From the M. E. Müller Institute, University of Bern, CH-3010 Bern, Switzerland
Received for publication, October 6, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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The N terminus of chicken collagen XIV is subject
to alternative splicing. The longer isoform contains a fibronectin type III (F3) domain at its N terminus, whereas the shorter isoform is
lacking this domain. Alternative splicing of the F3 domain is
developmentally regulated. At early embryonic stages, both isoforms are
expressed, whereas after hatching only the longer isoform is expressed.
When immobilized on plastic dishes, the recombinant F3 domain promotes
the adhesion of mesenchymal cells. Attachment to this domain is
specifically inhibited by heparin but not by other glycosaminoglycans.
Molecular modeling studies illustrate that the first F3 domain harbors
a positively charged groove, which may accommodate the negatively
charged heparin chain. Site-directed mutagenesis of a single lysine
residue within this groove abolishes the cell binding activity but does
not affect the heparin binding activity. Cell binding and heparin
binding are therefore two functionally distinct properties shared by
the N-terminal F3 domain. When full-length collagen XIV polypeptides that either contain or lack the first F3 domain are tested on heparin-Sepharose, a pronounced difference in their relative affinity is observed. Thus, alternative splicing of the N-terminal F3 domain influences the interaction of this FACIT (fibril-associated collagens with interrupted triple helices) collagen with cells and with glycosaminoglycans.
The biochemical properties of interstitial collagen fibrils are
modulated by a distinct family of adapter proteins, which add new
functional domains to the fibrils and which may control their precise
architecture. This family is represented by the fibril-associated
collagens with interrupted triple helices
(FACIT)1 and includes
collagens IX, XII, XIV, XVI, and XIX (for review see Refs. 1, 2).
We have been interested for some time in the elucidation of the
structure and function of collagen XIV (3, 4). This homotrimeric
protein is expressed in every tissue that contains type I collagen (1,
5, 6). It is associated with the surface of interstitial collagen
fibrils (7, 8), although a direct interaction with type I collagen
could not be demonstrated (1, 9, 10). The complete primary structure of
chicken collagen XIV has been established by cDNA cloning (4, 11). These studies revealed a protein with two short collagenous helices (COL1 and COL2) and three noncollagenous domains (NC1, NC2, and NC3).
The extended, N-terminal NC3 domain makes up the majority of the entire
protein (80%), whereas the two collagenous helices contribute only
14% to the molecular mass. The NC3 domain has a complex, modular
structure comprising eight F3 modules related to fibronectin, two VA
domains related to von Willebrand factor, and one TspN domain
related to thrombospondin. Undulin, a large glycoprotein isolated from
human placenta, has recently been identified as the human homologue of
chicken collagen XIV (3, 7, 12, 13).
Collagen XIV interacts with a broad palette of extracellular matrix
molecules and a few of these interactions have been assigned to a
particular domain of the polypeptide chain. Collagen XIV binds to
collagen VI (9). This interaction seems to be accomplished by the
collagenous domain, because the triple helices of collagens VI and XIV
have about the same length, allowing a lateral aggregation. Collagen
XIV binds to procollagen N-proteinase (14). In this way the proteinase
is immobilized in close vicinity to type I collagen fibrils, where it
could control the processing and incorporation of newly synthesized
molecules. Collagen XIV also interacts with glycosaminoglycans and
proteoglycans (9). Two binding sites have been described for heparin,
one in the C-terminal NC1 domain and one at the opposite end in the
N-terminal NC3 domain. Although the interaction with the NC1 domain has
been characterized in great detail (15, 16), not much is known about
the interaction with the NC3 domain except that it is facilitated by a
stretch of basic amino acids located between the first F3 repeat and
the adjacent VA domain (17). Collagen XIV also interacts with the small
proteoglycan decorin (18, 19), and it is likely that the two binding
sites identified for heparin are also involved in this interaction.
Collagen XIV has the potential to bind to cell surfaces. In
vitro, it promotes the adhesion of various mesenchymal and
epithelial cells to plastic dishes. For human fibroblasts, a
chondroitin/dermatan sulfate form of CD44 has been proposed as a cell
surface receptor (20). Another study demonstrated adhesion of
hematopoietic cells to collagen XIV and suggested a heparan sulfate
proteoglycan as cell surface receptor (21). In contrast to these
reports, an earlier study did not find any interaction between collagen
XIV and nine different cell lines (9).
The apparent discrepancies between different reports about the
interaction of collagen XIV with cells might be explained, at least in
part, by the existence of different protein isoforms. Several isoforms
that are likely to be generated by alternative splicing have been
described for chicken collagen XIV. We reported the existence of two
splice variants that possess distinct C-terminal domains (4). These
variants differ by the inclusion or skipping of 93 nucleotides encoding
31 amino acids of the NC1 domain. Another splice variant concerns the
sixth F3 repeat in the center of chicken collagen XIV. This F3 repeat
either contains or lacks an insert of the three amino acids Val-Arg-Thr
(22). Two additional transcripts with alternative 5'-regions have been
identified by Gerecke et al. (11). Because these transcripts
differ only in the 5'-untranslated region, they encode the same polypeptide.
