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INTRODUCTION |
SC1 was originally cloned from a rat brain expression library by
screening with a polyclonal antibody raised against synaptic junction
glycoproteins (1). Its mRNA is expressed widely in the brain and
could be detected in many types of neurons. In immunoblots of brain
proteins SC1 was detected as a doublet band of 116 and 120 kDa, whereas
the molecular mass predicted from sequence was 70.6 kDa (1). A
homologous cDNA called hevin was also cloned from a library derived
from human high endothelial venule cells (2). Human high endothelial
venule cells form specialized postcapillary vascular sites (high
endothelial venules) that mediate extensive extravasation of
circulating lymphocytes from the blood into lymphoid organs and sites
of chronic inflammation. The high sequence identity suggests that hevin
is the human SC1 ortholog. SC1/hevin is expressed in many tissues as a
secreted glycoprotein of the extracellular matrix.
Sequence analysis showed that SC1/hevin belongs to the
BM-40/SPARC/osteonectin family of proteins and is composed of a highly acidic domain I without homology to any known protein, a
follistatin-like domain (FS) and an extracellular calcium-binding
domain (EC) (Fig. 1). The presence of the FS and the EC domain is the
hallmark of the BM-40/SPARC/osteonectin family, which consists of
BM-40/SPARC/osteonectin, SC1/hevin, QR1, tsc36/flik, the testicans, and
the SMOCs (3).
Functional assays suggest that SC1/hevin may serve as an antagonist of
cell adhesion, an effect also studied extensively for SPARC (4). Hevin
was shown to inhibit the attachment and spreading of endothelial cells
on fibronectin substrates, and it was suggested that hevin modulates
the high endothelial venule cell adhesion and phenotype, thus
facilitating lymphocyte transendothelial migration (5). The effects of
SC1/hevin on cell adhesion are of particular interest due to the fact
that SC1/hevin is down-regulated in many types of cancer cells and may
serve as a negative regulator of cell growth and proliferation (6-8).
Furthermore, expression of SC1/hevin is associated with the migration
of myotomes during somitogenesis in early mouse embryos and undergoes a
rapid down-regulation just before myotome emigration from the somitic
environment (9). In another line of work, SC1/hevin was shown to bind
to B-lymphocyte precursors (10) and to augment B cell lymphopoiesis
(11, 12), effects that are mediated by the N-terminal acidic domain I. Gene targeting of SC1 in mouse did not show any obvious phenotype (13), a finding that is not too surprising considering the number of members
of the BM-40 family that may play a compensatory role.
The major objective of this investigation was to characterize SC1/hevin
at the protein level, to investigate the functionality of its predicted
calcium-binding EC domain, and to identify mechanisms by which it can
be integrated into the extracellular matrix. For this purpose we
expressed full-length SC1 as well as fragments with domain-specific
truncations. These were analyzed for structural features as well as for
the ability to bind calcium and collagen fibrils. The purified SC1
FS-EC domain pair was used to raise an antibody with which we could
demonstrate a colocalization of SC1/hevin with collagen I in the
extracellular matrix of SAOS-2 cells and determine the distribution of
SC1 in many tissues also outside the central nervous system.
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MATERIALS AND METHODS |
Construction of Expression Vectors for Full-length and Truncated
Forms of SC1--
The cDNA clone for mouse SC1 was kindly supplied
by Dr. Peter J. McKinnon. Four cDNA fragments, representing the
full-length SC1, the pair of I and FS domains, the pair of FS and EC
domains, and the EC domain, were generated by PCR using the following
primers: forward primer-1, 5'-GCCCCGCTAGCCCCGACAAGTACAAGGTTC,
reverse primer-2, 5'-CAATGACTGCGGCCGCTCAAAAGAGGAGGTTTTCAT, forward
primer-3, 5'-GCCCCGCTAGCCTCTTGCACGAACTTCCAAT, forward primer-4,
5'-GCCCCGCTAGCCCCTGCTTGTACGGACTTT, and reverse primer-5,
5'-CAATGACTGCGGCCGCTCAAATAGATTTGCAAGCTCCG. Primers 1 and 2 were
used for full-length SC1, primers 1 and 5 were used for the pair of I
and FS domains, primers 3 and 2 were used for the pair of FS and EC
domains, and primers 4 and 2 were used for the EC domain. The primers
introduced new restriction sites (NheI, NotI) and
a stop codon. The NheI/NotI-restricted PCR
products were purified and cloned into the corresponding restriction
sites of pCEP-Pu/BM40 (14) to obtain the final expression vectors pCEP-Pu-SC1, pCEP-Pu-SC1-I-FS, pCEP-Pu-SC1-FS-EC, and pCEP-Pu-SC1-EC. The cDNA fragment of I-FS was additionally cloned in the modified expression vector pCEP-Pu with an N-terminal tag consisting of six
histidines, a Myc epitope, and an enterokinase recognition site for
cleavage of the tag (15). The eukaryotic expression vector pRcCMV
(Invitrogen) was used to produce a modified vector pRcCMV/BM40 (5.5 kilobases). Therefore, a HindIII/NotI fragment containing the signal peptide sequence and some of the 5'-untranslated region of BM-40 (105 bp) together with the full-length SC1 sequence (1.9 kilobases) was cleaved from pCEP-Pu-SC1, purified, and ligated to
the same restriction sites in pRcCMV. The resulting vector was
designated pRcCMV-SC1. The correct insertion and sequences of all
amplified fragments were verified by cycle sequencing of both
strands using an ABI Prism 377 Automated Sequencer (PE Biosystems).
