INTRODUCTION

Decorin, a small chondroitin sulfate proteoglycan is a ubiquitous component of the extracellular matrix of many tissues (1, 2). The decorin mRNA encodes a prepro-core protein of 360 amino acids (3, 4). This core protein can be divided into several distinct structural domains comprising: 1) a short signal sequence of 16 amino acids that targets the protein to the rough endoplasmic reticulum; 2) a propeptide of 14 amino acids of unknown function; 3) the glycosaminoglycan (GAG)¹ acceptor region with the chondroitin/dermatan sulfate chain substituted at the Ser-4 residue of the mature core protein (5); 4) a hypervariable cysteine globular domain; 5) a leucine-rich domain comprising a major portion of the core protein and containing three N-linked oligosaccharide attachment sites; and 6) a carboxyl-terminal globular domain. The mature decorin molecule that is recovered from tissues lack the propeptide domain. Decorin belongs to a growing family of proteoglycans with one or more GAG chains, that are characterized by core proteins of about 40 kDa containing 8-12 homologous leucine-rich repeats (LRR) of 20-29 residues with leucines in conserved positions (3, 6). Similar LRR motifs are found in other proteoglycans like biglycan (7), fibromodulin (8), and lumican (9), and numerous other apparently unrelated molecules such as the porcine ribonuclease inhibitor (10, 11). In the ribonuclease inhibitor, each LRR contains an alternating  sheet- helix structural unit, with the short -strand and the -helix approximately parallel to each other (12). The leucine-rich sequences of some of these proteins have been postulated to mediate protein-protein (13) and protein-membrane (14) interactions. The LRR repeats in the extracellular matrix proteoglycans are flanked by disulfide loops located at conserved positions near the NH₂ and COOH termini and thus may comprise a specialized subgroup within the LRR superfamily (3, 6). Although biglycan, fibromodulin, lumican, and decorin are similar in their general structural features they differ markedly in their gene regulation, pattern of expression, and functional interactions.

Most of the biological functions ascribed to decorin are believed to relate to complex formation with other molecules via the GAG or core protein domains. Thus decorin has been demonstrated in vitro to bind to several proteins including fibronectin (15), transforming growth factor- (16), and membrane receptors required for its endocytosis (17). In addition, decorin binds to certain types of fibrillar collagens including type I (18), type II (19), and type VI (20) and retards fibrillogenesis (19). Decorin also interacts with the collagen-like molecule C1q, a subcomponent of the C1 complex, the first component of the classical pathway of complement activation (21, 22). However, without exception these studies have utilized decorin extracted from tissues under harsh denaturing conditions which are likely to alter the secondary structure of this molecule. In fact, we demonstrate in this study that decorin purified under native conditions has an extensive secondary structure that is severely and irreversibly disrupted when treated by standard extraction techniques (23, 24). Binding of denatured decorin with other matrix molecules may not reflect biologically relevant interactions.

In the present study we have successfully utilized the vaccinia virus/T7 phage expression system to produce chemical amounts of differentially glycosylated forms of decorin that encompass the range of species described to date for tissue extracts and cell culture systems. We have purified the decorin glycoforms under native conditions and analyzed their glycosylation patterns. Preliminary investigation of the secondary structure of the decorin and biglycan extracted under denaturing and non-denaturing conditions is presented. The affinity of these recombinant native forms of decorin for collagenous extracellular matrix molecules is likely to better represent interactions that may occur in vivo.

EXPERIMENTAL PROCEDURES

All experimental procedures are as described in the accompanying paper (34), except for the following.

First-strand cDNA synthesis was done (Gene Amp kit, Perkin Elmer) with specific downstream oligonucleotide primers (0.5 µg/reaction) and 1-2 µg of RNA in a final reaction volume of 20 µl. cDNA extension was at 42 °C for 60 min, and the reaction terminated by incubation at 100 °C for 5 min. PCR cDNA amplification was done using the entire 20-µl first-strand cDNA reaction as a template in a final reaction volume of 100 µl containing upstream oligonucleotide primer (0.5 µg) and 2.5 units of Pfu DNA polymerase.

