(Received for publication, August 9, 1996, and in revised form, October 25, 1996)
From the A recombinant system was used to prepare human
type II procollagen containing the substitution of Cys for Arg at
Collagen II is the major structural component of cartilage, in
that it accounts for about 30% of the dry weight of developing cartilage and about 60% of the dry weight of adult articular cartilage (1, 2). The fibrils of collagen II are distended by the presence of
water, proteoglycans, and other matrix components such as collagens IX
and XI that bind to the surface of collagen II fibrils (3, 4). Like
other fibrillar collagens, collagen II is first synthesized as a
procollagen in which the three One special class of mutations found in the human COL2A1
gene are single base substitutions that converted codons for Arg residues in the Y position of the repeating
-Gly-X-Y- sequence of the triple helix to codons
for Cys. The first Arg to Cys substitution was seen in a family with
early onset generalized osteoarthritis together with features of a mild
chondrodysplasia probably best classified as a spondyloepiphyseal
dysplasia (15). Subsequently, four additional families with a similar
phenotype were seen with the same mutations in the codon for
Arg- Previously, we expressed the human COL2A1 gene in HT-1080
cells, a human tumor cell line that synthesizes the collagen IV found
in the basement membrane but not any fibrillar collagen (22, 23). As
was shown previously, these cells are a very useful tool for producing
properly folded and posttranslationally modified procollagen II
(24).
Here we have prepared recombinant human procollagen II containing the
substitution of Cys for Arg at Previously we
prepared recombinant procollagen II by using a
COL1A1/COL2A1 hybrid construct in which
expression of the COL2A1 gene was driven by a promoter of
the COL1A1 gene (25). To obtain a fragment of the
COL2A1 gene coding for Cys at To assemble a COL1A1/COL2A1 hybrid construct
containing the mutated For the first step of assembly, the Sphl site in the 5 To express the normal and mutated
COL1A1/COL2A1 genes, the constructs were stably
transfected into the human cell line HT-1080 by calcium phosphate
precipitation, and clones were selected with G418 for expression of the
co-transfected neomycin resistance gene (see Refs. 24 and 31).
Transfected clones were screened for secretion of the recombinant
protein by a Western blot assay using a polyclonal antibody specific
for the C-telopeptide of collagen II (see Ref. 25). Positive clones
were grown in 175-cm2 culture flasks in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum. To
prepare larger amounts of the recombinant protein, about 3 × 108 cells were seeded into six separate intercommunicating
stacks of culture flasks (6,000 cm2 each; Cell
FactoryTM, Nunc). After reaching confluency, the cell layer
was washed twice with phosphate-buffered saline. To harvest the
recombinant procollagen, the cells were incubated in Dulbecco's
modified Eagle's medium supplemented with L-ascorbic acid
phosphate magnesium salt n-hydrate (Wako, Osaka, Japan)
without fetal calf serum for 24 h on each of six successive days.
In a few experiments, 0.8 or 0.2 µCi/ml 14C-labeled amino
acids (DuPont NEN) were added to the medium to obtain
14C-labeled proteins. The 36 liters of medium from the six
24-h collections was concentrated 10-fold using a counterflow
filtration system with a 100-kDa cutoff limit (PREP-SCALETM
TFF cartridge; Millipore). Proteins in the medium were further concentrated by precipitation with 176 mg/ml ammonium sulfate. Procollagen was then purified by chromatography on two anion exchange columns as described previously (24, 31).
Total RNA was extracted
from cells expressing the mutated COL1A1/COL2A1
hybrid gene with a selective resin (RNeasy; Qiagen). The RNA was
reverse transcribed with random primers (First Strand cDNA
synthesis kit; Pharmacia Biotech Inc.) and amplified by PCR with the
primer pairs SW-13 (AGAGGTGCTCCCGGAAAC) and SW-12 (TCTCGCCAGGCATTCCCTG) directed to sequences spanning the region from 1321 to 2053 base pairs
in the COL2A1 cDNA. The PCR product was separated by
electrophoresis on a 2.5% agarose gel, extracted, and sequenced by
cycle sequencing with fluorescently labeled dNTPs on an automated
sequencer (Applied Biosystems, Inc.).
