(Received for publication, January 31, 1995; and in revised form, May 24, 1995)
From the
In the hyaluronan binding region (HABR) peptide of aggrecan,
there is a marked increase in the level of keratan sulfate (KS) during
aging. To determine the sites of KS attachment, KS-containing peptides
were prepared from HABRs from immature and mature bovine articular
cartilage by digestion with trypsin or papain followed by carbohydrate
analysis and peptide sequencing. KS is attached to Thr within loop A in mature, but not in immature, HABR. Within loop B
KS is N-linked to Asn
in both HABRs, but in the
immature HABR the chains are shorter. Asn
in loop B` of
mature HABR is substituted either with a KS chain or with an
oligosaccharide of the complex type. In immature HABR this site does
not carry KS. In the interglobular domain, 2 threonine residues within
the sequence TIQTVT are substituted in both calf and steer, and in
steer further substitution occurs within the sequence NITEGEA, which
contains a major catabolic cleavage site (Sandy, J., Neame, P. J.,
Boynton, R., and Flannery, C. R.(1991) J. Biol. Chem. 266,
8683-8685). The extreme polydispersity of mature HABR was
investigated by preparing four subfractions of increasing molecular
size which had essentially the same protein core, i.e. Val
-Arg
or
Val
-Arg
. The smaller species lacked the
KS chains attached to loop A. These results show that KS substitution
occurs within each of the disulfide-bonded loops of the HABR, that the
KS may be either N- or O-linked, and that variations
in the addition of KS are responsible for the polydispersity of mature
HABR.
Aggrecan is the main proteoglycan component in the extracellular
matrix of cartilage. It contains at its N terminus two globular domains
referred to as G1 and G2 which are separated by a 90-residue
interglobular domain (IGD). ()The G1 domain mediates the
binding of aggrecan to hyaluronan (HA), thus allowing the formation of
multimolecular complexes which absorb water and contribute to the
resilience of the tissue. The G1 domain has a structure consisting of
three disulfide-bonded loops. Loop A has structural characteristics
common to members of the IgG family (Perkins et al., 1989;
Bonnet et al., 1986) and is, by analogy with link protein,
involved in the interaction between aggrecan and link protein
(Périn et al., 1987). Loops B and B` are
involved in binding to HA (Yang et al., 1994). They form a
structural motif referred to as the proteoglycan tandem repeat which
appears to be unique to HA-binding proteins. Aggrecan is heavily
substituted with both chondroitin sulfate (CS) and keratan sulfate (KS)
and the size, distribution, and degree of sulfation of the KS chains
increase with age. KS is a glycosaminoglycan occurring widely in
connective tissues. It consists of a Gal(
1-4)GlcNAc
repeating disaccharide structure and is sulfated on the 6-position of
either the galactose or the glucosamine or both. It occurs in O-linkage to serine and threonine residues in aggrecan (Meyer et al., 1953).
The distribution of KS along the protein core of aggrecan from bovine articular cartilage has been studied by Heinegand Axelsson(1977). They showed that a large proportion of the KS chains are located within the KS domain and in proximity to the N terminus, with the remainder being found in the CS attachment region. The KS domain consists of a series of hexapeptide repeats, each of which contains a KS-substituted serine (Antonsson et al., 1989). Apart from this domain, however, little is known about the specific protein sequences that are post-translationally modified by the addition of KS. Furthermore, the extent to which specific amino acid residues are variably glycosylated in immature and mature cartilage is poorly understood.
Tryptic digestion of aggregate
yields a mixture of heavily glycosylated peptides, including the
hyaluronan binding region (HABR) peptide. This peptide, consisting of
Val-Arg
or
Val
-Arg
(Rosenberg et al.,
1993), contains the complete G1 domain and a portion (one-third) of the
IGD. HABR isolated from aggregate from mature bovine articular
cartilage represents a polydisperse population of molecules which are
heavily substituted with KS. HABR from immature cartilage, on the other
hand, is a more monodisperse population of lower molecular size. The
differences in structure between the two which account for these
observations have not been defined.
