From the Institute of Experimental Medicine,
Friedrich Alexander University, Erlangen, Germany,
§ Department of Biochemistry and Molecular Biology, Faculty
of Chemical Sciences, Universidad Complutense, Madrid, Spain, and
¶ Max Planck Institute of Biochemistry, D-82152
Martinsried, Germany
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ABSTRACT |
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Type X collagen is a short-chain, network-forming
collagen found in hypertrophic cartilage in the growth zones of long
bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human 1(X)
cDNA in the human embryonal kidney cell line HEK293 and the
fibrosarcoma cell line HT1080. Stable clones were obtained secreting
recombinant human type X collagen (hrColX) in amounts of 50 µg/ml
with
1(X)-chains of apparent molecular mass of 75 kDa. Pepsin
digestion converted the native protein to a molecule migrating as one
band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin
digestion. Polyclonal antibodies prepared against purified hrColX
reacted specifically with type X collagen in sections of human fetal
growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin
digestion experiments of hrColX at increasing temperatures indicated
triple helical molecules with a reduced melting temperature of 31 °C
as a result of partial underhydroxylation. Ultrastructural analysis of
hrColX by rotary shadowing demonstrated rodlike molecules with a length
of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of
apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even
after reduction and heat denaturation, and gave rise to monomers of
18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains
prepared by collagenase digestion comigrated with NC-1 domains prepared
as recombinant protein in HEK293 cells, both in the multimeric and
monomeric form. These studies demonstrate the potential of the pCMVsis
expression system to produce recombinant triple helical type X
collagens in amounts sufficient for further studies on its structural
and functional domains.
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INTRODUCTION |
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Type X collagen is a short chain collagen with a triple helical
portion half the length of fibril-forming collagens, flanked by
globular, noncollagenous
(NC)1 domains at the amino
terminus (NC-2) and the carboxyl terminus (NC-1) (for reviews, see
Refs. 1-3). It is primarily expressed in hypertrophic cartilage of
epiphyseal growth plates of long bones, ribs, and vertebrae (4-6), but
also in bone fracture callus (7) and in osteoarthritic cartilage
(8-10). A substantial body of information on the structure and
molecular assembly of type X collagen is available from studies on type
X collagen isolated from hypertrophic cartilage (6, 7, 11-15) and from
cell cultures of chicken, rabbit, and bovine hypertrophic chondrocytes
(16-20). Electron microscopic studies indicate that type X collagen
molecules form fine pericellular filaments in vivo in
association with type II collagen (21), or assemble into a hexagonal
meshwork in vitro (22). Rotary shadowing data show that type
X collagen molecules aggregate primarily through their COOH-terminal,
nontriple helical NC-1 domains, which are highly hydrophobic (20, 23).
Conflicting data, however, have been reported concerning the size of
type X collagen extracted from hypertrophic cartilage or isolated from the culture medium of hypertrophic chondrocytes, ranging from 58 to 82 kDa for the intact chains (15-20). Thus, the issue of whether there is
processing of type X collagen following biosynthesis and secretion has
not been settled. Two studies provide evidence for the secretion of a
70-kDa procollagen form and processing in the culture medium of chicken
chondrocytes (24-26), while another study did not support processing
of type X collagen in cartilage organ cultures within 4 h (19).
Here we show that intact, recombinantly expressed human 1(X) chains
migrate with an apparent molecular mass of 75 kDa significantly above
that of
1(X) chains extracted from chondrocytes cultured in
alginate, supporting the notion that type X collagen may be processed
by chondrocytes but not by HEK293 cells.
A number of functions have been proposed for type X collagen. Its role
in the structural integrity of the hypertrophic cartilage has become
evident from the genetic analysis of patients affected with Schmid type
metaphyseal chondrodysplasia (SMCD), a mild autosomal disorder
associated with growth plate abnormalities, short stature, and waddling
gait (27-30). All mutations detected have been located in the NC-1
domain of type X collagen and include single amino acid substitutions,
deletions, and premature termination mutations. Furthermore, an
essential role for type X collagen in endochondral ossification of
hypertrophic cartilage is supported by a study on a mouse made
transgenic with a shortened chicken Col101 gene. Coexpression of the mutant type X collagen with the endogenous type X
collagen resulted in severe alterations in the cartilage growth plate
and a hunchback, probably owing to the formation of unstable hybrid
molecules (31).