Thermal
stability of the collagen triple helix was assayed by brief protease
digestion at varying temperatures with a trypsin and chymotrypsin
mixture as described previously (24, 31, 32). To assay for
intramolecular disulfide bonds, 14C-labeled procollagen was
digested with the mixture of trypsin (100 µg/ml) and chymotrypsin
(250 µg/ml) at 25 °C for 5 min. The resulting collagen was assayed
for the presence of protease-resistant About 2 mg of purified
procollagen II was digested by procollagen N-proteinase (EC 3.4.24.14)
purified from chick embryo tendons (33) for 6 h at 32 °C in
0.15 M NaCl, 5 mM CaCl2, 0.05% Brij, and 0.01% NaN3 in 50 mM Tris-HCl buffer,
adjusted to pH 7.8 at 20 °C. The reaction was stopped by addition of
0.1 volume of 250 mM EDTA and 0.02% NaN3 in 50 mM Tris-HCl buffer, pH 7.4 at 20 °C. The pC-collagen II
was concentrated on a membrane filter (Amicon YM-100) under a pressure
of 7 p.s.i. and purified with a gel filtration column
(HiLoadTM SuperdexTM 200 column; Pharmacia) in
a fast protein liquid chromatography system (Pharmacia). The column was
equilibrated, and proteins were eluted with 0.4 M NaCl, 50 mM EDTA, and 0.01% NaN3 in 0.1 M
Tris-HCl buffer, pH 7.4, with flow rate of 0.5 ml/min. Fractions that
contained pC-collagen II were combined and concentrated on a membrane
filter (Amicon YM-100).
For
the studies on fibril formation, mutated pC-collagen II was
14C-labeled, but normal pC-collagen II was not. The
pC-collagens and C-proteinase were dialyzed separately against fibril
formation buffer (34) and then stored in parafilm-sealed tubes under an atmosphere of 10% CO2 and 90% air at For dark-field light microscopy,
mixtures of pC-collagen and C-proteinase (40 µl final volume) were
placed in sealed chambers formed by gluing a plastic ring to a
microscopic slide and capping the chamber with a coverslip (35).
Fibrils were photographed using a light microscope (model 9901;
Zeiss).
For the electron microscopy, a hanging drop system for fibril assembly
was used to minimize aggregation of the fibrils (36). Aliquots of 5 µl of a mixture containing pC-collagens and C-proteinase were
transferred onto a Teflon-insulated wire mounted inside a screw cap to
form a hanging drop. Then the drop on the wire was suspended above a
small amount of fibril formation buffer, and the tube was tightly
closed. The tube was submerged in the water bath at 37 °C and
incubated for 24 h. The whole drop was then transferred to a
carbon-coated electron microscopic grid and stained negatively for 1 min with 1% phosphotungstate. The grids were examined with a
transmission electron microscope (model 7000; Hitachi) at a
magnification of × 3,000-50,000.
Molecular modeling
was performed on a Silicon Graphics (Onyx) computer system using the
SYBYL software package, version 6.1 (Tripos, Inc., St. Louis, MO).
Models of the collagen II triple helix were created as described by
Chen et al. (37). The parent model was generated with a
36-amino acid peptide in which the Arg at To obtain a
construct of the mutated COL2A1 gene, a 12-kb
SphI-SphI fragment was isolated from a cosmid
library prepared with genomic DNA from a proband previously found to
have primary generalized osteoarthritis associated with a mild
chondrodysplasia (15). The proband was heterozygous for a single base
mutation that converted the codon for Arg at
To obtain adequate amounts of the recombinant mutated procollagen II
for functional assays, 36 liters of medium from cultures of the clone
were concentrated, and the protein was purified to homogeneity by ion
exchange chromatography. The protein was shown to be homogeneous by
electrophoresis on a SDS-polyacrylamide gel stained with colloidal
Brilliant Blue G (not shown).
To assay the
thermal stability of the triple helix of the mutated collagen II, the
protein was assayed by brief digestion with mixture of chymotrypsin and
trypsin. As indicated in Fig. 2, there was no apparent
difference in thermal stability between the mutated collagen II and
normal collagen II.
