(Received for publication, May 23, 1996, and in revised form, January 28, 1997)
From the Osteometer BioTech A/S, Herlev Hovedgade
207, DK-2730 Herlev, Denmark and the ¶ Department of Clinical
Biochemistry, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-2100, Copenhagen, Denmark
The heterogeneity of urinary degradation products
of C-terminal telopeptides derived from the 1 chain of human type I
collagen was investigated and characterized. The urinary fragments
characterized in this study consisted of two cross-linked
(X) amino acid sequences derived from the C-terminal
telopeptide (
1) of type I collagen. Fragments containing the
sequence EXAHDGGR, with a DG site being either
nonisomerized (Asp-Gly) or
-isomerized (
Asp-Gly), were identified. Pyridinoline was detected among the pyridinium cross-links, but there was a dominance of deoxypyridinoline and a cross-link containing pyridinoline having a molecular weight identical with that
of galactosyl pyridinoline. A nonfluorescent cross-link was also found.
The concentration of fragments derived from the C-terminal telopeptide
region of type I collagen containing the sequence Asp-Gly (
CTX)
and/or
Asp-Gly (
CTX) was measured by enzyme-linked immunosorbent
assays in urine and in collagenase digests of trabecular and cortical
bone of young and old origin. It was shown that the urinary ratio
between such fragments,
CTX/
CTX, was higher in children compared
with adults and that the ratio decreased with increasing age of bone.
The results indicated that the C-terminal telopeptide fragments derived
from type I collagen excreted into urine originated mainly from bone.
In conclusion, it is demonstrated for the first time that the
C-terminal telopeptide
1 chain of type I collagen contains an
Asp-Gly site prone to undergo
-isomerization and that the degree of
-isomerization of this linkage apparently increases with increasing
age of bone. These findings indicate that the ratio
CTX/
CTX might
be clinically important in diagnosing metabolic bone diseases.
The rate of bone turnover has for years been estimated by measurement of different markers reflecting bone formation and bone resorption in urine and blood (1). The traditional bone-resorption markers are urinary calcium and hydroxyproline. Hydroxyproline, however, is not specific for bone (2), and its urinary excretion is influenced by diets rich in hydroxyproline (3), renal clearance, and metabolization (4, 5).
The pyridinium cross-links pyridinoline (Pyr)1 and deoxypyridinoline (Dpyr) are trifunctional cross-links that together with other di-, tri-, and tetrafunctional cross-links stabilize the collagen structure within the extracellular matrix (6). Pyr is found mainly in bone and cartilage (7, 8) but also in collagen from most other connective tissues, whereas Dpyr is present mainly in bone and dentin (7). Although the pyridinium cross-links are not entirely bone-specific, they appear to be superior markers of bone resorption compared with hydroxyproline (1).
The pyridinium cross-links occur at two intermolecular sites, one
linking two C-terminal telopeptides to residue 87 of the triple helix
1 or
2 chain and the other linking two N-terminal telopeptides to
residue 930 of the triple helix
1 chain (9). The C-terminal
telopeptide
1 chain of type I collagen consists of a 26-amino acid
sequence (10) and contains an Asp-Gly sequence that is a potential site
for
-isomerization in proteins/peptides prone to
-isomerize (11,
12). In the case of
-isomerization, the peptide backbone is
transferred from the aspartyl
-carboxyl group to the side chain
-carboxyl group, resulting in a perturbation of the peptide backbone
(11) (Fig. 1).
-Isomerization is generally believed
to be associated with aging of proteins and peptides (13, 14).
Recently, new assays measuring urinary degradation products derived
from the C-terminal telopeptide region of type I collagen have been
developed (15-17). It has been shown that measurements of such
fragments in urine reflects the rate of bone resorption in a number of
metabolic bone diseases (15-19). However, little is known about these
fragments, their size, and their possible relation to the degree of
osteoclastic activity. So far, it is uncertain whether the fragments
are further metabolized in the circulation, in the liver, or in the
kidneys. Neither is it clear to what extent the fragments are
cross-linked, what types of cross-links are dominant, and if certain
fragments can be related to degradation of different connective tissues
containing type I collagen. Finally, it is unknown whether the
C-terminal telopeptide Asp-Gly sequence is prone to undergo
-isomerization in type I collagen from bone and other connective
tissues.