Several lines of evidence indicate that type X collagen may be involved in mineralization of cartilage (3). In the developing growth plate and in chondrocyte cultures, type X collagen expression precedes calcification of hypertrophic cartilage (32-35). Furthermore, type X collagen synthesis is altered in chondrocytes from the rachitic chicken (36, 37), and recently it was shown that purified type X collagen regulates calcium uptake by matrix vesicles isolated from hypertrophic cartilage (38, 39). The putative role of type X collagen in mineralization and endochondral ossification of hypertrophic cartilage is supported by mice with a Col10A1 null mutation showing alterations in the endochondral bone trabecular meshwork in the growth plate, as well as a shift of matrix vesicles from the hypertrophic to the resting zone (40).
A striking feature of type X collagen and their isolated NC-1
domains is the unusual stability of the trimeric molecules which migrate as trimers in SDS-PAGE even after denaturation under reducing conditions (20, 41, 42). However, when pre-1(X) collagen chains are
synthesized with SMCD mutations in the NC-1 domain by cell-free
translation and transcription, they are no longer able to from stable
trimers in vitro (41, 42). Accordingly, when Col10A1 genes
with mutations in the NC-1 domain were transfected into eukaryotic
cells, no corresponding proteins were secreted from the cell nor
expressed as intracellular protein, indicating failure of the mutated
chains to assemble to triple helical molecules, which causes
intracellular degradation (42). Mutant collagens lacking the
amino-terminal NC-2 domain or containing in-frame deletions in the
triple helix, however, were secreted as trimeric molecules (42).
The mechanism of the assembly of 1(X) subunits into trimeric
molecules and extracellular meshworks as well as the sequences within
the NC-1 and NC-2 domains involved in these events are of central
importance for our understanding of the structure-function relationship
of type X collagen in hypertrophic cartilage. The question remains,
however, what the structural requirements (e.g. degree of
prolylhydroxylation, completeness of the globular domains) are for the
assembly of
1(X) chains to triple helical molecules in
vivo, and which mutations would be tolerated for assembly under less dissociating conditions than those applied prior to SDS-gel electrophoresis (41, 42). Such studies require the production of
sufficient amounts of normal and mutated type X collagen molecules or
their NC-1 domains, respectively. Here we describe the preparation of
human recombinant type X collagen in amounts sufficient to investigate
structure and assembly of this collagen. We show that the recombinant
protein expressed in HEK293 or HT1080 cells migrates on SDS-PAGE with
an apparent molecular mass of 75 kDa, while
1(X) extracted from
chondrocyte cultures or from cartilage migrate with an apparent mass of
60 kDa. Although the 75-kDa human recombinant type X collagen (hrColX)
is partially underhydroxylated, it is secreted, and triple helical
molecules with a melting temperature of 31 °C were obtained that
form aggregates in vitro via their globular NC-1 domains.
Furthermore, we compare the NC-1 domains released by collagenase
digestion of hrColX with NC-1 domains prepared as recombinant proteins
in HEK293 cells. Both migrate as aggregates of apparent mass of 40 kDa
in SDS-PAGE and are extremely stable and resistant to heat denaturation
and reduction in the presence of detergent and urea, but dissociate
into monomers of approximately 20 kDa after acid treatment.
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MATERIALS AND METHODS |
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Construction of the Type X Collagen Expression Vectors
pCMV-ColX and pCMV-NC-1--
A 265-bp
HindIII-XhoI fragment corresponding to position
+31 to +295 (EMBL no. X68952) of human type X collagen cDNA (43) was cloned into the pBluescript SK vector as described
previously (44). The XbaI-XhoI insert was combined with a 2080-bp XhoI-SspI fragment
(position 295-2383, EMBL no. X68952) from the human COL10A1
gene and cloned into the XbaI-HpaI restricted
vector pCMVsis (45, 46), resulting in the expression vector pCMV-ColX.
The 2352-bp insert contains the complete coding sequence together with
65 bp of a 5'-untranslated region and 249 bp of a 3'-untranslated
region.
Cell Cultures and Transfection-- The human kidney epithelial cell line HEK293 and the human fibrosarcoma cell line HT1080 were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 5% fetal calf serum. One million cells/10-cm culture dish were transfected using the calcium phosphate method with 20 µg of the expression vectors pCMV-ColX or pCMV-NC1, mixed with 1 µg of a selection plasmid pSV2pac containing a puromycin resistance gene according to Nischt et al. (46). Puromycin-resistant HEK293 or HT1080 clones were selected and tested for type X collagen expression by precipitating the culture medium with 10% trichloroacetic acid, analyzing the precipitate by SDS-PAGE, and immunoblotting with rabbit anti-human type X collagen (48). NC-1-expressing clones were screened by Western blotting of the supernatants using the monoclonal antibody X34 specific for the native NC-1 domain (48).
