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(Received for publication, December 11, 1995; and in revised form, March 1,
1996) From the
An efficient expression system for recombinant collagens would
have numerous scientific and practical applications. Nevertheless, most
recombinant systems are not suitable for this purpose, as they do not
have sufficient amounts of prolyl 4-hydroxylase activity. Pro-
The collagens are a family of closely related but distinct
extracellular matrix proteins. At least 19 proteins representing more
than 30 gene products have now been defined as collagens, and at least
another 10 proteins contain collagen-like domains. All collagen
molecules consist of three polypeptide chains, called Recombinant expression of collagens has proven
difficult to achieve, as their biosynthesis requires processing by up
to eight specific posttranslational
enzymes(1, 6, 11) . Nevertheless, only one of
these enzymes, prolyl 4-hydroxylase, which hydroxylates about 100
proline residues in each of the Baculoviruses have proven to be very efficient
expression vectors for the large scale production of various
recombinant proteins in insect cells. The proteins produced in this
expression system are usually correctly processed, properly folded, and
disulfide-bonded(16, 17) , but as described in this
paper, our initial experiments indicated that recombinant collagen
polypeptide chains produced in a baculovirus system do not form triple
helices that are stable at 37 °C. A recent success in producing a
fully active human prolyl 4-hydroxylase
Figure 1:
Analysis of the
expression of recombinant human type III procollagen in Sf9 and High
Five cells by SDS-PAGE under reducing conditions. Sf9 and High Five
cells were infected with a recombinant baculovirus coding for the
pro-
The level of
pro- One possible
explanation for the low level of expression of pepsin-resistant type
III collagen could be that insect cells have insufficient amounts of
prolyl 4-hydroxylase activity. In order to study this possibility,
insect cells were coinfected with three recombinant baculoviruses, one
of them coding for the pro-
Figure 2:
Analysis of the expression of recombinant
human type III procollagen by SDS-PAGE under reducing conditions in
insect cells also expressing the
The efficiency of multiple baculovirus infection was
assessed by immunocytochemical staining of the insect cells. Sf9 cells
were coinfected with two recombinant viruses coding for the
Figure 3:
Analysis of the efficiency of expression
of the
A considerable variation was found in the
values obtained in different experiments, as shown in Table 2,
but a number of conclusions can still be made from the results. First,
the amount of 4-hydroxyproline formed was distinctly higher in the
cells infected with the prolyl 4-hydroxylase-coding viruses than in
their absence in all the experiments. Second, the level of expression
obtained in the High Five cells was consistently higher than that
obtained in the Sf9 cells. Third, the level of type III collagen
produced in the cells coinfected with the prolyl 4-hydroxylase-coding
viruses was always higher when calculated from the 4-hydroxyproline
values than from the radioimmunoassay values, suggesting either that
some of the N-propeptides of type III procollagen had been degraded or
that some of the fully 4-hydroxylated pro- Experiments were also performed in which High Five cells
were grown in suspension in spinner or shaker flasks. A similar effect
of the prolyl 4-hydroxylase-coding viruses was seen as above. The
highest expression levels found in such experiments were approximately
40 mg of cellular type III collagen/liter of culture in 72 h, about
80-90% of the total collagen produced being found in the cell
pellet and 10-20% in the medium (details not shown).
Figure 4:
Nonreducing SDS-PAGE analysis of trimer
formation of the pro-
The thermal stability of
the type III collagen expressed under different cell culture conditions
was studied using digestion with a mixture of trypsin and chymotrypsin
after heating to various temperatures(22) , ascorbate being
either added to the cell culture medium daily as usual or omitted
during infection. The Triton X-100-soluble proteins were first digested
with pepsin for 1 h at 22 °C to convert the type III procollagen to
type III collagen(22) , and the trypsin/chymotrypsin digestion
was then performed for aliquots of the pepsin-treated samples. When the
pro-
Figure 5:
Analysis of the thermal stability of the
recombinant human type III collagen produced in insect cells by a brief
protease digestion. High Five cells were infected with viruses coding
for the pro-
The purified type III collagen was analyzed by 5%
SDS-PAGE under reducing (Fig. 6, lane 2) and
nonreducing (Fig. 6, lane 3) conditions. No
contaminants were seen in the Coomassie-stained gel, and the
Figure 6:
SDS-PAGE analysis of purified type III
collagen under reducing and nonreducing conditions. The reduced type
III collagen sample is shown in lane 2, and the nonreduced
sample is shown in lane 3. Molecular weight markers were run
in lane 1. The gel was stained with Coomassie Brilliant Blue.
