From the Section on Connective Tissue Disorders,
HDB/NICHD, National Institutes of Health, Bethesda,
Maryland 20892, § Section on Physical Biochemistry, NICHD,
National Institutes of Health, Bethesda, Maryland 20892, ¶ Laboratory of Connective Tissues Research, University of Liege,
Liege, Belgium, and
Department of Human Genetics, MCV/VCU,
Richmond, Virginia 23298
Received for publication, December 9, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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The majority of collagen mutations
causing osteogenesis imperfecta (OI) are glycine substitutions that
disrupt formation of the triple helix. A rare type of collagen mutation
consists of a duplication or deletion of one or two
Gly-X-Y triplets. These mutations shift the
register of collagen chains with respect to each other in the helix but
do not interrupt the triplet sequence, yet they have severe clinical
consequences. We investigated the effect of shifting the register of
the collagen helix by a single Gly-X-Y triplet
on collagen assembly, stability, and incorporation into fibrils and
matrix. These studies utilized a triplet duplication in COL1A1 exon 44 that occurred in the cDNA and gDNA of two siblings with lethal OI.
The normal allele encodes three identical Gly-Ala-Hyp triplets at aa
868-876, whereas the mutant allele encodes four. The register shift
delays helix formation, causing overmodification. Differential scanning
calorimetry yielded a decrease in Tm of 2 °C for
helices with one mutant chain and a 6 °C decrease in helices with
two mutant chains. An in vitro binary co-processing assay
of N-proteinase cleavage demonstrated that procollagen with the triplet
duplication has slower N-propeptide cleavage than in normal controls or
procollagen with pro Osteogenesis imperfecta
(OI)1 is an autosomal
dominant disorder of connective tissue. Its most significant clinical
feature is skeletal fragility, causing the bones of affected
individuals to be susceptible to fracture from minimal trauma or
nontraumatic impact (1). Other symptoms of OI include short stature,
blue sclerae, joint laxity, dentinogenesis imperfecta, and hearing loss
(2). The severity of OI varies widely, ranging from perinatal lethal to
barely detectable, as delineated by the Sillence classification (3).
The full clinical spectrum of OI is caused by defects in the structure
or synthesis of type I collagen, the most abundant protein of the
extracellular matrix of bone, skin, and tendon (4, 5). Defects in
COL1A1 that result in the synthesis of half the normal amount of
collagen cause the mildest form of the disease (OI type I) (6). The
clinically significant forms of OI (OI types II, III, and IV) are
caused by structural defects in either the An additional rare and interesting group of mutations consists of
deletions or duplications of the codons for one or two
Gly-X-Y triplets. Only 10 cases of single triplet
deletion or duplication have been reported (11-14), including five
deletions and three duplications in the We report here a single triplet duplication in Clinical Cases--
The probands were the male and female
offspring of a 22-year-old gravida II para1 (G2P1) mother and a
25-year-old father, born at 32 and 37 weeks gestation, respectively.
Prenatal ultrasound at 18-22 weeks gestation detected a short-limbed
skeletal dysplasia in each child. The male child was delivered
vaginally with forceps due to breech presentation. Weight was
appropriate for age (2013 g), but crown to heel length was 38 cm (50%
for 28 weeks gestation). At delivery, he had a soft skull with an
anterior laceration, draining blood and cerebrospinal fluid, a narrow
chest, and bowed extremities. He died 1 h after birth. The female
child was born by spontaneous vaginal delivery. Birth weight was
appropriate for age (2770 g), but length was short (43 cm; 50% for 32 weeks gestation). Deformities noted at birth included a soft cranium with mineralized bone only on lateral portions of the skull, blue sclerae, a high arched palate, and a narrow chest. Extremities had
rhizomelic shortening and bowing and were abducted into an extreme
frog-legged position. Radiograms showed multiple fractures of ribs and
all long bones. The infant died at age 1 month of respiratory insufficiency.
Fibroblast Culture--
Cultures established from dermal
biopsies were grown in Dulbecco's modified Eagle's medium, with 10%
serum and 2 mM glutamine. For large scale procollagen
preparation, confluent cells of Proband 1, father, control, and Mutation Detection and Sequencing--
Fibroblast RNA was
isolated using Tri-Reagent (Molecular Research Center, Cincinnati, OH)
(18).
For RT-PCR analysis, cDNA amplification used a sense primer
in exon 43 (5'-CCTGGACGAGACGGTTCTCCTGGCGCCAAG-3') and an antisense primer complementary to exon 45 (5'-GCCGACAGGACCGGCGGGACCAGCAGGACC-3'). PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for
1 min, 69 °C for 30 s, and 72 °C for 30 s; and finally
72 °C for 7 min.