The aim of the present study was to investigate the nature of degradation products derived from the C-terminal telopeptide region of type I collagen excreted into urine.
Materials
All chemicals were of analytical grade and obtained from Sigma or Merck. Acetonitrile was from Rathburn, heptafluorobutyric acid was from Aldrich, and trifluoroacetic acid was from Applied Biosystems (Foster City, CA). Synthetic peptides were from Schäfer-N (Copenhagen, Denmark). The CrossLapsTM ELISA was from Osteometer BioTech A/S (Herlev, Denmark), CNBr-activated SepharoseTM 4B was from Pharmacia Biotech Inc., and the Sep-Pak® C18 cartridges were from Waters (Milford, MA).
Subjects
Urine samples were collected as second morning void between 8 and 10 a.m. from 36 children and 42 postmenopausal women aged 6-11 and 45-54 years of age, respectively. The urine used for immunoaffinity purification was first morning void from an 11-year-old boy. Informed consent was obtained from all participants according to the Helsinki Declaration of 1975, as revised in 1983, and the investigation was approved by the ethical committee of Copenhagen County.
MAbA7 ELISA
The MAbA7 ELISA is a competitive assay employing a
peroxidase-labeled monoclonal antibody (MAbA7) reactive with peptide
fragments derived from degradation of the C-terminal telopeptide (1)
region of human type I collagen. The assay was performed as described previously (16).
CrossLaps ELISA
The CrossLaps ELISA is a competitive assay based on a polyclonal
antibody reactive with degradation products derived from the C-terminal
telopeptide region (1) of human type I collagen. The assay was
performed as recommended by the manufacturer.
Amino Acid Analysis
Amino acid composition analysis of EKAHDGGR, EKAHDGGR, and
urinary fragments derived from type I collagen was performed after acid
hydrolysis by ion exchange chromatography combined with post-column derivatization as described previously (20).
Peptide Sequence Analysis
N-terminal sequencing was performed on a 494A protein sequencer with an on-line 120 A analyzer (Applied Biosystems) and chemicals recommended by the manufacturer.
Mass Spectrometry
Freeze-dried material was redissolved in 20 µl of 30%
acetonitrile (v/v) containing 0.1% trifluoroacetic acid (v/v). A
0.5-µl aliquot was mixed with 0.5 µl of saturated
-cyano-4-hydroxycinnamic acid as matrix in the same solvent. The
mixture (1 µl) was analyzed in a Biflex instrument in linear mode at
15 kV (Bruker-Franzer Analytic). Spectra were averaged from 40-100
laser-beam shots. Using the synthetic peptide PYDRISNSAFSDF-NH2
(1517.6 Da) and melittin (2846.5 Da) as external calibrators, the
inaccuracy of the method was lower than 0.1%. The molecular mass of
each molecule was determined as the mean value of several measurements
estimated using different external standard curves.
HPLC Equipment
The chromatographic system consisted of two 510 pumps, a 717 autosampler, a scanning fluorescence detector, and an extended wavelength module equipped with a 214-nm cutoff filter (all from Waters). Purification of synthetic EKAHDGGR, EKAHDGGR, and urinary fragments derived from type I collagen was performed at room
temperature using a reverse-phase Delta Pak C18 column (3.9 mm × 150 mm; particle size, 5 µm; pore size, 30 nm; Waters). Data were
collected on a personal computer and evaluated using the Maxima
software from Waters. In the following, the effluents were monitored
for peptide bonds at 214 nm and for fluorescent molecules at 380 nm
(emission) using 297-nm light for excitation.
Characterization of MAbA7 ELISA and CrossLaps ELISA Binding Specificity
Preparations of synthetic EKAHDGGR were contaminated with its
corresponding -isomerized form, EKAH
DGGR. Prior to evaluation of
the MAbA7 ELISA and the CrossLaps ELISA binding specificity, EKAHDGGR
and EKAH
DGGR were separated by reverse-phase HPLC using an isocratic
gradient of 0.5% acetonitrile (v/v) containing 0.1% trifluoroacetic
acid (v/v) at a flow rate of 1 ml/min. The effluent was monitored for
peptide bonds, collected in 1-ml fractions, freeze-dried, redissolved
in PBS (1 ml), and measured in the two assays (Fig.
