(Received for publication, January 6, 1997, and in revised form, March 10, 1997)
From Osiris Therapeutics Inc., Baltimore, Maryland 21231, the
Johns Hopkins University School of Medicine, Departments
of Oncology and Molecular Biology & Genetics, Baltimore, Maryland
21205, and the § Department of Cell and Molecular Biology,
Lund University, S-221 00 Lund, Sweden
Analysis of the carboxymethylated subunit of human cartilage oligomeric matrix protein (COMP) by matrix-assisted laser desorption time-of-flight mass spectrometry indicated a protonated molecular mass of 86949 ± 149 Da, compared with 83547.0 Da calculated from the sequence. Treatment with N-glycanase caused a reduction in mass of 3571 ± 219 Da, but there was no loss of mass after treatment with O-glycanase or neuraminidase. Peptides containing two putative sites of N-glycosylation were purified and characterized. Analysis of the masses of these after N-glycanase treatment indicated that one was substituted at Asn-101 with an oligosaccharide of mass 1847.2 ± 6.6 Da, and the other was unsubstituted at Asn-124. The remaining site of attachment, at Asn-721, was, therefore, also substituted with an oligosaccharide of mass 1724 ± 226 Da. Analysis of the total monosaccharide content by chemical methods indicated that there were no additional oligosaccharide substituents. The MALDI-TOF mass spectra of COMP from bovine fetal and adult cartilage were compared, indicating a more heterogeneous pattern of substitution at Asn-101 in the fetal form. Since COMP is distributed throughout the pericellular and territorial environments in developing cartilage but occupies the interterritorial zone in mature cartilage, these changes in glycosylation may allow for different intermolecular interactions.
Cartilage oligomeric matrix protein
(COMP)1 is a pentameric glycoprotein
consisting of identical monomeric subunits of apparent molecular weight
100,000 (1). It is found in the extracellular matrix of tracheal,
nasal, and articular cartilage and is also synthesized by Swarm rat
chondrosarcoma cells (2). In tendon, it is distributed around and
within tendon bundles (3). The sequences of rat (4) and human (5) COMP
have been determined from cDNA clones. The rat and human sequences
contain several regions that show a high homology with the
thrombospondins, having a series of four contiguous type 2 epidermal growth factor (EGF) repeats followed by a series of seven
type 3 calcium binding domains. There is also an RGD cell-binding motif
in human, but not in rat COMP. This is also present in bovine COMP, for
which a partial sequence is available (4). The human COMP gene is
located on chromosome 19p13.1 (5). The interactions between the COMP
subunits that give rise to the pentameric structure are mediated by an -helical portion of the protein close to the N terminus.
Oligomerization is independent of disulfide bond formation although
these interactions stabilize the assembled structure (6). Recently (6)
the crystal structure of the oligomerization domain of COMP was
described. This structure consists of a bundle of five
-helical
strands, each 46 residues in length, that form a parallel coiled coil. The resulting structure is a pore that may act as an ion channel, but the hydrophilic outer surface of this structure indicates that it
is not a transmembrane domain (6).
The function of COMP in cartilage is unknown, but a good deal of information has been obtained about its distribution in immature and mature tissues. During development of the rat femoral head, COMP shows a predominant pericellular and territorial distribution around chondrocytes in the cartilage extracellular matrix and in the growth plate (7). As the secondary center of ossification forms, COMP protein disappears from the calcified tissues but persists in the growth plate. In the mature articular cartilage, COMP is prominent both in its mRNA levels, as studied by in situ hybridization, and in protein abundance. Interestingly, the protein is primarily found within the interterritorial matrix compartment at this stage (7). This observation may indicate different roles of COMP in immature and adult tissue. During limb chondrogenesis in mice, COMP expression begins at an early stage, where it is seen in the peripheral region of the developing humerus as the 100 kDa subunit protein (8). Later, it is more uniformly distributed throughout the cartilaginous layer. These observations suggest that COMP, because of its early expression during cartilage development, may play a role in the assembly of the extracellular matrix.
The importance of the role that COMP plays in cartilage development is underscored by the observation that mutations in the COMP gene give rise to pseudoachondroplasia (9) and multiple epiphyseal dysplasia (10), conditions characterized by short stature and cartilage abnormalities. These mutations occur within the type 3 calcium-binding repeats, suggesting an essential role for COMP in calcium-mediated interactions.