Of note was that the band below To assay the mutated procollagen for the presence of intramolecular
disulfide bonds within the triple helix, the purified protein was
digested with trypsin and chymotrypsin at 25 °C to remove the N- and
C-propeptides. The resulting collagen was then assayed by
electrophoresis in polyacrylamide gels in SDS under nonreducing
conditions. No dimers of
To explore why the Arg-
Distances measured between atoms in models of the collagen II triple
helix
Department of Biochemistry and Molecular
Biology,
1-519 found in three unrelated families with early onset
generalized osteoarthritis together with features of a mild
chondrodysplasia probably best classified as spondyloepiphyseal
dysplasia. In contrast to mutated procollagens containing Cys
substitutions for obligatory Gly residues, the Cys substitution at
1-519 did not generate any intramolecular disulfide bonds. The
results were consistent with computer modeling experiments that
demonstrated that the
carbon distances were shorter with Cys
substitutions for obligatory Gly residues than with Cys substitutions
in the Y position residues in repeating -Gly-X-Y- sequences of the collagen triple
helix. The mutated collagen did not assemble into fibrils under
conditions in which the normal monomers polymerized. However, the
presence of the mutated monomer in mixtures with normal collagen II
increased the lag time for fibril assembly and altered the morphology
of the fibrils formed.
chains of the monomer are extended
by the presence of N- and C-propeptides. The processing of procollagen
II to collagen II requires a procollagen N-proteinase to cleave the
three chains of the NH2-terminal propeptide and a separate
procollagen C-proteinase to cleave the three chains of the C-propeptide
(5). After processing of the procollagen by the two proteases, the
resulting collagen II spontaneously forms fibrils. Over 50 mutations in
the gene coding for collagen II (COL2A1) have been reported
in patients with the heterogeneous cartilage disorders referred to as
chondrodysplasias (5-10). Also, transgenic mice expressing mutated
collagen II genes develop phenotypes similar to human chondrodysplasias
(11-14). About half of the mutations in the COL2A1 gene
causing human chondrodysplasias are single-base substitutions that
convert codons for obligate glycine residues in the repeating
-Gly-X-Y- sequence of the collagen triple helix to codons for amino acids with bulkier side chains (6, 9, 10). The
remaining mutations are premature termination codons, RNA-splicing
mutations, partial gene deletions, sequence insertions, or sequence
duplications.
1-519 (16). Of the total of five families with the
Arg-
1-519 mutations, three may be related through an early
Icelandic founder, and the other two are apparently unrelated (17).
Three additional probands with similar phenotypes were reported with a
Cys for Arg substitution at
1-75 (18, 19). In contrast, a similar
Cys for Arg substitution at
1-789 was found in two unrelated
probands with a severe phenotype of spondyloepiphyseal dysplasia (20,
21). The Cys for Arg substitutions in collagen II are of special
interest because they are the first amino acid substitutions in the
Y position of the repeating -Gly-X-Y-
sequences of a collagen shown to cause a phenotype. They are also of
interest because they are among the most frequently recurrent mutations
in collagen genes among the more than 200 such mutations now reported
(6-10). In addition, since the Arg-
1-519 and Arg-
1-75
mutations produce relatively mild phenotypes, they may be
representative of mutations that cause relatively common disorders of
connective tissue.
1-519 found in three unrelated
families with a mild cartilage disorder (15-17). In contrast to
mutated procollagens containing Cys substitutions for obligatory Gly
residues (6-10), the Cys in the Y position at
1-519 in
collagen II did not generate any intramolecular disulfide bonds. Also, the mutated collagen did not assemble into fibrils under conditions in
which the normal monomers readily polymerized. However, the presence of
the mutated monomer in mixtures with normal type collagen II increased
the lag time for fibril assembly and altered the morphology of the
fibrils formed.
The COL1A1/COL2A1 Hybrid Gene Construct
1-519, genomic DNA was
isolated from cultured skin fibroblasts (26) from a proband with
primary generalized osteoarthritis associated with a mild
chondrodysplasia (15). The genomic DNA was partially digested by
SphI and used to prepare a cosmid library as described earlier (27). A cosmid clone that contained the COL2A1 gene was digested with SphI, and two
SphI-SphI fragments of 14 and 12 kb1 were isolated. The 12-kb fragment
contained a single base mutation that converted the codon -CGT- for Arg
at position
1-519 to -TGT-, a codon for Cys.