For genomic PCR, DNA was isolated from parental leukocytes and proband
fibroblasts with the Puregene DNA Isolation Kit (Gentra Systems,
Minneapolis, MN). Reactions used 500 ng of DNA and 1.0 units of
Amplitaq. The sense primer corresponded to nt 13240-13269 of
COL1A1 intron 43 genomic sequence
(5'-TGACCCATATTCCCCTGCTCTCCCCGCCAG-3'), and the antisense primer was
complementary to nt 13378-13407 (5'-GGTACAGGGAACTGGAGCCCAGCTACTTAC-3') in intron 44. PCR conditions were 94 °C for 5 min; then 30 cycles of
94 °C for 1 min, 65 °C for 30 s, and 72 °C for 30 s;
and finally 72 °C for 7 min. RT-PCR and genomic PCR products were
electrophoresed on 2% agarose gels and then visualized with ethidium bromide.
The exon 43-45 RT-PCR and intron 43-44 gDNA PCR products were
subcloned and sequenced by the dideoxy chain termination method (21)
with the Sequenase 2.0 kit (Amersham Biosciences). The sequencing
primer for cDNA subclones corresponded to cDNA nt 3189-3218 in
Matrix Deposition--
Confluent fibroblasts were stimulated
every other day for 9 days with 100 µg/ml ascorbic acid and then
incubated for 24 h with 260 µCi/ml [3H]proline in
serum-free medium. Procollagens in media were precipitated with
ammonium sulfate. Matrix collagens were serially extracted, as
described (22). In brief, newly synthesized collagens were extracted
for 24 h with neutral salt (0.15 M NaCl in 50 mM Tris-HCl, pH 7.5). Collagens with acid-labile
cross-links were extracted for 24 h with 0.5 M acetic
acid. Collagens with mature cross-links were extracted by pepsin
digestion (0.1 mg/ml) for 24 h. All matrix fractions were
precipitated with 2 M NaCl.
Matrix Chase--
Confluent fibroblasts were stimulated every
other day for 9 days with 100 µg/ml ascorbic acid, incubated for
48 h with 260 µCi/ml [3H]proline in serum-free
medium, and then chased with fresh Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum and 10 mM nonradioactive
proline. Individual cultures were harvested at 24-h intervals for 5 days, and the matrix layer was processed with protease inhibitors, as
described (22). Matrix extracts were resuspended in 0.5 M
acetic acid and digested overnight with pepsin. Collagens were
precipitated with 2 M NaCl.
Preparation of Fluorescent Labeled Procollagen--
Ammonium
sulfate protein precipitates were redissolved in 0.1 M
sodium carbonate, 0.5 M NaCl (pH 9.3) at 0.2 mg/ml collagen concentration. Cy2 and monoreactive Cy5 dyes (Amersham Biosciences) (23, 24) were dissolved in 1 ml of anhydrous dimethylformamide, according to product directions. Procollagen was added to lyophilized dye aliquots, 100 µl of protein solution/10 µl of dye aliquot for
Cy5, and 100 µl of protein solution/50 µl of dye aliquot for Cy2
labeling, shaken for 30 min at room temperature, analyzed by SDS-PAGE
for labeling efficiency, frozen on dry ice, and stored at
N-proteinase Cleavage--
Labeled procollagen was transferred
into 50 mM Tris, 0.5 M NaCl, 4 mM
CaCl2, 0.5 mM phenylmethylsulfonyl fluoride,
2.5 mM N-ethylmaleimide, 0.02% Brij 35 (pH 8)
on AutoSeq G-50 microspin columns (Amersham Biosciences). Collagen
concentration was adjusted to 0.1 mg/ml. All possible binary mixtures
of Cy2- and Cy5-labeled procollagens were prepared (e.g.
C-Cy2/C-Cy5, C-Cy2/OI-INS-Cy5, C-Cy5/OI-INS-Cy2, and
OI-INS-Cy2/OI-INS-Cy5 were made) to compare cleavage kinetics of
C and OI-INS. N-proteinase (25) was added on ice, and mixtures were
placed at 34 °C. Sample aliquots were collected at different times
after the start of the reaction, mixed with a lithium dodecyl
sulfate gel sample buffer (Invitrogen) with added dithiothreitol
and EDTA, and rapidly frozen. Samples were analyzed on precast 6%
Tris/glycine and 7% Tris acetate minigels (Invitrogen). The gels were
scanned on a FLA3000 fluorescence scanner (Fuji Medical Systems,
Stamford, CT) at 50 × 50-µm resolution. Fractions of the
cleaved proteins were determined from band intensities using PeakFit
software (SPSS Inc., Chicago, IL), corrected for the cleaved protein in
the initial mix and for the effect of the fluorescent label on the
cleavage by using control mixtures (e.g. C-Cy2/C-Cy5 and
OI-INS-Cy2/OI-INS-Cy5 for the C versus OI-INS experiment).
The effect of the fluorescent label did not exceed 10%. Experiments
were repeated in triplicate.