The positions of the trimeric
Figure 7:
Circular dichroism analysis of the
denaturation of purified type III collagen. Panel A, melting
curve of recombinant type III collagen as determined by CD. Normalized
ellipticity of type III collagen at 221 nm was plotted against the
temperature (1 is triple-helical collagen, and 0 corresponds to totally
denatured collagen). Panel B, first derivative of the melting
curve. The T
The data reported here indicate that it is possible to
achieve large scale expression of native-type triple-helical human
collagens in insect cells. The High Five cells gave consistently higher
production rates than the Sf9 cells, the highest rates seen in High
Five cells when cultured in monolayers being about 80 µg of
cellular recombinant human type III collagen/5 Prolyl 4-hydroxylase plays a central role
in the biosynthesis of all collagens, as 4-hydroxyproline residues are
essential for the folding of the newly synthesized polypeptide chains
into triple-helical molecules(6, 12, 26) .
When the pro- Previous experiments had demonstrated that a fully
active human prolyl 4-hydroxylase tetramer can be produced in insect
cells by infecting them with two recombinant baculoviruses, one of them
coding for the The A major difference between the insect
cell system studied here and human fibroblasts is that most of the
triple-helical collagens produced by the insect cells were found to be
retained within the cells, whereas collagenous molecules are rapidly
secreted from human fibroblasts after formation of their triple
helices(27) . The low rate of secretion of triple-helical
recombinant collagens from insect cells may be related to the finding
that such cells often secrete secretory proteins poorly in comparison
with many vertebrate cells(33, 34, 35) . The properties of the purified human type III collagen produced in
insect cells were found to be very similar to those of type III
collagen extracted from various
tissues(1, 2, 3, 4, 5, 6) .
In particular, the 4-hydroxyproline content and the T The insect cell system studied here should allow
various recombinant human collagens to be produced for use in medical
applications. Furthermore, the insect cell system should make it
possible to produce large quantities of various collagens that are
present in tissues in amounts too small to be characterized at the
protein level.
Volume 271,
Number 20,
Issue of May 17, 1996 pp. 11988-11995
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
PRODUCTION OF A PROTEIN WITH A STABLE TRIPLE HELIX REQUIRES
COEXPRESSION WITH THE TWO TYPES OF RECOMBINANT PROLYL 4-HYDROXYLASE
SUBUNIT (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1
chains of human type III collagen expressed in insect cells by a
baculovirus vector are reported here to contain significant amounts of
4-hydroxyproline and to form triple-helical molecules, although the T
of the triple helices was only about 32-34
°C. Coexpression of the pro-1(III) chains with the and
subunits of human prolyl 4-hydroxylase increased the T to about 40 °C, provided that ascorbate was
added to the culture medium. The level of expression of type III
procollagen was also increased in the presence of the recombinant
prolyl 4-hydroxylase, and the pro-1(III) chains and 1(III)
chains were found to be present in disulfide-bonded molecules. Most of
the triple-helical collagen produced was retained within the insect
cells and could be extracted from the cell pellet. The highest
expression levels were obtained in High Five cells, which produced up
to about 80 µg of cellular type III collagen (120 µg of
procollagen) per 5 
10
cells in monolayer culture
and up to 40 mg/liter of cellular type III collagen (60 mg/liter
procollagen) in suspension. The 4-hydroxyproline content and T of the purified recombinant type III collagen
were very similar to those of the nonrecombinant protein, but the
hydroxylysine content was slightly lower, being about 3 residues/1000
in the former and 5/1000 in the latter.
chains,
that are coiled around one another into a triple-helical conformation.
In some collagen types all three chains of the molecule are
identical, while in others the molecule contains two or three different
chains. The most abundant collagens form extracellular fibrils
and are hence known as fibril-forming collagens, while others form
supramolecular aggregates of other kinds (for recent reviews on
collagens, see (1, 2, 3, 4, 5, 6) ).
Nevertheless, many of the recently discovered collagens are present in
tissues in such small quantities that it has not been possible to
isolate them for characterization at the protein level, and thus many
important questions concerning their structure and organization are
still
open(1, 2, 3, 4, 5, 6, 7) .