2).
Production of Monoclonal Antibody
The monoclonal antibody MAbA7, which is reactive with peptide
fragments from the C-terminal telopeptide (1) region of type I
collagen, was produced, hybridomas were propagated, and monoclonal antibodies were purified as described previously (16).
Immunoaffinity Chromatography
Monoclonal antibody MAbA7 was coupled to CNBr-activated
Sepharose 4B according to the manufacturer's instructions. Urine was diluted 1:3 (v/v) in PBS, the pH was adjusted to 8.0 with 1 M NaOH, and 800 ml of diluted urine was recirculated on a
MAbA7-Sepharose column (14 cm3) for 24 h at 4 °C at
a flow rate of 0.8 ml/min. After washing the column with 200 ml of PBS,
pH 8.0, bound material was eluted using 20 ml of 50% saturated
(NH4)2SO4 containing 1%
trifluoroacetic acid (v/v). The eluted molecules were desalted using a
C18 Sep-Pak cartridge conditioned with 20 ml of 80% methanol (v/v) and
equilibrated with 20 ml of 1% trifluoroacetic acid (v/v). Bound
material was washed with 20 ml of 1% trifluoroacetic acid (v/v) and
eluted with 40% acetonitrile containing 0.1% trifluoroacetic acid
(v/v), freeze-dried, and stored at 20 °C.
Purification of Immunoaffinity Purified Material
The purification of immunoaffinity purified material was carried out using three consecutive reverse-phase HPLC steps.
Step 1Freeze-dried material extracted from urine by
immunoaffinity chromatography was redissolved in 0.1%
heptafluorobutyric acid (v/v) and eluted from a Delta Pak C18 column
with a 16-24% acetonitrile gradient containing 0.1%
heptafluorobutyric acid (v/v) over 90 min at a flow rate of 1 ml/min.
Two-ml fractions were collected and stored at 20 °C. An aliquot
(25 µl) from each fraction was freeze-dried, redissolved in PBS (200 µl), and assayed in the MAbA7 ELISA and the CrossLaps ELISA.
Selected fractions from Step 1 containing high
amounts of CTX and
CTX as determined by CrossLaps ELISA and MAbA7
ELISA were freeze-dried, redissolved in 0.1% trifluoroacetic acid, and
further purified using a Delta Pak C18 column. The fragments were
eluted with a 0 to 12% acetonitrile gradient containing 0.1%
trifluoroacetic acid (v/v) over 80 min at a flow rate of 1 ml/min.
Two-ml fractions were collected and stored at
20 °C. From each
fraction an aliquot of 50 µl was freeze-dried, redissolved in PBS
(200 µl), and assayed in the MAbA7 ELISA and CrossLaps ELISA.
Selected fractions from Step 2 containing high
amounts of CTX and
CTX as determined by CrossLaps ELISA and MAbA7
ELISA were purified once more on a Delta Pak C18 column. Freeze-dried
material was redissolved in 0.1% trifluoroacetic acid (v/v) and eluted with a 1-5% acetonitrile gradient containing 0.1% trifluoroacetic acid (v/v) over 50 min at a flow rate of 1 ml/min. Fractions (240 µl)
were collected, and an aliquot (10 µl) from each fraction was
freeze-dried, redissolved in PBS (100 µl), and assayed in the MAbA7
ELISA and CrossLaps ELISA.
Determination of Pyridinium Cross-Links
The possible presence of the pyridinium cross-links Pyr and Dpyr was evaluated in fractions containing high levels of fragments reactive in the MAbA7 ELISA and CrossLaps ELISA by their fluorescence after acid hydrolysis and separation by reverse-phase HPLC (21).
