From the
The thrombospondins (TSPs) are a family of 5 distinct gene
products designated TSP1, -2, -3, -4, and COMP, for cartilage
oligomeric matrix protein. TSP1, the prototypical member, is a trimeric
extracellular matrix molecule implicated in cell migration and
development. TSP1 trimer formation is mediated by interchain disulfide
linkage involving two NH
Thrombospondin 1 (TSP1),
The two PCR products were directionally subcloned
into pCEP4 using the unique restriction sites
NheI/NotI for TSP2 and HindIII/NotI
for TSP1. The resulting constructs were fully sequenced to ensure
fidelity of PCR amplification.
The four truncated molecules were subcloned into the cytomegalovirus
promoter-based mammalian expression vector pcDNA3 (InVitrogen, San
Diego, CA) using the unique sites HindIII/NotI.
Finally, the two cysteine residues (at positions 245 and 248) were
altered to serine residues in the native full-length TSP3 molecule by
site-directed mutagenesis using the ``Altered Sites'' kit
(Promega, Madison, WI) according to the manufacturer's
instructions. Two point mutations were introduced at nucleotides 797
and 806 (TGT to TCT and TGC to TCC, respectively; indicated in bold),
using the following antisense mutagenic primer:
5`-CTCGTGGAAACCGGACACCTGAGACTCCATGATGGT-3`.
To confirm that Cys-245 and Cys-248 were involved in oligomerization
of the intact native TSP3 molecule, these two residues were altered to
serine by site-directed mutagenesis of a full-length TSP3 cDNA.
Following subcloning into the pcDNA3 expression vector and transient
transfection of 293T cells, the expression of native and mutant TSP3
was monitored by metabolic labeling and immunoprecipitation in the
absence and presence of reductant (Fig. 5).
These results confirmed the critical role played by Cys-245 and
Cys-248 in oligomerization of native TSP3 and also suggested that the
remaining 45 cysteines were not involved in interchain disulfide
linkage but rather intrachain linkage or free sulfhydryls.
Whereas the similarities
in the sequences around the interchain disulfides may provide the
common basis for multimerization, the differences may result in TSP1
and TSP2 forming trimers and TSP3, TSP4, and COMP forming pentamers. In
the former group, there are three amino acids between the cyteines that
form the interchain disulfides and in the latter group there are two
amino acids. If these regions are helical, then both cysteines are on
the same side of the helix with either spacing. However, the additional
residue between the interchain disulfides of TSP1 and TSP2 will change
the relative positions of the cysteine residues. In addition, the
cysteines that form the interchain disulfides are near the
NH
We gratefully acknowledge Karen O'Rourke for
invaluable advice and technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal cysteines. TSP3, a recent
addition to the family, is a developmentally regulated heparin binding
protein that is similar in sequence to the COOH terminus of TSP1 but
has a distinct NH
terminus. This has raised the question of
the oligomeric nature of TSP3 and identification of the cysteine
residues involved in oligomer formation. We demonstrate, using a
combination of deletional and site-directed mutagenesis and rotary
shadowing electron microscopy, that TSP3, like TSP4 and COMP, is a
pentameric molecule. TSP3 is held together by interchain disulfide
linkage involving just two cysteine residues, Cys-245 and Cys-248.
(
)
the first
described and best studied member of the TSP family, is a large
trimeric heparin binding protein that is synthesized by a variety of
cell lines and incorporated into the extracellular matrix where it
differentially modulates cell adhesion in a cell type-specific manner
(see Refs. 1-3 for reviews). The function of the other TSPs
(TSP2, -3, -4, and COMP, for cartilage oligomeric matrix protein) at
present is unclear, although it is evident that they are all highly
expressed in vertebrate
development
(4, 5, 6, 7, 8) .
TSP3, for example, is predominantly expressed in the developing
lung
(6) , whereas COMP expression is limited to cartilage
following the initial phase of chondrogenesis
(8) . The TSPs are
modular proteins and both TSP1 and TSP2 possess NH
-terminal
heparin binding domains
(3) . Two cysteine residues (Cys-252 and
Cys-256 in TSP1 and TSP2) that are involved in interchain disulfide
linkage and trimer assembly are just distal to the heparin binding
domain
(9) . Conservation of these cysteines in TSP1 and TSP2,
coupled to their high degree of overall homology, allows for the
expression of heterotrimers composed of both polypeptides
(10) .