In this study we examine the distribution of KS chains in the HABR peptides from immature and mature bovine articular cartilage. We show that the mature HABR has KS attachment sites within each of the disulfide-bonded loops of the G1 domain. In two of these the KS chains are in N-linkage to asparagine residues, whereas the other is O-linked. In immature HABR these sites are substituted at a much lower level. Within the IGD there are several KS chains in both HABRs at sites previously shown to be occupied by KS in HABR from adult pig laryngeal aggregate (Barry et al., 1992). However, the mature HABR has additional KS attached to residues in close proximity to the aggrecanase cleavage site. We also show that the extreme polydispersity of populations of HABR peptide from mature cartilage can be accounted for by differences in the levels of KS substitution.
The purpose of this study was to examine the distribution of KS chains within the HABR of aggrecan from immature and mature bovine articular cartilage. In order to assess the purity of the HABR preparations used, N-terminal sequence analysis was carried out. A single sequence (VEVSEPDNSL) was detected in each case which is identical to the N terminus of pig HABR (Barry et al., 1992) and very similar to the N-terminal sequences of rat (Doege et al., 1987) and human (Doege et al., 1991) aggrecans. The glycosaminoglycan content was estimated to be 171 and 444 µg/mg protein, respectively, for the calf and steer HABRs. Both HABRs were eluted from Superose 6 under dissociative conditions, and the profiles (Fig. 1A) showed the steer and calf HABRs to be approximately 160 and 100 kDa, respectively. On SDS-PAGE (Fig. 1B) the steer HABR peptide appeared as a diffuse band ranging from about 73 to 198 kDa. The calf HABR ranged from 78 to 158 kDa. Digestion of both HABRs with a mixture of keratanase and keratanase II gave rise to a product which appeared on SDS-PAGE as two bands of molecular size 68 and 73 kDa (Fig. 1B). This indicated that both HABRs had the same or similar protein cores. One of the characteristics of the steer HABR, therefore, is the addition of KS chains which are longer or which occupy new sites. The monosaccharide content of each was analyzed (Table 1) showing that the steer HABR had 2.7-fold more glucosamine (78.4 mol/mol of protein) compared with the calf HABR (29.5 mol/mol of protein). Estimation of the molecular size of the KS chains on calf and steer HABR was made by chromatography on a Bio-Sil SEC 30XL column (Fig. 2) and showed that they were 2,600 and 7,700 respectively. These correspond to KS chains of about 3 and 15 disaccharides, respectively. The value obtained for the steer KS chains is in full agreement with estimates made by Heinegand Axelsson(1977) and Swann et al. (1985) for bovine articular cartilage. These chains are twice as long as the KS chains on aggrecan from bovine nasal cartilage (Heineget al., 1979; Stuhlsatz et al., 1989).
Figure 1:
Characterization of the HABR peptide
from calf and steer articular cartilage aggrecan. A,
chromatography of the HABR peptide from calf (closed squares)
and steer (open squares) articular cartilage aggrecan on a
Superose 6 (1 30 cm) column, eluted with 4 M guanidinium hydrochloride, 20 mM Tris-Cl, pH 6.8. The arrows show the elution position of globular protein
standards. B, electrophoresis on 5-20% gradient
polyacrylamide gels of the HABR peptide from steer and calf aggrecan
before (lanes 1 and 3, respectively) and after (lanes 2 and 4, respectively) digestion with
keratanase. The numbers refer to the size, in kDa, of molecular mass
standards (left lane).
Figure 2:
Chromatography of KS chains released by
alkaline -elimination. The KS chains released from steer (top) and calf (bottom) HABR by alkaline
-elimination were chromatographed on Bio-Sil SEC 30XL, using 0.2 M NaCl as eluting solvent. The V
and V
were estimated by applying samples of
bovine serum albumin and NaCl, respectively. The flow rate was 0.5
ml/min, and fractions of volume 0.25 ml were collected. Each fraction
was measured for reaction with antibody 5D4 by ELISA (absorbance at 450
nm).
In order to gain some information about the distribution of
KS chains between the IGD and G1 portions of steer HABR, it was
digested with stromelysin. This enzyme specifically hydrolyses the
Asn-Phe
in aggrecan (Fosang et
al., 1991) and therefore removes most of the IGD portion. The
digested material was applied to a column of HA-Sepharose. Collected
fractions were monitored for absorbance at 280 nm and reaction with 5D4
by ELISA. Two fractions eluted from the column (Fig. 3): the
first with 1 M NaCl and the second with 4 M guanidinium hydrochloride. The former had the N-terminal sequence
FFGVGEEDI, indicating that it represented the IGD portion of the HABR.