Purification and Collagenase Digestion of Type I Collagen from Bone
Trabecular and cortical bone was obtained from bovine femurs from an embryo and a cow. Soft tissue surrounding the femur was removed, and the bone was cut into pieces. Trabecular bone was obtained from the femoral neck and cortical bone from the midshaft of the bones. The bone samples were washed extensively at 4 °C with 0.14 M NaCl, 0.05 M Na2(SO4), pH 7.5, and subsequently extracted overnight at 4 °C with 4 M guanidine HCl, 0.05 M Tris, pH 7.5. After rinsing with water the bone was lyophilized and processed into fine powder using a Spex freezer mill (Spex Inc., Edison, NJ). The bone powder was defatted with acetone for 2 h, lyophilized, and demineralized in 0.5 M EDTA, 0.05 M Tris, 1% NaN3 (w/v), pH 7.5, in Spectrapor-4 dialysis tubing (Spectrum, Los Angeles, CA) for 72 h. The sample was centrifuged at 3000 × g for 30 min. The pellet was rinsed in cold water, reconstituted in 0.05 M Tris, 0.036 M CaCl2, pH 7.5, and digested with 500:1 (w/w) bacterial collagenase 1A (Sigma C-9891) overnight at 37 °C. The collagenase-treated collagen was centrifuged for 30 min at 3000 × g, and the supernatant was assayed for reactivity in the MAbA7 ELISA and in the CrossLaps ELISA.
The monoclonal antibody
MAbA7 was coupled to CNBr-activated Sepharose and used to purify
urinary CTX fragments by immunoaffinity chromatography. All urinary
molecules reactive in the MAbA7 ELISA and the CrossLaps ELISA were
retained on the MAbA7-Sepharose column even though synthetic
EKAHDGGR could not be detected in the MAbA7 ELISA (Fig. 2 and data
not shown). This indicates a relatively high cross-reactivity between
the MAbA7 antibody and
CTX under the chromatographic conditions
employed.
The urinary CTX fragments extracted by immunoaffinity chromatography
were further separated by three consecutive reverse-phase HPLC steps,
all employing acetonitrile as eluent. In HPLC Step 1, heptafluorobutyric acid was used as ion-pairing agent. The urinary
extract consisted of a variety of different CTX molecules as shown by
the elution profiles in Fig. 3. All fractions were assayed for CTX and
CTX by MAbA7 ELISA and CrossLaps ELISA, respectively (Fig. 3). The immunological
CTX and
CTX profiles, however, were quite different. Only fractions from HPLC Step 1 containing high levels of
CTX and/or
CTX were subjected to HPLC Step 2 employing trifluoroacetic acid as ion-pairing agent. Fig. 4 and Fig. 5 show the reverse-phase and
immunological elution profiles of fractions F1 and F4, respectively,
from HPLC Step 1. Both fractions F1 and F4 contained several different
molecules as shown by the UV absorbance and fluorescence elution
profiles. The molecules responsible for the immunological response
given in fraction F1 and F4 were nonfluorescent (Fig. 4,
F1*) and fluorescent (Fig. 5, F4*), respectively.
In HPLC Step 3, selected fractions from HPLC Step 2 were subjected to a
final purification prior to further analysis (data not shown). To
ensure high purity of the CTX fragments, each peak was divided into
three to five fractions. Again, fractions were assayed for their
content of
CTX and
CTX fragments. The recovery was better than
95% in each of the three HPLC steps as evaluated by both MAbA7 ELISA
and CrossLaps ELISA (data not shown). In addition, no destruction or
conversion of synthetic EKAHDGGR or native
CTX fragments to their
corresponding
-isomerized forms was seen, indicating that the
purified urinary
CTX fragments were generated in vivo
(data not shown).
Determination of Fragment Purity
The purity of the fragments
separated by HPLC was evaluated by mass spectrometry and by
determination of the cross-links Pyr and Dpyr after acid hydrolysis.
Fractions containing only one detectable mass were regarded pure if
they contained only Pyr, Dpyr, or none of the pyridinium cross-links.