The heparin binding domain is followed by a short stretch of sequence
with homology to procollagen, and by three distinct repeating motifs
designated Types 1, 2, and 3. There are 3 copies of the Type 1 repeat
in TSP1 and TSP2. The putative melanoma cell adhesive sequence, VTCG
(11), and a novel heparin binding sequence, WSXW
(12) , that
mediates binding of certain cells to TSP are included in the Type 1
repeats. The Type 2 repeats have homology to epidermal growth factor
and 3 copies are present in TSP1 and TSP2
(3) . The Type 3
repeats are enriched in aspartic acid and constitute the calcium
binding domain that is capable of mediating significant conformational
alterations in the molecule, some of which, like the unmasking of a
reactive thiol in TSP1, may have functional consequence
(13) . In
stark contrast to TSP1 and TSP2, the other TSPs (TSP3, -4, and COMP)
have a distinct structure. Notably, they possess a distinct NH
terminus, no procollagen or Type 1 repeats, and contain an extra
Type 2 repeat
(3) . Previous studies have shown TSP4
(14) and COMP to be pentamers
(15) , and the two cysteines
involved in COMP interchain disulfide linkage have been identified as
Cys-68 and Cys-71
(16) . These cysteines are in an equivalent
position to Cys-252 and Cys-256 of TSP1 since COMP lacks the
approximately 200 amino acid NH
-terminal domains that are
found in the other thrombospondins
(17) . TSP3 was shown to be a
disulfide-linked higher order oligomer that was larger than a trimer.
However, its precise oligomeric nature was undefined. Using deletional
and site-directed mutagenesis and rotary shadowing electron microscopy,
we show that TSP3, like TSP4 and COMP, is a pentameric molecule. In
addition, the two cysteine residues involved in disulfide linkage are
unambiguously identified.
Construction of Expression Plasmids
A
full-length coding HindIII/NotI TSP3 cDNA fragment
(6) was directionally subcloned into the Epstein-Barr virus
based episomal expression vector pCEP4 that also contains a hygromycin
resistance cassette (InVitrogen, San Diego, CA). TSP1 and TSP2 cDNAs
lacking both the 5`- and 3`-untranslated regions were obtained by PCR
employing previously cloned mouse TSP1 and TSP2 cDNAs as
template
(10) , Vent DNA polymerase (New England Biolabs,
Beverly, MA), and upstream primers with an ATG start codon (bold) and
downstream primers containing a stop codon (italics). For ease of
cloning, the primers contained custom restriction sites (underlined).
TSP1 (upstream primer), 5`-GCACAAAGCTTCCACCATGGAGCTCCTGCGGGGACTA
3`, HindIII; TSP1 (downstream primer),
5`-TGATTGGCAGCGGCCGCTTAGGAATCTCGACACTCGTATTT-3`,
NotI; TSP2 (upstream primer),
5`-CACAGCTAGCGCCACCATGCTCTGGGCACTGGCCCTG-3`, NheI; TSP2
(downstream primer),
5`-AGCTGGAGCGCGGCCGCCTAGGCATCTCTGCACTCATACTT-3`,
NotI.
Transfections
293T cells, a human renal epithelial
cell line, were stably transfected with plasmid expression constructs
encoding mouse TSP1, TSP2, or TSP3 using the calcium phosphate
coprecipitation method as described previously
(6, 10) .
Following hygromycin selection (100 µg/ml), individual colonies
were subcloned and tested for expression of each TSP by metabolic
labeling and immunoprecipitation.
Expression and Purification of Mouse TSP1, -2, and
-3
293T cells expressing recombinant TSP were grown in complete
Eagle's minimum essential medium (BioWhittaker, Walkersville, MD)
supplemented with 10% bovine calf serum for 24 h, following which the
medium was replaced with serum-free insulin, transferrin, and
selenium-supplemented Eagle's minimum essential medium
(Collaborative Biomedical Products, Bedford, MA) for 72 h. Conditioned
medium was mixed with a 50% slurry of heparin-Sepharose and incubated
for 8-12 h at 4 °C on a rotating shaker. The
heparin-Sepharose slurry was poured into a 1-ml column and washed
extensively with 0.15 M NaCl, 20 mM Tris-HCl (pH
8.0), and bound thrombospondin step-eluted by the application of 0.5
M NaCl in the same buffer. 500-µl aliquots were collected
and analyzed by SDS-acrylamide gel electrophoresis and Coomassie Blue
staining.