1-519 codon, the complete gene was assembled
from four fragments: a 1.9-kb SphI-HindIII
fragment from the 5
-end of the human COL1A1 gene (28-30);
the 12-kb SphI-SphI fragment containing the
mutated
1-519 codon; and two additional
SphI-SphI fragments of 14 and 3.5 kb (25). The
construct contained 476 base pairs of the promoter, the first exon, and
most of the first intron of the COL1A1 gene extending up to
+1,445 base pairs. The COL1A1 fragment was linked to 29.5 kb of
sequences of the human COL2A1 gene that extended from the
SphI site in the 3
-end of the second intron of the gene at
+1,249 base pairs to about 3.5 kb beyond the major polyadenylation
signal of the gene.
-end
of the 1.9-kb SphI-HindIII fragment and the
SphI site in the 3
-end of the 3.5-kb
SphI-SphI fragment were converted to
SalI sites. The HindIII site in the 3
-end of the
1.9-kb SphI-HindIII fragment was converted to an
SphI site. The two SalI-SphI and
SphI-SalI fragments were then assembled into a
SalI site in a modified cosmid vector by three-way ligation
(27). In the second step, the 14-kb SphI-SphI
fragment with the wild type sequence and the 12-kb
SphI-SphI fragment with the mutated
1-519
codon were inserted by three-way ligation into the SphI site
of the construct obtained in the first step.
1(II) chains by
electrophoresis in a 7.5% polyacrylamide gel in SDS under nonreducing
conditions. Intermolecular disulfide bonds were assayed with the same
procedure after the 14C-labeled pC-collagen II was
converted to collagen by digestion with C-proteinase under the
conditions used in the experiments on assembly of collagen II fibrils
(see below).
20 °C. To
initiate fibril formation, pC-collagen and C-proteinase were mixed at
4 °C in a 250-µl polypropylene tube in a total volume of 20 µl
to give a final concentration of 150 µg/ml pC-collagen and 50 units/ml C-proteinase. To study fibril formation in the presence of
mutated collagen, normal and mutated pC-collagens II were mixed to
provide solutions in which the concentration of normal pC-collagen II was 150 µg/ml but mutated pC-collagen II was added to give ratios of
normal to mutated that were 1:1, 2:1, and 4:1. Therefore, the total
concentrations of pC-collagen varied from 188 to 300 µg/ml. The
concentration of C-proteinase was increased so that the time required
for complete cleavage was about 60 min. After preparing the mixtures,
the tubes were briefly flushed with water-saturated 10%
CO2 and 90% air. The tubes were capped and incubated from 0.5 to 24 h at 37 °C. The formed fibrils were separated by
centrifugation at 13,000 × g for 10 min. The
supernatant was removed and transferred to a separate tube. The pellet
was resuspended in 20 µl of fibril formation buffer. Concentrated
electrophoresis sample (5 ×) buffer was added to each sample to give a
final concentration of 2% SDS, 2% glycerol, and 0.01% bromphenol
blue in 62 mM Tris-HCl buffer, pH 6.8 at 20 °C. The
samples were heated at 100 °C for 3 min and separated by
electrophoresis on a 7.5% polyacrylamide gel in SDS. The gels were
stained with colloidal Brilliant Blue G (Sigma), destained, and air-dried between two sheets of cellophane. Relative amounts of stained
1(II) chains were assayed with a laser
densitometer (Ultrascan XL; Pharmacia). With 14C-labeled
samples, the gels were also assayed with a phosphor storage plate
(PhosphorImager 400S; Molecular Dynamics) for the pixel counts in the
protein bands. The assay made it possible to detect as little as 0.1 µg of
1(II) chains in either the supernatant or pellet
fractions.
1-519 was in the middle
of the wild type sequence: GFP GER GSP GAQ GLQ GP
GLP GTP
GTD GPK GAS GPA. To minimize end group effects, the amino terminus was
substituted with an N-acetyl group and the carboxyl terminus with
NHCH3. The parent molecule was then modified to generate
three mutated molecules in which a Cys residue was substituted at
1-518,
1-519, and
1-520. All the models were energy
minimized using a conjugate gradient method and subject to repeating
cycles of molecular dynamics using Kollman force fields and united
atoms (38). Two hundred-picosecond dynamic trajectory runs were saved
and analyzed for low energy conformers.