Some of the fibril-forming collagens are now in medical use, in
applications ranging from biomaterials and drug delivery systems to
trials for the potential of type II collagen as an oral
tolerance-inducing agent for the treatment of rheumatoid
arthritis(8, 9, 10) . The collagens used in
these applications have been isolated from animal tissues, and it is
thus obvious that an efficient large scale recombinant system for
expressing collagens would have numerous scientific and practical
applications. chains in the case of the
fibril-forming collagens, is an absolute requirement, as
4-hydroxyproline-deficient polypeptide chains cannot form triple
helices that are stable at 37 °C(6, 12) . No
attempts have previously been reported regarding the production of
triple-helical collagens in insect cells, bacteria, or yeast, as insect
cells are likely to have insufficient levels of prolyl 4-hydroxylase
activity, and bacteria and yeast do not contain this enzyme at all.
Recombinant collagens have recently been produced in mammalian cells,
however(7, 13, 14, 15) , as such
cells do have adequate levels of prolyl 4-hydroxylase, at least for
experiments in which the aim has not been to obtain a very high
expression level.
tetramer in insect cells by coinfection with two recombinant
baculoviruses, one coding for the subunit and the other for the
subunit(18) , suggested that it might be possible to
produce collagens with stable triple helices by using three
baculoviruses, one of them coding for the recombinant collagen
polypeptide chains and two coding for the and subunits of
prolyl 4-hydroxylase. The present paper reports on the special features
of such a system with human type III collagen as the test collagen and
on the characterization of the recombinant collagen produced.
Construction of the Baculovirus Transfer Vector and
Generation of the Recombinant Virus
A BglII site was
created 16 base pairs upstream of the translation initiation codon to a
full-length cDNA for the pro-1 chain of human type III procollagen (19) by polymerase chain reaction, and the cDNA was digested
with the BglII and XbaI restriction enzymes. The BglII-XbaI fragment was then ligated to pVL1392
(Invitrogen). The recombinant pVL construct was cotransfected into Spodoptera frugiperda Sf9 insect cells with a modified Autographa californica nuclear polyhedrosis virus DNA using
the BaculoGold transfection kit (Pharmingen), and the resultant viral
pool was collected, amplified, and plaque-purified(17) . The
recombinant virus, termed rhproCIII, was checked by a polymerase chain
reaction-based method(20) .Analysis of Recombinant Proteins in Insect Cell
Cultures
Insect cells (Sf9 or High Five; Invitrogen) were
cultured in TNM-FH medium (Sigma) supplemented with 10% fetal bovine
serum (BioClear) or in a serum-free HyQ CCM3 medium (HyClone), either
as monolayers or in suspension in spinner or shaker flasks at 27
°C. To produce recombinant proteins, insect cells seeded at a
density of 5-6 10
/ml were infected with the
rhproCIII virus and with the viruses for the subunit (virus
59) and subunit of human prolyl 4-hydroxylase(18) ,
the rhproCIII virus being used in a 5-10-fold excess over the
other two. Ascorbate (80 µg/ml) was added to the culture medium
daily. The cells were harvested 48-120 h after infection, washed
with a solution of 0.15 M NaCl and 0.02 M phosphate,
pH 7.4, homogenized in a 0.3 M NaCl, 0.2% Triton X-100, and
0.07 M Tris buffer, pH 7.4, and centrifuged at 10,000 g for 20 min. The remaining cell pellet that was insoluble in
the homogenization buffer was further solubilized in 1% SDS and
analyzed by SDS-PAGE. (
)The cell culture medium was
concentrated 10 times in an ultrafiltration cell (Amicon) with a PM-100
membrane. Aliquots of the supernatants of the cell homogenates and the
concentrated cell culture medium were analyzed by denaturing SDS-PAGE,
followed by staining with Coomassie Brilliant Blue or Western blotting
with an antibody to the N-propeptide of human type III procollagen.
Other aliquots were studied by a radioimmunoassay for the trimeric
N-propeptide of human type III procollagen (Farmos Diagnostica) and a
colorimetric method for 4-hydroxyproline(21) . Still further
aliquots were digested with pepsin for 1 h at 22 °C(22) ,
and the thermal stability of the pepsin-resistant recombinant type III
collagen was measured by rapid digestion with a mixture of trypsin and
chymotrypsin (22) . Prolyl 4-hydroxylase activity was assayed
by a method based on the hydroxylation-coupled decarboxylation of
2-oxo[1-
C]glutarate(23) .Double Immunostaining of Sf9 Cells
Sf9 cells were
grown on glass slides and fixed in 100% ethanol at -20 °C.
