 library. The predicted amino acid sequence indicates that the thrombospondin-4 mRNA encodes for a peptide of 106,326 daltons. It is distinct from thrombospondin-1 and -2 in that it lacks the procollagen homology region and the type 1 repeats and has an additional type 2 repeat. The amino acid sequences indicate that thrombospondin-3 and -4 have a similar composition of structural motifs (Bornstein et al., 1993; Qabar et al., 1994). Cartilage oligomeric matrix protein (COMP) (^1)is also similar but lacks the NH(2)-terminal domain of approximately 200 amino acids that is present in all of the thrombospondins that have been sequenced to date (Oldberg et al., 1992).

We have recently shown that thrombospondin-4 is present in the human genome (Lawler et al., 1993b). Evolutionary comparison of the thrombospondins by progressive sequence alignment using parsimony-based algorithms indicates that the primordial genes that gave rise to the modern thrombospondin genes diverged over 900 million years ago (Lawler et al., 1993b). Subsequent gene duplications have given rise to the known members of the thrombospondin gene family, thrombospondin-1, -2, -3, and -4, and COMP.

The structural and functional properties of thrombospondin-1 have been extensively characterized (for reviews see Adams and Lawler (1993a) and Bornstein(1992)). Thrombospondin-1 is composed of 140,000-dalton polypeptides that migrate with an apparent molecular weight of 185,000 on a discontinuous SDS-polyacrylamide gel system (Lawler and Hynes, 1986). The thrombospondin-1 polypeptides assemble to form a trimeric molecule with a molecular weight of 420,000. Thrombospondin-1 and -2 form homo- and heterotrimers (O'Rourke et al., 1992). COMP is a 100,000-120,000-dalton polypeptide that has been isolated from bovine cartilage (Hedborn et al., 1992). In the absence of reducing agent, the protein migrates with a molecular weight that is consistent with a pentameric structure (Hedborn et al., 1992; Morgelin et al., 1992). This conclusion is consistent with electron microscopic images that reveal a structure with five subunits that are similar in appearance to the thrombospondin-1 subunits but that have some differences in dimensions (Morgelin et al., 1992).

In this paper, we report on the cloning, sequencing, and expression of human thrombospondin-4. A full-length cDNA construct has been used to express human thrombospondin-4 in mouse fibroblast and myoblast cell lines. The recombinant protein has a subunit molecular weight of 140,000. Electron microscopy and SDS-PAGE indicate that the thrombospondin-4 peptides assemble to form pentamers. Whereas the structure of thrombospondin-4 is distinct from thrombospondin-1, both molecules bind heparin and calcium.


MATERIALS AND METHODS

Cloning and Sequencing of Human Thrombospondin-4

A cDNA clone, designated hTSP4-9, that contains the 3` end of the human thrombospondin-4 sequence was isolated from a human heart library and was described previously (Lawler et al., 1993b). This sequence was identified as the human homolog of Xenopus laevis thrombospondin-4 using two phylogenetic analysis programs (Lawler et al., 1993b). A 400-base pair EcoRI to BamHI fragment from the 5` end of this clone was used to rescreen the human adult heart cDNA library (the generous gift of Dr. Paul Allen). The probe was labeled with digoxigenin-dUTP, and hybridization was performed using the Genius kit following the supplier's protocols (Boehringer Mannheim). Positive plaques were taken through successive rounds of screening with the same probe at progressively lower plaque densities. Because the library was constructed in the ZAP II vector, pBluescript II SK clones that contained the inserts were excised with helper phage and grown up directly following the supplier's protocols (Stratagene). One of the twelve clones, designated hTSP4-11, contained sequence that extended beyond the 5` end of the signal sequence based on similarity to the X. laevis sequence (Fig. 1) (Lawler et al., 1993a). This clone and the original clone were used to determine the sequence on both strands using the chain termination method of Sanger et al.(1977) with Sequenase reagents (U. S. Biochemical Corp.).


Figure 1: Restriction map of the human thrombospondin-4 clones.