Cell Culture and Transfection--
The human embryonic kidney
cell line EBNA-293 (Invitrogen), which constitutively expresses the
EBNA-1 protein from Epstein-Barr virus was used for transfection with
pCEP-Pu-SC1-I-FS, pCEP-Pu-SC1-FS-EC, and pCEP-Pu-SC1-EC and selected
with puromycin as described (14). The fibrosarcoma cell line HT1080
(kindly provided by Dr. M. Aumailley) was transfected with pRcCMV-SC1.
Two days after transfection the cells were split 1:10, and selection
was started by adding 800 µg/ml G418 (Invitrogen). Resistant clones
were isolated after 4-5 weeks and then screened for expression of SC1
by immunoblotting with an antibody against the N-terminal portion of
SC1 (16). Conditioned serum-free media were stored at
80 °C.
Purification of Full-length SC1--
Conditioned serum-free
medium was dialyzed against 50 mM BisTris, pH 7.0, containing 0.5 mM phenylmethylsulfonyl fluoride and passed
at 4 °C over a DEAE-Sepharose (Amersham Biosciences) column
equilibrated in the same buffer. Fractions that eluted between
0.26-0.33 M NaCl showed immunoreactivity with the antibody to SC1. These were dialyzed against 50 mM BisTris, pH 7.0, and applied to a Q-Sepharose (Amersham Biosciences) column equilibrated in the same buffer. The recombinant protein was eluted at 0.35-0.41 M NaCl. Further purification was carried out on a HiTrap
heparin column (Amersham Biosciences) equilibrated in 10 mM
sodium phosphate, pH 7.0. The protein was eluted at 0.21 M
NaCl. After concentration on a Resource Q (Amersham Biosciences) column
equilibrated in 20 mM BisTris, pH 6.0, the protein was
further purified by gel filtration on Sephadex G-75 (Amersham
Biosciences) in 0.3 M NaCl, 50 mM Tris-HCl, pH
7.4. The SC1 pool was dialyzed against 20 mM BisTris, pH
5.9, and final purification was performed on a Resource Q column
equilibrated in the same buffer. The pure SC1 eluted at 0.32-0.35
M NaCl (linear gradient 0-1 M NaCl) and was
dialyzed against 5 mM Tris-HCl, pH 7.4.
Purification of the I-FS Domain Pair--
Conditioned serum-free
medium was dialyzed against 0.1 M NaCl, 50 mM
sodium phosphate, pH 8.2, and passed over an affinity column with
cobalt Talon Metal Affinity Matrix (Clontech). The protein was eluted at a concentration of 20-70 mM
imidazole. I-FS-containing fractions were further purified by gel
filtration on a column of Sephadex G-75 equilibrated in 50 mM Tris-HCl, pH 7.4. The final pool was dialyzed against 5 mM Tris-HCl, pH 7.4.
Purification of the FS-EC Domain Pair--
Conditioned
serum-free medium was dialyzed against 50 mM Tris-HCl, pH
8.6, and passed over a column of DEAE-Sepharose equilibrated in the
same buffer at 4 °C. The recombinant protein was found in the
flow-through and was, after dialysis against 50 mM sodium acetate, pH 4.9, applied to a column of SP-Sepharose (Amersham Biosciences). The FS-EC protein eluted at 0.35-0.77 M NaCl
and was, after concentration by ultrafiltration (YM-10, Amicon),
further purified by gel filtration on a column of Sephadex G-75
equilibrated in 0.2 M NaCl, 50 mM Tris-HCl, pH
8.6. Fractions containing the FS-EC-protein were dialyzed against 20 mM MES,1 pH 6.0, and subjected to chromatography on Resource S matrix, where it eluted
at 0.25 M NaCl. The final pool was dialyzed against 5 mM Tris-HCl, pH 7.4.