A striking variation is also observed at the N terminus of the two
sequences published for chicken collagen XIV. Compared with our
cDNA sequence (4), the sequence reported by Gerecke et
al. (11) lacks a portion of ~350 bp coding for an F3 domain. So
far it is not known whether this difference is simply a cloning artifact or whether it occurs under physiological conditions. We
therefore set out to investigate whether the two variants are the
result of alternative splicing and whether the encoded polypeptides have distinct biological properties.
RT-PCR--
Poly(A)+ RNA was isolated from various
embryonic and adult chicken tissues by the guanidinium thiocyanate
method as described previously (22). The RNA was transcribed into
cDNA with avian myeloblastosis virus reverse transcriptase
(Roche Molecular Biochemicals) and primer P1 (see Table I). The
single-stranded cDNA was amplified by PCR through 36 cycles of 1'
at 95 °C, 2' at 50 °C and 3' at 72 °C using Pfu polymerase
(Stratagene) and primers P2 and P3. The products were resolved on a 1%
agarose gel stained with ethidium bromide. Bands of interest were
subcloned into the plasmid pcDNA3.1( Expression of GST Fusion Proteins--
The cDNA sequences
coding for the domains F3, VA, F3+VA, as well as the fifth and eighth
F3 domain of chicken collagen XIV were amplified by PCR using 1 ng of
template and a pair of strand-specific primers as outlined in Table I.
The reaction products were subcloned into the expression vector pGEX-5X
(Amersham Pharmacia Biotech) in the desired reading frame downstream of
the GST gene. For this purpose, suitable restriction sites had
been introduced into the PCR primers. Orientation and reading frame of
the final products were verified by DNA sequencing. The plasmids were
transfected into competent host cells (Escherichia coli
BL21) and expressed after induction with
isopropyl- Gel Electrophoresis and Immunoblotting--
Purity and size of
the expressed proteins were analyzed on SDS-polyacrylamide gels
according to the method of Laemmli (23). For particularly small
proteins, Tricine-SDS-polyacrylamide gels were used (24). After
transfer to nitrocellulose by electroblotting, the proteins were
detected with the GST detection module (Amersham Pharmacia Biotech)
using goat anti-GST antibodies, followed by alkaline-phosphatase-conjugated secondary antibodies (Sigma Chemical Co.). The color reaction was performed with bromochloroindolyl phosphate and nitroblue tetrazolium substrate.
Site-directed Mutagenesis--
The codons for individual amino
acids of the first F3 domain were mutated by the ExSite PCR-based
mutagenesis method (Stratagene). The forward primer harbored the
desired mutation, while the reverse primer was phosphorylated at its
5'-end and selected in a way that it annealed directly adjacent to the
5'-end of the forward primer (Table I).
To improve the efficiency of amplification, the template cDNA was
cloned into pBluescript SK+ and all mutations were performed in this
relatively small plasmid. After amplification through 21 cycles of 1'
at 95 °C, 2' at the optimal annealing temperature of the primer pair
used (54-62 °C), and 8' at 72 °C, the maternal DNA was removed
by digestion with the restriction enzyme DpnI (Roche
Molecular Biochemicals). The ends of the linear products were joined by
ligation with T4 DNA ligase (Roche Molecular Biochemicals), and the
circular, nicked plasmids were transfected into competent bacteria
(E. coli XL-1 blue). Authenticity and reading frame of all
mutated clones were verified by DNA sequencing. Finally, the inserts
with the desired mutation were subcloned into the
EcoRI/XhoI site of the expression vector pGEX-5X
and expressed in E. coli BL21 as described above.
Cell Culture--
Cell lines were purchased from the American
Type Culture Collection (ATCC, Manassas, VA): U266 (TIB-196), U937
(CRL-1593.2), KG-1a (CCL-246.1), HT1080 (CCL-121). Primary chicken
fibroblasts (CTF) and primary chicken smooth muscle cells (CSM)
were prepared from tendons and gizzards of 17-day-old chicken embryos
with the help of collagenase (Roche Molecular Biochemicals) as
described previously (25). All hematopoietic cells (U266, U937, KG-1a) were cultivated in RPMI 1640 supplemented with 10% fetal bovine serum,
100 units/ml penicillin, and 100 µg/ml streptomycin. All adhesive
cells (HT1080, CTF, CSM) were propagated in Dulbecco's modified
Eagle's medium containing the same supplements.
Cell Adhesion Experiments--
Two different assays were used to
determine the cell binding activity of GST fusion proteins. For the
method of Klein et al. (21), water droplets (5 µl)
containing 10 pmol of fusion protein were spotted onto virgin plastic
plates (Falcon 1007, 60-mm diameter) and air-dried. Residual binding
sites of the plates were blocked with bovine serum albumin (10 mg/ml in
RPMI 1640 medium). Cells were seeded onto the washed plates
(107 cells/plate) and allowed to attach for 1 h at
37 °C. Nonadherent cells were removed by three gentle washing steps
with phosphate-buffered saline, and adherent cells were inspected under
the microscope.