COMP has three sites of potential N-glycosylation (4, 5), asparagines 101, 124, and 722 (the residue numbers refer to human COMP), but the extent to which they are substituted has not been determined. The role of carbohydrate substituents in modulating the interactions and stability of extracellular proteins has been well described. In some cases, the nature and degree of substitution varies with the developmental state. For example, aggrecan, which is an abundant proteoglycan in cartilage, has several sites of N-glycosylation within the N-terminal globular G1 domain that become substituted with sulfated polylactosamine chains during maturation (11). In this report, the results of studies on the distribution and structure of carbohydrate substituents in bovine and human COMP, using chemical analysis and mass spectrometry, are described. It is shown that there are some important maturation-related differences in the structure of the N-linked sugars.
COMP was purified from mature bovine articular cartilage obtained from the metacarpalphalangeal joints by dissociative extraction (1). Briefly, this involved cesium chloride density gradient centrifugation of the 4 M guanidine hydrochloride (GdnHCl) cartilage extract. The fraction of lowest buoyant density (1.35 g/ml), representing one-fourth of the total volume, was collected, dialyzed against water, and concentrated by drying. The concentrate was applied to a Superose 6 column (Pharmacia Biotech Inc.) and eluted with 4 M GdnHCl, 20 mM Tris-HCl, pH 7.4. The material recovered from the void volume peak was dialyzed and applied to a Mono-Q HR 5/5 column (Pharmacia) equilibrated in 10 mM Tris-Cl, 7 M urea, 0.15 M NaCl, pH 7.4. Elution was with a linear gradient of NaCl from 0.15 M to 1.5 M. Human COMP was prepared from femoral head cartilage from a 35-year-old individual using the same procedure. COMP was also prepared from steer fetlock cartilage and from third trimester bovine fetuses.
Enzyme DigestionsCOMP was reduced and alkylated by standard methods (12). Digestion with lysine-C proteinase (Wako BioProducts, Richmond, VA) or with trypsin (Boehringer Mannheim) was carried out at 30 °C using an enzyme:substrate ratio of 1:50 or at 37 °C using an enzyme substrate ratio of 1:100, respectively, in 0.1 M Tris-Cl, pH 8.5. Peptides were separated by chromatography on a Vydac (Hesperia, CA) 2.1 × 250 mm C18 column on a Hewlett-Packard (Palo Alto, CA) 1090L HPLC system. The column was equilibrated in 0.1% trifluoroacetic acid (TFA) in water. Elution was with a gradient of acetonitrile (0-55% solvent B in 55 min, 55-100% solvent B in 20 min; solvent B was 70% acetonitrile, 0.85% TFA). Digestion with recombinant N-glycanase (Genzyme, Inc., Cambridge MA) was carried out at 37 °C in 10 mM ammonium bicarbonate, pH 8.0, using 250 milliunits of enzyme and 20 pmol peptide.
Monosaccharide AnalysisFor monosaccharide analysis, samples were hydrolyzed in the liquid phase in the presence of either 2 M TFA or 4 M HCl at 100 °C for 3 and 5 h, respectively. To check for losses of monosaccharides during hydrolysis, fetuin was used as a control and was hydrolyzed under conditions identical to those used for COMP. Analysis was carried out on a Dionex (Sunnyvale, CA) LC500 system using a PA-1 column (13). Detection was by pulsed amperometry using a Dionex ED-40 system.
MALDI-TOF Mass SpectrometryThis was carried out using a
Hewlett-Packard G2025A instrument in the positive ion mode. 10 µl of
matrix solution was mixed with 1 µl of sample, and a 0.7-µl aliquot
was dried under vacuum. Crystal formation was monitored using the
Hewlett-Packard G2024A sample prep station. For intact proteins, the
matrix used was sinapinic acid (Hewlett Packard), approximately 100 shots were summed and the total laser irradiance was 5-6 µJ. For
peptides derived from proteolytic digests, -cyano-4-hydroxycinnamic
acid (Hewlett Packard) was used as matrix, approximately 30 shots were summed, and the total laser irradiance was 0.6-1.0 µJ. An
alternative method for sample preparation was carried out as follows.
Sample and matrix were applied as already described and allowed to dry, and then 0.7 µl of a formic acid solution (water:acetonitrile:formic acid 100:100:35) was overlaid and allowed to
dry.2
Protein sequencing was carried out using a Hewlett-Packard G1000A protein sequencer using Routine 3.0 methods. For lectin blotting, COMP was reduced and carboxymethylated, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 10% gel (14), and electrotransferred onto polyvinylidene difluoride (PVDF) membrane. The membrane was probed with lectins SNA (15) and MAA (16) using the CHO kit (Boehringer Mannheim), with bovine fetuin as the standard.
