(Received for publication, March 26, 1997, and in revised form, June 6, 1997)
From the Collagen Research Unit, Biocenter and
Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu,
Finland and the § Institute for Medical Molecular Biology,
Medical University of Lübeck, D-23538 Lübeck, Germany
Insect cells coinfected with a baculovirus coding
for the pro1(I) chain of human type I procollagen and a double
promoter virus coding for the
and
subunits of human prolyl
4-hydroxylase produced homotrimeric [pro
1(I)]3
procollagen molecules. The use of an additional virus coding for the
pro
2(I) chain led to the formation of a heterotrimeric molecule with
the correct 2:1 ratio of pro
1 to pro
2 chains of type I
procollagen (pro
1(I) and pro
2(I) chains, respectively), unless
the pro
1(I) chain was expressed in a relatively large excess.
Replacement of the sequences coding for the signal peptide and the N
propeptide of the pro
1(I) chain with those of the pro
1(III) chain
increased level of expression of the pro
1(I) chain, whereas no
similar effect was found when the corresponding modification was made
to the virus coding for the pro
2(I) chain. Molecules containing such
modified N propeptides were found to be processed at their N terminus
more rapidly than those containing the wild-type propeptides. The
Tm of the type I collagen homotrimer was
similar to that of the heterotrimer, both values being about
42-43 °C when determined by circular dichroism. The wild-type
pro
2(I) chain formed no homotrimers. Replacement of the C propeptide
of the pro
2(I) chain with that of the pro
1(I) chain or pro
1
chain of type III procollagen (pro
1(III) chain) led to the formation
of homotrimers, but the
2(I) chains in such molecules were
completely digested by pepsin in 1 h at 22 °C. The data thus
suggest that, in addition to control at the level of the C propeptide,
other restrictions may exist at the level of the collagen domain that
prevent the formation of stable homotrimeric [pro
2(I)]3 molecules in insect cells.
The collagen superfamily now includes at least 19 proteins
formally defined as collagens and more than 10 additional proteins with
collagen-like domains (for reviews, see Refs. 1-6). Type I collagen is
the most abundant member of this family and was the first to be
characterized. Its molecule is a heterotrimer consisting of two
identical 1(I) chains and a slightly different
2(I) chain that
are coiled around one another into a triple-helical conformation. The
molecule is synthesized in the form of a precursor in which the
pro
1(I)1 and pro
2(I)
chains have propeptide extensions at both their N- and C-terminal ends.
In addition to this heterotrimer, several tissues contain small amounts
of a molecule with a chain composition of [
1(I)]3
known as the type I collagen homotrimer (1). In fact, early
renaturation experiments with individual
chains of type I collagen
indicated that they are able to form both
[
1(I)]2
2(I) heterotrimers and
[
1(I)]3 homotrimers although the former were favored,
and the Tm of the latter was slightly lower than
that of the former (7). Homotrimers with the structure of
[
2(I)]3 were also obtained at low temperatures, but
their yield was much lower and the Tm was only
about 20-24 °C (7).
Type I collagen is now used in many medical applications as a biomaterial and as a delivery system for certain drugs (8-10). The collagen used in these applications has been isolated from animal tissues and is liable to cause allergic reactions in up to 3% of human subjects (11). It is obvious, therefore, that an efficient large-scale recombinant system for the production of type I collagen would have many practical applications.
We recently reported that insect cells provide an excellent system for
the large-scale expression of native triple-helical human type III
collagen, a homotrimer consisting of three identical 1(III) chains
(12). Nevertheless, coexpression with the
and
subunits of human
prolyl 4-hydroxylase, an
2
2 tetramer (6, 13, 14), was required for the production of molecules with stable
triple helices (12). The properties of the recombinant type III
collagen were very similar to those of the corresponding nonrecombinant
protein, and the highest expression levels obtained in suspension
cultures were up to 40 mg/liter type III collagen, corresponding to 60 mg/liter type III procollagen.
The purpose of the present work was to study whether coexpression of
the pro1(I) and pro
2(I) chains of human type I procollagen in
insect cells that also express human prolyl 4-hydroxylase will lead to
the formation of heterotrimeric molecules with the correct 2:1 chain
ratio. The association of type I procollagen chains begins with
interactions among the C propeptides, and it has been regarded as
likely that their structural features favor the formation of
heterotrimers rather than [pro
1(I)]3 homotrimers and
prevent the formation of [pro
2(I)]3 homotrimers (1, 6,
15). We therefore also studied whether pro
2(I) chains in which the C propeptide has been replaced with that of the pro
1(I) or
pro
1(III) chain will form homotrimeric molecules or whether
additional restrictions exist at the level of the collagen domain, as
suggested by the early renaturation experiments with isolated
2(I)
chains (7).