Bovine fetal chondrocytes were prepared from epiphyseal growth plates (49, 50) and cultured in alginate beads as described in Guo et al. (51) and Häuselmann et al. (52) for 1 week in Ham's F-12 medium containing 10% fetal calf serum and 50 µg/ml ascorbate-phosphate. For type X collagen analysis, the alginate beads were dissolved in 10 mM EDTA, pH 7.5, and the released chondrons were dissolved in sample buffer for SDS-PAGE. Type X collagen production was detected by immunoblotting with a polyclonal antibody prepared against recombinant human type X collagen (see below).Preparation of Recombinant Human Type X Collagen-- Two HEK293 clones (5/16, 6/16) and one HT1080 clone (7/7), which secreted up to 50 µg/ml hrColX were expanded to mass culture. Partial purification of hrColX was achieved by dialysis of the serum-free supernatant against phosphate-buffered saline or against 0.15 M NaCl, 0.05 M Tris-HCl, pH 7.4, at 4 °C. Purification to more than 95% homogeneity was achieved by dialysis against 0.02 M NaCl, 0.05 M Tris-HCl, pH 7.5, and chromatography on DEAE-cellulose, equilibrated in the same buffer. The material not bound to DEAE-cellulose was further purified by carboxymethylcellulose chromatography in 50 mM sodium acetate, pH 4.8, at room temperature. Type X collagen was eluted with a linear NaCl gradient between 0 and 1000 mM NaCl.
Immunological Techniques-- Rabbit antisera against human type X collagen were obtained by subcutaneous immunization with 3 × 0.2 mg of purified hrColX. The antisera were tested for reactivity against hrColX and lack of cross-reactivity with human types I and II collagen by enzyme-linked immunosorbent assay and immunoblotting as described previously (15). The antisera R239 and R244 revealed a titer of 1:60,000 in immunoblotting against hrColX. The titers against type I and II collagen were 104-fold lower. The specificity of the antibody for type X collagen was verified by the selective staining of the hypertrophic zone of human epiphyseal growth cartilage (see Fig. 4).
Circular Dichroism Analysis-- Circular dichroism spectra of purified hrColX, dissolved in 50 mM Hepes, 50 mM NaCl, pH 7.4, at 0.25 mg/ml, were recorded at 20 °C on a Jaso J-715 dicrograph using 0.01- or 0.05-cm path length thermostated cuvettes. Melting curves were recorded by measuring molar ellipticity at different wavelengths (198, 222, or 225 nm) between 15 and 70 °C, increasing the temperature by 30 °C/h. The protein concentration was determined by amino acid analysis in triplicate samples.
Enzyme Digestion-- For trypsin/chymotrypsin digestion, serum-free hrColX containing cell culture supernatants were heated to various temperatures up to 50 °C for 30 min, rapidly cooled to 20 °C, and then digested with 1 mg/ml trypsin (2 × crystallized, Boehringer Mannheim, bovine pancreas, EC 3.4.4.4) and 2.5 mg/ml chymotrypsin (Boehringer, EC 3.4.4.5) for 1 h at 20 °C (53). For pepsin digestion, type X collagen was dialyzed against 0.5 M acetic acid and incubated with 0.2 mg/ml pepsin (swine stomach, 2 × crystallized, Serva, Heidelberg, EC 3.4.4.1) for 18 h at 4 °C. For collagenase digestion, purified type X collagen or serum-free cell culture supernatant was dialyzed against 0.15 M NaCl, 50 mM Tris, pH 7.4, containing 5 mM CaCl2 and 1 mM N-ethylmaleimide. The material was heat denatured at 45 °C for 30 min and digested with highly purified collagenase (Clostridium histolyticum microbial collagenase, EC 3.4.4.19, Advanced Biofacturers, Lynnbrook, NY) for 4 h at 37 °C. After enzyme digestion, the material was either precipitated with 10% trichloroacetic acid, or concentrated by vacuum centrifugation and analyzed by SDS-gel electrophoresis on 12% polyacrylamide-SDS gels.