Production of Antiserum

The COOH-terminal 14 amino acids (FQEFQTQNFDRFDN) of the predicted amino acid sequence of human thrombospondin-4 was synthesized with a cysteine at the amino-terminal for coupling to keyhole limpet hemocyanin and bovine serum albumin (BSA) as protein carriers (Immuno-Dynamics Inc., La Jolla, CA). Two rabbits were immunized interdermally with peptide-conjugated keyhole limpet hemocyanin (2.5 mg) emulsified in Freund's complete adjuvant. Booster injections were made intramuscularly with conjugate emulsified in Freund's incomplete adjuvant. Production of antibody was monitored by enzyme-linked immunosorbent assay with the peptide conjugated to BSA and absorbed to plastic. Antisera, designated 1258 and 1259, from both rabbits gave a strong response at dilutions up to 1:100,000 by enzyme-linked immunosorbent assay. Both antibodies also stained thrombospondin-4 protein in Western blot protocols (see ``Results'').

Production of Fusion Protein

The clone hTSP4-9 was ligated into the EcoRI site of the bacterial expression vector pGEX1. This clone contains the 3` end of the human thrombospondin-4 sequence and includes the peptide that was used to produce the antiserum. The glutathione S-transferase control and the fusion protein of glutathione S-transferase and thrombospondin-4 were induced with IPTG and purified on glutathione-agarose (Sigma) as described previously (Adams and Lawler, 1993b).

Transfection and Tissue Culture

A cDNA clone that contains the complete coding sequence of human thrombospondin-4 was constructed by cloning an EcoRV fragment of clone hTSP4-9 between EcoRV sites in clone hTSP4-11 in the coding region and the polylinker (Fig. 1). A mixture of the thrombospondin-4 expression vector (pTSP4, 5-10 µg) and pSV2neo (0.5-1.0 µg) was transfected into NIH3T3 cells using the Lipofectin (Life Technologies, Inc.) protocol. Subsequent cell culture and selection of clones was performed as described previously (Lawler et al., 1992). To produce culture supernatant for analysis and purification of thrombospondin-4, the cells were grown to confluence in four T75 flasks in Dulbecco's modified Eagle's medium containing 3% fetal calf serum and 0.5 mg/ml G418. Fresh medium was placed on the cells, and the cells were grown for 48 h. The conditioned medium was removed, diisopropyl fluorophosphate was added to 1 mM and phenylmethylsulfonyl fluoride was added to 5 mM. After several hours at 0 °C, the culture supernatants were frozen and stored at -20 °C.

Purification of Recombinant Thrombospondin-4

Culture supernatant (800-1200 ml) was fractionated on a column (4-6 ml) of heparin-Sepharose (Pharmacia Biotech). After application of the sample, the column was eluted stepwise with 15 mM Tris-HCl (pH 7.6) containing 0.02% NaN(3), 2 mM CaCl(2), and 0.15 M NaCl, 0.25 M NaCl, or 0.55 M NaCl. The fractions that were eluted with 0.55 M NaCl were pooled and treated with 1 mM diisopropyl fluorophosphate and then applied to a column (2 ml) of antibody 1259 immunoglobulins conjugated to Affi-Gel HZ (Bio-Rad). The immunoglobulins were purified from the antiserum 1259 by fractionation on protein A-Sepharose (Pharmacia). The immunoglobulins were eluted from the column with pH 3.0 citrate/citric acid buffer and collected into tubes containing 0.1 ml of 1.0 M Tris (pH 9.5). The immunoglobulins were dialyzed into phosphate-buffered saline and coupled to Affi-Gel HZ according to the supplier's protocols (Bio-Rad). The thrombospondin-4 was eluted with glycine buffer (pH 2.5) containing 1 mM CaCl(2) and immediately dialyzed into 15 mM Tris-HCl (pH 7.6), 0.15 M NaCl, 0.02% NaN(3) (TBS) containing 2 mM CaCl(2).

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

One- dimensional SDS-PAGE was carried out using the discontinuous system of Laemmli(1970) as described previously (Lawler et al., 1985). The electrophoresed proteins were transferred to nitrocellulose (Bio-Rad) or Immobilon-P (Millipore) membranes and probed with the polyclonal antibodies 1258 and 1259 as described previously (Lawler et al., 1985). The bound antibody was detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cooper Biomedical Inc., West Chester, PA).