Generation of a Polyclonal TSP3 Peptide Antibody
A
synthetic peptide, RLRGPSRPSPC, from the NH-terminal end of
TSP3 corresponding to amino acid residues 107-116 was synthesized
and covalently conjugated to keyhole limpet hemocyanin as a carrier
protein via the free -SH group of the carboxyl-terminal cysteine
residue (Multiple Peptide Systems, San Diego, CA). 2 mg of the
conjugated peptide was emulsified with 500 µl of TiterMax (Vaxcel
Inc., Norcros, GA) and injected into New Zealand White female rabbits
subcutaneously. Subsequent injections were carried out using incomplete
Freund's adjuvant.
Construction of TSP3 Truncations and
Mutagenesis
Four TSP3 truncations were derived by PCR using a
common 5`-oligonucleotide primer containing an ATG start codon (bold)
and a custom HindIII restriction site (underlined),
5`-AGTGAAGCTTCTAAGCGGCATGGAGAAGCCG-3`, and four
different downstream oligonucleotide primers (antisense strand) having
an in-frame TAA stop codon (italics) and custom NotI
restriction site (underlined). The truncations and downstream primers
were designated according to the identity of the COOH-terminal amino
acid (Glu-696, Asp-437, Cys-268, and Gly-249): Glu-696 primer:
5`-AAGTCTGTGCGGCCGCCTTCTGCACTTTACTCCGGGCA-3`,
NotI; Asp-437 primer:
5`-TGGTCCGGGCGGCCGCCTATATCTTAGTCAGGCCCACA-3`;
Cys-268 primer:
5`-TAGCCCGGGCGGCCGCACACTTCTTAGAAGTCCACGCC-3`;
Gly-249 primer:
5`-CTGCAGTGGCGGCCGCGCTCGTGTTAACCGCACAC-3`.
Expression of Truncated Molecules
293T cells were
transiently transfected with each truncation/mutant construct and
expression detected by metabolic labeling and immunoprecipitation
essentially as described in previous
publications
(6, 10) , using the TSP3 peptide antibody.
Electron Microscopy
Thrombospondin-3 in TBS
containing 2 mM CaCl was diluted 1:5 or 1:10 with
70% glycerol, 0.15 M ammonium acetate, and 0.2 mM
CaCl
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
. The
samples were sprayed and rotary shadowed with platinum from an angle of
6° to the horizontal by the method of Tyler and
Branton
(18) . 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°.
Expression and Purification of Recombinant Mouse TSP1,
TSP2, and TSP3
Recombinant TSP was purified from three day
serum-free 293T cell-conditioned medium transfected with the
appropriate TSP expression construct. 293T cells have previously been
shown not to synthesize any endogenous TSP and are therefore ideal for
the expression of recombinant TSPs
(10) . On average, conditioned
medium from ten 10-cm diameter dishes allowed for the purification of
5-10 µg of TSP3 and 1-5 µg each of TSP1 and TSP2.
The purification process consisted of application of the serum-free
medium to a heparin-Sepharose column which, after extensive washing,
was step eluted in a high salt (0.5 M NaCl) buffer. The
predominant protein in the resulting eluate was the recombinant TSP as
evidenced by SDS-polyacrylamide gel electrophoresis and Coomassie Blue
staining (Fig. 1).
Figure 1:
Purified recombinant TSPs. Recombinant
mouse TSP1, -2, and -3 proteins were purified from serum-free
conditioned medium of transfected 293T cells by heparin-Sepharose
affinity chromatography. Purified TSP was resolved on a 7.5%
SDS-polyacrylamidegel and stained with Coomassie Blue. The
predominant protein in each sample is the recombinant TSP. Molecular
mass markers in kilodaltons are shown to the
left.
Characterization of TSP1, -2, and -3 Antibodies
To
establish the specificity of each TSP antibody, an immunoblot analysis
of the recombinant TSPs was carried out (Fig. 2). Production of
rabbit polyclonal antisera against the divergent NH terminus of TSP1 and TSP2 using bacterially expressed recombinant
fusion protein as immunogen has been described previously
(10) .