Alternatively, cells in monolayer culture were detached, washed twice
with a solution of 0.15 M NaCl and 0.02 M phosphate,
pH 7.4 (washing solution), suspended in cold ethanol, and spread on
silanated (24) glass slides. They were then incubated with 1%
bovine serum albumin in 0.15 M NaCl and 0.02 M phosphate, pH 7.4, for 15 min, followed by incubation for 30 min
in a 1:50 dilution of a mouse monoclonal antibody to the subunit
(5B5, Dako) and a rabbit polyclonal antibody to the subunit of
human prolyl 4-hydroxylase in the above solution containing bovine
serum albumin. The cells were subsequently washed 4 times for 20 min
with the washing solution and incubated in a 1:10 dilution of a sheep
anti-mouse Ig-rhodamine F(ab)`2 fragment (Boehringer Mannheim) and a
sheep anti-rabbit IgG fluorescein F(ab)`2 fragment (Boehringer
Mannheim) in the bovine serum albumin solution for 30 min, washed with
the washing solution, rinsed with distilled water, and mounted in
Glycergel (Dako). The samples were photographed using a Leitz
Aristoplan microscope equipped with epi-illuminator and filters for
fluorescein isothiocyanate and tetramethyl rhodamine B isothiocyanate
fluorescence.Purification of Recombinant Type III
Collagen
Insect cells expressing the recombinant type III
procollagen were washed with a solution of 0.15 M NaCl and
0.02 M phosphate, pH 7.4, homogenized in a cold 0.2 M NaCl, 0.1% Triton X-100, and 0.05 M Tris buffer, pH 7.4
(20 10 cells/ml), incubated on ice for 30 min, and
centrifuged at 16,000 g for 30 min. Unless otherwise
mentioned, all the following steps were performed at 4 °C. The
supernatant was chromatographed on a DEAE cellulose column (DE-52,
Whatman) equilibrated and eluted with a 0.2 M NaCl and 0.05 M Tris buffer, pH 7.4, the void volume being collected. The pH
of the sample was lowered to 2.0-2.5, and the sample was digested
with a final concentration of 150 µg/ml of pepsin for 1 h at 22
°C. The pepsin was irreversibly inactivated by neutralization of
the sample followed by overnight incubation on ice. The recombinant
type III collagen was precipitated by adding solid NaCl to a final
concentration of 2 M and centrifuging at 16,000 g for 1 h. The pellet was dissolved in a 0.5 M NaCl, 0.5 M urea, and 0.05 M Tris buffer, pH 7.4, for 1 day,
and the sample was digested with pepsin as above a second time. The
sample was then chromatographed on a Sephacryl HR-500 gel filtration
column (Pharmacia Biotech Inc.), eluted with a solution of 0.2 M NaCl and 0.05 M Tris, pH 7.4, dialyzed against 0.1 M acetic acid, and lyophilized.Other Assays
The amount of purified type III
collagen obtained was determined by Sircol collagen assay (Biocolor).
Amino acid analysis of the purified type III collagen was performed in
an Applied Biosystems 421 amino acid analyzer. The melting curve was
determined in a Jasco J-500 spectropolarimeter, equipped with a
temperature-controlled quartz cell of 1-cm path length (Gilford). The
collagen concentration for the sample shown was 104 µg/ml in 0.05%
acetic acid. Thermal transition curves were recorded at a fixed
wavelength (221 nm) by raising the temperature linearly at a rate of 30
°C/h using a Gilford temperature programmer.
Expression of Recombinant Human Type III Procollagen in
Sf9 and High Five Cells
In order to ascertain whether it is
possible to produce full-length pro-1 chains of human type III
procollagen in insect cells and whether the pro-1(III) chains
produced in these cells can form triple-helical molecules, a
recombinant baculovirus coding for the pro-1(III) chains was
generated and used to infect Sf9 and High Five cells. The cells were
cultured in TNM-FH medium supplemented with 10% fetal bovine serum or
in a serum-free medium if the cell culture medium was to be analyzed,
harvested 72 h after infection, homogenized in a buffer containing 0.2%
Triton X-100, and centrifuged. Samples of the Triton X-100 soluble
protein fraction and the concentrated cell culture medium were then
analyzed by SDS-PAGE under reducing conditions, followed either by
Coomassie staining (Fig. 1A) or Western blotting with
an antibody to the N-propeptide of type III procollagen (Fig. 1B). Some of the samples were digested with
pepsin for 1 h at 22 °C before the SDS-PAGE (Fig. 1, A and B, lanes with odd numbers). The
triple helix of collagens is resistant to proteolytic enzymes, whereas
nontriple-helical pro-1(III) chains and the propeptides of
triple-helical procollagen molecules are digested.