Purification of the EC Domain--
Conditioned serum-free medium
was dialyzed against 50 mM Tris-HCl, pH 8.6, and passed
through a DEAE-Sepharose column at 4 °C. The pH of the flow-through
was adjusted to pH 9.0 and concentrated on a HiTrap Q column (Amersham
Biosciences) equilibrated in 50 mM Tris-HCl, pH 9.0. The EC
domain was eluted at 0.5 M NaCl. Final purification was
carried out by gel filtration on a column of Sephadex G-75 equilibrated
in 5 mM Tris-HCl, pH 7.4.
Verification of Recombinant Proteins--
The identity of all
proteins was verified by MALDI-TOF peptide mass fingerprinting, and in
the case of the FS-EC domain pair and the EC domain alone, also by
Edman degradation (model 473A, PE Biosystems).
N-Glycosidase F and Endoglycosidase H Digestion--
Purified
proteins (20 pmol) were reduced with 0.5% of mercaptoethanol and
denatured by heating at 100 °C for 5 min. After denaturation, the
proteins were incubated with 0.3 units of enzyme per mg of protein for
24 h at 37 °C in the buffers recommended by the supplier (Roche
Molecular Biochemicals). Digested and non-digested samples were
analyzed by MALDI-TOF mass spectrometry.
Mass Spectrometry--
Samples for MALDI-TOF mass spectrometry
were prepared as described (17) with
-cyano-4-hydroxycinnamic acid
(Sigma) as matrix. Detection and analysis of cations was performed
utilizing the high or low mass detector in the linear mode of a Bruker
Reflex III. The time of flight analyzer was calibrated using the
Mr of bovine serum albumin dimer (Sigma) of
132,859.0.
Electron Microscopy of Purified SC1--
Isolated full-length
SC1 (5 µg/ml) was visualized by negative staining with 0.75% uranyl
formate on carbon-coated grids rendered hydrophilic by glow discharge
(18). The samples were examined in a Zeiss E 902 electron microscope.
Circular Dichroism (CD) Spectroscopy--
CD spectra were
recorded in a Jasco model 715 CD spectropolarimeter at 25 °C in
thermostatted quartz cells of 1-mm optical path length. The molar
ellipticity [
] (expressed in
deg·cm2·dmol
1)
was calculated on the basis of a mean residue molecular mass of 110 Da.
The Ca2+ dependence of the CD spectrum was measured by the
addition of 2 mM CaCl2. Reversibility of the
conformational change was tested by subsequent addition of 4 mM EDTA. To improve the signal to noise ratio, five spectra
were accumulated. A base line with buffer (5 mM Tris-HCl,
pH 7.4) was recorded separately and subtracted from each spectrum.
Protein concentrations were determined from UV spectra using extinction
coefficients calculated according to Gill and von Hippel (19). The
program SELCON was used to estimate the content of secondary structure elements.
Fluorescence Spectroscopy--
Intrinsic fluorescence was
measured with a PerkinElmer LS50B spectrofluorometer in 10-mm path
length rectangular cells at 25 °C with excitation at 280 nm.
Emission spectra for the full-length SC1 (0.5 µM) and the
I-FS and FS-EC domain pairs (each 1 µM) were recorded in
5 mM Tris-HCl, pH 7.4, and for the EC domain (1 µM) in 0.15 M NaCl, 50 mM
Tris-HCl, pH 7.4. All spectra were recorded with 2 mM
CaCl2 and after the addition of 4 mM EDTA. The
percent change in fluorescence intensity was calculated as
F350 = 100 × (FCa
FEDTA)/FCa, with
FCa representing the fluorescence signal at
Ca2+ saturation, and FEDTA
representing the signal in the presence of excess EDTA.
Preparation of Antibodies against SC1--
The purified FS-EC
domain pair was used to immunize a rabbit. The antiserum was purified
by affinity chromatography on a column with the antigen coupled to
CNBr-activated Sepharose (Amersham Biosciences).
Binding of SC1 to Reconstituted Collagen I
Fibrils--
Solutions of highly purified chicken collagen I were
prepared in 0.4 M NaCl, 0.1 M Tris-HCl, pH 7.4. Samples were degassed under vacuum and then transferred to
microcuvettes (light path, 1 cm) and mixed with an equal volume of
distilled water at 4 °C. The cuvettes were sealed and placed into a
spectrophotometer (Beckman UV 640, equipped with a multicell holder
Micro Auto 12) connected to a water bath at 37 °C. Aggregation was
followed by monitoring turbidity at 313 nm. When a plateau was reached,
aliquots were spotted onto Parafilm sheets. Nickel grids coated with
Formvar/carbon were floated for 5 min on drops to allow adsorption of
fibrils. The grids were washed with PBS and treated for 30 min with 5% bovine serum albumin in PBS. The adsorbed fibrils were allowed to react
with full-length SC1 or the FS-EC domain pair in 0.15 M
NaCl, 50 mM Tris, pH 7.4, containing 10 mM
CaCl2 for 90 min. After washing 5 times with PBS, the grids
were incubated with the affinity-purified SC1 antibody in PBS
containing 0.2% (w/v) dried skim milk and, after renewed washing with
a suspension of colloidal gold particles (18 nm), coated with goat
antibodies to rabbit IgG (Jackson ImmunoResearch) in PBS containing
0.2% (w/v) dried skim milk. Finally, the grids were washed with
distilled water and negatively stained with 2% uranyl acetate for 10 min. In control experiments SC1 or the first antibody was omitted. Electron micrographs were taken at 80 kV with a Philips CM 10 electron microscope.