Analytical Procedures-- Amino acid analysis of hrColX after hydrolysis with 6 N HCl was performed on a Biotronic amino acid analyzer (courtesy of Dr. K. H. Mann, Max Planck Institute for Biochemistry, Martinsried, FRG). For amino-terminal sequencing, 20 µg of recombinant type X collagen were subjected to SDS-PAGE in 7% polyacrylamide gels, blotted to a Hybond® nylon membrane, and sequenced on an automatic solid phase amino acid sequencer (Applied Biosystems) (courtesy of Dr. R. Deutzmann, Department of Biochemistry, University of Regensburg, FRG).
Rotary Shadowing-- For electron microscopic examination, purified hrColX was dialyzed against 0.2 M ammonium hydrogen carbonate, mixed with an equal volume of glycerol, and sprayed onto mica discs. Rotary shadowing and electron microscopic analysis was kindly performed by H. Wiedemann at the Max Planck Institute for Biochemistry, Martinsried, as described in Kühn et al. (54).
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RESULTS |
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Preparation of Recombinant Human Type X Collagen-- A full-length human type X collagen cDNA was generated from cDNA and genomic clones (32, 33) and cloned into the eukaryotic expression vector pCMVsis. The resulting expression vector pCMV-ColX was stably transfected into human embryonal kidney cells HEK293 and fibrosarcoma cells HT1080. Puromycin-resistant clones were selected and tested for type X collagen production by SDS-PAGE and immunoblotting of the secreted proteins. Several stable clones were obtained secreting up to 50 µg/ml protein with a molecular mass of 75 kDa, which was not secreted by mock-transfected cells. The band reacted specifically with a rabbit antiserum against human type X collagen (15). The immunoreactive band was purified to homogeneity from serum-free conditioned medium of the HEK293 6/16 clone or from the HT1080-7/7 clone by DEAE-cellulose chromatography, followed by CM-cellulose chromatography, both under native conditions (Fig. 1b). Pepsin digestion of purified hrColX reduced its size to 62 kDa (Fig. 1c).
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Immunolocalization of Type X Collagen with Antibodies against
hrColX--
For preparation of antibodies, rabbits were immunized with
purified hrColX, and the antisera were tested by enzyme-linked immunosorbent assay and immunoblotting. In the immunoblot, the antisera
reacted specifically with the 75-kDa 1(X) chain, as well as with the
pepsin- and trypsin/chymotrypsin-resistant parts of hrColX (Fig.
3). It cross-reacts with murine, bovine,
and canine type X collagen (not shown), but not with pepsin-extracted
porcine type X collagen.3
When applied to sections of fetal human epiphyseal cartilage, it
stained exclusively the zone of hypertrophic cartilage (Fig. 4). hrColX was also used to produce a
panel of monoclonal antibodies with different epitope specificities,
recognizing hrColX in the native, in the denatured, and in the
pepsin-digested form, or the native NC-1 domain (48).
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Pepsin and Trypsin/Chymotrypsin Digestion of Recombinant Human Type X Collagen-- To obtain information on the size and the proteolytic susceptibility of the noncollagenous domains and the protease-resistant, triple helical domain of the recombinant type X collagen, hrColX was digested for 24 h with pepsin at 4 °C, or with trypsin/chymotrypsin at 20 °C for 1 h. Interestingly, a pepsin-resistant fragment of type X collagen of 62 kDa was obtained (Figs. 1 and 3), while after trypsin/chymotrypsin digestion of hrColX, four fragments in the molecular mass range between 62 and 36 kDa were retained (Fig. 3). By comparison, pepsin digestion of human or bovine type X collagen extracted from hypertrophic cartilage or chondrocyte cultures generates a triple helical fragment of 45 kDa (6, 7, 50). Minor amounts of a fragment of this size were obtained after trypsin digestion of hrColX (Fig. 3a).
Circular Dichroism and Thermostability of hrColX-- The CD spectrum of purified of hrColX at 20 °C revealed a slight peak at 225 nm, as expected for triple helical collagens, and a minimum at 198 nm (Fig. 5a). The melting curve resulting from ellipticity determinations at 198 nm indicated a point of inflection at 31 °C, and further conformational changes at higher temperatures (Fig. 6). This finding is consistent with the results obtained after trypsin/chymotrypsin digestion of hrColX at various temperatures between 4 and 50 °C. Triple helical fragments of 50 and 65 kDa, and smaller, were resistant against enzyme digestion up to 30-31 °C, while minor fractions of the 50- and 65-kDa bands were stable up to 39 °C (Fig. 6).