Mass spectra of the fragments P1 and P4 (Table I)
responsible for the major immunological reactivity in fractions F1 and
F4 (Fig. 3, Fig. 4, F1*, Fig. 5, F4*) are shown
in Fig. 6. The molecular mass was 2039.8 Da for the nonfluorescent
fragment P1 from fraction F1* (Fig. 4) and 2036.9 Da for the
fluorescent fragment P4 from fraction F4* (Fig. 5). Consecutive
fractions with the same molecular mass and an equal CTX/
CTX ratio
as determined by ELISA were pooled prior to analysis for amino acid
composition and sequence analysis.
|
Characterization of Purified CTX Fragments
Table I summarizes
the analytical results for some of the purified molecules. The
fragments P4, P5, and P6 all contained Pyr and had a molecular mass of
2036.9 Da. However, the three fragments differed with
respect to retention time and CTX/
CTX ratio. The triplet pattern
evident for P4, P5, and P6, showing increasing retention times followed
by increasing
CTX/
CTX values, was also seen for the triplets P1,
P2, and P3 and for the triplets P7, P8, and P9. The molecules P7, P8,
and P9, with a molecular mass of 1859.7 Da, all contained the
pyridinium cross-link Dpyr. The fraction containing P8 was slightly
contaminated with molecules having a molecular mass of 2036.9 Da
containing Pyr. In accordance with their lack of fluorescence, the
fragments P1, P2, and P3, all with a molecular mass of 2039.8 Da, did
not contain pyridinium cross-links. These results indicate that the
three fragments in each of the three triplets consisted of two
cross-linked CTX fragments: either two
CTX fragments (P1, P4, and
P7), one
CTX and one
CTX fragment (P2, P5, and P8), or two
CTX
fragments (P3, P6, and P9).
Amino acid composition analysis of the fragments revealed that they
apparently all consisted of the same six amino acids (Ala, Asp, Glu,
Gly, His, and Arg) in the ratio 1:1:1:2:1:1. The amino acid
compositions of P4, P5, P6, and synthetic EKAHDGGR and EKAHDGGR are
given in Table II. Except for lysine (K), the amino acid
composition was consistent with the sequence EKAHDGGR specific for a
part of the C-terminal telopeptide
1 chain in type I collagen (10). Sequence analysis of the fragments P2, P3, P5, P6, P8, and P9 confirmed
the identity of the fragments as being degradation products derived
from the C-terminal telopeptide
1 chain of type I collagen. Sequence
data for P4, P5, P6, EKAHDGGR, and EKAH
DGGR are given in Table
III. The blank run in the second cycle indicates the
presence of a cross-link in P4, P5, and P6. This is consistent with
previous findings showing that the C-terminal telopeptide lysine
residue (K) is a site for intermolecular cross-linking between mature type I collagen molecules (9, 22, 23).
|
|
N-terminal sequencing of the fragments P1, P4, and P7 stopped after the
N-terminal His (H) residue, suggesting the presence of an alteration in
the peptide backbone at the Asp-Gly (DG) site to preclude further steps
in Edman degradation. As demonstrated by amino acid composition
analysis using synthetic EKAHDGGR and EKAHDGGR,
-isomerization of
the Asp-Gly (DG) linkage did not result in an altered amino acid
composition (Table II), nor was the mass of the sequence altered. In
the case of
-isomerization of the Asp-Gly sequence, however, the
peptide backbone was transferred from the
-carboxyl to the side
chain
-carboxyl group, resulting in a significant structural
perturbation of the peptide backbone, making it resistant to Edman
degradation (Table III) (11). Based on the immunological
characterization (Table I), molecular weight, and the result of the
Edman sequence analysis, we suggest that the fragments P1, P4, and P7
consist of two cross-linked
CTX fragments, also derived from type I
collagen. Likewise, the fragments P3, P6, and P9 are proposed to
consist of two cross-linked
CTX fragments, since they were
immunologically reactive in the MAbA7 ELISA only and further revealed a
full peptide sequence (EXAHDGGR). Immunologically, the
fragments P2, P5, and P8 were all reactive in both the MAbA7 ELISA and
the CrossLaps ELISA, indicating that the fragments consist of a
cross-linked
CTX sequence and a
CTX sequence. Sequence data for
P5 and P6 supported this assumption, since the relative yield of Asp
(D) and Gly (G) was significantly higher in P6 containing two
CTX
fragments than in P5 (Table III). It has been shown in several studies
that
-isomerized peptides elute prior to their corresponding
nonisomerized forms when separated by reverse-phase HPLC employing
conditions similar to those used in this study (Fig. 2) (24, 25). The
reverse-phase HPLC elution pattern seen for the triplets with
CTX-X-
CTX fragments eluting before
CTX-X-
CTX fragments, again followed by
CTX-X-
CTX fragments, is consistent with these
findings.