A full-length cDNA for the human
pro1(I) chain (16) was digested with XbaI and ligated to
pVL1392 (Invitrogen). A full-length cDNA Hp2010 (17) for the human
pro
2(I) chain was cloned as a blunt-ended fragment into the
EcoRV site of pSp72 (Promega), generating pSp72-C1A2. A
BglII site was created 9 bp upstream of the translation
initiation codon by PCR, and the full-length cDNA was digested with
BglII and BamHI and ligated to pVL1392. The pVL
constructs were cotransfected into Spodoptera frugiperda Sf9
cells with a modified Autographa californica nuclear
polyhedrosis virus DNA using the BaculoGold transfection kit
(Pharmingen), and the resultant viral pools were collected, amplified,
and plaque-purified (18). The recombinant viruses were termed C1A1 and
C1A2, respectively.
The sequences coding for the signal peptides and N propeptides of the
human pro1(I) and pro
2(I) chains were replaced with those coding
for the corresponding regions of the human pro
1(III) chain by PCR. A
construct pSp72-C1A1 was created by cloning the full-length cDNA
for the pro
1(I) chain into the XbaI site of pSp72. Two
fragments, the first including a 5
BglII site 16 bp upstream of the translation initiation codon and the sequences coding
for the pro
1(III) chain up to the N propeptide cleavage site, and
the second starting from the codon for the first amino acid in the N
telopeptide of the pro
1(I) chain and continuing up to an internal
DraIII site, were generated by PCR. These fragments were
ligated into BglII-DraIII digested pSp72-C1A1,
and the resultant construct was termed pSp72-C1A1NproIII. The
full-length C1A1NproIII was digested with BglII and
XbaI and ligated to pVL1392. For the generation of a
corresponding pSp72-C1A2NproIII construct, two fragments, the first as
above and the second starting from the codon for the first amino acid
in the N telopeptide of the pro
2(I) chain and continuing to an
internal SacII site, were generated by PCR. These fragments
were ligated into BglII-SacII digested pSp72-C1A2, and the resultant construct was termed pSp72-C1A2NproIII. The full-length C1A2NproIII was digested with BglII and
SmaI and ligated to pVL1392. The pVL constructs were
cotransfected into Sf9 cells as above, and the recombinant viruses were
termed C1A1NproIII and C1A2 NproIII, respectively.
To replace the sequence coding for the C propeptide of the pro2(I)
chain with that coding for the C propeptide of the pro
1(III) chain,
two fragments were generated by PCR, the first extending from an
internal AvrII site of the cDNA coding for the
pro
2(I) chain to the codon for the last amino acid of the C
telopeptide and the second from the codon for the first amino acid of
the C propeptide of the pro
1(III) chain to a BamHI site
created 51 bp downstream of the translation stop codon. These fragments
were ligated into AvrII-BamHI digested
pSp72-C1A2, and the resultant construct was termed pSp72-C1A2CproIII.
Another modified C1A2 construct coding for both the C telopeptide and
the C propeptide of type III procollagen was also generated. Two
fragments were made by PCR, the first extending from the internal
AvrII site of the cDNA for the pro
2(I) chain to the
codon for the last amino acid of the triple-helical region, and the
second from the codon for the first amino acid of the C telopeptide of
the pro
1(III) chain to a BamHI site created 51 bp
downstream of the translation stop codon. These fragments were ligated
into pSp72-C1A2 as above, and the resultant construct was termed
pSp72-C1A2Ctelo-proIII. The C telopeptide and C propeptide of the
pro
2(I) chain were also replaced by those of the pro
1(I) chain.
Two fragments were generated by PCR, the first one being the same as
used for the construction of pSp72-C1A2Ctelo-proIII and the second
extending from the codon for the first amino acid of the C telopeptide
of the pro
1(I) chain to a BamHI site created 20 bp
downstream of the translation stop codon. These fragments were ligated
into pSp72-C1A2 as above, and the resultant construct was termed
pSp72-C1A2Ctelo-proI. The full-length C1A2CproIII, C1A2Ctelo-proIII,
and C1A2Ctelo-proI were digested with BglII and
BamHI and ligated to pVL1392, the pVL constructs being
cotransfected into Sf9 cells as above. The recombinant viruses were
termed C1A2CproIII, C1A2Ctelo-proIII, and C1A2Ctelo-proI,
respectively.