For the second method described by Ehnis et al. (20), the
wells of a 96-well microtiter plate were coated with fusion proteins (1 µg/100 µl) for 4 h at 37 °C. Remaining sites were blocked
with bovine serum albumin (10 mg/ml in RPMI 1640 medium). Approximately 105 cells in 100 µl of RPMI 1640 medium were added to
each well and, after an incubation period of 1 h at 37 °C,
nonadherent cells were washed off with RPMI 1640 medium. The wells were
subsequently incubated at 37 °C with 50 µl of a solution
containing 3.75 mM p-nitrophenol-N-acetyl-
In some experiments the coated plastic plates were incubated with
various glycosaminoglycans (0.01-100 µg/ml, heparin, heparan sulfate, and decorin from Sigma; chondroitin sulfate and hyaluronic acid from Fluka) prior to the addition of cells.
Molecular Modeling--
The three-dimensional structure of
several F3 domains was modeled with the help of a computer using the
Swiss-Model Version 1.1 program (26). The tenth F3 domain of
fibronectin was used as template (27). Homology modeling, superposition
and energy minimization were done as previously described (22).
Circular Dichroism and Fluorescence Spectroscopy--
The
circular dichroism spectrum of purified F3 domains was recorded over a
range of 190-250 nm in 50 mM sodium phosphate buffer, pH
7.5 (10 µg/100 µl). For fluorescence measurements, the proteins (5 µg/ml) were analyzed in a luminescence spectrometer set at 280-nm
excitation and 290- to 400-nm emission. These experiments were kindly
performed by Drs. T. Kiefhaber and O. Bieri (University of Basel).
Full-length Constructs--
Two different full-length constructs
were prepared using the original cDNA clones 113, 8, and 9 (3-4)
as well as the two XbaI/EcoRI fragments that
either contain or lack the sequence of the first F3 domain as outlined
above (RT-PCR). The final constructs harbored the following fragments
in pcDNA3.1( Heparin Binding Studies--
Bacterially expressed fusion
proteins as well as in vitro translated full-length
constructs were tested for their interaction with heparin-Sepharose
CL-6B (Amersham Pharmacia Biotech) in 20 mM Tris-HCl, pH
7.4. The protein solutions (100 µg of fusion protein or 75 µl of
translation mixture) were applied at room temperature to the
equilibrated column (bed volume 500 µl) and eluted stepwise with Tris
buffer containing 0, 100, 200, 300, 400, 600, 800, 1000, 1500, and 2000 mM NaCl. The NaCl concentration of the effluent was
determined with a CDM3 conductivity meter (Radiometer, Copenhagen), and
the radioactivity was monitored with a Tri-Carb liquid scintillation counter (Canberra Packard). Proteins in the effluent were analyzed by
SDS-polyacrylamide gel electrophoresis. The gels were either processed
for immunoblotting with anti-GST antibodies or dried and exposed to
Kodak MR x-ray film (Rochester, NY).
Alternative Splicing of the N-terminal F3 Domain--
Comparison
of the two cDNA sequences available for chicken collagen XIV
revealed a striking difference at the 5'-end. The sequence of
Wälchli et al. (4) contained a segment of 348 bp
inserted after the sequence corresponding to the signal peptide (Fig.
1). This segment coded for 116 amino
acids forming an F3 domain and a short linker. In the sequence of
Gerecke et al. (11) this insert was missing and the region
of the signal peptide was directly followed by the VA domain.
Because the inserted segment corresponded to a complete protein module
and the reading frame was maintained, it seemed likely that the two
forms were generated by an alternative splicing event. We therefore
analyzed the exon/intron structure of the collagen XIV gene at this
region. So far the chicken gene has not yet been isolated and
characterized, but the human gene has been sequenced to a great extent
in the course of the human genome project. We therefore analyzed the
exon/intron structure of the human gene. This gene is located on
chromosomal band 8q23 (28), which is covered to a large extent by a
BAC clone (GenBankTM accession number AC020603) and by several
GSS tags (GenBankTM accession numbers AQ308761, AQ466836, and
AQ700646). The unrefined sequences of these clones demonstrated
that the first F3 domain is encoded by two separate exons that are
followed by a short exon for the linker and by four exons for the VA
domain (Fig. 1C and Table II).
All splice boundaries conform to the ag/gt rule. The 348-bp insert exactly spans exons 2, 3, and 4, lending strong support to the
notion that the two isoforms are the result of alternative splicing.
All introns in this region of the gene have a length of 1-4 kb, except
for the intron separating exons 4 and 5. This intron is nearly 30 kb
and may contain regulatory sequences required for alternative splicing.
In an effort to verify these results for the chicken collagen XIV gene,
genomic PCR was performed utilizing exon specific primers and genomic
DNA from chicken fibroblasts (not shown). Introns 2 and 3 were
successfully amplified, and their DNA sequences confirmed the location
of the splice boundaries deduced from the human gene. Intron 4, however, could not be amplified, suggesting that this intron must also
be extremely large in the chicken gene.