Immunofluorescence Microscopy of SaOS-2 Cell Layers--
The
human osteosarcoma cell line SaOS-2 (DSMZ, Braunschweig) was cultivated
in McCoy's 5A medium supplemented with 10% of fetal calf serum, 2 mM L-glutamate, and optionally, 50 µg/ml
sodium ascorbate. For immunofluorescence microscopy cells were plated onto plastic chamber slides, cultivated until they reached confluency, fixed in 2% paraformaldehyde in PBS for 10 min, and blocked in 1%
bovine serum albumin. Immunolabeling for SC1 was done by consecutive treatment for 1 h with the affinity-purified rabbit antibody to SC1 followed by a diaminotriazinylaminofluorescein (DTAF)-conjugated goat antibody against rabbit IgG and for collagen with a mouse monoclonal antibody (Sigma) followed by CyTM3-conjugated
affinity-purified goat antibody against mouse IgG (Jackson
ImmunoResearch). Pictures of the cell layers were taken with a
fluorescence laser scanning microscope (Leica).
Immunoblotting of Tissue Extracts--
Mouse tissues were
homogenized and extracted in 5 volumes (ml/g) 0.15 M NaCl,
50 mM Tris-HCl, pH 7.4, containing a protease inhibitor
mixture (Complete®, Roche Molecular Biochemicals). The homogenates
were centrifuged, and aliquots of the supernatants were submitted to
SDS-PAGE. For immunoblotting, proteins were transferred
electrophoretically to a nitrocellulose filter and developed with the
affinity-purified antibody against SC1, followed by
peroxidase-conjugated swine anti-rabbit IgG (DAKO) and the enhanced
chemiluminescence reagent (Amersham Biosciences). In inhibition
experiments the first antibody was preincubated with 50 µg of the
FS-EC domain pair for 2 h at room temperature and then applied to
an immunoblot of kidney extract and recombinant SC1 protein.
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RESULTS |
Recombinant Production of Full-length and Truncated SC1
Proteins--
Eukaryotic cells were used for protein expression to
ensure proper folding and posttranslational modifications. The use of the BM-40 signal peptide gave targeting through the endoplasmic reticulum and Golgi apparatus and secretion into the medium from which
recombinant proteins could be purified. The human embryonic kidney cell
line EBNA-293 was initially used for expression of all four constructs,
corresponding to full-length SC1, the I-FS and FS-EC domain pairs, and
the EC domain (Fig. 1). Although the shorter constructs were produced at acceptable levels, expression of
full-length SC1 was low in this cell line. As an alternative we
transfected the human fibrosarcoma cell line HT1080 with a plasmid
coding for full-length SC1 and selected stably transformed clones. One
of these showed good SC1 expression and only slight degradation of the
protein and was used for recombinant production of the full-length
protein. In SDS-PAGE full-length SC1 migrated above the 94-kDa marker,
although the calculated peptide mass is
only 70.9 kDa (Fig. 2, Table I). The I-FS
domain pair was cloned with a His-Myc tag and could be purified by
cobalt affinity chromatography. In SDS-PAGE, the protein gave a single
band with an apparent molecular mass of 95 kDa (Fig. 2), in contrast to the calculated mass of 57.6 kDa. The purified FS-EC domain pair gave a
single SDS-PAGE band at 33 kDa, whereas the EC domain migrated as a 17 kDa protein (Fig. 2), in both cases in reasonable agreement with their
predicted molecular masses. Taken together, the results show an
unexpectedly low mobility in SDS-PAGE of the larger proteins, which may
be caused by posttranslational modifications within the I and FS
domains and/or the highly acidic nature of the I domain.

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Fig. 1.
Domain composition of recombinant full-length
and truncated SC1 proteins. FS denotes the
follistatin-like domain, and EC denotes the globular
extracellular calcium binding domain. The black bars within
the EC domain indicate two EF-hand motifs. The open box
represents the acidic N-terminal domain I. Signal peptides as well as
the tag on the I-FS protein have been omitted. fl,
full-length.
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Fig. 2.