1(III) chains, harvested 72 h after infection, homogenized in
a buffer containing 0.2% Triton X-100, and centrifuged. Aliquots of the
Triton X-100 soluble protein fraction and the concentrated cell culture
medium were then analyzed either without pepsin treatment or after
treatment with pepsin for 1 h at 22 °C. The samples were
electrophoresed on 8% SDS-PAGE under reducing conditions and analyzed
by Coomassie staining in panel A and by Western blotting using
an antibody to the N-propeptide of human type III procollagen in panel B. Lane 1, molecular weight markers; lanes
2 and 3, cell extracts; lanes 4 and 5,
media from Sf9 cell cultures; lanes 6 and 7, cell
extracts; lanes 8 and 9, media from High Five cell
cultures. The samples in the odd numbered lanes were digested
with pepsin. Because the antibody used in the Western blotting reacts
only with the N-propeptide of type III procollagen, it does not
recognize pepsin-digested samples. The arrows indicate the
pro-1(III) and 1(III) chains. The intense band of about 64
kDa seen on lanes 4 and 8 in panel A probably represents bovine serum albumin. The bands with
mobilities higher than that of the pro-1(III) chains in panel
B probably represent degradation products of these
chains.
1(III) chain expression was too low for these to be detected
in the Coomassie-stained SDS-PAGE (Fig. 1A, lanes
2, 4, 6, and 8), but they could be seen
by Western blotting in samples of the Triton X-100 soluble proteins (Fig. 1B, lanes 2 and 6) and cell
culture media (Fig. 1B, lanes 4 and 8) in the case of both the Sf9 and High Five cells. After the
pepsin digestion, 1 chains of type III collagen were seen in the
High Five cells in the Coomassie-stained gel (Fig. 1A, lane 7). The pepsin-resistant 1(III) chains were not
detected in the Western blot (Fig. 1B, lanes
3, 5, 7, and 9), since the antibody
used reacts only with the N-propeptides of pro-1(III) chains,
which were apparently digested by the pepsin.1(III) chain as above, and the other
two coding for the and subunits of human prolyl
4-hydroxylase. The cells infected with the three viruses were then
cultured, harvested, and analyzed as above, the Coomassie-stained
SDS-PAGE being shown in Fig. 2A and the Western blot in Fig. 2B. The addition of prolyl 4-hydroxylase-coding
viruses increased the amount of pro-1(III) chains present (Fig. 2, A and B, lanes 2 and 6) and that of pepsin-resistant 1(III) chains (Fig. 2A, lanes 3 and 7) in both the
Sf9 and High Five cells. No pro-1(III) chains (Fig. 2A, lanes 4 and 8) or
1(III) chains (Fig. 2A, lanes 5 and 9) could be detected in the medium samples in the
Coomassie-stained gel, but a minor amount of pro-1(III) chains was
seen in the Western blot (Fig. 2B, lanes 4 and 8).
and subunits of
recombinant human prolyl 4-hydroxylase. Sf9 and High Five cells were
coinfected with three recombinant baculoviruses, one of them coding for
the pro-1(III) chains and the other two coding for the and
subunits of human prolyl 4-hydroxylase. The cells were harvested
72 h after infection and analyzed as described in the legend to Fig. 1. The samples were electrophoresed on 8% SDS-PAGE and
analyzed by Coomassie staining in panel A and by Western
blotting in panel B. The antibody used in this experiment was
the same as in Fig. 1. Lane 1, molecular weight
markers; lanes 2 and 3, cell extracts; lanes 4 and 5, media from Sf9 cell cultures; lanes 6 and 7, cell extracts; lanes 8 and 9, media from
High Five cell cultures.
and
subunits of prolyl 4-hydroxylase and immunostained with
antibodies to these two subunits (Fig. 3). When the analysis was
performed 48 h after infection, 87% of the cells were found to express
at least one of the two types of subunit, with 90% of the cells
expressing one type of subunit also expressing the other.
and subunits of recombinant human prolyl
4-hydroxylase in Sf9 cells. Sf9 cells were coinfected with two
recombinant viruses coding for the and subunits of human
prolyl 4-hydroxylase, and analyzed 48 h after infection. The cells were
double-immunostained with a rabbit polyclonal antibody to the
subunit and a mouse monoclonal antibody to the subunit of prolyl
4-hydroxylase. Fluorescein-conjugated sheep antibody to rabbit IgG and
rhodamine-conjugated sheep antibody to mouse IgG were used as secondary
antibodies. Cells expressing both the and subunits are yellow. Cells expressing the subunit only are indicated
by closed arrows, while cells expressing the subunit
only are indicated by open arrows. Negative cells are labeled
with asterisks.