Preparation of Full-length Collagen by N- and C-proteinase
Cleavage--
Ammonium sulfate procollagen precipitate was doped with
10% Cy5-labeled procollagen. The mixture was chromatographed on two 1.6 × 5-cm columns of DEAE-cellulose (DE52; Whatman) as described (26-28). First, the mixture was loaded in 2 M urea, 0.15 M NaCl, 0.1 M Tris-HCl (pH 7.4) and eluted with
the same buffer. Second, the mixture was loaded in 2 M
urea, 0.1 M Tris-HCl (pH 8.6) and eluted with an NaCl
gradient. Fractions containing procollagen on SDS-PAGE were pooled and
concentrated by pressure ultrafiltration through an Amicon YM30
membrane. Procollagen was transferred into 50 mM Tris, 0.5 M NaCl, 4 mM CaCl2, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 mM
N-ethylmaleimide, 0.02% Brij 35 (pH 8) and simultaneously digested by N- and C-proteinase at 32 °C. C-proteinase was the generous gift of Prof. K. E. Kadler (University of
Manchester, Manchester, UK). Cleavage was monitored by SDS-PAGE.
Complete digestion was observed at 40-70 h, and the reaction was
stopped by the addition of EDTA to a final concentration of 20 mM. Collagen was precipitated twice by 0.6 M
NaCl in 0.5 M acetic acid and analyzed for purity by
SDS-PAGE.
Differential Scanning Calorimetry--
DSC scans from 10 to
50 °C were performed at 0.125 to 1 °C/min heating rates in a Nano
II DSC instrument (Calorimetry Sciences Corp., American Fork, UT) as
described (29). Pepsin digestion (~1:10 pepsin/collagen) of ammonium
sulfate precipitates in 0.5 M acetic acid at 4 °C
overnight resulted in complete removal of N- and C-propeptides. To
prevent fibrillogenesis, 0.1-1.2 mg/ml procollagen, full-length
collagen, or pepsin-treated collagen solutions in either 2 mM HCl (pH 2.7) or 0.2 M sodium phosphate, 0.5 M glycerol (pH 7.4) were used. The denaturation temperature (Tm) in phosphate/glycerol buffers was used to
extrapolate Tm to physiological conditions (29), but
better resolution of mutant collagen forms was achieved in 2 mM HCl.
In Vitro Fibrillogenesis--
At 5 °C, full-length collagen
in 2 mM HCl (pH 2.7) was mixed 1:1 with 0.26 M
NaCl, 6 mM sodium phosphate (final pH 6.9), transferred into a precooled quartz cuvette (1-cm path length), and placed into a
V-560 spectrophotometer (Jasco Inc., Easton, MD) equipped with a
thermoelectric temperature controller. Fibrillogenesis was initiated by
a temperature jump to 36.6 °C and monitored by turbidity measurement
(optical density at 350 nm, A350) (30). After
A350 reached saturation, fibers were pelleted (5 min at 10,000-14,000 × g) and redissolved in 2 mM HCl for subsequent analysis by DSC. Supernatant was
dialyzed against 2 mM HCl for DSC analysis. Collagen
concentration before fibrillogenesis and in supernatant after
fibrillogenesis were measured by Sircol assay (Biocolor Ltd., Belfast,
Northern Ireland). Aliquots from fibers and supernatant were labeled
with Cy5 and analyzed on 3-8% Tris acetate or 4-12% Bis-Tris
minigels (Invitrogen).
Collagen Protein Analysis--
The type I collagen produced by the
cultured dermal fibroblasts of both probands displayed identical
electrophoretic abnormalities on SDS-urea-PAGE. The Mutation Identification in cDNA and gDNA--
The
RT-PCR screening of
Sequencing of subclones of proband cDNA exons 43-45 and proband
and mother gDNA intron 43-intron 44 revealed the same relatively unusual type of collagen mutation, confirming the mother as a mosaic
carrier. The mutant allele has a 9-bp insertion (5'-GGT GCT CCT-3'),
coding exactly for an extra Gly-Ala-Hyp triplet (Fig. 3). The insertion is a duplication in a
highly repetitive region. The normal allele has two identical 9-bp
sequences coding for aa 868-873 and an adjacent 9 bp that differs by
only one nucleotide. The mutant allele has three of the identical 9-bp
units.
Effect of Register Shift on Collagen Thermal
Stability--
Differential scanning calorimetry thermograms of
proband collagen (OI-INS) were done at acidic and neutral pH (Fig.