The fragment eluting with 4 M guanidine was the G1 portion of
the HABR peptide, and on SDS-PAGE (not shown) it was reduced in size
compared with the intact HABR. Both fragments showed about equal
reaction with antibody 5D4 (Fig. 3), indicating that they were
both substituted with KS. This result therefore confirms that KS chains
are attached at sites within the G1 portion of steer aggrecan, as well
as within the IGD.
Figure 3: Affinity chromatography of stromelysin-generated fragments on HA-Sepharose. Separation of the fragments generated by digestion of steer HABR with stromelysin on a column of HA-Sepharose. The column was eluted with 20 ml of 0.05 M sodium acetate and then with 20 ml of 1 M NaCl in the same buffer, followed by 20 ml of 4 M guanidinium HCl, 20 mM Tris-Cl, pH 6.8. The arrow indicates the starts of the sodium chloride elution and of the subsequent guanidine HCl elution. The absorbance at 280 nm (A) and reaction with antibody 5D4 (by peroxidase-based ELISA) were measured (absorbance at 450 nm, B).
To gain further information about the location of the KS chains, the HABR peptides from calf and steer were reduced, carboxymethylated, digested with trypsin, and applied to a column of immobilized antibody 5D4. This antibody specifically recognizes a highly sulfated octasaccharide sequence on KS (Mehmet et al., 1986). Most (>90%) of the detectable reaction with 5D4 was in the fraction that bound to the column and was recovered after elution with 1 M NaCl (pool II, Fig. 4, A-D). The material which did not bind to the column was referred to as pool I. The peptides in pools I and II were separated by reverse-phase chromatography (Fig. 4, E-J). In the case of steer HABR, five peaks were recovered in pool II. All of these eluted from the reverse-phase column as broad, poorly resolved peaks, behavior which is typical of heavily glycosylated peptides (Fig. 4G). Sequence data were obtained from all of the material recovered (Table 2), with the exception of the peak eluting at 18.2 min, for which no sequence was obtained. Those peptides for which sequences were obtained were referred to as T-1 to T-5 (Fig. 4G). Peptides T-2 and T-5 are derived from loops B and B`, respectively, and peptide T-3 encompasses part of the N-terminal peptide and part of loop A. Peptide T-1 is derived from the IGD portion and peptide T-4 spans part of loop B` and part of the IGD. Fig. 8A shows the distribution of these peptides.
Figure 4: Affinity chromatography of tryptic fragments on 5D4-agarose. HABR was reduced, carboxymethylated, and digested with trypsin and the peptides applied to the column, equilibrated in 0.01 M Tris-Cl, pH 7.2. The sample was eluted with 10 ml of the same buffer, followed by 10 ml of 1 M NaCl, 0.01 M Tris-Cl, pH 7.2 (arrow). Collected fractions (1 ml) were assayed for protein (absorbance at 280 nm, A and C) and for reaction with antibody 5D4 in an ELISA assay (absorbance at 450 nm, B and D). The profiles of steer (A and B) and calf (C and D) HABR tryptic peptides at 280 and 450 nm, respectively, are shown. Pools I and II from the 5D4-agarose chromatography were applied to a Vydac C18 microbore reverse-phase column. E shows the whole tryptic digest of steer HABR, and F and G show the steer pools I and II, respectively. The whole digest of calf HABR (H), calf pool I (I), and pool II (J) are also shown. Tryptic peptides from steer HABR which bound to the 5D4-agarose (T-1 to T-5) were collected and sequenced. In E-J absorbance at 220 nm was monitored.
Figure 8: Distribution of KS chains in the HABR peptide from immature and mature bovine aggrecan. A, location of the tryptic and papain peptides which contain KS and which are described in Table 2. The diagram shows the disulfide-bonded loop arrangement of the G1 domain. Loops A, B, and B` are indicated. The locations of the KS chains (wavy lines) and of the N-linked oligosaccharides in calf (B) and steer (C) HABR are shown.