Prolyl 4-Hydroxylase Activity in the Insect
Cells
The 0.2% Triton X-100 extracts of the cell homogenates
were analyzed for prolyl 4-hydroxylase activity with an assay based on
the hydroxylation-coupled decarboxylation of
2-oxo[1-C]glutarate(23) . As reported
previously(25) , a significant level of prolyl 4-hydroxylase
activity was found in both the Sf9 and High Five cells, that in the
High Five cells being distinctly greater than that in the Sf9 cells (Table 1). Infection of the cells with a virus coding for the
pro-1(III) chains had only minor effects on this activity, whereas
the activity in the cells infected with the virus coding for the
pro-1(III) chains together with viruses coding for the two types
of subunit of human prolyl 4-hydroxylase was markedly higher (Table 1).
Effect of Recombinant Prolyl 4-Hydroxylase on the Level
of Type III Procollagen Expression
Sf9 and High Five cells were
infected with the virus coding for the pro-1(III) chains either
with or without viruses coding for the two types of subunit of prolyl
4-hydroxylase (Table 2). The level of expression of total type
III procollagen was measured with a radioimmunoassay for the trimeric
N-propeptide, and the amount of 4-hydroxyproline formed in the cells
was determined by a colorimetric assay. Both values were used to
calculate the amount of type III collagen produced, assuming that all
the pro-1(III) chains formed triple-helical molecules and that all
the hydroxylatable proline residues in the pro-1(III) chains had
been converted to 4-hydroxyproline. Starting out from the known
structure of type III procollagen and the amount of 4-hydroxyproline in
type III collagen, the amount of type III collagen in the samples was
calculated by multiplying the N-propeptide values by 7 and the
4-hydroxyproline values by 8. If all the pro-1(III) chains are
present in fully hydroxylated, triple-helical molecules, both assays
should give identical values, whereas if some of the type III
procollagen is converted to type III collagen and the propeptides are
subsequently degraded, then the values calculated from the
4-hydroxyproline assay should be higher. All these measurements were
made 72 h after infection.
1(III) chains had
remained nontriple-helical. The highest type III collagen expression
values were seen in the High Five cells that also expressed prolyl
4-hydroxylase, the amount of cellular type III collagen in these cells
being about 41-81 µg/5 10 cells (Table 2). The amount of type III collagen found in the culture
medium, as measured with the radioimmunoassay, was about 25-50%
of the total in the Sf9 cells and about 10-30% that in the High
Five cells.Time Course for the Synthesis and Secretion of Type III
Procollagen in High Five Cells
Since the level of expression of
type III collagen in the High Five cells was found to be about
3-10-fold relative to that in the Sf9 cells, High Five cells were
selected for the subsequent experiments. The time course for the
expression of hydroxylated type III procollagen was studied in High
Five cells infected with the three recombinant viruses. The cells were
harvested at 48, 72, 96, and 120 h after infection, and the amount of
type III collagen produced was calculated from the radioimmunoassay and
4-hydroxyproline values as above. In three complete experiments the
amount of cellular type III collagen was found to be highest at about
72 h, the value decreasing distinctly at 96 and 120 h (details not
shown). The amount of prolyl 4-hydroxylase activity in the cell
homogenate was likewise highest 72 h after infection (details not
shown). The radioimmunoassay showed the amount of type III procollagen
in the medium to increase continuously, being about 30-45% of the
total at 120 h. The amount of 4-hydroxyproline in the culture medium
increased even more, but evidently most of that was present in collagen
degradation products, as no band corresponding to the 1 chains of
type III collagen was seen at 96 or 120 h by SDS-PAGE analysis (details
not shown). Since the amount of cellular type III collagen appeared to
be highest 72 h after infection, this time point was used in the
subsequent experiments.Conformational Integrity of the Recombinant Type III
Collagen
Association of the pro-1(III) chains into trimers
was studied by SDS-PAGE analysis under nonreducing conditions.