Analysis by MALDI-TOF mass spectrometry of reduced and
carboxymethylated COMP from adult human articular cartilage indicated a
protonated subunit molecular mass of 86949 ± 149 Da (the average of three measurements, Fig. 1 upper
spectrum). The predicted value was 83547.0 Da (5), with the mass
difference of 3402 ± 149 Da indicating a low level of
post-translational addition. The carboxymethylated subunit protonated
molecular mass of COMP prepared by associative extraction from adult
bovine articular cartilage was 86560 ± 163 Da (Fig. 1,
lower spectrum), close to the value obtained for human COMP.
There is a high degree of homology between the bovine, rat, and human
sequences (4, 5) in the region where comparisons may be made (residues
299-737 of human COMP, or 59% of the sequence). It was assumed that
there is an equally high homology between the sequences in the regions
where a direct comparison is not possible. Therefore, many of the
mapping studies described here were carried out using bovine COMP and
related to predictions made by comparison with the human and rat
sequences.
Bovine COMP was digested with lysine-C proteinase, and the peptides
were separated by reverse-phase HPLC (Fig.
2A). Several peptides were collected and
identified by N-terminal sequencing, MALDI-TOF mass analysis, or both.
These are referred to as peptides K-1 through K-17 (Table
I). Of particular interest was peptide K-17, with a
protonated mass of 11744.3 ± 3.3 Da. The N-terminal sequence was
identified as LTVRPLSQCRPGFCFPGVAXTXT by Edman degradation. When
aligned with the human COMP sequence (5), this peptide corresponds to
residues 62-155. This peptide carries two putative N-glycosylation sites, at Asn-101 and Asn-124. The
protonated mass was reduced to 9897.1 ± 3.3 Da by digestion with
N-glycanase, a difference of 1847.2 ± 6.6 (Fig.
2B). This peptide was digested with trypsin and fractionated
by HPLC (Fig. 3A). The tryptic peptides, referred to as T-1 through T-4, were identified by N-terminal sequencing, and their protonated masses were determined by MALDI-TOF MS, with and without treatment with N-glycanase. The results
for peptides T-1 and T-3 are shown in Fig. 3, B and
C and are summarized in Table I. One of these peptides
(peptide T-1, with sequence CGPCPEGFTGXGSHCADVNECXAHP ... , corresponding to residues 91-121 of the human sequence, see Table I)
was reduced by 1849.8 Da after N-glycanase treatment. The
mass of peptide T-3, which contains the other putative
N-linkage site with sequence
CINTSPGFRCEACPPGFSGPTHEXV ... , corresponding to residues 122-152
of human COMP, agrees with the calculated mass and was unchanged by
N-glycanase treatment. These results indicate that, of the
two possible sites of N-glycosylation on peptide K-17,
Asn-101 is glycosylated and Asn-124 is not.
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Monosaccharide analysis of adult bovine COMP was carried out as described under "Experimental Procedures." An identical amount of sample was subjected to amino acid analysis to gain an accurate measurement of the quantity of protein applied. The result (Table II) showed that adult COMP had approximately 14 mol glucosamine and 9 mol mannose/mol protein.
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The MALDI-TOF MS spectrum of the intact fetal COMP after reduction and
carboxymethylation showed a double peak indicating protonated mass
values of 84661 ± 126 and 86240 ± 46 Da as well as some
fragment ions at 59578 and 48171 Da and a doubly charged intact ion
(Fig. 4). This mass spectrum differed somewhat from that
obtained for adult bovine COMP (Fig. 1) and suggested that the fetal
protein may carry a different substitution pattern. Both adult and
fetal bovine COMP were digested with lysine C-proteinase and separated
by reversed phase HPLC. The chromatograms, shown in Fig.
5, indicate two areas where a difference can be seen
between the adult and fetal samples. These are marked with bold arrows. The adult COMP digest showed an additional peak, at 32.65 min, not
present in the fetal sample; furthermore, there was a side-peak at 46 min, unique to the fetal digest. Fraction 1 was separated using the
same HPLC column and shown by peptide sequencing to contain two
peptides, 270-305 and 426-492 (Table III, numbering refers to the human COMP sequence). The C-terminal portions of the
peptides was extrapolated using the measured protonated mass for each
and the sequence of bovine COMP, which is known in these regions (4).
These derive from repeats 1 and 6, respectively, of the calcium binding
domain of COMP. The MALDI-TOF MS spectrum of the adult COMP fraction
(Fig. 6A) showed an ion of protonated mass
3821.8 ± 0.8 Da, which was absent in the fetal COMP fraction. It
is likely that this peptide is a variant of peptide 270-305.