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Ultrastructure of hrColX-- Electron microscopic imaging of hrColX by rotary shadowing revealed threadlike molecules with an average length of 130 ± 5 nm, corresponding to that reported for chick type X collagen (20, 22), and a large globular domain at one end, corresponding to the NC-1 domain. In some molecules a small globular domain, probably the NC-2 domain, was detectable at the other end of the molecule. hrColX molecules tended to form aggregates ranging from dimers to multimers by assembling at their NC-1 globules (Fig. 7).
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Assembly of hr1(X) Chains--
Recombinant hrColX migrated with
the approximate mass of a trimer (around 220 kDa) when loaded onto
SDS-gels in nondenaturing sample buffer (Fig.
8c). Trichloroacetic acid
precipitation resulted in complete dissociation into monomers (Fig.
8e), while partial dissociation to monomers and dimers was
achieved by heat denaturation in the presence of 8 M urea
and 3% SDS at 20 and 60 °C (Fig. 8). Under these conditions,
addition of 5%
-mercaptoethanol caused complete dissociation of
dimers into monomers (Fig. 8, a and e). These
observations indicate that (i) the dimers and trimers formed by the
hr
1(X) chains are less stable than the dimers formed by the
NC-1 domains (see below) and (ii) interchain disulfide bonds contribute
less to the stability of hr
1(X) to trimers and dimers than do
hydrophobic bonds and other noncovalent bonds susceptible to
trichloroacetic acid treatment.
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Aggregation of the NC-1 Domain-- Digestion of recombinant type X collagen with purified bacterial collagenase gave rise to a noncollagenous domain, which migrated on SDS-gel electrophoresis with an apparent mass of 38.5-40 kDa (Fig. 9). The complex was resistant to heat denaturation up to 100 °C, even in the presence of 8 M urea, 3% SDS, and 5% mercaptoethanol (Fig. 10), conditions that have been shown to dissociate NC-1 domains prepared from chicken type X collagen (20). Only by precipitation of the material with 10% trichloroacetic acid, in the presence of Triton X-100, did the NC-1 multimer dissociate into a monomer, migrating on SDS-gels with an apparent mass of 18-20 kDa (Figs. 9 and 10).
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DISCUSSION |
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Here we report for the first time on the preparation of triple helical recombinant type X collagen in an eukaryotic expression system in amounts sufficient for structural and functional studies. By stable transfection several HEK293 and HT1080 clones were obtained that secreted up to 50 µg/ml intact hrColX as the predominant protein into the culture medium. hrColX was purified to homogeneity under native conditions using DEAE- and CM-cellulose chromatography. The recombinant type X collagen, although underhydroxylated, was secreted into the medium and isolated as a triple helix with a Tm of 31 °C. With the electron microscope it appeared as threadlike molecules after rotary shadowing with an average length of 130 nm, flanked by a large and a small globular domain at both ends, similar to chick type X molecules described by Schmid and Linsenmayer (20), Schmid et al. (55), and Kwan et al. (22). The material was used to produce high titered polyclonal and monoclonal antibodies (48). The affinity-purified antibodies stained specifically hypertrophic cartilage in the growth plate of human fetal epiphyseal cartilage. Monoclonal antibodies prepared against hrColX (38) confirmed our previous finding of enhanced production of type X collagen in osteoarthritic articular cartilage (9, 48, 56).
The recombinant human 1(X) chains consistently migrated on SDS-PAGE
with an apparent molecular mass of 75 kDa (compared with globular
standards), while in the same gel
1(X) chains extracted from
chondrocyte cultures in alginate migrated with an apparent mass of 65 kDa, suggesting processing to shorter forms in the alginate cultures.
Retarded migration of hrColX due to overhydroxylation or
overglycosylation can be excluded according to the results of the amino
acid analysis showing underhydroxylation, and from treatment with
glycosidases showing no differences in migration (results not shown).
The approximately 75 kDa of the full-length
1(X) chains synthesized
in HEK293 and HT1080 cells are in accordance with the size of the
1(X) chains synthesized in cell-free systems in vitro
(31, 57). The appearance of shorter forms of
1(X) of 58-65 kDa in
cartilage extracts (14, 15, 24, 26) corroborates two previous reports
on the processing of a 70-kDa procollagen chain synthesized by chick
hypertrophic chondrocytes in vitro to a 59-kDa form
(24-26). In another study using cartilage organ cultures, however, no
processing of a 82 kDa
1(X) chain to smaller forms was observed
within a 4-h pulse-chase period (19).