Human skin fibroblast RNA was used to generate a first-strand cDNA encoding the mature form of the decorin core protein (i.e. amino acids 31-359). First-strand cDNA was synthesized using the downstream primer, DCN-M2 5-GCCAGGTTATAAAAATGAGGGGTCTTGAGA-3 (base pairs 1163-1194). A cDNA encoding the mature decorin core protein was then PCR amplified using the upstream primer, DCN-M1 5-GGCTTATTTGACTGCATTAGAAGATGAGGC-3 (base pairs 148-179). An SphI site was created by site-specific changes in both oligonucleotide primers (bold). A shuttle vector, pAH1 containing a factor Xa protease cleavage sequence was digested with SphI. The decorin cDNA was cloned into pAH1 at the SphI site to yield pAH1-DCN. The pAH1-DCN plasmid was used as a template for PCR amplification using the following synthetic oligonucleotide primers: upstream primer, P3 5-CGGGATCCGCCATCGAGGGTAGGGGCATG-3; and downstream primer, P4 5-CGGAAACTATAAGTAATTCTCTGCAGGGA-3. An EcoRI site at the 5 end (primer 3) and a SacII site at the 3 end (primer 4) (bold) were created. These sites were used to clone the decorin cDNA into a polyhistidine vaccinia expression vector, pBGN4 (34). pBGN4 was digested with EcoRI and SacII to create sites for insertion of the decorin cDNA fragment. The resulting polyhistidine fusion vector, pDCN1 was sequenced to ensure no rearrangements occurred during PCR reactions.

A 41-mer synthetic peptide spanning amino acid residues 180-220 of human decorin was coupled to KLH (Immunogen Conjugation kit, Pierce), and used to generate a rabbit polyclonal antiserum (under contract with Cocalico Inc., Reamstown, PA). The antiserum, PR2 demonstrated specificity toward decorin and in separate experiments (not shown) exhibited immunoreactivity similar to the widely used LF-30 antiserum (kindly provided by Dr. Larry Fisher, National Institutes of Health). Five µl of the cell-free translation reaction mixture was preadsorbed with normal rabbit serum and protein A-Sepharose for 2 h at 4 °C. The supernatant was incubated with PR2 coupled to protein A-Sepharose and bound antigen released from the beads by incubation with SDS-PAGE sample buffer (see below).

The decorin expression plasmid, pDCN1 was used to generate a recombinant vaccinia virus by homologous recombination. This recombinant virus containing the polyhistidine-decorin fusion protein sequence under control of the T7 promoter, vDCN1, was propagated, amplified, and titered by standard methods (25).

Proteins were resolved on a 7.5% SDS-PAGE gel, and subsequently transferred to a nitrocellulose membrane. The membrane was blocked with 1% BSA, washed, and then incubated with PR2 antisera (1:3500 dilution) followed by goat anti-rabbit alkaline phosphatase conjugate (1:2000 dilution). The membrane was developed with 0.3 mg/ml nitro blue tetrazolium and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂, pH 9.5.

A Jasco J720 spectropolarimeter was calibrated with a 0.1% (w/v) 10-d-camphor sulfonic acid solution and CD spectra measured at room temperature in a 2-mm path-length quartz cell. Purified protein was exchanged into 10 mM Tris, 1 mM EDTA, pH 8.0, and protein concentration calculated by theoretical molar extinction coefficient (26). Estimation of secondary structure content was done according to Sreerama and Woody (27).

Decorin proteoglycan and core protein were radiolabeled (28) and separated from free [¹²⁵I]iodine by chromatography on PD-10 (Pharmacia) columns equilibrated with phosphate-buffered saline containing 0.5 mM MgCl₂, 0.9 mM CaCl₂ (PBS⁺), 0.1% BSA. Specific activity of the [¹²⁵I]iodine-labeled preparations were ~3 × 10⁷ cpm/µg. Immulon 1 wells were incubated overnight with 2 µg of C1q (20 µg/ml) or 50 µl of 1% BSA. Wells were washed with PBS⁺ and blocked with 1% BSA for 1 h at 37 °C. Non-radiolabeled competitor (see text) was added at 0-100 µg/ml for 1 h and [¹²⁵I]decorin (5 × 10⁴ cpm/well) was added and allowed to incubate for 3 h at room temperature. Wells were washed 3 times and the amount of bound radiolabel measured. All determinations were done in triplicate (mean ± S.D.).