The TSP3 antibody was raised against a peptide corresponding to
residues 107-116 that are unique to TSP3. Fig. 2clearly
shows that the predominant protein recognized by each antibody is the
corresponding TSP. Also of note (see Fig. 1and Fig. 2) are
the relative molecular masses of the three TSPs in the presence of
reductant, with TSP2 being the largest (
190 kDa), followed by TSP1
(
180 kDa), and TSP3 being the smallest (
160 kDa). In
contrast, in the absence of reductant (Fig. 3), native TSP3
migrates slower than either native TSP1 or TSP2 which are both
trimeric. Since the apparent subunit molecular mass of TSP3 is smaller
than that of TSP1 and TSP2, it follows that in the native nonreduced
state TSP3 must be larger than a trimer.
Figure 2:
Specificity of TSP1, TSP2, and TSP3
antibodies. Conditioned medium from 293T cells transfected with each
TSP construct was resolved by SDS-polyacrylamide gel electrophoresis in
the presence of reductant, transferred to nitrocellulose, and probed
with TSP1, TSP2, or TSP3 antisera. The antibodies recognize the
corresponding TSP (arrow).
Figure 3:
Migration of recombinant TSP1, -2, and -3
in the absence of reductant. Transfected cells were metabolically
labeled with [S]cysteine and methionine and
labeled proteins in the medium immunoprecipitated with the
corresponding antibody. Samples were resolved by SDS-polyacrylamide gel
electrophoresis and subjected to autoradiography. TSP3 is the slowest
migrating species, followed by TSP2, and then
TSP1.
Expression of Truncated Forms of TSP3
The deduced
amino acid sequence of native TSP3 contains 47 cysteine
residues
(6) , most of which are presumably involved in
intrachain disulfide linkages. To identify the cysteine
residues involved in interchain disulfide linkages and
oligomerization, four COOH-terminal truncated molecules, each having a
defined number of cysteine residues, were generated by PCR, subcloned
into a mammalian expression vector (pcDNA3), and expressed by transient
transfection in 293T cells. Metabolic labeling and immunoprecipitation
using TSP3 NH-terminal polyclonal antiserum revealed that
each of the four truncations (Glu-696, Asp-437, Cys-268, and Gly-249)
was synthesized and present in both the cell layer and medium
compartments (Fig. 4A). In the presence of reductant,
the apparent molecular mass of each species corresponded to the number
of amino acid residues expressed, i.e. Glu-696 was
75
kDa, Asp-437 was
47 kDa, Cys-268 was
29 kDa, and Gly-249 was
27 kDa. In the absence of reductant (Fig. 4B), all
four species migrated substantially slower, consistent with the
formation of oligomers. The two shortest truncations (Gly-249 and
Cys-268) migrate with an apparent molecular mass of
150 kDa
consistent with the formation of a pentameric species. The two larger
truncations (Glu-696 and Asp-437) also migrate with appreciably reduced
mobility, barely entering the gel system, presumably due to oligomer
formation.
Figure 4:
Expression of TSP3 truncations in the
presence and absence of reductant. 293T cells transfected with the
indicated constructs were metabolically labeled with
[S]methionine and cysteine and subjected to
immunoprecipitation with the TSP3 peptide antibody. Medium and cell
layer samples were resolved by SDS-polyacrylamide gel electrophoresis
in the presence of reductant (A) or in the absence of
reductant (medium alone, panel B). In the presence of
reductant, TSP3 truncations of the predicted size are found in both the
cell layer and medium. In the absence of reductant, there is a clear
shift of all the truncations to a higher molecular mass oligomeric
form. Also shown (panel C) is a schematic representation of a
monomeric chain of TSP3 with cysteine residues numbered and truncations
indicated by the identity of the COOH-terminal residue. The four
cysteine residues present in the Gly-249 truncation are
circled.