Electron Microscopy

Thrombospondin-4 in TBS containing 2 mM CaCl(2) was diluted 1:5 or 1:10 with 70% glycerol, 0.15 M ammonium acetate, and 0.2 mM CaCl(2) just prior to spraying. A parallel sample was adjusted to 5 mM in EDTA prior to mixing with 70% glycerol and 0.15 M ammonium acetate prepared without CaCl(2). The samples were sprayed and rotary-shadowed with platinum from an angle of 6° to the horizontal by the method of Tyler and Branton(1980). A shadow thickness of approximately 12 Å was used. Replicas were coated with a 100-Å thick supporting film of carbon from an angle of 90°.


RESULTS

The Sequence of Human Thrombospondin-4

The nucleotide and amino acid sequence of human thrombospondin-4 is shown in Fig. 2. An open reading frame that is 961 amino acids long is predicted from the nucleotide sequence. The 3`-untranslated region is approximately 150 nucleotides long and contains a consensus polyadenylation sequence (AATAAA). Based on a message size of 3.4 kilobases, the 5`-untranslated region is approximately 350 base pairs long. The first 21 amino acids appear to comprise a signal sequence. The predictive algorithm of von Heijne(1986) indicates that the signal sequence would be cleaved after alanine(-1) and that alanine(+1) is the NH(2)-terminal of the mature human thrombospondin-4 peptide (Fig. 2). The mature human thrombospondin-4 peptide has a calculated molecular weight of 106,326.





Figure 2: The sequence of human thrombospondin-4. The predicted site for signal sequence cleavage is indicated (open triangle). The termination codon TAA and the polyadenylation signal AATAAA are underlined. The RGD cell binding sequence is boxed. The sites for potential N-linked glycosylation are circled, and the sites for potential beta-hydroxylation are starred. Note that asparagine 322 could be either glycosylated or beta-hydroxylated. This sequence is available from EMBL/GenBank/DDBJ under accession number Z19585.



The human sequence is 73.2% identical with the X. laevis sequence. Like other members of the thrombospondin gene family, the level of identity is lower at the NH(2)-terminal (52.3% for residues 1 to 220) than at the COOH-terminal (92.3% for residues 721 to 940). The human sequence contains three potential sites for N-linked glycosylation although one of these also fits the consensus sequence for beta-hydroxylation within epidermal growth factor repeats (Hubbard and Ivatt, 1981; Stenflo et al., 1988). The second and third type 2, epidermal growth factor-like repeats of both human and X. laevis thrombospondin-4 contain potential sites for beta-hydroxylation.

Human thrombospondin-4 contains an RGD sequence in the third type 3 repeat (Fig. 2). The only other members of the thrombospondin gene family that have an RGD sequence in this position are human and bovine COMP (Morgelin et al. 1992; Newton et al., 1994). However, the RGD sequence that is present in all of the thrombospondin-1 and -2 proteins is in an equivalent position in the seventh type 3 repeat (Lawler et al., 1993b).

Expression of Recombinant Thrombospondin-4

The antibodies 1258 and 1259 reacted with the COOH-terminal peptide in an enzyme-linked immunosorbent assay (data not shown). On Western blots, these antibodies bind specifically to a fusion protein that contained the COOH-terminal globular domain of human thrombospondin-4, but do not bind to the glutathione S-transferase control (Fig. 3). The preimmune sera from both antibodies did not produce any staining (Fig. 3).


Figure 3: Immunoblot of fusion proteins with antibody 1259. The human thrombospondin-4 fusion protein (lanes b, d, and f) or the glutathione S-transferase control (lanes c, e, and g) were electrophoresed on a 3-10% gradient gel. A portion of the gel (lanes a;enc) was stained with Coomassie Blue, and the remainder (lanes d-g) was transferred to Immobilon-P. The transfer was divided, and lanes d and e were probed with the preimmune serum from rabbit 1259 at a 1:1000 dilution, and lanes f and g were probed with immune serum from rabbit 1259 at a 1:1000 dilution. The molecular weight standards (lane a) are: 1, myosin, 200,000; 2, beta-galactosidase, 116,000; 3, phosphorylase b, 97,400; 4, BSA, 66,200; and 5, ovalbumin, 45,000.