Curiously, after pepsin digestion of hrColX an 1(X) chain of 62 kDa
was retained, while pepsin digestion of type X collagen isolated from
chondrocyte cultures or from hypertrophic cartilage leaves a fragment
of 45 kDa (6, 12-18). Trypsin/chymotrypsin digestion of the native
hrColX gave rise to smaller fragments, one of them, 45-50 kDa, the
size of the pepsin-resistant fragment of chondrocyte-derived type X
collagen.
Whether and how processing of type X collagen occurs after a prolonged
time of type X collagen in the extracellular cartilage matrix in
vivo or in vitro, however, needs to be clarified in cartilage cell or organ culture. If there is processing of type X
collagen in cartilage, the question remains at which end of the
molecule this occurs. Amino-terminal sequencing of chick-type X
collagen isolated from chick cartilage cultures revealed an intact
amino end, including the complete NC-2 domain (19), even after a 4-h
pulse-chase period. However, there are substantial sequence differences
between bovine, human, and chick NC-2 domains, so that processing in
bovine chondrocytes cultured in 48-h alginate cultures would not
necessarily be in conflict with the results described by Summers
et al. (19). However, COOH-terminal processing is also
possible. Although Chan et al. (42) have shown that truncation of the Col10A1 gene at the carboxyl terminus by 50 amino
acid residues gives rise to 1(X) chains that are unable to assemble
to trimers during biosynthesis and thus are degraded intracellularly,
extracellular processing of intact type X collagen molecules following
secretion may occur, which may not affect sites in the NC-1 domain
responsible for intracellular, intermolecular aggregation.
Circular dichroism studies of the purified hrColX revealed a minimum in the molar ellipticity at 198 nm and a slight peak at 225 nm, indicating a triple helical molecule with contributions of large globular domains. Molar ellipticity at 198 nm measured at increasing temperatures indicated a Tm of 31 °C. Amino acid analysis revealed underhydroxylation with a hydroxyproline:proline ratio of 0.25:1, while type X collagen prepared from the culture medium of chick chondrocytes contains hydroxyproline and proline in a ratio of 0.7: 1 (16). Nevertheless, the triple helical domain of hrColX produced either in HEK293 or HT1080 cells was resistant to pepsin or trypsin/chymotrypsin digestion up to 31 °C, in agreement with the data obtained by circular dichroism.
The underhydroxylation of the recombinant type X collagen is very
likely a result of the high expression levels in the HEK293 or HT1080
cell clones due to the use of the strong CMV promotor. Even in HT1080
cells, the levels of endogenous prolylhydroxylase seem insufficient for
complete hydroxylation of the surplus of hr1(X) collagen chains in
the endoplasmic reticulum, although HT1080 cells produce endogenous
collagen, different from HEK293 cells. In contrast, recombinant type X
collagen and type X/II chimeric collagens cloned into the pCDNA3
vector and transfected into HEK293 cells have a Tm
of 42 °C, but are expressed at much lower levels (58). Similarly,
bovine type X collagen prepared by in vitro transcription
and translation of Col10
1 cDNA by using HT1080
microsomal membranes was thermally stable up to 42 °C (57), but
expression levels in that system were much lower. In addition, bovine
type X collagen contains 2 additional cysteine residues in the triple
helical part, thus stabilizing triple helix formation (7, 14, 50). In
HT1080 cells, human recombinant type II procollagen also was produced
in milligram amounts in fully hydroxylated and glycosylated form (59),
while no information is available on the degree of prolylhydroxylation of a recombinant, heterotrimeric type VI (60) and type IV (61) collagen. Large scale production of a fully hydroxylated type III
collagen in the baculovirus system was achieved by cotransfection with
the
- and
-units of prolylhydroxylase (62, 63). To achieve full
hydroxylation of hrColX in HEK293 cells, a similar experimental attempt
is in progress.4
The low melting temperature of hrColX of 31 °C suggests that during
biosynthesis only a minor part of the hr1(X) collagen chains are
folded into a triple helix within the cell. The level of hydroxylation
was, however, sufficient to allow secretion of trimeric hrColX
molecules into the medium where they seem to refold into a triple
helical structure at room temperature. Previous studies have shown that
complete inhibition of prolylhydroxylation by
,
'-dipyridyl
prevents secretion, with the unhydroxylated procollagen being retained
in the rough endoplasmic reticulum (64, 65). A reduced extent of
secretion has been reported for partially underhydroxylated type IV
collagen in cultures of lens epithelial cells (66). Satoh et
al. (67) have shown that chaperones such as HSP47 delay but do not
prevent the secretion of underhydroxylated collagen.