In the case of calf HABR some material bound to the 5D4-agarose column which was positive in an ELISA, but no peptides could be detected subsequently by HPLC (Fig. 4J). Presumably the level of KS substitution was too small to allow the peptides to be detected in this way. To gain some information about the location of these KS chains in calf HABR the tryptic peptides were separated on Superose 12 (Fig. 5), and the 5D4-reactive peptides was rechromatographed on a reverse-phase column. Two peptides were identified in this way (Table 2) which were identical to peptides T-4 and T-5 from steer HABR (see above).
Figure 5: Tryptic digestion of steer HABR after reduction and carboxymethylation. The peptides were separated on Superose 12, eluting with 4 M guanidine, 20 mM Tris-Cl, pH 6.8. Absorbance at 280 nm (closed squares) and reaction with antibody 5D4 (open squares) were measured.
The tryptic peptides shown above to be substituted with KS in steer HABR have several potential KS attachment sites. In order to determine the sites with greater precision shorter peptides were generated by digestion with papain. Two 5D4-reactive pools, A and B, were eluted from Superose 12 and were sequenced (Fig. 6). Three peptides were detected in the papain pool A (Table 2) and are referred to as P-1, P-2, and P-3. These are subfragments of tryptic peptides T-3, T-5, and T-4 respectively. Sequencing was continued for 15 sequencer cycles, and in all cases no amino acid derivatives were detected after 8 cycles. Each of the peptides P-1 to P-3 contains an unidentified residue, which corresponds to a glycosylated amino acid. Two peptides were obtained in pool B (Fig. 6) which were also identified by sequencing (P-4 and P-5, Table 2). The elution position of P-4 and P-5 from Superose 12 is similar to the N-glycopeptides isolated from pig laryngeal HABR (Barry et al., 1992). Peptide P-5 has the same sequence as P-2, so its appearance in both pools indicates that in a proportion of the HABRs the loop B site is substituted with either an N-linked oligosaccharide or a short KS chain. However this represents a minor portion of the total amount of this peptide recovered. Quantitatively this site is mostly occupied by a large KS chain.
Figure 6: Papain digestion of steer HABR. Chromatography of steer HABR, reduced and carboxymethylated and digested with papain, on Superose 12 with 4 M guanidinium hydrochloride, 20 mM Tris-Cl, pH 6.8, as eluting buffer. Fractions (0.5 ml) were monitored for absorbance at 280 nm (open squares) and for reaction with antibody 5D4 in an ELISA assay (closed squares). Pools A and B were desalted and applied directly to an ABI 477 sequencer. The peptide sequences that were obtained are shown in Table 1.
Within loop A there is at least one KS chain within the sequence TTAPSTAP, and it is attached to either the first threonine of the serine or both. This conclusion is based on the recovery from 5D4-agarose of peptide T-3 (Table 2) and also peptide P-1 from the 5D4-reactive fraction of the papain digest (Fig. 6). Interestingly, the motif TTAP or TAAP occurs frequently within the CS-1 domain of aggrecan: it is repeated once in each of the 19-amino acid repeats that comprise this domain (Doege et al., 1991). It is possible that this sequence represents a signal for the attachment of KS chains within the CS-1 domain. Since the motif occurs once in each 19-residue repeat where the sequence SG occurs twice, it would allow for one KS chain per two CS chains within this part of the molecule. The CS-2 domain lacks a repeating sequence and does not contain the TTAP motif (Doege et al., 1991). There are 146 SG pairs within the CS-1 and CS-2 domains of human aggrecan and 19 TTAP sequences. Assuming that all these sites were occupied with their respective GAGs, the total amount of KS would be similar to that estimated by Heinegand Axelsson(1977), who showed that an average of five CS chains and one KS chain could be linked to the same peptide derived from the CS domain.
Peptide T-5 (Table 2) was isolated as a tryptic peptide on
5D4-agarose. The papain-generated peptide P-2, which overlaps with T-5,
was recovered from the 5D4-positive fraction after size exclusion
chromatography. This peptide contains KS in N-linkage to the
asparagine, which is Asn in the human aggrecan sequence
(Doege et al., 1991). The conclusion that the KS is N-linked rather than O-linked to either Thr
or Thr
comes from analysis of the yield, in
picomoles, at each cycle during sequencing of the tryptic peptide
DTNET: D (14.6); T (20.2); N (not detected); E (15.9); T (20.2). The
high recovery of threonines indicates that they are unsubstituted.