When the culture supernatant from NIH3T3 or C2C12 cells that have been transfected with pTSP4 is transferred to Immobilon-P and probed with these antibodies, a polypeptide of 140,000 daltons is specifically stained (Fig. 4). In addition, a band with a molecular weight of 120,000 is variably observed (Fig. 4). The preimmune serum from both antibodies did not stain these bands (data not shown). Since the staining was more intense with antibody 1259, it was used for all subsequent studies. Neither the 140,000- nor the 120,000-dalton polypeptides are observed in the media (Fig. 4, lane a) or in the culture supernatant from NIH3T3 cells that are transfected with the pLEN-PT vector that did not contain an insert (Fig. 4, lane b). Equivalent staining patterns are observed when the expression construct is transfected into NIH3T3 (Fig. 4, lane c) or C2Cl2 (Fig. 4, lane d).


Figure 4: Immunoblot of recombinant thrombospondin-4. The culture supernatant from individual clones of NIH3T3 cells (lanes c and g) and C2C12 cells (lanes d and h) that were transfected with pTSP4 were electrophoresed in the presence (lanes c and d) and absence (lanes g and h) of reducing agent. Note that the C2C12-derived culture supernatant was diluted with an equal volume of TBS to reduce the serum content to 10%. Media containing 10% serum (lanes a and e) and the culture supernatant from NIH 3T3 cells that were transfected with pLEN-PT vector without an insert (lanes b and f) were electrophoresed in the presence (lanes a and b) and absence (lanes e and f) of reducing agent as a control. After electrophoresis, the proteins are transferred to Immobilon-P and probed with antibody 1259 followed by a peroxidase-conjugated goat anti-rabbit antibody. The molecular weights of the principal band are indicated on the right times10.



In the absence of reducing agents, bands with apparent molecular weights of 550,000, 125,000, and 90,000 are observed in the transfected NIH3T3 (Fig. 4, lane g) and C2C12 (Fig. 4, lane h) cells. A variable amount of a band with a molecular weight of 145,000 is also observed. The 125,000-dalton protein is also present at variable levels in the media (Fig. 4, lane e), and the culture supernatant from NIH3T3 cells that are transfected with pLEN-PT without an insert (Fig. 4, lane f). Since the NIH3T3 cells grew well in much lower serum than the C2C12 cells, the NIH3T3 transfectants were used for the subsequent studies.

Purification of Recombinant Thrombospondin-4

When the culture supernatants from cells that are expressing recombinant thrombospondin-4 are applied to a column of heparin-Sepharose, the 140,000- and 120,000-dalton bands are retained by the column (Fig. 5). The majority of the 140,000-dalton band is eluted in the 0.25 M and 0.55 M NaCl peaks. The 120,000-dalton polypeptide is present in all of the fractions. The ratio of the 140,000-dalton polypeptide to the 120,000-dalton polypeptide varies in the different pools. The protein that flows through the heparin-Sepharose column without binding is considerably enriched in the 120,000-dalton polypeptide (Fig. 5, lane b). The amount of the 120,000-dalton polypeptide varies in the different preparations, and less is observed when the protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride are included.


Figure 5: Heparin-Sepharose affinity chromatography of the culture supernatants from a NIH3T3 cell line that is expressing recombinant thrombospondin-4. The original sample (lane a), the flow-through (lane b), and the proteins that were eluted with buffer containing 0.15 M NaCl (lane c), 0.25 M NaCl (lane d), or 0.55 M NaCl (lane e) were electrophoresed on 3-10% polyacrylamide gradient gels. After electrophoresis, the proteins were transferred to Immobilon-P and probed with antibody 1259 followed by a peroxidase-conjugated goat anti-rabbit antibody. The molecular weights of the principal bands are indicated on the right times10.



When the protein that is eluted with 15 mM Tris-HCl (pH 7.6), 2 mM CaCl(2), and 0.55 M NaCl is applied to an antibody 1259-Affi-Gel column, the 140,000- and 120,000-dalton polypeptides are selectively retained and eluted with low pH (Fig. 6, lanes d and e). The ratio of the bands is again variable, and both bands bind antibody 1259 on Western blots (Fig. 6, lane e). In the absence of reducing agent, the material that is bound by the column electrophoreses as a single band with an apparent molecular weight of 550,000 (Fig. 6, lanes b and c). Purified thrombospondin-4 retained the ability to bind to heparin-Sepharose (data not shown).