High Five cells (H5,
Invitrogen) were cultured either as monolayers or in suspension in
TNM-FH medium (Sigma) or SF900IISFM medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (BioClear) at 27 °C. To
produce recombinant proteins, cells seeded at a density of 5-6 × 105 cells/ml in monolayers or 1-1.5 × 106 cells/ml in suspension were infected with different
combinations of viruses coding for the native or modified type I
procollagen chains together with a double-promoter virus
4PH2,3
coding for the
(19) and
(20) subunits of human prolyl 4-hydroxylase (21). The collagen-coding viruses were used in a
5-10-fold excess over the 4PH
virus (12). L-ascorbic acid phosphate (80 µg/ml) (Wako) was added to the culture medium daily. The cells were harvested 72 h after infection, washed with a
solution of 0.15 M NaCl and 0.02 M phosphate,
pH 7.4, homogenized in a solution of 0.3 M NaCl, 0.2%
Triton X-100, and 0.07 M Tris, pH 7.4, and centrifuged at
10,000 × g for 20 min. The Triton X-100 soluble
proteins were analyzed by SDS-PAGE, followed by staining with Coomassie
Brilliant Blue or Western blotting with a polyclonal antibody against
the N propeptide of type I (PINP) or type III procollagen (PIIINP)
(Farmos Diagnostica) or a monoclonal antibody 95D1A recognizing the
collagenous regions of various collagen chains.4 Aliquots of the
Triton X-100 supernatants were incubated with pepsin (0.2 mg/ml) for
1 h at 22 °C (22), and some samples were subsequently digested
with a combination of trypsin (0.1 mg/ml) and chymotrypsin (0.25 mg/ml)
for 2 min at various temperatures (22).
Two assays
were used to measure the level of expression of the wild-type or
modified type I collagen. The first was based on measurement of the
4-hydroxyproline content, assuming that all the hydroxylatable proline
residues in the 1(I) and
2(I) chains had been converted to
4-hydroxyproline. Aliquots of the Triton X-100 supernatants were
hydrolyzed at 110 °C for 16 h and studied by a colorimetric
method for 4-hydroxyproline (23). The second assay was based on
densitometry of the Coomassie-stained collagen
chain bands in
SDS-PAGE using known amounts of type I collagen (Chemicon) as a
standard. The amounts of the
1(I) and
2(I) chains were estimated
by densitometry of the Coomassie-stained bands using a Bioimage
instrument (Millipore Corp.).
The recombinant type I collagen was purified as described previously (12), with the exception that the collagen was precipitated with 4 M NaCl.
Other AssaysAmino acid analyses of the homotrimeric and heterotrimeric recombinant type I collagens were performed in an Applied Biosystems 421 or Beckman system 6300 amino acid analyzer. The melting curves were determined in a Jasco J-500 spectropolarimeter with a temperature-controlled quartz cell of path length of 1 cm (Gilford) (12).
A recombinant virus coding for the pro1(I)
chains was generated and used to infect High Five cells together with a
double-promoter virus coding for the
and
subunits of human
prolyl 4-hydroxylase. The cells were cultured either as monolayers or
in suspension, harvested 48 and 72 h after infection, homogenized
in a buffer containing 0.2% Triton X-100, and centrifuged. The Triton
X-100 soluble proteins of the cell homogenates were then digested with pepsin at 22 °C for 1-4 h. Samples were analyzed by SDS-PAGE under reducing conditions followed by Coomassie staining or Western blotting.
Two bands corresponding to the pro
1(I) and pN
1(I) chains were
seen in the Coomassie-stained gel of nonpepsinized samples 48 h
after infection (Fig. 1A,
lane 2) while only pN
1(I) chains were seen at 72 h
(Fig. 1A, lane 3). The presence of the pro
1(I)
and pN
1(I) chains was confirmed by Western blotting using the 95D1A
antibody against the collagenous regions of various collagens (Fig.
1B, lane 1) and the PINP antibody against the N
propeptide of human type I procollagen (data not shown). In the case of
the pepsin-digested samples, a major band corresponding to the
1(I)
chains was seen in the Coomassie-stained gel (Fig. 1A,
lane 4), and the same band was identified in the Western
blot using the 95D1A antibody (Fig. 1B, lane 2).