Mutagenesis of Cys-245 and Cys-248 in Native
TSP3
Since the shortest truncation (Gly-249) contained only four
cysteine residues (Cys-124, Cys-183, Cys-245, and Cys-248) and was
capable of oligomerization to a pentameric structure, it
followed that, at the very least, a pair of these cysteines had to be
involved in pentamer assembly. Given that COMP does not possess the
equivalent first pair of cysteines
(17) and that the second pair
of NH-terminal cysteine residues are involved in TSP1
trimer assembly
(9) , it appeared likely that this second
analogous pair of cysteines in TSP3 (Cys-245 and Cys-248) was
responsible for interchain disulfide linkage and pentamer assembly.
Figure 5:
Cys-245 and Cys-248 are critical for
oligomerization of native TSP3. Native TSP3 (Wt TSP3) or TSP3
with cysteine 245 and cysteine 248 altered to serine (Mut
TSP3) were expressed in 293T cells, metabolically labeled, and
immunoprecipitated with a TSP3 peptide antibody, resolved by
SDS-polyacrylamide gel electrophoresis either in the absence (-)
or presence (+) of reductant and visualized by autoradiography. In
the absence of reductant, there is a complete lack of oligomerization
of the mutant TSP3, implicating Cys-245 and Cys-248 in oligomer
assembly. A cartoon interpretation of the results is
shown.
Native TSP3, as
expected, migrated as an oligomer in the absence of reductant and a
monomeric species in the presence of reductant. Mutant TSP3, with both
Cys-245 and Cys-248 altered to serine, also migrated as a monomer in
the presence of reductant, but in the absence of reductant, there was a
complete absence of oligomerization. In fact, the only discernible
species had an electrophoretic mobility greater than the reduced
monomeric form, presumably due to intrachain disulfide linkage
producing a more compact, faster migrating form of the molecule.
Electron Microscopy of TSP3
Electron microscopy of
TSP3 revealed well defined globular structures that were connected by
thin-flexible regions of polypeptide (Fig. 6). In those molecules
where the globular structures are not overlapping and are clearly
defined, six globular domains are present. Five of these appear at the
ends of the thin-flexible regions and the sixth is near the site where
the thin connecting regions are connected to each other. Comparison
with the published images of TSP1, TSP4, and COMP indicates that the 5
globular domains at the ends of the thin-flexible regions are the
COOH-terminal and the single globular domain near the site where the
polypeptides are connected is formed by the NH-terminal
domains of all of the subunits in close
proximity
(15, 19) . Like TSP1 and TSP4, the globular
domains at the ends of the thin-flexible regions undergo a
conformational change when calcium is removed from the molecule
(Fig. 6), consistent with the presence of the calcium binding
Type 3 repeats at the COOH terminus of the molecule. In the presence of
calcium the globular domains are 10.8 ± 1.3 nm in diameter (all
dimensions are given as mean ± S.D. and are not corrected for
thickness of replicating metal). When calcium is removed from the
sample prior to electron microscopy, the diameter of these globular
domains decreases to 8.9 ± 1.1 nm and the length of the
thin-flexible region increases. These data are consistent with a
pentameric model of TSP3 in which each subunit is composed of a single
small globular domain at the NH
-terminal, a site where the
subunits are connected, a thin-flexible region of extended polypeptide,
and five globular domains containing the calcium sensitive Type 3
repeats at the COOH-terminal.
Figure 6:
Electron microscopy of purified
recombinant TSP3. Samples were prepared for rotary shadowing in the
presence (A) or absence (B) of 200 µM
CaCl. Bar = 50
nm.
It has been proposed that the cysteine
residues that form the interchain disulfides of TSP1 and COMP are
included in -helical segments of
polypeptide
(9, 16) . These
-helices reportedly
assemble to form three- or five-stranded bundles that drive trimer or
pentamer formation of TSP1 or COMP, respectively. Deletion of
polypeptide that includes this region results in defective trimer
formation of TSP1, even though the cysteine residues are still
present
(20, 21) . The data presented here and the
similarity of the COMP and TSP3 sequences around the corresponding
cysteine residues indicates that a similar mechanism may result in
pentamer formation in TSP3. In the TSP1, TSP2, and TSP3 genes these
regions are included in a single
exon
(22, 23, 24) .
-terminal of the predicted
-helix in TSP1 and TSP2,
and they are near the COOH-terminal of the predicted
-helix in
TSP3, TSP4, and COMP. We hypothesize that these differences result in
the
-helices packing together to form bundles of either three or
five strands.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.