SDS-PAGE of purified recombinant SC1
proteins. The purified full-length (fl) SC1
(lanes 1 and 5), I-FStag (lanes
2 and 6), FS-EC (lanes 3 and 7),
and EC (lanes 4 and 8) were submitted to SDS-PAGE
under non-reducing (lanes 1-4) and reducing (lanes
5-7) conditions. The 12% polyacrylamide gel was stained with
Coomassie Brilliant Blue. The molecular mass of marker proteins are
given at the left margin.
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Molecular Mass and Glycosylation of Recombinant Full-length and
Truncated SC1 Proteins--
In an attempt to resolve the cause for
these discrepancies all proteins were analyzed by MALDI-TOF mass
spectrometry either directly or after digestion with
N-glycosidase F or endoglycosidase H (Table I). All
proteins, with the exception of the EC domain, carried
N-glycosidase F-sensitive N-linked glycans.
Full-length SC1 contained about 4.8 kDa of N-glycans, of
which 2.7 kDa were contributed by domain I and about 2.1 kDa by the FS
domain. Potential N-glycosylation sites are located at
Asn-12, -131, and -151 in domain I and at Asn-444 in the FS domain. The
mass of endoglycosidase H-sensitive high mannose/hybrid glycans in SC1
was about 2.4 kDa, with these structures mostly found on the FS domain
(Table I). The masses determined for N-glycosidase
F-digested full-length SC1 and domain I-FS were about 12 kDa higher
than that predicted. Because the mass of the N-glycosidase
F-digested FS-EC domain pair was as expected from sequence, these
12-kDa posttranslational modifications of full-length SC1 and domain
pair I-FS are located in domain I.
Electron Microscopic Appearance of Full-length SC1--
The
purified full-length SC1 was visualized by negative staining electron
microscopy (Fig. 3). SC1 was seen as a
monomeric particle with a length of about 25 nm composed of a globule
with a diameter of 9.5 nm attached to a thread-like structure with a
length of about 15 nm. Because the x-ray structure of the FC-EC domain
pair from the homologous protein BM-40 shows a rather compact fold
(20), the globule is likely to correspond to the FS and EC domains that
make up the C-terminal part of SC1, whereas the thread-like structure
is contributed by the highly acidic N-terminal domain I.

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Fig. 3.
Electron microscopic visualization of
recombinant SC1 after negative staining. Homogeneity of the sample
is demonstrated in the overview (top panel). Selected
particles are shown at higher magnification (middle panels),
and schematic drawings (bottom panels) emphasize the shape
of the particles. The bar corresponds to 75 nm for the
overview (top) and to 25 nm for the enlarged single
molecules (middle).
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Conformation and Calcium Binding of Full-length and Truncated
SC1--
The conformations of the recombinant proteins were analyzed
by circular dichroism in the far-ultraviolet region. Spectra of full-length SC1 and the I-FS domain pair exhibited a marked negative band at 200 nm, indicating a large content of unordered structures (Fig. 4). In contrast the FS-EC domain
pair and the isolated EC domain displayed shoulders at 222 nm typical
for
-helices. Analysis of the CD spectra (Table
II) suggested that the EC domain has the
highest content of
-helix followed by the FS-EC domain pair, which
also shows a high content of
-sheet. Domain I consists largely of
unordered structures, which it contributes to full-length SC1 and the
I-FS domain pair. All four proteins gave similar CD spectra when
equilibrated in 2 mM CaCl2 or 4 mM
EDTA (Fig. 4).

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Fig. 4.
Circular dichroism spectra of recombinant
full-length and truncated SC1. Far UV spectra were recorded at a
protein concentration of 1.97 µM for full-length
(fl) SC1 (A), 2.6 µM for
I-FStag (B), and 7.2 µM for FS-EC
(C) in 5 mM Tris-HCl, pH 7.4. The EC domain was
dissolved in 0.15 M NaCl, 50 mM Tris-HCl, pH
7.4, at a concentration of 5.8 µM (D). The
spectra were recorded after the addition of 2 mM
CaCl2 (dashed line) and subsequent addition of 4 mM EDTA (solid line).
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Table II
Secondary structure of calcium-saturated SC1 proteins calculated from
circular dichroism spectra
Values were calculated using the SELCON software by which fractions are
not normalized to 100% and negative values are permitted. The small
deviation from 100% indicates that the spectra of the SC1 are well
represented by known secondary structure elements (30). fl,
full-length.
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The potential calcium dependence of SC1 conformation was also studied
by intrinsic fluorescence. Upon excitation at 280 nm all proteins
showed emission maxima between 339 and 351 nm that were not changed by
the addition of 2 mM CaCl2 (Fig.
5). However, calcium addition led to a
decrease in fluorescence intensity for all proteins containing the EC
domain, i.e. full-length SC1 (24%), the pair of FS and EC
domains (64%), and the single EC domain (97%) itself. The
conformational change was reversible when EDTA was added in excess. In
contrast, the fluorescence signal of the I-FS domain pair was not
influenced by calcium (results not shown).