Essentially all the pro-1(III) chains synthesized were found as
disulfide-bonded trimers, judging from the disappearance of the band
corresponding to monomeric pro-1(III) chains and the appearance of
a protein band of high molecular weight (Fig. 4, lane
2). After pepsin digestion, the band corresponding to the
recombinant type III procollagen was converted to one corresponding to
type III collagen, and the protein remained in the form of the trimer,
thus indicating the existence of disulfide bonds between the
1(III) chains (Fig. 4, lane 3). Virtually all the
type III procollagen expressed was soluble in the homogenization buffer
containing Triton X-100, as no band corresponding to type III
procollagen was seen in the Triton X-100-insoluble, SDS-soluble
fraction (Fig. 4, lane 4).
1(III) chains expressed in High Five insect
cells. High Five cells were coinfected with viruses coding for the
pro-1(III) chains and the and subunits of human prolyl
4-hydroxylase. The cells were harvested 72 h after infection,
homogenized in a buffer containing 0.2% Triton X-100, and centrifuged,
and the remaining cell pellets were further solubilized in 1% SDS.
Aliquots of the Triton-soluble proteins were treated with pepsin for 1
h at 22 °C. The samples were electrophoresed on 5% SDS-PAGE under
nonreducing conditions and analyzed by Coomassie staining. Lane
1, molecular weight markers; lane 2, cell extract; lane 3, cell extract digested with pepsin; lane 4,
proteins soluble in 1% SDS. The positions of the trimeric
pro-1(III) and 1(III) chains are shown by arrows.
1(III) chains were expressed in the absence of prolyl
4-hydroxylase and ascorbate, the T of the type III
collagen was found to be about 32-34 °C (Fig. 5A). The presence of either ascorbate or prolyl
4-hydroxylase without the other caused virtually no increase in thermal
stability (Fig. 5, B and C), but when the
pro-1(III) chains were produced in the presence of both, the T was increased considerably, being about 40
°C (Fig. 5D).
1(III) chains and the and subunits of
human prolyl 4-hydroxylase. The cells were harvested 72 h after
infection, homogenized in a buffer containing 0.2% Triton X-100, and
centrifuged. The Triton-soluble proteins were digested with pepsin for
1 h at 22 °C, and the samples were subsequently treated with a
mixture of trypsin and chymotrypsin at temperatures between 27 and 42
°C as described(22) , the digestion being terminated by the
addition of soybean trypsin inhibitor. The samples were electrophoresed
on 8% SDS-PAGE and analyzed by Coomassie staining. Panel A,
cells infected only with the virus coding for the pro-1(III)
chains, ascorbate omitted from the culture medium; panel B,
cells infected only with the virus coding for the pro-1(III)
chains, ascorbate present in the culture medium as usual; panel
C, cells coinfected with viruses coding for the pro-1(III)
chains and the and subunits of prolyl 4-hydroxylase,
ascorbate was omitted from the culture medium; panel D, cells
infected with the three viruses, ascorbate present in the culture
medium. Lane P shows a sample digested with pepsin without
subsequent trypsin/chymotrypsin digestion, lanes 27-42 show samples treated with the trypsin/chymotrypsin mixture at the
temperatures indicated. The arrows show the positions of the
1(III) chains.
Purification and Characterization of Recombinant Type III
Collagen
Type III procollagen was expressed in High Five cells
cultured either as monolayers or in suspension in shaker flasks. The
cells were harvested 72 h after infection, homogenized in a buffer
containing 0.1% Triton X-100, and centrifuged, and the supernatant of
the cell homogenate was passed through a DEAE cellulose column to
remove nucleic acids. The flow-through fractions containing the type
III procollagen were pooled and digested with pepsin. This converted
the type III procollagen to type III collagen and digested most of the
noncollagenous proteins. The type III collagen was then concentrated by
salt precipitation, solubilized, and treated with pepsin as above. The
type III collagen was finally separated from pepsin and other remaining
contaminants by gel filtration on a Sephacryl S500-HR column. The
fractions containing the type III collagen were pooled, dialyzed, and
lyophilized.1(III) chains were disulfide-bonded. Amino acid and CD spectrum
analyses were performed on the purified type III collagen. The amino
acid composition corresponded well with that reported for the human
protein (Table 3), although the 4-hydroxyproline content was
slightly lower. A distinct difference was found in the amount of
hydroxylysine, which was about 3 residues/1000 amino acids in the
recombinant type III collagen rather than 5/1000 amino acids in the
authentic human type III collagen. The T of the
recombinant type III collagen was 40.8 ± 1.3 °C (±
S.D., n = 4), and virtually all of the recombinant
protein was stable at 37 °C (Fig. 7).
1(III) chains and the monomeric
1(III) chains are shown by arrows.
value determined from the maximum of
the differentiated curve is 42.3 °C.