Tissue Expression of the Two Isoforms--
The expression of the
two isoforms was studied by PCR using cDNA transcribed from poly(A)
RNA of various chicken tissues. The forward primer corresponded to a
sequence immediately preceding the signal peptide, the reverse primer
corresponded to a sequence within the second F3 repeat (see Fig. 1). A
PCR product of 1115 bp was expected for the longer splice variant,
whereas a product of 767 bp was expected for the shorter splice variant
lacking the F3 domain and the linker. A prominent band of ~1100 bp
indicative of the longer variant was obtained with RNA from eight
different embryonic tissues, including skeletal muscle, heart, gizzard, skin, liver, brain, sternum, and calvaria (Fig.
2, left). Most of the samples
also contained a band of ~800 bp indicative of the shorter splice
variant. However, the intensity of this band was considerably weaker
and varied among the samples investigated. It was prominent in the
sample from brain but barely detectable in the samples from sternum and
calvaria. For a detailed analysis, the two PCR products were subcloned
and sequenced. The sequence of the larger band was identical with that
published by Wälchli et al. (4), the sequence of the
shorter fragment was identical with that published by Gerecke et
al. (11). Thus, the difference between the two published cDNA
sequences reflects the result of inclusion or skipping of three
exons.
Striking differences were observed when the relative amount of the two
isoforms was analyzed at different developmental stages (Fig. 2,
right). In skeletal muscle and gizzard of embryonic day 10, the shorter isoform was expressed at nearly the same level as the
longer isoform. In samples from adult animals, however, the shorter
variant was not detected at all. Thus, the expression of the shorter
variant without N-terminal F3 repeat is developmentally regulated.
Cell Adhesion to the N-terminal F3 Repeat--
To search for
functional differences between the two splice variants, the N-terminal
F3 repeat (residues 28-124), the N-terminal VA domain (residues
146-344) as well as the combined F3+VA domains (residues 28-344) were
expressed in bacteria as GST fusion proteins. The affinity-purified
fusion proteins were tested for their activity to promote cell
adhesion. Two different cell binding assays previously described in the
literature were employed (17, 21). For a qualitative assay, the fusion
proteins were spotted onto virgin plastic plates and air-dried. Control
experiments demonstrated that all the three fusion proteins adsorbed
equally well to the plastic surface. Cells were seeded onto the coated
plastic dishes and allowed to attach. Nonadherent cells were removed by
several washing steps, and adherent cells were inspected under the
microscope. For a more quantitative assay, a multiwell plate was coated
with the fusion proteins and a cell suspension was applied to the
coated wells. After a short incubation period, nonadherent cells were washed off and adherent cells were quantified by measuring the activity
of the endogenous enzyme hexosaminidase. Although the amount of protein
bound to the plastic surface may differ substantially between the two
methods, both assays yielded very similar results.
In a first set of experiments, six different cell types were tested,
namely, chicken tendon fibroblasts (CTF), chicken smooth muscle cells
(CSM), human fibrosarcoma cells (HT-1080), human plasmocytoma cells
(U266), human myeloblastic cells (KG-1a), and human promonocytic cells
(U937). Four of these cell types (CTF, HT-1080, U266, and KG-1a)
readily adhered to the F3 fusion protein, whereas two (CSM and U937)
did not bind at all (Fig. 3). The same four cell types also adhered to the F3+VA protein, but none of the
cells bound to the VA fusion protein alone. Thus, the alternatively spliced F3 domain promotes attachment of several hematopoietic and
fibroblastic cells. Adhesion to the F3 fusion protein was dependent on
the correct conformation of the protein, because it was abolished when
the fusion protein was heat-denatured prior to application to the
plastic surface (not shown). No adhesion was observed to the GST
protein alone that was lacking the F3 domain. Furthermore, the ability
to promote cell adhesion was specific for the first F3 domain of
collagen XIV. It was not detected with control proteins containing
either the fifth or the eighth F3 domain of chicken collagen XIV fused
to GST. These results demonstrate that fibroblasts and some
hematopoietic cells possess specific cell surface receptors that
interact with the alternatively spliced F3 domain of collagen XIV.
To test whether cell binding was modulated by glycosaminoglycans, we
examined the effect of heparin, heparan sulfate, chondroitin sulfate,
hyaluronic acid, and decorin. Heparin completely inhibited the adhesion
of U266 cells to the F3 and the F3+VA fusion proteins when applied to
the precoated dishes prior to the addition of cells (not shown). The
inhibitory effect was concentration-dependent. Half-maximal
inhibition was observed at ~0.03 µg/ml with U266 cells and the F3
fusion protein. Heparan sulfate, chondroitin sulfate, and hyaluronic
acid showed no effect up to a concentration of 50-100 µg/ml.