The level of expression of the human type I collagen homotrimer was
about 10-20 mg/liter, which is lower than the figure of up to 40 mg/liter obtained for type III collagen in the same cells (12). As in
the case of type III collagen expression (12), only a minor fraction of the total type I collagen homotrimer produced in High Five cells was
found in the culture medium (data not shown).
Sequences at the 5 ends of DNA constructs influence the expression
level of many polypeptides in the baculovirus system (18). Because the
expression level obtained for type I homotrimer was less than that
obtained for type III collagen, we decided to study whether the level
of expression of the former can be increased by replacing the sequences
coding for the signal peptide and the N propeptide of the pro
1(I)
chain with those of the pro
1(III) chain. A new virus, C1A1NproIII,
was generated and High Five cells were infected and analyzed as above.
A faint band corresponding to the hybrid pro
1(I) chains and a major
band corresponding to the hybrid pN
1(I) chains were seen in
Coomassie-stained SDS-PAGE 48 h after infection (Fig.
1A, lane 5), and these bands were also stained in
Western blotting using the 95D1A (Fig. 1B, lane
3) and PIIINP (data not shown) antibodies. A major band
corresponding to the hybrid pN
1(I) chains and a minor band with the
mobility of fully processed
chains were seen 72 h after
infection (Fig. 1A, lane 6). The latter band
could not be stained by the PIIINP antibody, suggesting that it indeed
represented fully processed
chains. In the case of the
pepsin-digested samples, a major band corresponding to the
1(I)
chain was seen both in the Coomassie-stained SDS-PAGE (Fig.
1A, lane 7) and in the Western blot stained by the 95D1A antibody (Fig. 1B, lane 4). About a
3-fold increase was obtained in the level of expression of the type I
collagen homotrimer using the C1A1NproIII virus, the level ranging up
to about 60 mg of collagen/liter, which corresponds to about 90 mg of
procollagen.
The thermal stability of the type I collagen homotrimer was studied by digestion with a mixture of trypsin and chymotrypsin at various temperatures. These experiments indicated that the Tm of the type I collagen homotrimer was about 40 °C, a value which remained unaffected by replacement of the type I N propeptide (data not shown).
Purification of the Recombinant Type I Collagen HomotrimerThe type I procollagen homotrimer was expressed in
High Five cells in suspension in shaker flasks using either the C1A1 or the C1A1NproIII virus together with the 4PH virus. The
recombinant type I collagen was purified as described previously for
the recombinant type III collagen (12), with minor modifications. The
purified type I collagen homotrimer was studied by amino acid and CD
spectrum analyses. The amino acid composition agreed with that reported for the human
1(I) chains (24), except that the hydroxylysine content was about 80% of that in the nonrecombinant protein (Table I). The Tm of the
recombinant type I collagen homotrimer was 42.8 ± 1.2 °C
(determined from four individual samples) (Fig. 2).
|
Expression of Recombinant Human Type I Procollagen Heterotrimer in High Five Cells
To study whether collagens consisting of more
than one type of chain can be assembled in insect cells, a
recombinant virus C1A2 coding for the pro
2(I) chain was generated
and used to infect High Five cells either with or without the C1A1
virus but in the presence of the 4PH
virus. Equal MOI amounts of
the C1A1 and C1A2 viruses were used in a 5-10-fold excess over the
4PH
virus. The cells were cultured and homogenized as above, and
the samples were analyzed by SDS-PAGE under reducing conditions
followed by Coomassie staining. When the pro
1(I) and pro
2(I)
chains were coexpressed, bands corresponding to the
1(I) and
2(I)
chains with an approximate ratio of 2 to 1 (1.89 ± 0.20 to 1, determined from the integrated densitometry values of four individual
samples) were seen in Coomassie-stained SDS-PAGE of pepsinized samples (Fig. 3, lane 2). When the
pro
2(I) chains were expressed alone, no pepsin-resistant band was
seen (Fig. 3, lane 3). Thus, the pepsin-resistant
2(I)
chains seen in lane 2 must have been present in
heterotrimeric molecules. As the ratio of the
1(I) to
2(I) chains
was about 2:1, essentially all the
1(I) chains must likewise have
been present in heterotrimers. The highest expression levels obtained
for the type I collagen heterotrimer were found to be about 20 mg/liter.