Figure 6: Electrophoresis of immune affinity-purified recombinant thrombospondin-4. The lanes are: a, human platelet thrombospondin-1 electrophoresed in the absence of reducing agent; b and c, human recombinant thrombospondin-4 electrophoresed in the absence of reducing agent; d and e, human thrombospondin-4 electrophoresed in the presence of reducing agent; and f, molecular weight markers electrophoresed in the presence of reducing agent. Lanes a, b, d, and f were stained with Coomassie Blue. Lanes c and e were transferred to Immobilon-P and probed with antibody 1259 followed by a peroxidase-conjugated goat anti-rabbit antibody. The standard proteins are: 1, myosin (200,000); 2, beta-galactosidase (116,000); 3, phosphorylase b (97,400); 4, BSA (66,200); and 5, ovalbumin (45,000).



Limited Tryptic Digestion of Thrombospondin-4

Binding of calcium to thrombospondin-1 results in a conformational change that can be detected by an increased susceptibility to tryptic digestion and by differences in electron microscopic appearance (Galvin et al., 1985; Lawler et al., 1982, 1985). When human recombinant thrombospondin-4 is digested with TPCK-trypsin at 0 °C for 20 h in the presence of calcium, a broad spectrum of proteolytic fragments is produced (Fig. 7). A considerable amount of mass is associated with high molecular mass bands of 115,000, 107,000, 97,000, 71,000, and 64,000 daltons. When the digestion is performed in the absence of calcium, the majority of mass is associated with bands of 56,000 and 42,500 daltons (Fig. 7).


Figure 7: Limited tryptic digestion of thrombospondin-4. Human platelet thrombospondin-1 (lanes a and b) or recombinant thrombospondin-4 (lanes c and d) was digested in the presence (lanes a and c) or absence (lanes b and d) of calcium with TPCK-trypsin at an enzyme to substrate ratio of 1:100. The bands were visualized by staining with Coomassie Blue (lanes a and b) or by Western blotting with antibody 1259 (lanes c and d). The molecular weights are given on the left for the thrombospondin-1 experiment (lanes a and b) and on the right for the thrombospondin-4 experiment (lanes c and d) which were run on different gels. The molecular weights of the principal bands are indicated on the right times10.



Electron Microscopy of Thrombospondin-4

Electron microscopy of recombinant human thrombospondin-4 revealed well-defined globular structures connected by thin flexible regions of polypeptide (Fig. 8). Like thrombospondin-1, thrombospondin-4 displays a calcium-dependent conformational change as evidenced by an increase in the length of the thin-connecting regions when the molecules are treated with EDTA (Fig. 8b). In the presence of EDTA, the thin-connecting regions of thrombospondin-4 are 28.3 ± 2.9 nm in length and terminate in globular regions that are 8.5 ± 1.2 nm in diameter (all dimensions are given as mean ± S.D. and are not corrected for thickness of replicating metal). In those images in which the thin-connecting regions are splayed out and readily resolvable, five thin-connecting regions and globular terminal domains are observed most frequently (Fig. 8, a and b). However, in the images of some of the molecules, only four thin-connecting regions with globular regions at the end are observed (Fig. 8c). A globular region is also observed in the area where the thin-connecting regions appear to be connected to each other.


Figure 8: Electron microscopy of purified recombinant thrombospondin-4. Thrombospondin-4 was prepared for electron microscopy in the presence (a) or absence (b and c) of 200 µM CaCl(2). Whereas, in some molecules, five thin-connecting regions and the associated globular structures are resolved (a and b), some molecules appear to have only four thin-connecting regions and associated globular domains (c). Bar = 50 nm.




DISCUSSION

Thrombospondin-4 was first identified from clones that were isolated from a Xenopus laevis stage 42 embryo cDNA library (Lawler et al., 1993a). A subsequent study identified human thrombospondin-4 and established that this protein is most closely related to thrombospondin-3 and more distantly related to thrombospondin-1 and -2 (Lawler et al., 1993b). Thrombospondin-4 has apparent molecular weights of 140,000 and 550,000 in the presence and absence of reducing agents, respectively. Coomassie Blue-stained gels detect the 140,000- and 120,000-dalton polypeptides as the reduced subunits of the 550,000-dalton protein. Since both of these bands react with the antibody to the COOH-terminal peptide (antibody 1259), the difference in molecular mass is probably due to proteolytic cleavage of a 20,000-dalton fragment from the NH(2)-terminal of thrombospondin-4.