4). Since mutant collagen has three
species ( Propagation of Register Shift Affects N-propeptide
Processing of Mutant Procollagen--
We compared the kinetics of
N-propeptide cleavage in the insertion mutation (OI-INS), paternal
control collagen (C-F), unrelated control collagen (C), and
three-glycine substitution mutations in the region of
In order to detect small differences in cleavage kinetics, we analyzed
all possible binary combinations in a co-processing assay. In each
pair, two proteins with different fluorescent labels were processed in
the same test tube by N-protease under identical conditions,
co-electrophoresed on SDS-PAGE, and distinguished by fluorescence
scanning. This assay yielded reproducible detection of differences in
cleavage kinetics as small as 5-10%. The results of N-propeptide
co-processing of different mutant procollagen pairs is shown in Fig. 1,
C-F. The triplet insertion causes substantially slower In Vitro Fibrillogenesis of Mutant Collagen--
Classical
fibrillogenesis kinetics, a lag phase followed by rapid fiber growth
(30), was observed for both mutant and wild type collagens (Fig.
5A). Mutant collagen formed
fibrils more slowly than control and required a higher initial
concentration to achieve a similar extent of fiber formation. Virtually
all control collagen formed fibers, since collagen was not detected in
the fibrillogenesis supernatant. In contrast, over 20% of molecules remained in the supernatant after fibrillogenesis of proband collagen. The fraction of mutant collagen that did not form fibrils contained helices with a greater extent of posttranslational overmodification (Fig. 5B) and, therefore, a higher content of molecules
containing mutant chains.
DSC thermograms (Fig. 5C) demonstrated that only molecules
with no or one mutant chain were incorporated into fibers in
vitro. The melting peak corresponding to helices with two mutant
chains is totally missing from thermograms of fibers; the shoulder
corresponding to helices with a single mutant chain is substantially
reduced. All molecules with two mutant chains and a significant
fraction of molecules with one mutant chain remained in the
supernatant, explaining the higher extent of posttranslational
modification in the supernatant collagen. By circular
dichroism,3 we found that
many of the molecules with two mutant chains were irreversibly
denatured during fibrillogenesis because of their extreme instability.
The main peak of the supernatant thermogram is a mixture composed
mostly of molecules with a single mutant chain and a small fraction of
molecules with no mutant chain.
Matrix Deposition--
The incorporation of proband and control
collagen into matrix was compared by serial extractions of the matrix
deposited by cultured cells (Fig. 6). The
proband overmodified chains are equally abundant in the media and the
neutral salt extract (fraction 1), the later containing helices that
are not cross-linked with other matrix molecules. The mutant chains are
also efficiently incorporated into the immaturely cross-linked fraction
(fraction 2). However, they are substantially less abundant in the
maturely cross-linked fraction extracted with pepsin (fraction 3),
which has predominantly normally migrating Matrix Chase--
A pulse-chase experiment examined the stability
of collagen deposited in matrix by cultured proband and control cells.
Matrix stability was not significantly altered (Fig.
7). The probands' normal and
overmodified We have described here a novel single triplet duplication in the
type I collagen The mutant COL1A1 allele has a 9-bp duplication in exon 44, which has a
highly repetitive sequence. In the normal allele, there are two
consecutive 5'-GGT GCT CCT-3' units at nt 3255-3272, followed by 9 bp
that differ by a single nucleotide, 5'-GGT GCC CCT-3'.
These 27 bp code for three consecutive Gly-Ala-Hyp collagen triplets,
at aa 868-876. In the mutant allele, a duplication of one 5'-GGT GCT
CCT-3' unit results in an extra Gly-Ala-Hyp triplet. The repetitive
sequence of exon 44 has made it a hot spot for single triplet deletions
and duplications. Nine of the 11 known triplet deletions or
duplications in Triplet duplication and deletion mutations form a very interesting and
relatively unusual set of mutations causing OI. The more prevalent
glycine substitution mutations disrupt the otherwise uninterrupted
Gly-X-Y triplet repeats of type I collagen.
Accommodation of the substituting amino acid in the internal aspect of
the helix delays helix formation. Triplet mutations do not interrupt
the Gly-X-Y sequence; they shift the
"register" of the chains with respect to each other. The severe to
lethal phenotype of all cases of OI with this type of mutation
indicates the significance of the register shift for the structure and
interactive functions of the collagen helix. Functional studies on type
I collagen helix formation and structure have been published for two
single triplet deletions in exon 44 (11, 12). Both cases have
equivalent triplet deletions, with two consecutive Gly-Ala-Hyp triplets
present, rather than the usual three.
All triplet deletion and duplication cases, including the one described
here, are associated with significant overmodification. This suggests
that accommodation of shifted register interactions between different
X and Y residues along the helix is causing delay
in helix formation. For the duplication presented here, our findings
support propagation of the register shift toward the N-terminal end of
the helix rather than formation of a loop accommodating the extra triplet.
The global register shift may affect the stability of the entire helix.