Expression of the Mutated Procollagen II
-519 to a codon for Cys
and that was co-inherited with the phenotype in his large family. The
12-kb SphI-SphI fragment was assembled into a
COL1A1/COL2A1 hybrid construct in which expression of the
COL2A1 gene was driven by a COL1A1 promoter (see Ref. 25). The construct was assembled in two steps, each of which required a
three-way ligation. The gene construct was then used to prepare stable
transfectants of the mammalian cell HT-1080 (24, 31, 36). A clone
secreting the mutated procollagen II was isolated and used for
preparation of the recombinant protein. To confirm the presence of the
mutation in the expressed gene, total RNA was isolated from the clone,
the RNA was reverse-transcribed, the resulting cDNA was amplified
with a primer paired specific for the region containing the mutation,
and the PCR product was sequenced. As indicated in Fig.
1, the results demonstrated that the codon of -CGT- for
Arg at
1-519 was converted to -TGT-, a codon for Cys.
Fig. 1.
Analysis of a PCR product by automated cycle
sequencing. The antisense strand was sequenced. The deduced sense
strand and the amino acids encoded (COOH- to NH2-terminal)
are shown above. The mutation converted the codon of -CGT- for Arg at
1-519 to a codon of -TGT- for Cys.
[View Larger Version of this Image (47K GIF file)]
Fig. 2.
Thermal stability of collagen II. Normal
collagen II and 14C-labeled mutated collagen II were
digested by trypsin and chymotrypsin at the temperatures indicated, and
the products were electrophoresed in an SDS-polyacrylamide gel.
Upper panel, polyacrylamide gel of the digested normal
collagen II stained with colloidal Brilliant Blue () and phosphor
storage plate image of a separate polyacrylamide gel of the digested
14C-labeled mutated collagen II (
). Lower
panel, plot of the data from the upper panel.
, normal collagen
II;
, mutated collagen II containing a Cys for Arg substitution at
1-519. The band below
1(II) chains is a product of the cleavage
of the NH2-terminal region of the
1(II) chains (see
Refs. 40 and 41).
[View Larger Version of this Image (24K GIF file)]
1(II) chains seen previously with
the sample of recombinant normal collagen II digested with a mixture of
chymotrypsin and trypsin (39) was also present in mutated recombinant
collagen II. The same band was seen after digestion of collagen II from
chick sterna cartilage (not shown) with the chymotrypsin/trypsin
mixture. A similar band after digestion of collagen II was also seen by
Sokolov et al. (40, 41), who presented evidence that the
band arose from cleavage of an NH2-terminal fragment in
20-30% of the molecules.
1(II) chains were detected (not shown).
Under the same conditions, a series of mutated procollagens I
containing cysteine substitutions for obligate glycine residues were
previously shown to form interchain disulfide bonds (see Ref. 42).
Although no interchain disulfide bonds were detected in the mutated
collagen II, a small fraction of disulfide-linked
1(II) chains was
detected in the supernatant of samples that were incubated at 37 °C
for 20 h or longer (Fig. 3, B and
C). The relative migration of the protein bands without
reduction indicated that they were dimers of
1(II) chains. Assay of
the 14C-labeled pro
1(II) chains by pixel counts
indicated that the disulfide-bonded dimers accounted for about 3% of
the total
1(II) chains.
Fig. 3.
Assay of formation of disulfide bonds with
collagen II containing the Cys for Arg substitution at 1-519.
Both proteins were labeled with 14C-amino acids, and
SDS-polyacrylamide gels were assayed with a phosphor storage plate.
A, pellet (P) and supernatant (S) from samples of normal pC-collagen II (120 µg/ml) and C-proteinase (40 units/ml) that were incubated at 37 °C for 24 h. B,
similar sample with pC-collagen II containing the Cys for Arg
substitution at
1-519. C, mixture of normal pC-collagen
II (120 µg/ml) and pC-collagen II containing the Cys for Arg
substitution at
1-519 (120 µg/ml) incubated with C-proteinase (80 units/ml) for 24 h. C1, C-propeptide;
S-S,
(II) dimers.