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Fraction 2 from the adult bovine COMP digest was identified as peptide K-17 (Table I) and has already been described (Fig. 2B). The corresponding peptide isolated from fetal COMP showed a cluster of peaks ranging in protonated mass from 11134 to 11714 Da (Fig. 6B).
To confirm that COMP contained sialic acid, its reactivity with lectins
SNA and MAA was determined. These lectins show specificity for
oligosaccharides that carry terminal 2-6-linked and
2-3-linked sialic acids, respectively (15, 16). Reduced and carboxymethylated adult bovine COMP was separated by SDS-polyacrylamide gel
electrophoresis and electroblotted onto PVDF (Fig. 7).
Reaction was detected with SNA and not with MAA, indicating the
presence of
2-6-linked sialic acid.
In these mapping experiments, the peptide carrying the third potential
N-glycosylation site at Asn-722 was not isolated. One possible explanation for this would be that the protein core in the
COMP preparations used was truncated and did not posses an intact
C-terminal end. This could lead to the loss of Asn-722, which is
located within 16 residues of the mature C terminus. An experiment was
therefore conducted to establish that the COMP preparations used in
this study had the predicted C terminus. Reduced and carboxymethylated
human COMP was digested with aspartate-N proteinase and the unseparated
digest was analyzed by MALDI-TOF MS. One of the peptides identified had
a protonated mass of 1814.6 ± 0.5 Da (Fig. 8),
consistent with the C-terminal peptide (DTIPEDYETHQLRQA), with
predicted mass of 1816.92 Da. This indicated that the C terminus was not in fact truncated.
Analysis of human COMP by MALDI-TOF MS following treatment with a
variety of glycosidases (Fig. 9) was carried out.
Treatment of the reduced and carboxymethylated monomer with
N-glycanase led to a reduction in mass of 3571 ± 219 Da. Further treatment with neuraminidase and O-glycanase did
not lead to any significant reduction in mass, indicating the absence
of O-linked oligosaccharides. The mass difference resulting
from N-glycanase treatment, however, was consistent with the
presence of 2 N-linked oligosaccharides. Since Asn-101
carries a substituent of average mass of 1847.2 ± 6.6 Da, and
Asn-124 is unsubstituted, the additional N-linked oligosaccharide resides on Asn-722.
We have used MALDI-TOF and chemical analysis to obtain detailed information on the structure and distribution of carbohydrate moieties on COMP. On the basis of the data presented, it is concluded that in adult COMP there are two sites of N-glycosylation at Asn-101 and Asn-721. The third N-linkage site (Asn-124) is unoccupied. The difference between the measured mass and that predicted from the sequence of human COMP indicated that the mass of all post-translationally added groups was 3402 ± 149 Da. Analysis of the total neutral monosaccharide and hexosamine content (Table II) indicated that carbohydrate would account for all of these substituents since they contribute an additional mass of 3700-4300 Da, depending on the degree of acetylation of hexosamine residues, to the mass of the COMP subunit. The mass of the N-oligosaccharide at Asn-101 was estimated to be 1847.2 ± 6.6, and this is consistent with the expected mass of a high mannose oligosaccharide such as that indicated in Table IV, with the structure (HexNAc)2-(Man)8-(Fuc)1. The oligosaccharide on Asn-721 had a mass of 1723.8 ± 225 Da. The error associated with this measurement means that a number of interpretations can be made, but the structure must contain sufficient glucosamine and sialic acid to account for the presence of these sugars, as measured by monosaccharide analysis and lectin blotting.
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In fetal COMP, the oligosaccharide substituent at Asn-101 exists in several structural forms (Fig. 6B). The most likely structural arrangements of oligosaccharide substituents that would give rise to this cluster are shown in Table IV. As in the case of the adult structure, the masses are consistent with a core structure of (GlcNAc)2-(Man)3 and additional substitutions that contain fucose, sialic acid, glucosamine, and mannose residues. Addition of these outer chain structures occurs in the trans region of the Golgi prior to secretion (17), and the final oligosaccharide structure depends on the rate at which the protein core moves through the Golgi apparatus. It may be that in fetal tissue there is an additional level of tight regulation relating to the rate of movement of the protein through the Golgi. This may in turn regulate exactly where the molecule is located in the extracellular matrix since changes in glycosylation may influence binding to other matrix components. COMP has been shown by immunohistochemical analysis to be distributed throughout the pericellular and territorial environments in developing cartilage and the interritorial compartment in mature cartilage (7). These age-related changes could also reflect differences in the content or binding affinity of COMP binding molecules in these compartments. It is very likely that the changes in glycosylation of COMP described here allow for different intermolecular interactions.
We thank Dr. Mary Murphy for helpful discussions and for reviewing the manuscript.