Previous studies have shown that the NC-1 domain is critical for the assembly of trimeric type X collagen molecules (41) as well as for intermolecular aggregation (20), leading possibly to hexagonal meshworks (22). Strong hydrophobic interactions had been reported for the NC-1 domains of chick and bovine type X collagen (20, 22, 23) and for the human NC-1 domain (42), which contains among others 11 tyrosine residues per chain (43, 44). The NC-1 domain obtained after cleavage of hrColX migrated in SDS-PAGE with apparent molecular mass of 38.5-40 kDa, similar to that obtained after collagenase digestion of chick type X collagen (20). Treatment with 10% trichloroacetic acid resulted in a complete and irreversible dissociation into monomers of 20 kDa. In contrast to the chick NC-1 domain (20), the 40-kDa complexes were stable, even under heat denaturation at 100 °C, in reducing SDS-sample buffer and resistant to disulfide-reducing agents.
Although the NC-1 domains prepared by collagenase digestion of hrColX migrate in SDS-PAGE with an apparent mass of 40 kDa, suggesting a dimer, it is likely that the band represents a trimer with a compact conformation, which is unfolded after trichloroacetic acid treatment. Significant degradation of the NC-1 domain by bacterial collagenase was excluded as it comigrates with intact NC-1 prepared recombinantly in an eukaryotic expression system both in multimeric and in the monomeric form.
In contrast to NC-1, the intact human recombinant pro-1(X) chains
form both trimers and dimers under native conditions. However, pro-
1(X) trimers seem less stable than NCI trimer and dissociate to
dimers and monomers under denaturing conditions in SDS. Strong bimolecular interactions of NC-1 domains involving NC-1 domains of
adjacent molecules would be compatible with a role of the NC-1 domain
in intermolecular aggregation of type X molecules in the course of
meshwork formation, in addition to their role in intermolecular assembly. Consistent with this role in intermolecular assembly is the
presence of only one cysteine residue per subunit in chicken and human
1(X), which permits the stabilization of the hexagonal networks by
intermolecular disulfide bridges, but does not allow the formation of
stoichiometric intramolecular disulfide bridges in a molecule involving
all three
-chains. It remains to be elucidated whether the same
domains and sequences in NC-1 are responsible for intramolecular and
intermolecular assembly.
In conclusion, the recombinant expression of type X collagen in eukaryotic cells opens new possibilities for the study of the mechanism of type X collagen processing and assembly, in particular by including mutations and deletions in the various domains of the molecule. Furthermore, the recombinant system should be useful to elucidate sites of type X collagen interactions with Ca2+ (38) or other extracellular components such as annexin V (68), type II collagen (21), or proteoglycans.
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ACKNOWLEDGEMENT |
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We thank Hanna Wiedemann, Max Planck Institute of Biochemistry, Martinsried, for expert performance of the rotary shadowing and electron microscopy experiments; Dr. R. Deutzmann, University of Regensburg, Department of Biochemistry, for amino acid sequencing; Dr. K. H. Mann, Max Planck Institute of Biochemistry, for amino acid analysis, and Prof. K. Kühn, Max Planck Institute of Biochemistry, for stimulating discussion and helpful advice.
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FOOTNOTES |
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* This work was financially supported by the Deutsche Forschungsgemeinschaft (Ma 534-10).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: Institute of
Experimental Medicine, Friedrich Alexander University, Schwabachanlage 10, D-91054 Erlangen, Germany. Tel.: 49-9131-85 9100; Fax: 49-9131-85 6341; E-mail: kvdmark{at}expmed.uni-erlangen.de.
1 The abbreviations used are: NC, noncollagen; hr, human recombinant; ColX, type X collagen; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; bp, base pair(s).
2 K. Wagner, E. Pöschl, T. Pihlajaniemi, and K. von der Mark, manuscript in preparation.
3 G. Rucklidge, personal communication.
4 K. Wagner, J. M. Baik, and K. von der Mark, manuscript in preparation.
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REFERENCES |
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