Peptide T-2, which is derived from loop B`, was identified as containing KS by virtue of its binding to 5D4-agarose. This peptide is also recovered from steer HABR in high yield as an N-linked oligosaccharide-substituted peptide using standard peptide purification protocols (Neame et al., 1987; Barry et al., 1992). Therefore KS must represent a minor substituent at this site. This KS is also N-linked, because analysis of the sequencer yields indicates that the threonines are unsubstituted.
Other KS chains are
attached to the interglobular domain portion of the HABR, based on the
recovery of peptides T-1 and T-4 (Table 2). The latter has also
been specifically identified in pig HABR as the major site of KS
attachment (Barry et al., 1992), where the chains are O-linked to the first and third threonines within the sequence
GVGGEEDITIQTVT (this sequence begins at residue 352 of human aggrecan;
Doege et al., 1991). The recovery of T-1 as a KS-substituted
peptide in steer aggrecan is, however, a novel finding which has
significance in relation to the degradation of aggrecan catalyzed by
aggrecanases. It has been shown (Sandy et al., 1991; Ilic et al., 1992) that catabolic processing of aggrecan in
articular cartilage involves proteolytic cleavage at, among other
sites, the Glu-Ala
bond, which is within
the sequence XIXEGEA. This site is not substituted
with KS in immature aggrecan. The presence of KS adjacent to this site
could influence the binding and catalytic activity of degradative
enzymes and therefore influence the catabolism of aggrecan which
increases with age.
To investigate the polydispersity of the HABR
peptide from steer aggrecan, it was divided into four subfractions of
uniform molecular size by chromatography on Sephacryl S-300. The modal
value calculated for the molecular mass of HABRs 1-4 ranged from
70.2 to 108.8 kDa (Fig. 7A). Analysis of each of these
by peptide mapping indicated that they had similar protein cores,
Val-Arg
(Rosenberg et al.,
1993) or Val
-Arg
(this study). Each of
the HABR subfractions was digested with stromelysin (Fig. 7B). Digestion of HABR 1 (70.2 kDa) and HABR 2
(76.5 kDa) gave two products of 54 and 43 kDa (Fig. 7B). HABR 3 (87 kDa) and HABR 4 (108.8 kDa), on
the other hand, appeared after digestion as diffuse bands which stained
lightly with Coomassie. When HABRs 1 and 2 were digested with
stromelysin and with keratanases, little change was observed in the
mobility of the 54- and 43-kDa bands, although they were sharpened (Fig. 7C). Both bands were transferred to Immobilon for
sequence analysis. The 43-kDa band had the sequence VEVSEP, which is
the N terminus of mature bovine HABR. No sequence was obtained for the
54-kDa band, but on the basis of observations described below, it is
probably a more heavily N-glycosylated variant of the 43-kDa
fragment. Digestion of HABRs 3 and 4 with stromelysin and keratanases
also caused the appearance of two bands at 43 and 54 kDa (Fig. 7, B and C). Additional bands were seen
at 30 and 20 kDa after digestion of HABRs 1-4 with stromelysin (Fig. 7B). These became more prominent after treatment
with keratanases (Fig. 7C). Both bands were transferred
to Immobilon for sequence analysis: no data were obtained for the
30-kDa band, but the 20-kDa band had the N-terminal sequence TYGIR,
arising from cleavage of the Arg
-Thr
bond
within loop B. The 30-kDa band presumably represents the C-terminal
fragment released by cleavage of the Asn
-Phe
bond by stromelysin. Its abnormal mobility on electrophoresis may
be due to the presence of numerous KS stubs. The 20-kDa band may arise
from cleavage at the additional site by stromelysin, but this has not
been documented in other reports (Fosang et al., 1991). This
fragment may also have arisen as a result of cleavage by trypsin used
during the preparation of the HABRs. However it is not seen on SDS-PAGE
of the intact HABRs under reducing conditions (Fig. 7A). It may be that the presence of large amounts
of KS on the peptide prevent its detection on the Coomassie-stained
gel.