The Tm of the collagen with a triplet deletion in
exon 44 was found to be decreased by 0-1 °C (11, 12). In our
triplet duplication mutation, the Tm of helices with one mutant chain was reduced 2 °C, whereas two mutant chains reduced Tm by 6 °C. Although Tm was
determined in the deletion cases by the less sensitive
trypsin-chymotrypsin digestion method, the 6 °C drop in
Tm with two mutant chains should not have been
missed and probably represents a more deleterious effect of the
duplication. Deletions and duplications result in register shifts of
opposite direction and may cause unequal effects on
Tm. Alternatively, the deletion mutations may
compensate by local looping out. Also, note that the thermal stability
of Furthermore, the difference between the thermograms of the triplet
duplication mutation at neutral and acidic pH also supports a longer
range effect of the duplication on helix folding. The change in
relative stability of mutant and normal forms at different pH suggests
that intramolecular salt bridges are different in these species. Since
the mutation occurs in a stretch containing no salt bridges, this
indicates that the register shift propagates at least three triplets
beyond the mutation to the first potential salt bridge in the direction
of the N-terminal end.
Propagation of the register shift for the entire length of the helix
may affect the kinetics of N-propeptide cleavage (32-34). The
duplication mutation showed a decrease in cleavage rate with respect to
two control collagens. We demonstrated that the delay was not simply
the result of overmodification along the length of the helix by
comparing the duplication with glycine substitution mutations in the
same region of The triplet duplication also alters fibril formation in
vitro and in culture. Collagen molecules with two mutant chains
are not incorporated into fibrils in vitro, and those with
one mutant chain are poorly incorporated. In addition, the critical
concentration for fibrillogenesis is higher for proband than for normal
collagen. Improper helix melting may be one of the reasons for poor
incorporation of mutant molecules into fibrils. At both the 36.6 °C
of in vitro fibrillogenesis and the 37.5 °C of body
temperature, molecules with two mutant The matrix deposition studies show that collagen molecules with a
single mutant chain are incorporated less efficiently than normal
helices. Mutant chains are present proportionately in the media and the
non-cross-linked fraction of matrix. They are relatively retained in
the immaturely cross-linked fraction, with slow progression into the
maturely cross-linked pepsin extract. Delayed cross-linking of mutant
molecules is probably related to misalignment of residues on opposite
molecules, which would be expected from propagation of the register
shift. The delay in cross-linking of the triplet duplication is not
seen with Proband mature matrix has a turnover that is comparable with normal,
reflecting both its predominantly normal collagen composition and also
the stability of the cross-links formed by helices with one mutant
chain that have become fully incorporated. Collagen helices containing
mutant Although a register shift mutation does not disturb the uninterrupted
Gly-X-Y triplet repeat of collagen, the altered
alignments of X and Y residues along the chain
have profound effects. Collagen formation is delayed, and its stability
is decreased along the entire length of the helix. Double mutant and
most single mutant helices are not incorporated into collagen fibrils,
because of a combination of extreme instability and interference of the
register shift with mature cross-linking. In addition to the deficiency of matrix that results from decreased fibrillogenesis, it is likely that misalignment of the collagen chains along the full helical region
will also disrupt many of the interactions of collagen with
noncollagenous molecules in matrix. Recent studies of a short synthetic
heterotrimer containing the integrin-binding epitope of type IV
collagen showed a strong effect of chain register on helix conformation
(38) and integrin binding (39), supporting the proposal that register
shifts may alter functionally significant side chain interactions. The
combination of changes in helix stability, interchain X and Y position
alignment, fibrillogenesis, and cross-linking results in the severe
clinical phenotype of these mutations.
1(I) G832S, G898S, or G997S substitutions,
showing that the register shift persists through the entire helix. The
register shift disrupts incorporation of mutant collagen into fibrils
and matrix. Proband fibrils formed inefficiently in vitro
and contained only normal helices and helices with a single mutant
chain. Helices with two mutant chains and a significant portion of
helices with one mutant chain did not form fibrils. In matrix deposited
by proband fibroblasts, mutant chains were abundant in the
immaturely cross-linked fraction but constituted a minor fraction
of maturely cross-linked chains. The profound effects of shifting the
collagen triplet register on chain interactions in the helix and on
fibril formation correlate with the severe clinical consequences.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I) or
2(I) chains.
Over 250 such mutations have now been delineated in individuals with OI
(7, 8). The overwhelming majority (about 85%) are point mutations that
result in the substitution of the glycine residue in a typical collagen
tripeptide, Gly-X-Y, by another amino acid.
Substitution mutations are thought to exert a detrimental effect on
collagen function, because their side chains are larger than that of
glycine and cause local interference with the folding of the triple
helix (9). A smaller fraction (about 10%) of collagen mutations result
in single exon skipping. These mutations maintain the
Gly-X-Y triplet pattern but may cause local
looping out of chains in the triple helix (4). An even less common set
of structural mutations is located in the C-terminal propeptide. Since
the C-propeptide is cleaved from the mature collagen molecule before
incorporation into fibrils, the mutant region of the chain is not
incorporated into matrix. Instead, they are thought to exert their
effect by delaying the incorporation of the mutant chains into collagen
trimer (10).