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Fig. 5.
Fluorescence emission spectra of recombinant
full-length and truncated SC1. Fluorescence emission spectra were
recorded with excitation at 280 nm in 5 mM Tris-HCl, pH
7.4, for full-length (fl) SC1 (A, 0.5 µM) and FS-EC (B, 1 µM). The EC
domain (C, 1 µM) was measured in 0.15 M NaCl, 50 mM Tris-HCl, pH 7.4. The spectra
were recorded after addition of 2 mM CaCl2
(dashed line) and after the addition of 4 mM
EDTA (solid line).
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Binding of SC1 to Reconstituted Fibrils of Collagen I--
Earlier
studies performed with the homologous protein BM-40/SPARC/osteonectin
showed a distinct binding of this family member to collagen via its EC
domain (21, 22) and a loss of BM-40 from the extracellular matrix of
collagen I-deficient mice (23). Because the EC domain, which has in
BM-40 been shown to carry the collagen binding site, is conserved in
SC1, we decided to test for affinity to collagen I. As collagen I is
present in tissues in fibrillar form, we used reconstituted fibrils
prepared from highly purified collagen I to which SC1 was added.
Electron microscopy showed a marked binding of both recombinant
full-length SC1 and the FS-EC domain pair to such collagen I fibrils
(Fig. 6), whereas control experiments,
performed by omitting SC1 or the antibody to SC1 used for detection,
were negative. The fibrils showed the characteristic periodic banding
pattern and carried SC1 bound to their surface.

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Fig. 6.
Binding of SC1 to reconstituted fibrils of
collagen I. Full-length SC1 (A) and the FS-EC domain
pair (B) were incubated with reconstituted collagen fibrils
made from highly purified collagen I. Binding was detected by
immunogold electron microscopy using the affinity-purified antibody
against SC1. The bar corresponds to 200 nm.
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Colocalization of SC1 and Collagen I in the Extracellular Matrix of
SaOS-2 Osteosarcoma Cells--
To further analyze the relevance of the
interaction between SC1 and collagen I we studied the deposition of the
two proteins in the matrix produced by cultured human osteosarcoma
SaOS-2 cells by immunofluorescence microscopy. An antiserum was raised
in rabbit against the FS-EC domain pair, and specific antibodies were
affinity-purified by binding to the original antigen coupled to
CNBr-Sepharose. The purified antibodies against SC1 showed no
cross-reactivity to other members of the BM-40 protein family,
i.e. BM-40, testican-1, -2, -3, TSC-36, SMOC-1, either in
native or in SDS-denatured form (results not shown).
When ascorbate was present in the SaOS-2 cell culture medium, allowing
secretion of collagens, a filamentous extracellular network was
produced (Fig. 7, A-C).
Immunostaining revealed a colocalization of SC1 and collagen I in large
portions of this network. In the absence of ascorbate neither collagen
I nor SC1 could be detected in the surrounding space, showing that
collagen is required for the deposition of SC1 into the extracellular
matrix produced in these cultures (Fig. 7, D-E). Under
these conditions the intracellular staining for both proteins was
increased, which indicates that SC1 is retained in the secretory
pathway when collagen secretion is abolished due to the lack of
ascorbate.

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Fig. 7.
Immunofluorescence laser scanning microscopy
of SC1 and collagen I in monolayer cultures of SaOS-2 osteosarcoma
cells. Cells were cultivated in the presence (A-C) or
absence of ascorbate (D-F). In A and
D SC1 was detected with affinity-purified rabbit antibodies
raised against the purified FS-EC domain pair and a DTAF-conjugated
affinity-pure goat anti-rabbit IgG (green). In C
and F collagen I (Col. I) was detected with a
mouse monoclonal antibody followed by a Cy3TM-conjugated goat
anti-mouse IgG (red). B and E are
merged images. The bar corresponds to 40 µm.
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Distribution of Different Size Forms of SC1 between Mouse
Tissues--
The tissue distribution of SC1 was studied by immunoblot
analysis of extracts of adult mouse tissues (Fig.
8A). The strongest signals
were found in extracts of brain, cerebellum, and lung, giving a band
migrating at about 116 kDa. Weaker signals with the same apparent
molecular mass were detected in samples from heart, eye, and muscle.
Another prominent band at about 55 kDa was found in brain, cerebellum,
heart, eye, muscle, and kidney. In kidney this was the only form of SC1
detected. To further verify that this lower band represents a true SC1
fragment or variant, recombinant full-length SC1 protein and kidney
tissue extract were tested in an inhibition experiment. A dilution of
the affinity-purified antibodies was preincubated with the recombinant
FS-EC domain pair against which the antibodies were originally raised.