Likewise, decorin, which consists of a core protein (Mr 40,000), and one chondroitin/dermatan
sulfate chain (Mr 60,000) had no effect when
used at 1 µg/ml. At a higher concentration of 30 µg/ml, decorin
partially inhibited cell binding. The interaction of collagen XIV with
its cell surface receptor is therefore modulated by heparin or by a
heparin-containing proteoglycan.
Site-directed Mutagenesis of the F3 Domain--
To study the
interaction of the alternatively spliced F3 domain with cells in
detail, we decided to replace individual amino acids by site-directed
mutagenesis. To determine which residues to replace, the
three-dimensional structure of the N-terminal F3 domain with adjacent
linker was predicted by computer modeling studies using the tenth F3
domain of human fibronectin as template. These protein modeling studies
revealed a structure with four clusters of positively charged residues
that formed a prominent groove, which theoretically could accommodate a
negatively charged polysaccharide chain (Fig.
4). No negatively charged residues that
would repel the negatively charged polysaccharide chain occurred within
this region of the F3 repeat. A similar cradle-like structure has
previously been proposed to be involved in the heparin-binding activity
of the tenth F3 domain in fibronectin (29).
Four of the positively charged amino acids, namely Arg-35, Arg-37,
Lys-51, and Lys-54, were selected for site-directed mutagenesis. The amino acids were replaced either separately or in combination by
serine, which possesses an uncharged, hydrophilic side chain. The
mutated fusion proteins were expressed in bacteria and tested for their
cell binding activity as described above. Mutation of Lys-51 to Ser
completely abolished binding of the F3 protein to U266 cells (Fig.
5). In contrast, none of the other three
single amino acid mutations (Arg-35
To exclude the possibility that the mutated F3 domain was inactive in
cell binding, because it had adopted an incorrect conformation, circular dichroism and fluorescence spectroscopy experiments were performed. The wild type and the Lys-51 Binding of the N-terminal F3 Domain to Heparin--
Because
heparin inhibits the interaction of the F3 domain with cells, we also
examined a direct binding of the F3 domain to heparin. The GST fusion
protein as well as its mutated form (Lys-51 Full-length Constructs of Collagen XIV--
To verify our results
obtained with the isolated F3 domain in the context of the entire
collagen XIV polypeptide, we prepared full-length cDNA constructs.
Five of the original 19 overlapping cDNA clones for collagen XIV
were assembled to yield two full-length clones, one corresponding to
the longer splice variant as described by Wälchli et
al. (4), the other corresponding to the shorter variant lacking
the F3 domain as described by Gerecke et al. (11). The two
full-length constructs were expressed in a coupled
transcription/translation system in the presence of radioactively
labeled methionine. In both cases, a prominent polypeptide was obtained
that migrated on a polyacrylamide gel with an apparent mobility of
~200 kDa (Fig. 7). The variant lacking
the N-terminal F3 repeat (predicted molecular mass of 190 kDa)
migrated slightly faster than the variant containing this repeat
(predicted molecular mass of 200 kDa). Thus, the full-length
polypeptides can successfully be prepared in vitro in
analytical amounts. So far, however, we have not been able to prepare
chemical amounts of these polypeptides, either in a prokaryotic or in a
eukaryotic expression system. Curiously enough, the full-length
cDNA clones appeared to be unstable even in a prokaryotic
expression system.
The two full-length polypeptides translated in vitro were
applied to a heparin-Sepharose column and eluted with a stepwise salt
gradient as outlined above. As shown in Fig. 7, both polypeptides bound
to the column but they exhibited clearly different elution profiles.
The longer splice variant with the N-terminal F3 domain eluted as a
broad peak at an ionic strength corresponding to ~500 mM
NaCl. The shorter splice variant without N-terminal F3 domain eluted as
a sharp peak at ~150 mM NaCl. Thus, the longer splice variant has a significantly higher affinity for heparin than the shorter variant.
Finally, we tried to demonstrate binding of the full-length
polypeptides to cells. For this purpose, radiolabeled translation products were incubated with a suspension of U266 cells. After washing,
the radioactive material bound to the cells was solubilized in SDS
sample buffer and analyzed on a polyacrylamide gel. Unfortunately, these experiments did not yield any conclusive results. The cells did
not bind significantly more radioactivity than background controls, and
the bound material, when analyzed on polyacrylamide gels, did not
migrate any more in the 200-kDa region, suggesting that unspecific
degradation had occurred.
Several recent reports provide compelling evidence that chicken
collagen XIV occurs in many differentially spliced variants, each of
which may fulfill subtly different functions. We have studied here two
variants that differ at their N-terminal end. The longer variant
contains an F3 domain and is expressed in virtually all tissues and at
every developmental stage investigated. This isoform seems to represent
the major form of collagen XIV. Another variant lacking the N-terminal
F3 repeat and starting with a VA domain is primarily expressed in
embryonic connective tissues but not in cartilage and bone. It shows a
pronounced down-regulation during development and is virtually
undetectable after hatching.
Together with other studies, our results demonstrate that chicken
collagen XIV contains at least three regions, which are subject to
alternative splicing: the first F3 domain at the N terminus (4, 11, this report), a 3-amino acid segment in the center of the molecule
(22), and a 31-amino acid segment in the C-terminal NC1 domain (4, 30).