1(I) chain and 2 deletions
in the
2(I) chain. There are also four cases involving deletion or
duplication of two triplets, all in the
2(I) chain (14, 15). These
mutations are of special interest because they must disrupt collagen
functioning by a mechanism quite different from that initiated by
glycine substitutions. A priori, one might have expected
mild functional defects from mutations that shift the
Gly-X-Y register of the collagen helix by a
single triplet unit rather than interrupt helix folding in the manner
of a glycine substitution. In fact, small register shifts cause a
lethal or severe phenotype. Determinations of helix stability and
procollagen processing were reported for two lethal cases with deletion
of one of the three Gly-Ala-Hyp triplets at aa 868-876 in
1(I) exon
44 (11, 12). These deletions decreased collagen helix
Tm by only 0-1 °C. Processing of proband
collagen by pericellular enzymes and purified N-proteinase was
indistinguishable from normal, as was cleavage by vertebral collagenase. The processing data led investigators to propose that
there was limited propagation of the register shift toward the
N-terminal end of the procollagen trimer.
1(I)E44 in siblings
with lethal type II OI. Determinations of thermal stability and
N-protease cleavage indicate that the register shift is propagated the
full length of the collagen helix. In vitro fibrillogenesis and matrix deposition studies demonstrate that presence of the register
shift impairs incorporation into fibrils and cross-linking into matrix.
These studies provide new insight into the mechanisms of register shift
mutations in collagen disorders.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I)
G832S, G898S, and G997S were treated with 50 µg/ml ascorbate in
serum-free Dulbecco's modified Eagle's medium. Medium was harvested
at 24-h intervals for 2 days and brought to 100 mM
Tris-HCl, pH 7.4, 250 mM EDTA, 0.2% NaN3, 1 mM phenylmethylsulfonyl fluoride, 5 mM
benzamidine, and 10 mM N-ethylmaleimide.
Ammonium sulfate-precipitated procollagen was collected by
centrifugation. To label steady state procollagens, confluent cells
were incubated without serum for 2 h with 50 µg/ml ascorbic acid
and then 16 h with 260 µCi/ml 3.96 TBq/mmol
L-[2,3,4,5-3H]proline. Procollagens were
harvested, and collagen was prepared as described (16). Isolated
1(I) chains were digested with cyanogen bromide (17).
1(I) exon 41-49 cDNA was obtained by reverse
transcription polymerase chain reaction (RT-PCR) using 1 µg of RNA,
20 units of murine leukemia virus reverse transcriptase, and oligo(dT).
The cDNA was amplified by PCR (19) using a sense primer
corresponding to nt 2961-2990
(5'-ACTCCCGGGCCTCAAGGTATTGCTGGACAG-3') and an antisense primer
complementary to cDNA nt 3696-3725 (5'-GGG CAG GAA GCT GAA GTC GAA
ACC AGC GCT-3') and 2.5 units of Amplitaq (Invitrogen). Cycling
conditions were 94 °C for 5 min; then 35 cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min; and finally 72 °C
for 7 min. 32P-Labeled antisense riboprobe was synthesized
for the aa 787-1173 region of
1(I) collagen (20). Riboprobe and
exon 41-49 cDNA were mixed for DNA:RNA hybrid analysis, as
described (20).
1(I) exon 43 (5'-CCTGGACGAGACGGTTCTCCTGGCGCCAAG-3'). The sequencing primer for genomic DNA subclones corresponded to COL1A1 nt 13240-13269 in intron 43 (5'-TGACCCATATTCCCCTGCTCTCCCCGCCAG-3').
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I) chain was
doubled in width, consisting of a normal and an electrophoretically
delayed form (Fig. 1A). The
2(I) band was slightly broadened with a delayed base line. The
abnormal collagen was secreted from the cell as efficiently as normal
collagen. All CNBr peptides from the
1(I) chain of the proband
showed both normal and electrophoretically delayed forms (Fig.
1B). Since the
1(I) chains were overmodified along their
full length, the mutation was localized to the COOH-terminal quarter of
either
chain. The mother had a lighter, less tightly resolved band
just above the
1(I) band in cell layer collagen (not shown). This
pattern is characteristic of a low percentage of
1(I) chains with
excess posttranslational modification. Given the clinical information
that the unaffected parents have two children with a dominant genetic
disorder, this finding prompted further investigation of the mother as
a likely mosaic carrier of the collagen mutation.
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Fig. 1.
Proband type I collagen and kinetics of
in vitro N-propeptide cleavage by N-protease.