[View Larger Version of this Image (23K GIF file)]
1-519 mutation did not
form intramolecular disulfide bonds, a series of modeling experiments
was carried out. For the modeling experiments, an amino acid sequence
containing 36 amino acids spanning the Arg at
1-519 was used to
define the normal conformation of the triple helix. Three mutated
versions of the model were then generated (Fig. 4). The
distances separating identical
-carbons were measured between pairs
of chains that are staggered by 1 amino acid in the collagen triple
helix (see Ref. 3). In the model with the normal sequence, the
-carbon distances between the Gly-
-520 residues were shorter than
the
-carbon distances between the Arg-
1-519 residues (Table
I). The
-carbon distances between the Gly-
-520
residues were also shorter than the
-carbon distances between the
Pro-
1-518 residues. With the three mutated sequences in which Cys
was placed in the Gly, X, and Y positions,
respectively, the
-carbon distances were
-520 <
1-519 <
1-518 when residues in chain 1 (most
NH2-terminal) were compared with residues in chain 2 (middle chain). All the
-carbon distances were greater when residues
in chain 2 (middle chain) were compared with chain 3 (most
COOH-terminal). Therefore, the results were consistent with the
conclusion that intramolecular disulfide bonds between two of the
chains (chains 1 and 2) can form more readily between Cys residues in
the obligate Gly position (
1-520) than Cys residues in the
Y position (
1-519). The S atom distances with the
Cys-
1-520 and Cys-
1-519 substitutions were essentially the same
(Table I), but these distances are more difficult to predict because of
the flexibility of the side chains.
Fig. 4.
Computer simulations of a 36-amino acid
peptide spanning the sequence found at 1-519. Only the peptide
backbone and the skeletons of side chains of Arg and Cys are shown. The
side chain nitrogens of the Arg residues are in blue. The
sulfhydryl groups of the Cys residues are in yellow.
Left panel, side view. Right panel, end-on view,
NH2- to COOH-terminal. A, normal sequence. B, same sequence with the substitution of Cys for Arg at
1-519. C, same sequence with the substitution of Arg for
Gly at
1-520. D, same sequence with the substitution of
Cys for Pro at
1-517.
[View Larger Version of this Image (139K GIF file)]
Collagen II
residue
-Carbon distance
S atom distance
Å
Å
Normal sequence
Pro
1-518
6.44
Arg
1-519
5.88
Gly
1-520
3.33
Mutated
sequence
Cys
1-518
6.35
8.07
Cys
1-519
4.63
6.06
Cys
1-520
3.81
5.97
To examine assembly of the mutated protein
into fibrils, the isolated procollagen II was converted to pC-collagen
II by digestion with procollagen N-proteinase, and the pC-collagen II
was reisolated on a gel filtration column. The pC-collagen II was then
used as a substrate for fibril assembly under the conditions that were previously used with both pC-collagen II and pC-collagen I (24, 31,
34-36). With samples containing 150 µg/ml pC-collagen and 50 units/ml C-proteinase, cleavage of both normal and mutated pC-collagen
II was complete in less than 1 h (24, 31, 36). There was no
apparent difference in the rate of cleavage of the normal and the
mutated proteins (not shown). As the C-propeptides are cleaved from the
protein, normal collagen II assembled into fibrils with a lag period of
about 1 h, and the assembly was complete in 7.5-10 h (Figs.
5A and 6). Under the same conditions, none of
the mutated collagen II assembled into fibrils that were recovered by
centrifugation (Fig. 5B). In additional experiments, it was found that the mutated collagen II did not assemble into fibrils even
when incubated in concentrations as high as 300 µg/ml (data not
shown).
To explore further the effects of the mutated collagen II on fibril
assembly, mixtures of normal and mutated pC-collagen II were prepared
and used as substrates for fibril assembly. As indicated in Figs.
5C and 6, the presence of mutated collagen II in the mixtures increased the lag time for the assembly of normal collagen II.
In the presence of 150 µg/ml of the mutated pC-collagen II, the lag
period for the assembly of normal pC-collagen II was increased from 1 to about 5 h (Fig. 6). The minimal concentration of
the mutated collagen II that increased the lag time was about 35 µg/ml (Fig. 7). The presence of the mutated collagen
II had no effect on the propagation rate that remained 160 ng/ml/min
under the conditions used (Fig. 6). Although the mutated collagen II
increased the lag period for the assembly of normal collagen II, none
of the mutated protein was detected in the pellet fractions (Fig. 5C).
Morphology of the Fibrils
As reported previously (24, 31,
36), collagen II assembles into fibrils that form three-dimensional
arcades under the conditions used here (Fig.
8A). Although the mutated collagen II did not
assemble into fibrils, the presence of the protein in mixtures with
normal collagen II altered the morphology of the fibrils formed by
normal collagen II (Fig. 8B). The fibrils were less uniform
in diameter. Also, they were shorter, and some showed distorted
ends.