Theoretically, alternative splicing of these three regions could give
rise to eight different collagen XIV isoforms. Whether all these
isoforms are expressed simultaneously or whether there is some way of
coordinated regulation remains to be determined.
Collagen XIV is closely related to collagen XII. Collagen XII also
occurs in several isoforms (31, 32) that differ at the N terminus (NC3
domain) as well as the C terminus (NC1 domain). At the N terminus, a
large region encompassing eight F3 repeats and two VA domains is
subject to alternative splicing, whereas at the C terminus, a long NC1
domain with 74 amino acids or a short NC1 domain with 19 amino acids is
expressed. In this case, coordinated regulation of alternative splicing
has been suggested based on the observation that the expression pattern
of the longer NC1 variant is very similar to that of the longer NC3
variant (31). To perform these studies, the authors prepared
full-length cDNA molecules for collagen XII with the help of
specific primers for the two NC1 isoforms. Analogous experiments with
collagen XIV have not been successful. We have not been able to prepare full-length cDNA transcripts for collagen XIV that extended all the
way from the region of the NC1 domain to that of the first F3 domain
(4). In fact, our results suggested that the structure of the mRNA
at the region coding for the first F3 domain must be unusual, because
it could not be cloned from commercial cDNA libraries but required
cloning from a special primer extension library (4).
What is the physiological role of the different collagen XIV isoforms?
Because collagen XIV seems to interact with interstitial collagen
fibrils via its COL1 and NC1 domains, alternative splicing within the NC1 domain may affect this interaction. The longer NC1
isoform contains an uncharged region inserted into the otherwise highly
charged NC1 domain (4, 30). It was therefore speculated that the longer
NC1 variant may favor a strong interaction with the surface of the
collagen fibrils and their tapered ends, whereas the shorter isoform
may have a lower affinity (30). The function of the two isoforms
containing or lacking the three-amino acid insert is less clear.
Computer modeling studies suggested that the extra amino acids cause an
increase in a flexible loop connecting two More is known about the putative function of the isoforms containing or
lacking the first F3 domain. We as well as another research group (17)
could demonstrate that this F3 domain possesses cell binding
properties. The isoform without N-terminal F3 domain will therefore
lack the capability of interacting with cells. Cell binding to collagen
XIV has been observed with hematopoietic (21) as well as fibroblastic
cells (17, 20), but not with smooth muscle cells (this report). The
interaction is inhibited by heparin at concentrations as low as 0.03 µg/ml. It is also inhibited by other glycosaminoglycans but
considerably higher concentrations (1000 µg/ml for heparan sulfate,
30 µg/ml for decorin) are required to obtain half-maximal inhibition
(17, 20, this report). Heparin does not only inhibit cell binding, but
also interacts directly with the F3 domain and requires 500 mM NaCl for displacement. A second heparin-binding site has
been identified by Font et al. (18) at the opposite end of
the collagen XIV molecule in the NC1 domain. This site has been
analyzed in great detail. It adopts an We have evidence that cell binding and interaction with heparin
represent two functionally distinct properties shared by the first F3
domain. This notion is supported by site-directed mutagenesis experiments. Cell binding is completely abolished when a single amino
acid within the F3 domain (Lys-51) is mutated. In contrast, the same
mutation does not affect the interaction of the F3 domain with heparin.
Thus, the two activities can be distinguished, although they seem to be
exerted by spatially overlapping areas. Molecular modeling of the first
F3 domain indicates that the mutated Lys is situated within a
positively charged groove that resembles the three-dimensional
heparin-binding site proposed by Busby et al. (29).
Ehnis et al. (20) have proposed a chondroitin/dermatan
sulfate form of CD44 as a cell surface receptor for collagen XIV. This
membrane-bound proteoglycan was identified by affinity chromatography of cell surface proteins on collagen XIV immobilized to Sepharose. Based on our results, it does not seem likely that a
chondroitin/dermatan sulfate form of CD44 represents the major receptor
for collagen XIV. Chondroitin sulfate up to 100 µg/ml did not inhibit
binding of cells to the first F3 domain. Furthermore, we were not able to block cell binding to the F3 domain by two different antibodies against human CD44, although both antibodies have been demonstrated to
abolish the interaction of CD44 with
heparin.2 In addition, U937
and smooth muscle cells that also express CD44 on their surface did not
attach to the F3 domain. We therefore favor the idea that the major
receptor for collagen XIV is not a chondroitin/dermatan sulfate
proteoglycan, but we cannot exclude the possibility that CD44 is
involved in cell binding as a coreceptor.
It is possible that the cell binding activity of the first F3 domain is
responsible for several biological functions assigned to collagen XIV.
