©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
N- and O-Linked Keratan Sulfate on the Hyaluronan Binding Region of Aggrecan from Mature and Immature Bovine Cartilage (*)

(Received for publication, January 31, 1995; and in revised form, May 24, 1995)

Frank P. Barry (§) Lawrence C. Rosenberg (1) Joanne U. Gaw Janette U. Gaw Thomas J. Koob Peter J. Neame

From the Shriners Hospital for Crippled Children and Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, Florida 33612 and the Orthopedic Research Laboratory, Montefiore Medical Center, Bronx, New York, New York 10467

ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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^1-Arg or Val^1-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.


INTRODUCTION

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). (^1)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(beta1-4)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^1-Arg or Val^1-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.


MATERIAL AND METHODS

Reagents

Monoclonal antibody 5D4 was a kind gift from Dr. Bruce Caterson, University of North Carolina at Chapel Hill. Activated stromelysin was a kind gift from Dr. Michael Lark, Merck. Streptomyces hyaluronidase, chondroitinase ABC, keratanase (from Pseudomonas), and keratanase II (Bacillus sp.) were from Seikagaku. Sequencer grade trypsin and Staphylococcus aureus V8 proteinase were from Boehringer Mannheim, and papain (papaya latex) was from Sigma. All other reagents were as described previously (Barry et al., 1992).

Preparation of HABR

Fresh, wet articular cartilage was shaved from the metacarpalphalangeal joint from calf (1 week) or steer (1.5-2 years) and added immediately to ice-cold 4 M guanidinium hydrochloride, 0.05 M sodium acetate, pH 5.8, containing 0.1 M 6-aminohexanoic acid, 10 mM EDTA, 5 mM benzamidine HCl, 1 mM phenylmethylsulfonyl fluoride, and 10 mMN-ethylmaleimide as proteinase inhibitors. Extraction was continued with stirring for 24 h at 4 °C. The extract was filtered and dialyzed against 7 volumes of 0.05 M sodium acetate, pH 5.8, containing proteinase inhibitors. Equilibrium density gradient centrifugation under associative conditions was then carried out in 3.5 M CsCl at 100,000 g for 48 h. The A1 fraction was dialyzed for 48 h against a large excess of water followed by 0.5 M NaCl and then with water again. It was digested with chondroitinase ABC (3.5 units/g dry weight) for 1 h and then with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (2 mg/g) for 5 h at 37 °C in 0.1 M Tris acetate, pH 7.2. The aggregate, consisting of the HABR peptide, link protein, and hyaluronan, was isolated by gel filtration on Sepharose CL-6B (2.5 100 cm) in 0.5 M sodium acetate, pH 6.8. The void fraction containing the complex was dissolved in 4 M guanidinium hydrochloride in 20 mM Tris-Cl, pH 6.8, and eluted from Superose 12 with the same buffer.

Preparation of HABR Subfractions

A series of relatively monodisperse HABR fragments was prepared from bovine aggrecan as follows: fractions containing the complex of HABR, link protein and hyaluronan were dialyzed against 4 M guanidinium hydrochloride, 0.15 M sodium acetate, 5 mM EDTA, pH 6.3 and applied to a 3.7 228-cm column of Sephacryl S-300 equilibrated in the same solvent. Fractions from the column containing fragments of the same molecular weights were combined, recycled on the column under the same conditions, and the relatively monodisperse fragments were recovered. For this study, four subfractions were used. They were referred to as HABRs 1 through 4 and had average molecular mass values of 70.2, 76.5, 87, and 108.8 kDa respectively, estimated by SDS-PAGE.

Monosaccharide Composition Analysis

This was carried out using a Dionex BioLC system. For analysis of N-linked oligosaccharides, N-oligopeptides were prepared by digestion of the reduced and carboxymethylated HABR with either trypsin or V8 proteinase and purified as described previously (Neame et al., 1987; Barry et al., 1992). 50 pmol of each oligopeptide was hydrolyzed with either 2 M trifluoroacetic acid for 3 h at 100 °C or 4 M HCl for 5 h at 100 °C. After hydrolysis the samples were dried, dissolved in water, and chromatographed on a CarboPac PA1 column (4 250 mm), eluting with 16 mM NaOH. The eluted material was passed through a pulsed amperometric detector. Intact HABR peptides were analyzed in the same way. To estimate the proportion of fucose which was associated with the KS chains on steer HABR, samples were digested with a mixture of keratanase and keratanase II as described below and then spun through a Microcon column (Amicon Corp.). The proportions of released monosaccharides in the filtrate and retentate were measured.

Enzyme Digests

Reduction and carboxymethylation of HABR were carried out as described previously (Neame et al., 1987). Trypsin digestion was carried out in 0.1 M Tris-Cl, 10 mM EDTA, pH 8.0, using sequencer grade trypsin at an enzyme:substrate ratio of 1:100. For papain digestion, HABR was dissolved at a concentration of 2 mg/ml in 0.2 M sodium acetate, 5 mMDL-cysteine, 10 mM EDTA, pH 5.0. Papain was added to give a ratio of 1:50, and incubation was for 14 h at 37 °C. At this time an equal amount of enzyme was added and digestion continued for a further 10 h. Digestion with stromelysin was carried out in 0.02 M Tris-Cl, 10 mM calcium chloride, pH 7.5. 1 µg of activated stromelysin was added to 100 µg of HABR peptide and incubated at 37 °C for 12 h. A further 1 µg was then added and digestion continued for 6 h. Digestion with V8 proteinase was carried out for 12 h at 37 °C in 0.05 M ammonium bicarbonate with 1 µg of enzyme/100 µg of reduced and carboxymethylated HABR. Keratanase digests were carried out using 20 milliunits of keratanase (from Pseudomonas) and 1 milliunit of keratanase II (from Bacillus)/100 µg of glycosaminoglycan (as dimethylmethylene blue reactive material) in 0.02 M Tris-Cl, pH 7.2, for 16 h at 37 °C.