RESULTS

A recombinant expression vector, pDCN1 (Fig. 1a), was developed for optimum production and secretion of decorin in eukaryotic cells using the vaccinia virus/bacteriophage T7 polymerase expression system, essentially as described in the accompanying paper (34). The vector, pDCN1, contains a cDNA that encodes the mature decorin core protein and does not contain sequence encoding the endogenous signal sequence or the putative propeptide domain. pDCN1 was transcribed and translated in vitro in the presence of Trans³⁵S-label (Fig. 1b). The radiolabeled products migrated as a single predominant band with an apparent molecular mass ~ 46 kDa (lane 1) that was selectively immunoprecipitated with PR2 (lane 2) confirming the presence of the epitope in the translation product. Transcription/translation and immunoprecipitation of other fusion proteins did not cross-react with PR2 (data not shown).

The vector pDCN1 was used to generate a new recombinant vaccinia virus by homologous recombination utilizing thymidine kinase sequences that flank the decorin fusion protein construct shown in Fig. 1a. HT-1080 cells do not secrete endogenous decorin (data not shown) and were co-infected with vDCN1 and vTF7-3. Cultures were incubated in medium containing Trans³⁵S-label, the medium harvested and recombinant decorin purified by nickel chelating chromatography (Fig. 2a). Several peaks were observed, including a large peak of radioactivity in the unbound fraction which did not contain immunoreactive decorin. The material eluted from the column by stepwise increases in imidazole concentration was monitored by analysis of selected fractions (Fig. 2a, asterisks) by SDS-PAGE (Fig. 2b). A proteoglycan form of decorin migrating as a polydisperse smear centered at ~100 kDa eluted between fractions 30 and 40 (Fig. 2b, fractions 30, 33, 36, and 40), corresponding to an imidazole concentration of 60-80 mM (Fig. 2a). A mixed population containing decorin proteoglycan (median molecular mass ~ 90 kDa) and decorin core protein (molecular mass ~ 50 kDa) eluted at 80 mM (lane 46), with the majority of the radioactivity associated with the core protein form. Higher concentrations of imidazole, between 80 and 100 mM, were required to elute the remainder of the bound radioactivity (Fig. 2a, fractions 45-55) which was predominantly decorin core protein (Fig. 2b, fractions 46, 49, and 52). Identity of both the proteoglycan and core protein forms as decorin was confirmed by Western blot with PR2 antiserum (data not shown). The recombinant decorin was purified to greater than 90% by a single passage over the nickel column based on the absence of any other radiolabeled proteins co-purifying (Fig. 2b) and on the absence of any additional Coomassie staining bands (see below).

A sample containing both decorin proteoglycan and decorin core protein radiolabeled biosynthetically with Trans³⁵S-label and purified by nickel affinity chromatography was incubated with factor Xa protease and reapplied to a nickel column. Decorin that was not incubated with the factor Xa protease bound to the resin and was eluted with 200 mM imidazole, whereas after factor Xa digestion about 80% of the decorin (both proteoglycan and core protein) no longer bound to the resin and eluted in the unbound fraction (data not shown). The reason for incomplete cleavage is not clear, but may relate to enzyme inactivation. There was no apparent preferential cleavage of the proteoglycan versus the core protein forms indicating that the level of glycosylation of the fusion protein did not influence access of the protease to the cleavage site.

About 75% of the total recombinant protein purified from the culture supernatant was the proteoglycan form of decorin based on incorporation of ³⁵S-radiolabel (Cys and Met) before and after chondrotinase ABC digestion. In addition to the apparent high level of purity attained by the nickel column, we also noted that there was some resolution into subpopulations: the early eluting decorin proteoglycan was of larger molecular size compared to the later eluting proteoglycan (Fig. 2b, fractions 30 and 46, square brackets). The resolution of two subpopulations of proteoglycan was clearer when the proteins were labeled with [³⁵S]sulfate and [³H]glucosamine (see below). Although separation into subpopulations was more evident using a step elution procedure, it was still apparent during gradient elution procedures (data not shown). The core protein form of decorin also migrated on SDS-PAGE as a doublet, molecular mass ~ 53 and ~49 kDa, with the larger core protein eluting slightly ahead of the smaller form on the nickel column (Fig. 2b, fractions 46-52).