Figure 7: Analysis of HABR subfractions 1-4 from steer aggrecan. HABRs 1-4 were prepared as relatively monodisperse subfractions by size exclusion chromatography on Sephacryl S-300. Each subfraction was analyzed by SDS-PAGE on 5-20% gradient gels. HABRs 1-4 are shown before (A, lanes 1-4, respectively) and after (B, lanes 1-4) digestion with stromelysin. C (lanes 1-4) shows the HABR subfractions after digestion with stromelysin followed by a mixture of keratanase and keratanase II. D shows the HABR from adult bovine nasal cartilage (lane 1) digested with stromelysin (lane 2) and with N-glycanase (lane 3). The numbers on the left refer to the size, in kDa, of the molecular mass markers.
To examine the structural differences between the 43- and 54-kDa products generated by digestion with stromelysin, further treatment with N-glycanase was carried out. For this experiment HABR from adult bovine nasal cartilage was used. Digestion with stromelysin converted the intact HABR (69 kDa) into a product showing two bands on SDS-PAGE of similar size to those described above. When treated with N-glycanase the band of lower mobility disappeared (Fig. 7D), indicating that the appearance of two products was due to the variable addition of keratanase-resistant, but N-glycanase-sensitive oligosaccharide structures. This indicated that the two products that were generated by cleavage with stromelysin were differently N-glycosylated and probably had the same protein core.
HABRs 3 and 4, unlike HABRs 1 and 2, when treated with stromelysin, could not be resolved into discrete bands on SDS-PAGE (Fig. 7B). While stromelysin caused a significant reduction in molecular size, the products appeared as diffuse bands which stained lightly with Coomassie. This is typical behavior for heavily glycosylated molecules. When treated with keratanase, these diffuse bands were converted into a pair of products of 43 and 54 kDa (Fig. 7C), similar to those for HABRs 1 and 2. It appears, therefore, that HABRs 1 and 2 are less heavily substituted with KS in the globular portion and most of the KS chains are in the interglobular domain. HABRs 3 and 4 also carry KS chains in the interglobular domain, but they also have significant amounts of KS in the globular portion.
Papain digestion of the steer HABR subfractions 1 through 4 was carried out as described above. The peptides were separated by gel filtration on Superose 12, and the largest KS-substituted peptides were further chromatographed by reverse-phase HPLC (results not shown) and identified by sequence analysis. Peptides P-2 (GIRDTXE, from loop B) and P-3 (GEEDIT, from the IGD), but not P-1 (DPMHPV, from loop A, see Table 2), were detected in the KS-positive fractions prepared from HABR-2. In HABR-3 and 4, however, all three peptides were identified in the KS-positive fractions. Therefore all of the HABR subfractions were substituted with KS within the IGD sequence GEEDIT and the loop B` sequence GIRDTXE, but loop A is substituted only in HABRs 3 and 4.
Tryptic peptides and peptides derived from digestion with S. aureus V8 proteinase were purified from the calf and steer HABRs and identified by N-terminal sequence analysis, and those peptides carrying N-oligosaccharide substituents were subjected to monosaccharide analysis using a Dionex BioLC system (Table 3). Peptides were recovered from loops A and B`, but not from loop B. Peptide IQNLRSXDS (from loop A) showed a ratio of mannose/glucosamine of 3.1 for the calf peptide and 4.2 for the steer peptide. These data are consistent with the N-linked substituent on this peptide being of the high mannose type. Peptide TVYLHAXQT (from loop B`) had a ratio of mannose:glucosamine of 0.44 for calf and 0.45 for steer (Table 3). In addition both of these contained approximately 1 mol of fucose/mol of peptide. These results suggest that this oligosaccharide is of the complex type.
Nilsson et al.(1982) have shown that, in swarm rat chondrosarcoma proteoglycan, more than 70% of the N-linked oligosaccharides have a complex-type structure, with 60% of them carrying fucose. The ease with which the loop B` peptide is recovered in high yield from proteolytic digests of HABR suggests that this is the major glycosylated variant. However the fact that a peptide with the same sequence was bound to 5D4-agarose and eluted with 1 M NaCl ( Fig. 4and Table 2) indicates that this peptide is also KS-substituted, but addition of KS at this site most likely represents a minor post-translational modification.
