(Received for publication, October 25, 1994)
From the
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.
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) ()is also similar
but lacks the NH
-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.
Figure 1: Restriction map of the human thrombospondin-4 clones.
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 -hydroxylation
are starred. Note that asparagine 322 could be either
glycosylated or
-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-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
-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
-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).
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, -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 10
.
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.
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 10
.
When the protein that is eluted with 15 mM Tris-HCl (pH 7.6), 2 mM CaCl, 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, -galactosidase
(116,000); 3, phosphorylase b (97,400); 4,
BSA (66,200); and 5, ovalbumin
(45,000).
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 10
.
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. 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.
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-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.