Recombinant decorin was isolated from a large number of co-infected HT-1080 cells (2.5 × 10⁹) by a preparative scale nickel column. The concentration of purified samples were estimated by theoretical molar extinction coefficient and analyzed by 7.5% SDS-PAGE stained with Coomassie Blue (Fig. 3). Decorin proteoglycan was predominantly eluted in the 60 mM imidazole fraction, and 10, 5, and 1 µg were applied to the gel (Fig. 3, lanes 1-3, respectively). Decorin core protein was enriched in the 80 mM imidazole elution and an estimated 5 µg was loaded (Fig. 3, lane 4) which migrated as a doublet, with the larger form (molecular mass ~ 53 kDa) more abundant. No other contaminating protein bands were visualized on the gel, indicating purity of the sample. Based on these data, we are able to estimate that expression and purification of recombinant decorin under native conditions (i.e. in the absence of chaotropic solvents or detergents) yields about 10 mg of proteoglycan and 3.3 mg of core protein/10⁹ cells per 24 h. Recombinant decorin proteoglycan was also isolated and purified under denaturing conditions from HT-1080 culture supernatant in the presence of the chaotropic solvent 4 M GdnHCl and the detergent 0.5% Triton X-100. The use of this solvent throughout the extraction and purification protocol increased the final yield of proteoglycan 2-3-fold compared with the yield under native conditions. The significant increase in yield of recombinant protein in the presence of detergent was most likely a consequence of the hydrophobicity of native material.²

Samples of Trans³⁵S-labeled decorin proteoglycan and core protein were subjected to differential digestion with glycosidases to assess the nature of the carbohydrate substitutions. Undigested sample (Fig. 4, lane 1) shows the presence of the proteoglycan smear and a doublet core protein. Following digestion with chondroitinase ABC (Fig. 4, lane 2), the proteoglycan smear is no longer visible and all the radiolabel is coincident with the core protein doublet. Separate experiments showed that the proteoglycan was equally susceptible to chondroitinase ACII digestion indicating that the proteoglycan contained predominantly chondroitin sulfate. Chondroitinase ABC-treated sample was further incubated with N-glycosidase F (Fig. 4, lane 3) which resulted in the doublet core protein reducing to a single band with an apparent molecular mass ~ 43 kDa. Chondroitinase ABC digestion of proteoglycan generated a doublet core protein, indicating that both core protein forms contain GAG (data not shown). The core protein doublet appears to reflect differential N-glycosylation of the decorin core protein, most likely representing 3 and 2 N-linked oligosaccharides based on the shift in migration position (~10 and ~6 kDa, respectively). There was no evidence for secretion of unglycosylated decorin, or a proteoglycan form without N-linked oligosaccharides.

HT-1080 cells co-infected with vDCN1 and vTF7-3 were incubated in the presence of [³⁵S]sulfate and [³H]glucosamine. Radiolabeled macromolecules were applied to a nickel column, bound material eluted using the protocol described for Fig. 2a, and radioactivity of collected fractions monitored. Two distinct peaks of radioactivity were eluted (data not shown), one at 60 mM imidazole and the second at 70-80 mM imidazole, and were distinguished by the differential ratio of ³⁵S:³H which reflected the composition of the two pools. The early eluting peak (pool 1) and the later eluting peak (pool 2) were collected and aliquots subjected to gel filtration chromatography on Superose 6. Pool 1 eluted predominantly as a single broad peak (Fig. 5a) centered at K_d 0.40 (mass ~ 94 kDa, compared to globular protein standards), with the ³⁵S and ³H co-incident. Pool 2 resolved into a single broad ³⁵S-labeled peak centered at K_d 0.45 (mass ~ 80 kDa); and two distinct ³H-labeled peaks, one coincident with the ³⁵S label, and a second, sharp peak at K_d 0.60 (mass ~ 50 kDa) which consisted exclusively of ³H label (Fig. 5b). Based on the predicted composition of the two pools (see Fig. 2b), pool 1 represents decorin proteoglycan alone and pool 2 comprises a mixture of proteoglycan and core protein. The core protein from pool 2 is radiolabeled via ³H incorporation into N-linked oligosaccharides. The broad, high molecular weight peaks in both pools represent proteoglycan containing ³⁵S- and ³H-labeled chondroitin sulfate chains. This was confirmed by chondroitinase ABC digestion of pool 1 and pool 2 and elution on Superose 6 (Fig. 5, c and d). More than 95% of the ³⁵S label and 80% of the ³H label in pool 1 shifted to the V_t of the Superose 6 column after chondrotinase ABC digestion, leaving a small, sharp peak containing ³H label at K_d 0.60 (Fig. 5c, asterisk). Chondroitinase ABC digestion of pool 2 shifted more than 95% of the ³⁵S and 60% of the ³H label to the V_t, leaving a single, sharp peak containing primarily ³H label at K_d 0.60 (Fig. 5d). After chondroitinase ABC digestion, the Superose 6 resolves a core protein pool of molecular mass ~ 50 kDa, consistent with the core protein size determined on SDS-PAGE (~49 and ~53 kDa).