Nakagawa et al. (33) identified a chemotactic factor for
neutrophils, which turned out to be identical to an N-terminal
fragment of collagen XIV. This fragment attracted neutrophils in a
concentration-dependent manner, suggesting a role for
collagen XIV in neutrophil recruitment and inflammation. Akutsu
et al. (34) demonstrated a role of the NC3 domain from
collagen XIV in cell migration. When added to fibroblasts cultivated on
a reconstituted collagen gel, the isolated NC3 domain inhibited the
migration of these cells into the gel. In light of our results, it is
conceivable that attraction of neutrophils and inhibition of cell
migration are caused by the interaction of the first F3 domain from
collagen XIV with its cell surface receptor.
All our results have been obtained with collagen XIV from chick. Human
collagen XIV exhibits a highly homologous structure, suggesting that
some of the results may also hold true for human collagen XIV. Two
isoforms with different NC1 domains have in fact been described for
human collagen XIV, although their amino acid sequences and their way
of splicing differ considerably from those of the chicken isoforms
(12). Alternative splicing of the three-amino acid insert in the sixth
F3 domain of collagen XIV has not yet been found in the human protein
(12). Likewise, all cDNA clones described for human collagen XIV
correspond to the longer isoform containing the F3 domain at the N
terminus. In chicken tissues, however, the shorter isoform lacking the
N-terminal F3 domain is expressed exclusively before hatching. It
therefore remains to be demonstrated whether a similar isoform without
cell binding properties may be expressed in human tissues at an early developmental stage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (Invitrogen) employing the
XbaI restriction site of the forward primer P2 and the
internal EcoRI site of the PCR product (position 1283 according to Ref. 4). The DNA sequence of the insert was determined by
the dideoxy chain termination method with Sequenase 2.0 (U. S.
Biochemical Corp.).
-D-thiogalactopyranoside as suggested by the supplier of the GST gene fusion system (Amersham Pharmacia Biotech). The bacteria were collected by centrifugation and lysed by
sonication. Fusion proteins were purified from the lysates by affinity
chromatography on GSH-Sepharose. If required, the GST moiety was
released from the fusion protein by cleavage with endopeptidase factor
Xa (Roche Molecular Biochemicals) and removed by adsorption to
GSH-Sepharose.
Oligonucleotide primers used in this study
-D-glucoseaminide,
50 mM sodium citrate, 0.25% Triton X-100, pH 5. After
40-90 min, depending on the cell line used, the reaction was stopped
by the addition of 100 µl of 50 mM glycine, 5 mM EDTA, pH 10.4, and the absorbance was read at 405 nm.
): XbaI/EcoRI (positions 286-1283) derived from the two RT-PCR products;
EcoRI/BstEII (positions 1283-2655) from clone
113; BstEII/PstI (positions 2655-4944) from clone 9; PstI/EcoRI (linker) (positions
4944-6558) from clone 8D. The sequences of the constructs were
verified by DNA sequencing with the help of several synthetic primers.
The two constructs were expressed in a coupled
transcription/translation system (Promega) following the instructions
of the manufacturer. A typical reaction (total volume, 25 µl)
contained 12.5 µl of reticulocyte lysate, 1 µl of TnT reaction
buffer, 0.5 µl of T7 RNA polymerase (15 units/ml), 0.5 µl of amino
acid mix (1 mM), 2 µl of [35S]methionine
(1000 Ci/mmol, 10 mCi/ml), 0.5 µl of RNAsin (40,000 units/ml), and 2 µg of template DNA. After incubation at 30 °C for 2 h, the
reaction mixture was analyzed on an SDS-polyacrylamide gel.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
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Fig. 1.
Domain structure of the N-terminal region of
chicken collagen XIV. A, sequence published by
Wälchli et al. (4); B, sequence published
by Gerecke et al. (11); C, relative positions of
introns in the collagen XIV gene. Exons are numbered as in Table I.
Arrows show the position of primers used for RT-PCR.
S, signal peptide; F3, fibronectin type III
domain; L, linker; VA, von Willebrand factor A
domain.
Exon/intron structure of the human COL14A1 gene
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Fig. 2.
Expression of the two collagen XIV isoforms
in different tissues and at different developmental stages.
Poly(A) RNA from eight different tissues of 16-day old embryos
(left) and from five different developmental stages
(right) was transcribed into cDNA and amplified by PCR
utilizing the primers shown in Fig. 1. The PCR products were separated
on a polyacrylamide gel and stained with ethidium bromide.
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Fig. 3.
Adhesion of various cells to the recombinant
proteins F3, VA, and F3+VA. The purified proteins were dissolved
in water, spotted onto plastic plates, and allowed to air-dry. Residual
binding sites were blocked with bovine serum albumin. Cells in serum
free medium were added and allowed to attach. Nonadherent cells were
washed off. A, binding of three different cell types to the
F3 fusion protein; B, binding of U266 cells to the fusion
proteins F3, VA, and F3+VA.
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Fig. 4.
Three-dimensional structure of the first F3
domain from chicken collagen XIV as predicted by computer
modeling. Charged residues are indicated in red
(basic residues) and blue (acidic residues). The four
residues that were selected for site-directed mutagenesis are
highlighted again on the right-hand side.