The fluorescent fragments were shown to contain pyridinoline (P4, P5,
and P6) (Fig. 7) and deoxypyridinoline (P7, P8, and P9)
after acid hydrolysis (Table I). The theoretical molecular mass of
fragments consisting of two EXAHDGGR sequences cross-linked by Pyr or Dpyr at position X is 1874.9 and 1858.9 Da,
respectively. P7, P8, and P9 cross-linked by Dpyr were shown to have a
molecular mass of approximately 1859.7 Da, closely corresponding to the expected theoretical molecular mass of such a fragment.
The molecular mass of the fragments cross-linked by Pyr being 2036.9 Da, however, was not consistent with the theoretical molecular mass of such molecules (1874.0 Da), indicating that the Pyr cross-link has been further posttranslationally modified. Glycosylated Pyr derivatives can be present as galactosyl Pyr (Pyr-Gal) and glycosyl galactosyl Pyr (Pyr-Gal-Glu) (Fig. 7), dependent on whether the hydroxylysine residue from the triple helical domain involved in the Pyr cross-link originally was galactosyl hydroxylysine or glycosyl galactosyl hydroxylysine (26, 27). The theoretical molecular mass (2036.9 Da) of fragments consisting of two EXAHDGGR sequences cross-linked by Pyr-Gal at position X is identical with the molecular mass of P4, P5, and P6, approximately 2036.9 Da.
The fragments cross-linked by a nonfluorescent cross-link (P1, P2, and P3) had a molecular mass of approximately 2039.8 Da. This molecular mass did not correspond to the molecular mass of any known fragment consisting of two EXAHDGGR sequences cross-linked at position X by partly degraded nonfluorescent pyridinium cross-links or their glycosylated derivatives. A nonfluorescent cross-link, which is different from partly degraded pyridinium cross-links, containing a pyrrole ring has been described (22, 28, 29) (Fig. 7). The molecular mass of P1, P2, and P3, however, did not correspond to the theoretical molecular mass of any fragments cross-linked by intact pyrrole cross-links or their glycosylated counterparts.
Quantification ofThe
concentration of urinary CTX and
CTX fragments was evaluated in
urine from children and adults aged 6-11 and 45-54 years of age,
respectively, and in collagenase digests of young and old bovine bone
as well, by MAbA7 ELISA and CrossLaps ELISA. The
CTX/
CTX ratios
found in urine and in collagenase digests of bone are given in Fig.
8. Evaluated on a group basis, the ratio
CTX/
CTX
was approximately 3-fold higher in urine from children compared with
adults evaluated on a group basis (p < 0.001). In bone, the highest
CTX/
CTX value was found in young trabecular bone followed by young cortical bone (both of fetal origin), old trabecular bone, and cortical bovine bone. The urinary ratio
CTX/
CTX from adults being approximately 0.4 was almost comparable
to that found in old trabecular and cortical bone of bovine origin.
In this study, we identified an Asp-Gly sequence within the
C-terminal telopeptide 1 chain of type I collagen prone to undergo
-isomerization. The results presented here indicate that
-isomerization of this sequence takes place in vivo and
that the degree of
-isomerization increases with increasing age of
type I collagen molecules in bone.