To study whether the level of expression of the type I procollagen
heterotrimer can be improved by replacing the signal sequence and N
propeptide of the pro2(I) chain with those of the pro
1(III) chain, a new virus C1A2NproIII was generated. Several experiments were
performed to express the type I procollagen heterotrimer using
different combinations of equal MOI amounts of the viruses C1A1, C1A2,
C1A1NproIII, and C1A2NproIII, all in the presence of the 4PH
virus. Pepsin-resistant
2(I) chains were detected in all the samples
by Coomassie staining of SDS-PAGE, but significant differences were
found in the
1(I) to
2(I) chain ratios upon densitometry (Fig. 3,
lanes 4-7). When insect cells were coinfected with the C1A1
and C1A2 viruses coding for the wild-type pro
1(I) and pro
2(I)
chains, the ratio of pepsin-resistant
1(I) to
2(I) chains was
consistently 2 to 1 (Fig. 3, lane 2). However, when the C1A2
virus was used together with the C1A1NproIII virus, an excess of the
pepsin-resistant
1(I) chains was found in several experiments and
the
1(I) to
2(I) chain ratio varied, being about 2-5 to 1 (3.11 ± 1.09 to 1, determined from the integrated densitometry values of four individual samples) (Fig. 3, lanes 4-5). The
level of expression of type I collagen obtained by coinfection with the
C1A1NproIII and C1A2 viruses was about 40 mg/liter. When insect cells
were coinfected with the C1A1 or C1A1NproIII and C1A2NproIII viruses,
the formation of heterotrimeric type I collagen appeared to be very
inefficient, the ratio of the pepsin-resistant
1(I) to
2(I)
chains being about 5-10 to 1 (Fig. 3, lanes 6-7).
In further experiments, the ratio of viruses coding for the pro1(I)
and pro
2(I) chains was varied by keeping the amount of C1A2 virus
constant but using different amounts of either the C1A1 virus (Fig.
4, lanes 2-5) or C1A1NproIII
virus (Fig. 4, lanes 6-9) so that the MOI ratio of the C1A1
or C1A1NproIII virus to the C1A2 virus was 0.25 (Fig. 4, lanes 2 and 6), 0.5 (Fig. 4, lanes 3 and
7), 0.75 (Fig. 4, lanes 4 and 8), or
1.0 (Fig. 4, lanes 5 and 9). Pepsin-digested
samples were then studied by SDS-PAGE followed by Coomassie staining
and densitometry of the bands. The amount of pepsin-resistant
1(I)
chains increased with increasing amounts of the C1A1 or C1A1NproIII
virus as could be expected. In addition, the amount of pepsin-resistant
2(I) chains increased in a similar manner even though the amount of
C1A2 virus was kept constant (Fig. 4). The ratio of pepsin-resistant
1(I) to
2(I) chains varied with the original C1A1 and C1A2
viruses, from about 3:1 at a virus ratio of 0.25 (Fig. 4 lane
2) to about 2:1 with ratios of 0.75 and 1.0 (Fig. 4, lanes
4 and 5). When the C1A1NproIII virus was used instead
of the C1A1 virus, the 2:1 ratio was obtained with the MOI ratio 0.75, while a 3:1 ratio was obtained with a MOI ratio of 1.0 (Fig. 4,
lanes 8 and 9).
The Tm of the type I collagen heterotrimer when
studied by digestion with a mixture of trypsin and chymotrypsin at
various temperatures was about 40 °C (Fig.
5).
Purification of the Recombinant Type I Collagen Heterotrimer
The type I procollagen heterotrimer was expressed in
High Five cells in suspension using the C1A1 and C1A2 viruses together with the 4PH virus, and the recombinant protein was purified as
above. The amino acid composition of the purified recombinant collagen
corresponded well to that reported for the nonrecombinant human protein
(24) (Table I), with the exception that the hydroxylysine content was
only about 30%. The Tm for the purified
collagen when determined by CD analyses was 41.5 ± 0.8 °C
(determined from three individual samples) (Fig. 2).