Analysis of the monosaccharide composition of the intact HABRs (Table 1) indicates a 2.7-fold higher level of glucosamine in steer HABR compared with calf. Furthermore the ratio of glucosamine:galactose in steer HABR was 1.5, suggesting that a large proportion of the glucosamine in steer was in the form of KS chains. The level of glucosamine substitution in steer HABR (78.4 mol/mol of protein) is consistent with the estimate arrived at by the peptide mapping experiments described above that there are five major sites to which KS chains are attached and that the chains are on average 15 disaccharides in length (Fig. 2A). The glucosamine content of the calf HABR was 29.5 mol/mol of protein, and the ratio of glucosamine:galactose was 2.95, indicative of a mixture of short KS chains and N-linked oligosaccharides. This level of substitution would therefore be accounted for by the presence of three or four short KS chains of about three disaccharides each in addition to the N-linked oligosaccharides. The N-linked oligosaccharides on loops A and B` (Table 3) account for 14.8 and 46.1% of the total glucosamine on the steer and calf HABRs, respectively.
Steer HABR had 7.9 nmol of fucose/nmol of protein, whereas in the intact calf HABR fucose was not detected (Table 1). Further analysis of the N-substituted peptide from the calf loop B` (Table 3) indicated the presence of some fucose (1.5 mol/mol of peptide). A number of studies have shown that fucose residues are found in association with KS chains (Seno et al., 1965; Thornton et al., 1989). The proportion of fucose that was removed from steer HABR by digestion with keratanase II was estimated (Table 4) after filtration through a Microcon membrane. 35% of the fucose, 48% of the glucosamine, 45% of the galactose, and none of the mannose were recovered in the filtrate. These estimated were based on an overall recovery of monosaccharides between the filtrate and retentate of 60-70% (Table 4). Presumably the remainder was bound to the membrane. The amount of fucose that was released by keratanase II into the filtrate was about 10% of the glucosamine released. Assuming KS chains of average length 15 disaccharides this would suggest 1-2 fucose residues per chain, in good agreement with the estimate of 1 fucose per 12 disaccharides made by Tai et al.(1991).
One important question relates to the amount of non-5D4-reactive KS in the HABR peptides studied. The results described above for steer HABR indicate that there are five sites to which KS chains with an average length of 15 disaccharides are attached (Fig. 8B). These would account fully for the total glucosamine on steer HABR (78.4 mol/mol of HABR). In the case of calf HABR the N-linked oligosaccharides on loops A and B` (Table 3) account for about half of the total glucosamine content of 29.5 mol/mol of HABR. The three KS chains each of average length three disaccharides account for about 80% of the remaining glucosamine (Fig. 8C). The remaining glucosamine residues may be distributed among other structures that do not react with 5D4. However, these structures represent a minor portion of the total carbohydrate groups present on these HABRs.
These results provide much of the structural information needed to interpret the differences in molecular size between the HABR peptides from newborn and adult bovine articular cartilage and the variations in addition of KS that give rise to the polydispersity of adult HABR. Fig. 8summarizes the differences between the two HABR peptides in terms of addition of KS. During the post-translational processing of mature aggrecan KS chains are added to many sites, including several within the G1 domain. In at least two of these sites the KS chains are N-linked, whereas in others they are O-linked.
The three N-glycosylation sites within the HABR of mature aggrecan are processed differently. Loop A, which binds to link protein, has an N-glycosylation site that is never substituted with KS. Loops B and B`, which mediate binding to HA, both carry N-linked KS. The loop B site is always substituted with KS, and the loop B` site may carry either KS or an N-linked oligosaccharide. These regional differences in addition of N-linked KS may relate to the different functional roles of the subdomains. The finding that there is O-linked within loop A of mature HABR also provides information about the specific amino acid sequences around the KS attachment sites. The loop A site has the sequence T/STAP, which occurs at regular intervals within the CS-1 domain and may represent a consensus sequence for addition of KS in this region and elsewhere, although it differs markedly from the consensus sequence in the KS region. Furthermore, loop A in mature HABR is variably substituted and HABR subfractions of lower molecular size do not carry KS at this site. This accounts for the polydispersity of mature HABR. Finally, in mature HABR, KS is located in close proximity to the major catabolic site within the IGD and may exert an effect on the rate or specificity of cleavage.