Heparin binding is a common feature of the thrombospondins. Heparin binding consensus sequences are present in the first 90 amino acids of the thrombospondin-1 sequence. Mutagenesis of the basic amino acids in these sequences decreases the molecules ability to bind heparin (Lawler et al., 1992). Human thrombospondin-4 contains a heparin-binding consensus sequence (BBXB, where B is a basic and X is any amino acid) between amino acids 102 and 105. Whereas the data presented here indicate that thrombospondin-4 binds to heparin, we cannot preclude the possibility that a small protein that is tightly bound to thrombospondin-4 provides the heparin-binding activity. Future mutagenesis experiments will help to resolve this issue.

Table 1compares the molecular weight determinations of thrombospondin-4 with those of thrombospondin-1 and COMP. Whereas SDS-PAGE in the absence of reducing agent does not generally provide an accurate determination, the values that are obtained in this way for thrombospondin-1 and COMP agree well with values obtained by sedimentation equilibrium ultracentrifugation (Table 1). This suggests that the molecular weight of intact thrombospondin-4 is approximately 550,000. Upon reduction, a principal band of 140,000 daltons and a variable band at 120,000 daltons are observed. The subunit molecular weights of thrombospondin-1 and COMP as determined by SDS-PAGE are considerably higher than the molecular weights that are predicted from the amino acid sequence, indicating that the proteins electrophorese anomalously (Table 1). These proteins contain small percentages of carbohydrate that may contribute to this effect. In addition, the type 3 repeats contain a very high percentage of aspartic acid and may bind less SDS than other proteins that are used to calibrate the gel. Based on these considerations, the thrombospondin-4 subunit can be estimated to have a molecular weight of approximately 110,000. This value suggests that thrombospondin-4, like COMP, is a pentamer (Fig. 9). This conclusion is consistent with the electron microscopic images and the structural similarity to COMP (Efimov et al., 1994).




Figure 9: Schematic model of thrombospondin-4. Each subunit is depicted as having globular regions (open circles) at the ends and type 2 and 3 repeats in the middle.



Electron microscopy of recombinant human thrombospondin-4 is consistent with a pentameric structure. In some molecules, only four thin-connecting regions with attached globular domains can be resolved (Fig. 8c). Since we do not see evidence of different size molecular assemblies by SDS-PAGE in the absence of reducing agent, this is most likely due to the folding of one leg underneath the remainder of the molecule or due to partial proteolysis. However, we cannot rule out the possibility that a small percentage of molecules have fewer than five subunits. The sequence similarity of thrombospondin-4 to thrombospondin-1 and COMP suggests that the globular domains at the ends of the thin-connecting regions are equivalent to the COOH-terminal globular domains that are observed in electron microscopic images of thrombospondin-1 and COMP (Galvin et al., 1985; Lawler et al., 1985; Morgelin et al., 1992). The molecular dimensions and calcium-dependent conformational changes that are observed in this region are consistent with this hypothesis. The length of the thin-connecting region that we have determined for thrombospondin-4 is less than that of thrombospondin-1 and equivalent to that of COMP (Lawler et al., 1985; Morgelin et al., 1992). Like thrombospondin-1 and COMP, the thin-connecting regions display considerable flexibility.


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grants HL28749 and HL49081 from the NHLBI. 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.

(^1)
The abbreviations used are: COMP, cartilage oligomeric matrix protein; TBS, Tris-buffered saline (pH 7.6); TPCK-trysin, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.


ACKNOWLEDGEMENTS

We thank Dr. Paul Allen for providing the human heart cDNA library and Drs. Paul Johnson and Richard Hynes for providing the expression vector pLEN-PT. We also wish to thank Drs. Josephine Adams and Gail Newton for helpful discussions. The manuscript was typed by Pamela Caffey and Tracy Baker and edited by Sami Lawler.


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