Affinity Chromatography on 5D4-agarose

Monoclonal antibody 5D4 (4 mg) was covalently immobilized on agarose using the Immunopure IgG Orientation Kit (Pierce) by following the manufacturer's instructions. The efficiency of coupling, determined by measuring the amount of unbound antibody, was 84%. The immobilized antibody concentration was therefore 1.7 mg/ml gel. Reduced and carboxymethylated HABR (600 µg) digested with trypsin was applied to a 2 ml of 5D4-agarose column in three separate aliquots of 1 ml each. After 1 h the column was eluted with 10 ml of 10 mM Tris-Cl, pH 7.4, and then with 10 ml of 1 M NaCl/10 mM Tris-Cl, pH 7.4. The flow rate was 10 ml/h, and the collected fractions (1 ml) were monitored for absorbance at 280 nm and for reaction with the same antibody by enzyme-linked immunosorbent assay (ELISA).

Affinity Chromatography on HA-Sepharose

Fragments generated by digestion of HABR with stromelysin were separated on a 10-ml HA-Sepharose column prepared by coupling 100 mg of HA (rooster comb, from Sigma) to 12 ml of EAH-Sepharose (Pharmacia Biotech Inc.) and 0.23 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Pierce) according to the method of Tengblad(1979). Prior to coupling the HA was digested with 33 units of testicular hyaluronidase (Sigma) for 3 h in 100 ml of 0.15 M NaCl, 0.1 M sodium acetate, pH 5.0, to give fragments of average molecular weight 52,000 (Tengblad, 1979). No attempt was made to measure the efficiency of coupling of HA to the Sepharose, but when 400 µg of intact steer HABR was applied to the column, it showed essentially complete binding under the conditions used. For chromatography the samples were dissolved in 1 ml of 0.05 M sodium acetate, pH 5.8, applied to the HA-Sepharose column (12 ml), and recirculated overnight at 10 ml/h at room temperature. The column was then eluted with 20 ml of 0.05 M sodium acetate, pH 5.8, followed by 20 ml of 1 M NaCl in the same buffer and finally with 20 ml of 4 M guanidinium hydrochloride, 20 mM Tris acetate, pH 6.5. 1-ml fractions were collected and the absorbance at 280 nm of each was measured.

Peptide Mapping

Peptides generated by digestion with proteinases were separated on a Superose 12 column in 4 M guanidinium hydrochloride, 20 mM Tris-Cl, pH 6.8. Collected fractions were monitored for reaction with antibody 5D4 by ELISA. The 5D4-reactive peptides were subsequently separated on a Vydac microbore C18 or C4 reverse phase column using an Applied Biosystems 120A system. Chromatography conditions were 0-70% acetonitrile in 45 min at a flow rate of 0.25 ml/min and a temperature of 30 °C. Eluate was monitored at 220 nm, and peaks were collected manually. Purified N-glycopeptides were prepared in this way from either tryptic or V8 digests.

Analytical Methods

Sequence analysis was carried out by Edman degradation using an Applied Biosystems 477A or 473A sequencer. Released phenylthiohydantoin-derivatives were identified by analysis on a microbore HPLC (Applied Biosystems 120A). SDS-polyacrylamide gel electrophoresis was carried out using 5-20% gradient gels according to the method of Laemmli(1970). Glycosaminoglycan assays were done using N,N`-dimethylmethylene blue according to the method of Farndale et al.(1982). ELISA was carried out with monoclonal antibody 5D4 and peroxidase-conjugated goat anti-mouse IgG (Pierce). Alkaline beta-elimination was carried out in 0.05 M NaOH, 1 M sodium borohydride for 48 h at 45 °C. The size of the released KS chains was determined by size exclusion chromatography on a Bio-Sil SEC 30XL column (Bio-Rad) with 0.2 M NaCl as eluting solvent. The flow rate was 0.5 ml/min, and fractions of volume 0.25 ml were collected. The V(o) and Vwere measured using bovine serum albumin and NaCl, respectively. The column was calibrated by the method of Dickenson et al. (1990).


RESULTS AND DISCUSSION

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 beta-elimination. The KS chains released from steer (top) and calf (bottom) HABR by alkaline beta-elimination were chromatographed on Bio-Sil SEC 30XL, using 0.2 M NaCl as eluting solvent. The V(o) 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^1-Arg (Rosenberg et al., 1993) or Val^1-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.


FOOTNOTES

*
This work was supported by the Shriners of North America and by National Institutes of Health Research Grant 1526 AR35322 (to P. J. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Osiris Therapeutics Inc., 2001 Aliceanna St., Baltimore, MD 21231.

(^1)
The abbreviations used are: IGD, interglobular domain; CS, chondroitin sulfate; ELISA, enzyme-linked immunosorbent assay; HA, hyaluronan; HABR, hyaluronan binding region; KS, keratan sulfate; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.


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