Around 90% of the protein in bone is type I collagen (30). When bone is resorbed by osteoclasts, fragments of type I collagen are released into the circulation and to an unknown extent excreted into the urine. However, urinary degradation products of type I collagen might also be derived from nonskeletal sources, since type I collagen is widely distributed in most connective tissues throughout the body (30). So far, little is known about the origin and biochemical structure of such urinary collagen-degradation products. In this context, it is interesting that the MAbA7 ELISA and the CrossLaps ELISA appear exclusively to measure urinary degradation products of type I collagen derived from osteoclastic resorption of bone (15-19). A biological explanation for this phenomenon could be that certain fragments derived from mature type I collagen are protected against complete degradation by the liver and/or the kidney due to posttranslational modifications.
The MAbA7 ELISA and the CrossLaps ELISA both measure urinary
degradation products (CTX) derived from the C-terminal telopeptide 1
chain of type I collagen (10). The human C-terminal telopeptide
1
chain consists of a 26-amino acid sequence (Fig. 9) in
which the amino acid Lys (K) at position 16 is a site involved in
cross-linking of mature collagen (9, 22, 23). Using synthetic peptides, it has been shown that only CTX fragments containing the sequence from
residues 17-22 exposing a free Arg residue at position 23 are measured
in the MAbA7 ELISA (16). Likewise, the CrossLaps ELISA also determines
CTX fragments derived from the C-terminal telopeptide
1 chain of
type I collagen containing the amino acid sequence from residues 17-22
(15). Using synthetic EKAHDGGR and EKAH
DGGR, it is shown that the
MAbA7 ELISA and the CrossLaps ELISA measure nonisomerized and
-isomerized peptides, respectively (Fig. 2).
In the present study, the two immunoassays in addition to traditional
biochemical techniques were used to characterize the structure of type
I collagen-degradation products. It was demonstrated that the Asp-Gly
sequence (positions 19-20) within the human C-terminal telopeptide
1 chain of type I collagen may undergo
-isomerization.
-Isomerized CTX fragments were identified in collagenase digests of
type I collagen from bone and in urine. The majority of the urinary
fragments characterized were cross-linked and consisted of two
CTX
sequences, one
CTX and one
CTX sequence, or two
CTX
sequences.
The conversion of the Asp-Gly linkage to a -isomerized Asp-Gly
linkage is illustrated in Fig. 1. The attack by a peptide backbone
nitrogen on the side chain carbonyl group of an adjacent Asp residue
can result in the formation of a five-member succinimide ring (24, 31).
The succinimide ring is prone to hydrolysis, producing Asp-Gly and
Asp-Gly sequences (24). Succinimide intermediates can form and
undergo hydrolysis under physiological conditions (24). Several factors
are known to influence the formation rate of such succinimide
intermediates. Among these are the primary structure of the fragment,
the secondary, tertiary, and quaternary structures of proteins (11,
32), pH (33) and temperature (32, 33), and the protein
microenvironment.
The biological role of -isomerization is not known yet. It is
generally believed, however, that the degree of
-isomerization is
associated with aging of proteins (13). This hypothesis is based on
results showing that the degree of
-isomerization in proteins and
peptides prone to undergo this alteration increases with time (age)
in vivo and in vitro (12, 34, 35). In addition, studies evaluating the function of proteins and peptides have revealed
that increased
-isomerization is often accompanied by a decreased
physiological protein/peptide activity (36). Furthermore, the presence
of an intracellular methyltransferase repair system using
-isomerized molecules as substrate has been reported (25, 37),
whereas extracellular repair systems, such as the methyltransferase system present in cells, has not (38). It is therefore improbable that
a
-isomerized C-terminal telopeptide
Asp-Gly sequence will be
enzymatically repaired in vivo, since type I collagen is an extracellular protein.
Our results seem to confirm the suggested relationship between
-isomerization and tissue age, since the ratio
CTX/
CTX is high
in collagenase digests of young bone compared with those of old bone.
This relationship was further supported by our results showing that the
relative amount of
CTX in the above fractions (Fig. 8) was higher in
trabecular bone compared with cortical bone, two bone tissues
characterized by high and low bone turnover, respectively (39). We
therefore suggest that the degree of
-isomerization of the
C-terminal telopeptide Asp-Gly site of type I collagen in bone is
dependent on the age of the molecules in the bone tissue.