Electron microscopy of fibrils formed from mixtures of normal collagen
II and mutated collagen II (Fig. 9B) also
revealed distortions compared with fibrils of normal collagen II (Fig. 9A). The individual fibrils formed in the presence of
mutated collagen II appeared to be thinner and loosely packed into
thicker fibrils. Apparently because of loose packing of the structures, no D-period banding was apparent under the same conditions in which a
D-period banding pattern was readily demonstrated in the control
fibrils.
The unique conformation of the collagen triple helix places every
third amino acid in the restricted space at the center of the triple
helix where only glycine, the smallest amino acid residue, can be
accommodated (see Ref. 3). More than 150 naturally occurring mutations
that substitute codons for bulkier amino acids for the obligate
glycines of fibrillar collagens have now been reported (6-10). Among
the first Gly substitutions were single base changes that introduced
codons for Cys into collagen I, since monomers containing two Cys for
Gly-substituted 1(I) chains formed intramolecular disulfide bonds,
and the resulting dimers were readily detected by polyacrylamide gel
electrophoresis of collagen I synthesized by cultured skin fibroblasts.
Also, some of the intramolecular disulfide bonds introduced kinks in
the molecule that were detected by rotary shadowing electron microscopy
(43, 44). In assays for fibril assembly with four collagen I molecules
containing Cys for Gly substitutions, the mutated monomers were
incorporated into fibrils together with normal monomers from mixtures
of the two (42). However, the relative fraction of mutated to normal monomers in the fibrils varied, and there appeared to be a correlation between the severity of the phenotype produced by the mutation and the
extent to which the presence of the mutated monomer altered several
parameters of fibril assembly.
The results here demonstrate that the Cys at 1-519 did not form
intramolecular disulfide bonds in recombinant procollagen II molecules
in which all three chains contained the Cys substitution. A small
fraction of the protein formed disulfide bonds after processing of the
procollagen to collagen II and incubation at 37 °C for 24 h.
The disulfide bonds generated under these conditions were probably
intermolecular. The failure of the Cys-
1-519 to form intramolecular
disulfide bonds is apparently explained by the greater distance between
residues in the Y position than with substitutions for
obligate Gly residues in the collagen triple helix (Table I). An
incidental finding in the computer modeling carried out here is that
the distance between Gly residues is closer when measured between chain
1 (the most NH2-terminal chain) and chain 2 (the middle
chain) than between chain 2 and chain 3 (the most
NH2-terminal chain). Since Cys for Gly substitutions in the
pro-
1(I) chain of type I procollagen readily generate intramolecular
disulfide bonds (8-10), the model-building experiments suggest that
the still unresolved staggering order of
chains in type I collagen
(see Ref. 3) is
1,
1, and then
2.
The recombinant mutated collagen II did not assemble into fibrils even when incubated at concentrations five times the critical concentration for the assembly of fibrils for normal collagen II. However, the presence of mutated protein in mixtures with normal collagen II considerably increased the lag period for the assembly of the normal protein into fibrils. Also, the presence of the mutated protein altered the morphology of the fibrils formed by the normal collagen II. Therefore, although the monomers of the mutated collagen II did not have enough affinity to self-assemble into fibrils, their affinity for a nuclei formed by monomers of normal collagen II was apparently great enough to alter fibril assembly by the wild type protein. Although the assays used would have detected as little as 0.1 µg of the mutated monomers in pellet fractions, they probably would not have detected the relatively small numbers of monomers found in nuclei (see Ref. 34).
The five families with the Arg-1-519 mutations were heterozygous
for the mutated allele, and therefore, their tissues probably contained
normal collagen II monomers, heterotrimers comprising one or two
mutated chains, and homotrimers of mutated chains such as those
examined here. Cartilage from one patient demonstrated that some of the
Cys residues at
1-519 formed disulfide bonds linking two
1(II)
chains (45). On the basis of the results here, it is unlikely that the
disulfide bonds were intramolecular in homotrimeric monomers containing
the mutated
1(II) chains. Instead, they were probably present as
intermolecular bonds that formed after monomers containing one or two
mutated chains assembled into fibrils.
We are grateful to Dr. A. Sieron for help with screening of the clones, K. Sahm for preparation of N- and C-proteinases, Y. Bao for RNA isolation, and M. Kinnarney for sequencing of DNA.