This completely abolished antibody binding to both the full-length recombinant SC1 and to the 55-kDa band from kidney extracts, whereas a
control sample of the same antibody dilution without the addition of
inhibitor gave strong signals (Fig. 8B).

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Fig. 8.
Immunoblot analysis of SC1 in mouse tissue
extracts. A, mouse tissues were extracted with 5 volumes (ml/g) of 0.15 M NaCl, 50 mM Tris-HCl
containing 10 mM EDTA, and protease inhibitors. Aliquots
were applied to 12% SDS-polyacrylamide gels under reducing conditions,
and SC1 was detected by immunoblotting using an affinity-purified
antiserum to SC1. B, recombinant full-length SC1 and kidney
extracts were submitted to SDS-PAGE followed by immunoblotting as in
A. Inhibition of specific antibody binding was performed by
incubation of a dilution of the affinity-purified antiserum to SC1 with
recombinant FS-EC protein, the antigen against which the serum was
originally raised. The control lanes were developed with an identical
antiserum dilution that was not treated with inhibitor.
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DISCUSSION |
SC1 has been proposed to perform a number of important functions
in development and regeneration of the central nervous system (1, 16,
24-26) in the adhesion of high endothelial venule cells (2, 5), in
muscle differentiation (9), and in the maturation of B-lymphocytes
(10-12). However, the structural features of SC1 have not been
characterized beyond sequence interpretation (1, 2), and even though it
is described as an extracellular matrix protein, its mode of
integration into extracellular matrices has not been investigated. We
therefore undertook the systematic expression of full-length and
truncated SC1 proteins to be able to determine some of their important
structural features and to provide a basis for future mechanistic
studies of SC1 functions. Two important properties of BM-40 are the
functionality of the EC domain in calcium binding and the ability to
bind fibrillar collagens. Therefore, we investigated if both are
conserved in SC1. To determine whether a collagen binding activity can
be of physiological relevance, we studied SC1 to determine of it occurs in close association with fibrillar collagens and is expressed in
tissues rich in a collagenous matrix.
Full-length SC1 was recombinantly expressed in the human fibrosarcoma
cell line HT1080 and the truncated SC1 proteins (Fig. 1) in the human
embryonal kidney cell line EBNA-293. Both of these cell types have been
used frequently for the expression of other mammalian proteins and have
proven to reliably provide a correctly folded product with the right
pattern of disulfide bonds. We recently compared the glycosylation
pattern of human bone sialoprotein expressed in EBNA-293 cells with
that of the same protein purified from human bone and found that,
although differences were detected in both N- and
O-linked glycans, functional properties such as enhancement
of cell attachment and hydroxyapatite binding were retained in both
forms (15). Because there is no abundant tissue source for
purification, it has not been possible to perform an equally detailed
analysis of SC1. Nevertheless, the consistency of the mobilities of the
tissue-derived and the recombinant full-length protein in SDS-PAGE
(Fig. 8) indicate considerable similarities in processing.
Both full-length SC1 and the I-FS domain pair showed a lower mobility
in SDS-PAGE than expected from the molecular mass calculated from the
amino acid sequence. A part of the discrepancy could be explained by
the presence of N-glycans on both the I and the FS domain
(Table I). Digestion with endoglycosidase H indicated that the
N-glycans on the FS domain are mainly of high mannose or
hybrid type, whereas those on domain I are predominantly of complex
type. However, analysis of SC1 proteins that had been digested with
N-glycanase F with MALDI-TOF mass spectrometry indicated the
presence of a further ~12 kDa of posttranslational modifications. Likely candidates are O-glycosidically linked
oligosaccharides and, with an algorithm for prediction of
O-glycosylation sites (NetOGlyc) (27), eight potential
O-glycosylation sites encompassing six threonines and two
serines are predicted within domain I. As expected from the MALDI-TOF
analysis, no O-glycosylation sites are predicted for the
FS and the EC domains. The most likely explanation for the
discrepancies in measured molecular mass determined by SDS-PAGE (116 kDa) and MALDI-TOF mass spectrometry (88 kDa) is that the highly
negatively charged domain I (106 acidic residues of 400 residues)
causes an atypical binding of SDS to SC1, leading to an anomalous
electrophoretic behavior.
The predicted domain structure of SC1 could be partially confirmed by
electron microscopy of recombinant SC1. The full-length protein is seen
as a flexible tail of about 15-nm length connected to a globular
structure of 9.5-nm diameter (Fig. 3). The x-ray structure of the FS-EC
domain pair from BM-40 shows a comparatively compact fold and a close
interaction between these domains, yielding a roughly globular
structure of about the same dimensions as the SC1 globule seen by
electron microscopy (20). Accordingly, the flexible tail must be the
domain I, a conclusion that is also supported by the high content of
unordered secondary structure in this domain (see below). This domain
has been shown to increase the survival and proliferation of B lineage
lymphocytes and their precursors (10-12). Its extended flexible
structure will make it well available to cells also if SC1 is anchored
in the extracellular matrix. It may be that its high negative charge
density and potential O-linked glycosylation plays a role in
its receptor interactions.