In a previous study, Haley et al. (40) were not able to show
an increase in the number of -isomerized Asp-Gly sequences from
Achilles tendon per molecule of type I collagen with increasing age. In
the same study, Haley et al. (40) estimated the content of
-isomerized Asp-Gly sites to be between 0.8 and 1.9 per collagen molecule. The
1 and
2 chains of type I collagen both contain several Asp-Gly sites. Combined with our results, however, the number
of
-isomerized Asp-Gly sites as estimated by Haley et al.
indicate that the C-terminal telopeptide Asp-Gly sequence (
1)
probably is the only Asp-Gly sequence in type I collagen that is liable
to undergo
-isomerization.
Using synthetic peptides containing an Asp-Gly sequence, Gieger and
Clarke (24) showed that at equilibrium in the absence of the
methyltransferase repair system approximately 75% of the Asp-Gly
sequences were converted to their corresponding isoform, Asp-Gly. In
our study 50% of the CTX Asp-Gly sequences were
-isomerized in
urine from children, whereas 70-80% was
-isomerized in urine from
adults and in collagenase digests from old trabecular and cortical
bone. This seems to indicate that the C-terminal telopeptide Asp-Gly
sequence almost has reached equilibrium with respect to
-isomerization in old trabecular and cortical bone. Based on histological studies, it has been estimated that trabecular and cortical bone in adults are completely renewed within a period of 3 and
20 years, respectively (39). Accordingly, we suggest that the process
of
-isomerization of the C-terminal telopeptide Asp-Gly sequence of
type I collagen in mineralized tissues such as bone almost has reached
equilibrium within a period of 3 years.
Clinical results obtained using the MAbA7 ELISA and the CrossLaps ELISA
strongly indicate that both assays measure urinary fragments derived
from resorption of bone (15-19). This assumption was further supported
by the close resemblance between the various CTX/
CTX levels found
in urine and bone from young and old individuals, respectively.
Therefore, it seems reasonable to assume that the urinary ratio of
CTX/
CTX reflects the ratio in the resorbed bone. In this context,
it is interesting that the urinary ratio found in patients with
Paget's disease is markedly elevated compared with that of age-matched
healthy controls (16). Paget's disease is characterized by very high
bone turnover in restricted areas of the skeleton, resulting in a
disorganized mosaic of woven and lamellar bone at the affected sites
(41). If the hypothesis holds true that
CTX and
CTX reflect
degradation of young and old bone, respectively, apparently the
contribution of
CTX from the diseased areas of the skeleton is very
high compared with
CTX. This is in keeping with the notion that the
diseased areas in Paget's disease are characterized by a highly
increased rate of bone turnover, probably resulting in degradation of
relatively newly formed (young) bone.
The urinary CTX fragments identified in this study were cross-linked by either pyridinium cross-links or an unknown nonfluorescent cross-link. The ratio between Pyr and Dpyr in bone is 3.5:1, whereas the ratio is higher than 10:1 in almost all other connective tissues containing these cross-links (8). The pyridinium cross-links are normally excreted into urine in a Pyr/Dpyr ratio of 3.8-6.0, which is almost comparable to that found in bone. This indicates that the majority of the pyridinium cross-links in urine are derived mainly from bone. In the present study, the Pyr/Dpyr ratio was 3.8 in the mixture of CTX fragments extracted from urine by immunoaffinity, indicating that at least the fragments cross-linked by pyridinium cross-links were derived from bone (data not shown).
In conclusion, we have identified an Asp-Gly sequence within the
C-terminal telopeptide 1 chain of type I collagen prone to
-isomerization. The degree of
-isomerization at this site seems
to increase with increasing age of type I collagen molecules in bone.
The data further indicate that the degree of
-isomerization has
almost reached equilibrium in old trabecular and cortical bone. In
addition, our results seem to confirm previous clinical findings
(15-19) showing that urinary
CTX and
CTX are derived from bone
and that the urinary ratio
CTX/
CTX could be of clinical importance in assessing metabolic bone diseases.
We thank Inge Kolding for excellent technical assistance, Inger Byrjalsen, Christian Hassager, and Claus Christiansen for helpful discussions, and Brian J. Pedersen for a critical review of the manuscript.