A, a similar extent of posttranslational overmodification
was observed in the insertion mutation (OI-INS) and glycine
substitution mutations in the region of 1(I) surrounding the
insertion (OI-G832S, OI-G898S, and OI-G997S), as indicated by broad
1(I) bands on SDS-urea-PAGE. B, SDS-urea-PAGE of
1(I)
CNBr peptides of these mutant collagens showed closely matched
overmodification, especially for the N-terminal ends of OI-INS and
OI-G997 (note CB8 and CB8 + 5). C-F, binary
co-processing assays yielded slower N-propeptide cleavage in OI-INS
than in control and glycine substitution mutations. Each point on the
curve is an average of two co-processing experiments with inverted
fluorescent label (e.g. C-F show the average of data for
C-F-Cy2/OI-INS-Cy5, and C-F-Cy5/OI-INS-Cy2 binary mixtures).
Error bars indicate S.D. in these
experiments.
1(I) and
2(I) cDNA coding for the C-terminal quarter of the collagen
helix were examined by RNA:DNA hybrid analysis (20). Using RNase A, we
detected a mismatch in the cDNA coding for exons 41-49 of the
1(I) chain.
1(I) cDNA localized the mismatch to exon 44 (Fig. 2A). Both normal and
more slowly migrating products were detected in the probands'
cDNA. The more slowly migrating product was faintly visible in the
mother's sample. This electrophoretically slower product was shown to
be a heteroduplex of normal and mutant fragments. The small fraction of
mutant
1(I) transcripts in maternal cells can be easily visualized
because of the sensitivity of heteroduplex analysis for structurally
distinct products. The localization of the collagen mutation to exon 44 was confirmed by PCR amplification of genomic DNA (Fig.
2B).
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Fig. 2.
PCR screening of proband and parental
cDNA and genomic DNA. A, cDNA screening of
proband and parental fibroblasts. Normal and heteroduplex products were
observed in 1(I)E44 in the samples of both probands (lanes
4 and 5) and, to a lesser extent, in the samples of the
mother (lane 6). B, screening of genomic DNA from
control and parental leukocytes and proband fibroblasts. Normal and
heteroduplex products were obtained from the probands (lanes
4 and 5) and mother (lane 6).
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Fig. 3.
Sequencing of normal and mutant cDNA and
genomic DNA alleles. Sequencing of cDNA and genomic DNA
revealed a 9-bp duplication in COL1A1, which is also present in DNA
from the probands' mother. The normal allele encodes two identical
Gly-X-Y triplets (GGT GCT CCT) at amino acids
868-873, whereas the mutant allele contains three.
12
2,
1(ins)
1
2, and
1(ins)2
2), up to three different peaks may be
expected on thermograms. All three peaks are clearly visible at acidic
pH, compared with the single normal peak of the control (father (C-F),
Fig. 4). Based on relative intensity ratios determined by Gaussian
deconvolution, 0.25 (35 °C):0.5 (39 °C):0.25 (41 °C), these
peaks correspond to molecules with two, one, and no mutant
1 chains,
respectively. Only two peaks with the melting temperature
(Tm) difference of ~2 °C and a long low
temperature tail can be resolved at neutral pH. The difference between
thermograms at neutral and acidic pH and the change in the overall
Tm of the triple helix suggest that the register
shift propagates through the triple helix.
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Fig. 4.
Normalized DSC thermograms.
A, DSC of proband collagen (OI-INS (thick
line)) and normal control protein from his father (C-F
(thin line)) in 2 mM HCl, pH 2.7. B, DSC in 0.2 M sodium phosphate, 0.5 M glycerol, pH 7.4. Scans were performed at
0.125 °C/min. Phosphate/glycerol buffer was used to prevent
fibrillogenesis at neutral pH. Collagen Tm in this
buffer is 1.7 °C higher than in physiological solution (29).
1(I) around
the insertion, G832S (31), G898S, and
G997S.2 These Gly
Ser
substitutions should have post-translational modification similar to
that of the triplet duplication. By high pressure liquid
chromatography, we determined that lysine hydroxylation of all mutant
collagens was about double that of control: 21 ± 0.5% in C-F,
43 ± 1.5% in OI-INS, 40 ± 1.0% in G832S, 39 ± 0.5% in G898S, and 38 ± 1.0% in G997S. Gel migration of
1(I)
chains (Fig. 1A) indicated that all mutations had a similar
extent of lysine glycosylation. Most importantly, the N-terminal CB 8 + 5 peptide of OI-INS and OI-G997S had identical glycosylation
(Fig. 1B), so the effect of overmodification on cleavage of
N-propeptide from these mutant procollagens should be the same.
1
N-propeptide cleavage than from control or glycine substitutions with
equivalent overmodification. Thus, the decrease in kinetics of
N-propeptide cleavage in the insertion mutation is related to the
register shift per se. This supports the interpretation of
the DSC thermograms that the register shift persists through the entire helix.
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Fig. 5.
In vitro fibrillogenesis.