10 cells, which corresponds to about 120 µg of type III
procollagen. The largest amount of cellular type III collagen produced
when the High Five cells were cultured in suspension in spinner or
shaker flasks was about 40 mg/liter, corresponding to about 60 mg/liter
of type III procollagen.1 chains of type III procollagen were expressed in
insect cells alone, without recombinant prolyl 4-hydroxylase,
considerable amounts of 4-hydroxyproline were generated in the cells
and the pro-1 chains formed triple-helical molecules, as indicated
by the resistance of the collagenous domains of these chains to pepsin
digestion at 22 °C. However, the T of the
triple helices of such molecules was about 6-8 °C lower than
of those produced in the presence of the recombinant enzyme. Also, the
level of expression of type III collagen was lower in the absence of
recombinant prolyl 4-hydroxylase than in its presence, probably because
many of the partially hydroxylated polypeptide chains failed to form
triple-helical molecules even at 27 °C and were rapidly degraded.
The insect cell system was found to resemble human fibroblasts (27, 28) in that the presence of ascorbate in the
culture medium was necessary to produce collagen molecules with stable
triple helices. subunit of human prolyl 4-hydroxylase and the
other the subunit (18) . Nevertheless, the recombinant
enzyme had on all previous occasions been extracted from the insect
cell homogenates, and the assays had been performed in the presence of
the polypeptide substrate and the various cosubstrates in
vitro(18, 25, 29) . Although the
findings suggested that the enzyme may also be active in insect cells in vivo, the present data constitute the first demonstration
that this is indeed the case. Double-immunostaining experiments
demonstrated that about 90% of the insect cells expressing one of the
two types of subunit of human prolyl 4-hydroxylase also expressed the
other. subunit of prolyl 4-hydroxylase is a highly unusual
multifunctional
polypeptide(6, 12, 26, 30) , being
identical to the enzyme protein-disulfide
isomerase(31, 32) , which is regarded as the in
vivo catalyst of disulfide bond formation in the biosynthesis of
various secretory and cell surface proteins, including
collagens(6, 12, 30) . Although insect cells
have a small amount of endogenous protein-disulfide isomerase activity,
this activity is markedly increased when the cells are infected with a
recombinant baculovirus coding for the human protein-disulfide
isomerase/ subunit polypeptide(33) . In agreement with
this, the recombinant pro-1(III) chains produced in insect cells
expressing the two types of subunit of recombinant prolyl 4-hydroxylase
were found to be properly disulfide-bonded when studied by SDS-PAGE
under nonreducing conditions. of the triple helices, when determined by CD analysis, were found
to be very similar to those of nonrecombinant type III collagen.
Interestingly, the hydroxylysine content of the recombinant collagen
was found to be about 60% of that of type III collagen extracted from
various mammalian tissues even though no recombinant lysyl hydroxylase
was coexpressed. This indicates that insect cells must have a
considerable level of lysyl hydroxylase activity. We did not study the
level of glycosylation of the 3 hydroxylysine residues formed per
chain, as even the chains of type III collagen extracted from
various tissues contain only about 0.1 residue of
galactosylhydroxylysine and 0.8 of
glycosylgalactosylhydroxylysine(36) . However, as insect cells
appear to have relatively high levels of prolyl 4-hydroxylase and lysyl
hydroxylase activity, they may well also have relatively high levels of
collagen glycosyltransferase activities so that some of the
hydroxylysine present in the recombinant 1(III) chains may be
glycosylated.
)1(III) chain, pro-1 chain of type III procollagen, a
protein the molecules of which consist of 3 pro-1(III) chains;
1(III) chain, 1 chain of type III collagen, a protein
produced from type III procollagen by the cleavage of the N and C
propeptides.
We thank Anne Kokko, Eeva
Lehtimäki, and Riitta Polojärvi
for expert technical assistance and Dr. Eija-Riitta
Hämäläinen
for performing the amino acid analysis. We gratefully acknowledge Drs.
Leena Ala-Kokko, Helena Kuivaniemi, Gerard Tromp, and Darwin J. Prockop
(Department of Biochemistry and Molecular Biology, Jefferson Medical
College of the Thomas Jefferson University, Philadelphia, PA) for the
full-length cDNA for the pro-1(III) chains; Dr. Leila Risteli
(Department of Medical Biochemistry, University of Oulu) for the
antibody to the N-propeptide of human type III procollagen; and Dr.
Peter Fietzek (Institute for Medical Molecular Biology, Medical
University of Lübeck) for help in performing the CD
analysis.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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