Circular dichroism spectroscopy showed that the EC domain is rich in
-helix and that the FS domain has a comparatively high content of
-structure (Fig. 4, Table II). This is in good agreement with the
detailed knowledge of the three-dimensional structure of the homologous
domains in BM-40 (28, 20) and structure-based sequence alignments. On
the other hand, the domain I contains largely unordered secondary
structure, in agreement with its thread-like appearance in electron
microscopy. The circular dichroism spectra obtained in the presence of
saturating amounts of calcium or with excess EDTA were closely similar,
pointing to a rearrangement of secondary structure in SC1 upon calcium
binding that is too small to be detected by circular dichroism. In
contrast, large and reversible changes in intrinsic fluorescence were
detected for all SC1 proteins containing the EC domain when calcium was added and removed (Fig. 5). This demonstrates that the SC1 EC domain is
active in calcium binding, which is an agreement with the fact that
both EF-hands are conserved when compared with BM-40. EF-hand 2 is a
classic EF-hand as seen in multiple cytosolic EF-hand proteins in which
the acidic side chains of residues at position 1, 3, 5, and 12 within
the EF-hand loop mainly contribute to calcium binding. The EF-hand 1 of
BM-40 is of a non-classical type as it contains a one-amino acid
insertion and a proline in cis conformation in the EF-hand loop. This
leads to a structural rearrangement such that the carbonyl oxygen of
the proline coordinates the calcium ion instead of an oxygen from the
side chain of residue 3 (28). In SC1 the residues of EF-hand 1 including the insertion and the proline are strictly conserved compared
with BM-40; therefore, calcium coordination is most probably identical
to the one seen in BM-40. The fluorescence change upon calcium addition
further shows that the calcium binding induces a conformational change even though this could not be detected by circular dichroism. Calcium-induced conformational changes as well as a specific
proteolytic cleavage that may have related structural consequences have
been shown to enhance the binding of BM-40 to collagen (21, 22, 29).
Only the I-FS domain pair was unaffected by calcium binding, as
determined by intrinsic fluorescence, making it likely that these
domains do not bind calcium with high affinity or that calcium binding
does not strongly affect their fold.
BM-40 has been shown to bind strongly to several collagens (21, 22),
and Mov13 mice, which are deficient in collagen I, do not retain BM-40
in their extracellular matrix (23). We therefore determined if the
collagen affinity of BM-40 is conserved in SC1 and if this may indeed
be a mechanism by which SC1 is anchored in extracellular matrices.
Immunogold electron microscopy showed that both full-length SC1 and the
FS-EC domain pair bind to the surface of fibrils reconstituted from
purified collagen I (Fig. 6). Furthermore, when monolayers of SaOS-2
osteosarcoma cells, which express both SC1 and collagen I, were studied
by double immunofluorescence microscopy, a close colocalization between these proteins was seen in filamentous structures in the extracellular matrix (Fig. 7, A-C). When the same cell line was grown in
the absence of ascorbate, resulting in intracellular retention of collagen due to decreased activity of the prolyl hydroxylase, such
extracellular deposition of SC1 was lost in parallel with that of
collagen I (Fig. 7, D-F). Indeed, the intracellular
staining for both proteins was increased, creating the impression that SC1 was retained in compartments of the secretory pathway when collagen
I could not be processed and externalized. It may be that complexes
between collagen I and SC1 are formed already in the endoplasmic
reticulum or in the Golgi apparatus.
A screen performed by immunoblot of tissue extracts showed a broad
distribution of SC1, although the largest amounts were found in brain
(Fig. 8). The only two organs in which no SC1 could not be detected by
extraction and immunoblot was liver and adrenal gland, tissues
characterized by a high cellularity and a low collagen content. In
tissue extracts, SC1 was detected mainly as an SDS-PAGE band at 116 kDa, corresponding to the mature protein. However, some lower bands
were also specifically stained, and one of these, at 55 kDa, was the
major SC1 reactivity in muscle and the only band in kidney. There is no
evidence in the literature for alternative splicing of SC1 mRNA,
and it is, therefore, most likely that the 55-kDa band results from a
proteolytic cleavage that is particularly prominent in certain tissues.
Because the antibodies were raised against the purified FS-EC domain
pair, the fragment must contain epitopes from these domains.
Considering the flexible and unordered nature of domain I, it is
tempting to propose a cleavage in this domain, releasing a large part
of it from the rest of the molecule. Because domain I has been
implicated in interactions with B-lymphocyte precursor cells (11), it
may be that cellular interactions of SC1 may be modulated by such cleavage.