To study whether pro2(I) chains with a modified C
propeptide or C propeptide and C telopeptide are able to form
homotrimers, High Five cells were infected with any of the viruses
C1A2CproIII, C1A2Ctelo-proIII, or C1A2Ctelo-proI together with the
4PH
virus. In the virus C1A2CproIII, the sequences coding for the
C propeptide of the pro
2(I) chain had been replaced by those coding
for the C propeptide of the pro
1(III) chain, whereas in
C1A2Ctelo-proIII and C1A2 Ctelo-proI, the sequences coding for both the
C telopeptide and C propeptide of the pro
2(I) chain had been
replaced by those of the pro
1(III) or pro
1(I) chain,
respectively. When any of these viruses was used, a faint band
corresponding to the pro
2(I) chain with the modified C propeptide
was seen in SDS-PAGE after both Coomassie staining and Western blotting
using the 95D1A antibody (as shown for C1A2CproIII in Fig.
6A, lanes 2 and
4). A considerable portion of the modified pro
2(I) chains
was found in a Triton X-100 insoluble, 1% SDS soluble fraction (Fig.
6A, lanes 3 and 5), a result that
differs distinctly from those obtained in expression experiments
involving the pro
1(I) chain (data not shown).
The stability of the modified pro2(I) chains was studied by pepsin
treatment of the Triton X-100 soluble fraction. No pepsin-resistant
2(I) chains were seen in Coomassie-stained SDS-PAGE (as shown for
C1A2CproIII in Fig. 6A, lane 6) when the
digestion was performed for 1 h at 22 °C (as in the cases of
the [
1(I)]3 homotrimer and [
1(I)]2
2(I) heterotrimer, above). In contrast,
pepsin-resistant
2(I) chains were seen in the cases of all three
modified pro
2(I) viruses when the digestion was performed for 1 h at 4 °C (as shown for C1A2CproIII in Fig. 6B,
lane 1). Nevertheless, the pepsin-resistant
2(I) chains
dissappeared when the digestion at 4 °C was prolonged (Fig.
6B, lanes 2-4). Further experiments indicated
that no pepsin-resistant
2(I) chains were present when the cells
were expressing unmodified pro
2(I) chains, even when the digestion
was performed at 4 °C (Fig. 6C).
The data reported here indicate that coexpression of the
pro1(I) and pro
2(I) chains of human type I procollagen in insect cells leads to the formation of heterotrimeric molecules with the
correct 2:1 chain ratio. The data further indicate that expression of
pro
1(I) chains without pro
2(I) chains effectively leads to the
formation of homotrimeric molecules. The formation of the heterotrimers
was nevertheless clearly favored, as the ratio of pepsin-resistant
1(I) to
2(I) chains remained at 2:1 in the coexpression
experiments unless the pro
1(I) chains were expressed in a relatively
large excess. Replacement of the sequences coding for the signal
peptide and the N propeptide of the pro
1(I) chain with those of the
pro
1(III) chain increased the level of expression of the pro
1(I)
chain about 3-fold, whereas no corresponding effect was seen when a
similar modification was made to the pro
2(I) chain. The highest
expression level obtained for the type I collagen homotrimer with the
modified construct was 60 mg/liter, thus being slightly higher than
that previously obtained for type III collagen in High Five cells
(12).
The heterotrimeric and homotrimeric procollagen molecules produced in
High Five cells were found to be processed with time, as partial
conversion of the wild-type pro1(I) and pro
2(I) chains to
pN
1(I) and pN
2(I) chains (i.e. cleavage of the C
propeptides) was seen in all the expression experiments (although not
shown for the pro
2(I) chain in the coexpression experiments).
Although the N propeptides of the wild-type pro
1(I) and pro
2(I)
chains appeared to be stable, the N propeptide of the pro
1(III)
chain artificially transferred into the pro
1(I) chain was less so, as some of the modified pro
1(I) chains were processed to
(I) chains even in the case of homotrimeric molecules in which the three
type III N propeptides should be able to form a correctly folded
trimer. Similar processing of the N propeptide of the pro
1(III) chain in insect cells was also found when this propeptide was transferred to the pro
1(II) chain,3 whereas the
wild-type N propeptide of the pro
1(II) chain3 and the N
propeptide of the pro
1(III) chain (12) appeared to be quite stable
when present in their wild-type chains. The N propeptide is likely to
fold back on the triple helical collagen domain (25) and may thus
interact with sequences in it. The data obtained with the modified
constructs suggest that such an interaction may be lost or reduced when
the N propeptide is replaced with that of another pro
chain. As
procollagen proteinases are extracellular enzymes (1, 6, 15), the
processing of procollagen molecules found here was probably due to some
nonspecific intracellular proteinases.