Ser, Arg-37
Ser, Lys-54
Ser) had any influence on cell binding. However, cell binding was
completely abolished when two residues were mutated in combination
(Arg-35 plus Arg-37 or Lys-51 plus Lys-54). Similar results were
obtained with chicken fibroblasts and with KG-1a cells. Furthermore,
the droplet adhesion assay and the microtiter plate/hexosaminidase assay gave comparable results. Thus, Lys-51 plays a crucial role in
cell binding to the alternatively spliced F3 domain.
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Fig. 5.
Cell adhesion to mutated proteins. The
wells of a microtiter plate were coated with fusion proteins. Residual
binding sites were blocked with bovine serum albumin. U266 cells were
added in serum free medium and allowed to attach. Nonadherent cells
were removed by washing. Adherent cells were quantified by measuring
the activity of the enzyme hexosaminidase. The results are expressed in
relation to a positive control performed with the wild type F3 protein.
Each bar represents the mean and S.D. from three independent
experiments. F3(5), fifth F3 domain from chicken collagen
XIV; mut 35+37, first F3 domain with residues 35 and 37 mutated to serine.
Ser mutated fusion proteins were treated with endopeptidase Xa to release the GST moiety and the
circular dichroism and fluorescence spectra were recorded after
affinity purification. Within experimental error, identical spectra
were obtained with both protein preparations (not shown). These results
indicate that the Lys-51
Ser mutation did not cause any changes in
the conformation of the F3 domain, neither in the polypeptide backbone
(as revealed by circular dichroism) nor in the conformation of residues
in the vicinity of aromatic amino acids (as revealed by the
fluorescence spectrum).
Ser) were applied to a
heparin-Sepharose column and eluted in a stepwise fashion with an NaCl
gradient. As seen in Fig. 6, both forms
of the F3 domain bound specifically to heparin and eluted as broad
peaks at an ionic strength corresponding to ~500 mM salt. Within experimental error, the wild type and the mutated F3 domain showed the same elution profile. GST alone without fusion protein did
not bind to the column (not shown). Thus, the alternatively spliced F3
domain interacts with both heparin and cells. The mutated F3 domain,
which has lost its cell binding activity, still retains its heparin
binding activity. Cell binding and heparin binding are, therefore, two
functionally distinct features shared by the alternatively spliced F3
domain.
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Fig. 6.
Binding of the recombinant F3 domain to
heparin. The wild type F3 domain and its mutated form (mut51;
Lys-51 Ser) were applied to a heparin-Sepharose column and eluted
with a stepwise NaCl gradient. Individual fractions were analyzed on a
polyacrylamide gel stained with Coomassie Blue. The actual NaCl
concentration of the effluence was determined with a conductivity meter
and is shown at the bottom. B,
breakthrough.
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Fig. 7.
Binding of the two collagen XIV isoforms to
heparin. Collagen XIV containing (Col XIV + F3) or
lacking (Col XIV F3) the first F3 domain was
transcribed and translated in vitro from full-length
cDNA clones. The radiolabeled polypeptides were applied to a
heparin-Sepharose column and eluted with a stepwise NaCl gradient. The
radioactivity in the effluent was determined in a liquid scintillation
counter and is expressed in arbitrary units (Col XIV + F3
; Col XIV
F3
). Individual fractions were
analyzed on a polyacrylamide gel and exposed to x-ray film. An aliquot
of the translation mixture prior to application to heparin-Sepharose is
shown in lane T.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strands within the sixth
F3 domain (22). This increase might affect the arrangement of the
domain in the collagen XIV molecule, thereby modulating its
interactions with other matrix molecules.
-helical fold with a twisted
basic groove (16). It shows considerably lower affinity for heparin
than the first site, because only 250 mM NaCl is required
for displacement of the complex. This observation is in agreement with
our studies using full-length collagen XIV polypeptides translated
in vitro. The polypeptide containing the N-terminal F3
domain binds to heparin-Sepharose and requires 500 mM salt
for displacement. The polypeptide without N-terminal F3 domain also
interacts with heparin-Sepharose but requires only 150 mM
salt for displacement.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. T. Kiefhaber and O. Bieri (University of Basel) for their assistance with the recording of circular dichroism and fluorescence spectra. We thank C. Schild for a critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Swiss National Science Foundation (Grant 3100-061296.00) and by the Bernese Cancer Liga.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: M. E. Müller Institute, University of Bern, P. O. Box 30, CH-3010
Bern, Switzerland. Tel.: 41-31-632-8726; Fax: 41-31-632-4999; E-mail:
trueb@mem.unibe.ch.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M009148200
2 M. Imhof and B. Trueb, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: FACIT, fibril-associated collagens with interrupted triple helices; F3, fibronectin type III domain; NC1-3 domains, noncollagenous domains 1-3; VA domain, von Willebrand factor A domain; bp, base pair(s); RT-PCR, reverse transcriptase-polymerase chain reaction; GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CTF, chicken tendon fibroblasts; CSM, chicken smooth muscle cells; kb, kilobase(s).
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