In vitro fibrillogenesis of proband collagen (OI-INS) and
normal control protein from his father (C-F). A, kinetics of
fiber formation by collagen prepared with N- and C-proteinase monitored
by turbidity measurement. B, SDS-PAGE of total protein and
fibrillogenesis-capable (PELLET) and -incapable
(SUPER) fractions of OI-INS and C-F collagen. C,
DSC thermograms of redissolved pellets and supernatant after in
vitro fibrillogenesis. Each solution was dialyzed against 2 mM HCl (pH 2.7). The scans were performed in 2 mM HCl at 0.3 °C/min heating rate. Brackets
indicate expected positions of denaturation peaks for triple helices
containing no, one, and two mutant 1(I) chains, based on DSC
thermogram shown in Fig. 4A. The slightly higher
Tm values than seen in Fig. 4A relate to
faster scanning in this thermogram (29).
1(I) chains. Since
helices with two mutant chains are resistant to fibril incorporation in
the in vitro assay, the overmodified
1(I) chains in the
pepsin extracts are more likely derived from helices with one mutant
1(I) chain.
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Fig. 6.
Incorporation of proband collagen into
extracellular matrix. Sequential extraction of type I collagen
incorporated in matrix by control (3349) and proband
fibroblasts. Media collagen was digested with pepsin. Matrix was
extracted serially, first with NaCl to isolate newly incorporated
collagen without cross-links (matrix fraction 1) and then with acetic
acid for immaturely cross-linked collagen (matrix fraction 2) and
finally with pepsin to release fully cross-linked collagen (matrix
fraction 3). Fractions were analyzed by 6% SDS-urea-PAGE.
1(I) chains could not be quantitated separately, but an
equivalent proportion of overmodified
1(I) chains is visible in each
proband sample.
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Fig. 7.
Turnover of collagen incorporated into matrix
in culture. A, labeled collagen was allowed to incorporate
into extracellular matrix deposited in culture by postconfluent control
and proband fibroblasts. Matrices were collected at 24-h intervals, and
collagens were extracted by pepsin digestion. Samples were analyzed by
6% SDS-urea-PAGE. B, the experiment was done in triplicate
and quantitated by densitometry of autoradiograms.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I) chain and its functional consequences for helix
formation and fibrillogenesis. The mutation occurs in two siblings with
lethal type II osteogenesis imperfecta. Their mother is a mosaic
carrier with a low percentage of heterozygous fibroblasts and
leukocytes, 10 and 15%,
respectively.4 Her clinical
history and physical exam are entirely normal.
1(I) (11-14) have occurred in this region.
1(ins)2
2, in which the
2 chain is out of
register with two longer
1(ins) chains, is much lower than that of
1(ins)
1
2, in which one
1(ins) chain is out of register with
the remaining two chains. Proper register of the
2 chain appears to
be particularly important for type I collagen stability.
1(I). Although overmodification may delay propeptide
processing, our co-digestion assay demonstrated that propagation of the
register shift along the entire helix must also contribute to the
slower cleavage kinetics of the insertion mutation.
1 chains may have melted and
be unavailable for incorporation. Indeed, we observed a substantial
decrease in the circular dichroism signal characteristic of a collagen
triple helix (35) upon fibrillogenesis of the insertion mutation, in contrast to enhanced circular dichroism upon fibrillogenesis of control protein.
2(I)
E16 collagen (36), which causes a larger
six-triplet register shift and is more likely to realign by "looping
out" of the normal chains than to propagate the register shift along
the full helix.4
chains often have preferential intracellular breakdown and
decreased secretion and sometimes have decreased incorporation into
matrix. Matrix turnover of mutant collagen has been studied in only a
few additional cases and was reported to have a shorter half-life than
in control in a case of lethal OI with
1(I) G667R (22) and a
half-life comparable with controls in cases of severe type III OI with
1(I) G589S and
2(I) G586V (37).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Karl Kadler for the generous gift of C-proteinase. We acknowledge Michael Bonidie, M.D., and Patricia D. Gonzales, M.D., for their astute insistence that samples be obtained from the probands in their local hospital and for referral of the cases. We are grateful to the probands' mother for interest and continued participation in OI research efforts.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence and reprint requests should be addressed: Heritable Disorders Branch, Bldg. 10; Rm. 9s241, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-6683; Fax: 301-402-0234; E-mail: oidoc@helix.nih.gov.
Published, JBC Papers in Press, January 20, 2003, DOI 10.1074/jbc.M212523200
2 W. A. Cabral, H. Nishioka, and J. C. Marini, unpublished data.
3 E. Makareeva and S. Leikin, unpublished data.
4 W. A. Cabral and J. C. Marini, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: OI, osteogenesis imperfecta; RT-PCR, reverse transcription-PCR; nt, nucleotides; DSC, differential scanning calorimetry; aa, amino acids; gDNA, genomic DNA.
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REFERENCES |
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