The properties of the purified type I collagen heterotrimer were very
similar to those of the corresponding nonrecombinant protein. Although
early studies on the type I collagen homotrimer produced by
renaturation of individual chains suggested that the
Tm of the homotrimer may be slightly lower than
that of the heterotrimer (7), no lower Tm was
found here for the homotrimer whether studied by digestion of crude
cell extracts with a mixture of trypsin and chymotrypsin at various
temperatures or by CD spectrum analysis. The slightly lower
Tm found for the homotrimer produced by the
renaturation experiments (7) may thus be a property limited to that
special case.
The wild-type pro2(I) chain did not form any homotrimeric molecules,
as no pepsin-resistant
2(I) chains were seen in the absence of the
pro
1(I) chains. In agreement with this conclusion, the amount of
pepsin-resistant
2(I) chains obtained in the coexpression experiments increased with increasing expression levels of the pro
1(I) chains. Attempts were also made to produce homotrimeric molecules of pro
2(I) chains by replacing the wild-type C propeptide with that of the pro
1(III) or pro
1(I) chain. Previous data on the
formation of type III procollagen [pro
1(III)]3
homotrimers (12) and the present data on the formation of type I
procollagen [pro
1(I)]3 homotrimers clearly demonstrate
that the C propeptides used in the modified pro
2(I) constructs
become effectively associated in insect cells. Pepsin-resistant
2(I)
chains were found in these experiments only when the digestion was
performed at 4 °C, while the chains were completely digested at
22 °C. These data agree with those obtained in renaturation
experiments with individual
2(I) chains in which the
Tm of the [
2(I)]3 homotrimer
was only 22-24 °C (7). Evaluation of the potential interchain
interactions in type I collagen has likewise suggested that the
[
2(I)]3 homotrimer should have fewer stabilizing
interactions than the heterotrimer or [
1(I)]3
homotrimer (26). The present data differ from recent results obtained
in translation experiments in a rabbit reticulocyte lysate system
in vitro, in which truncated pro
2(I) chains with an
internal deletion of 830 amino acids in the collagen domain did form
triple helices with a Tm of 35 °C provided
that the C propeptide of the pro
2(I) chain had been replaced by that
of the pro
1(III) chain (27). This difference may be due to the large
deletion used in the pro
2(I) chain in the cell-free assembly experiments (27). Our data suggest that in addition to control at the
level of the C propeptide, additional restrictions may exist at the
level of the collagen domain of the full-length pro
2(I) chain, which
prevent the formation of stable homotrimeric molecules in cells.
Hsp-47 is regarded as a chaperone that may be specifically
involved in the assembly and/or secretion of collagens (28, 29). The
present data indicate, however, that formation of the type I
procollagen heterotrimers and homotrimers with stable triple helices
did not require the presence of any recombinant Hsp-47. The possibility
is not excluded that the cells may have markedly up-regulated
expression of an insect Hsp-47, and that this protein assisted folding
of the human procollagen molecules. This possibility does not seem very
likely, however, as baculovirus infection interferes with the synthesis
of cellular proteins (30). It is thus more likely that assembly of the
type I procollagen heterotrimers and homotrimers did not require
Hsp-47. The failure to obtain [pro2(I)]3 homotrimers
with stable triple helices from pro
2(I) chains having modified C
propeptides in insect cells and in renaturation experiments starting
from individual
2(I) chains in vitro (7) might be due to
the lack of Hsp-47 if this chaperone were especially important for
folding of the pro
2(I) chains. The current data on Hsp-47 (28, 31)
does not suggest any such specificity, however. The failure of insect
cells to secrete most of the type I and type III (12) procollagen
molecules might likewise be due to lack of Hsp-47, but it has been
reported previously that insect cells are also poor at secreting many
other recombinant secretory proteins (32-34). It thus seems more
likely that synthesis and assembly of recombinant heterotrimeric and
homotrimeric type I procollagen molecules in insect cells does not
require Hsp-47.
We thank Raija Juntunen, Anne Kokko, Eeva
Lehtimäki, Minta Lumme, and Liisa Äijälä for
expert technical assistance and Anne Snellman for the antibody 95D1A.
The authors gratefully acknowledge Drs. Helena Kuivaniemi and Gerard
Tromp, Center for Molecular Medicine and Genetics, Wayne State
University, Detroit, MI and Darwin J. Prockop, Center for Gene Therapy,
Allegheny University of Health Sciences, Philadelphia, PA for the
full-length cDNA clones for the pro1(I) and pro
2(I)
chains.