The molecular size difference between the decorin proteoglycan in pools 1 and 2 is illustrated by their different migration positions on SDS-PAGE (Fig. 6a). This size difference is largely due to differences in chondroitin sulfate chain size as determined from Superose 6 chromatography after alkaline hydrolysis. However, anomolous migration of proteoglycans on SDS-PAGE may be influenced by microheterogeneity in disaccharide composition which could affect the total charge density of the molecule. Disaccharide analysis of the chondroitin sulfate chains (summarized in Fig. 6b) show ratios of di-0S:di-4S:di-6S in the larger chondroitin sulfate chains (pool 1) of 2.7:1.0:10.6, markedly different for that observed for the shorter chains (pool 2) of 2.0:1.0:3.7. These two subpopulations partially resolve by nickel chelating chromatography and this may relate to both the size and different disaccharide composition of the chondroitin sulfate chains.

Recombinant decorin proteoglycan purified under native conditions was analyzed by CD spectroscopy. The far-UV spectra of the native proteoglycan showed greater than 70% secondary structure which was predominantly -sheet as indicated by a sharp minima at 218 nm (Fig. 7a, closed circles). Analysis of the CD spectra based on computer modeling (27) predicted the native proteoglycan to comprise 54% -sheet, 14% -turn, 12% -helix, and 20% random coil. Recombinant decorin proteoglycan was also isolated and purified under denaturing conditions from HT-1080 culture supernatant in the presence of the chaotropic solvent 4 M GdnHCl and 0.5% Triton X-100. After exchange back into physiological solvent, the CD spectra obtained for this material (Fig. 7a, open circles) revealed a sharp minima around 205 nm indicating an unordered conformation with no detectable secondary structure. Decorin extracted from bovine articular cartilage, representative of material utilized in numerous binding studies (29, 30), was also devoid of any secondary structure (Fig. 7a, open squares), and was similar to the spectra obtained for native decorin after thermal denaturation. The CD spectra for similarly produced recombinant biglycan (34) was identical to that for recombinant decorin; and exposure to denaturants resulted in disruption of the secondary structure. Consistent with these observations of significant secondary structure in the recombinant decorin proteoglycan, the PR2 antiserum selectively immunoprecipitated guanidine HCl-extracted decorin core protein (Fig. 7b, lane 1) but not the native recombinant decorin (Fig. 7b, lane 2), however, both denatured and native preparations of recombinant decorin were recognized by Western blot analysis (Fig. 7b, lanes 3 and 4). This is consistent with the epitope being inaccessible in the native conformation (i.e. conditions for immunoprecipitation), but available upon unfolding of the molecule (i.e. SDS treatment and transfer to nitrocellulose membrane).

Both the proteoglycan and core protein exhibit a high degree of hydrophobicity when purified under native conditions,² with this being more pronounced for the core protein preparation. This hydrophobicity may be a consequence of the secondary structure, since similar behavior was not apparent for GdnHCl-treated material. The presence of a polyanionic chondroitin sulfate chain on the decorin proteoglycan may play a role in maintaining a stable secondary structure in solution.

The binding of ³⁵S-labeled decorin proteoglycan and core proteins to collagen types I, II, III, V, VI, C1q or BSA was examined, and showed an identical pattern to that of biglycan (34). The binding of decorin to the fibrillar collagens was overshadowed by the much greater binding observed with C1q. Interaction with C1q appears to be mediated by the core protein of decorin, with binding up to 18-fold over the BSA control compared to 9-fold binding of intact proteoglycan. Krumdieck et al. (21) have shown that decorin proteoglycan and chondroitinase ABC-generated core protein from bovine articular cartilage also bind C1q.

The binding of ¹²⁵I-labeled proteoglycan and core protein to C1q was subjected to competition by the addition of unlabeled decorin proteoglycan and core protein (Fig. 8a, open and closed circles, respectively). Both decorin proteoglycan and decorin core protein inhibited binding of ¹²⁵I-labeled decorin to C1q. The IC₅₀ (concentration of competitor required for half-maximal inhibition) for the core protein was ~28 µg/ml (0.62 µM) and for intact proteoglycan the IC₅₀ value was ~18 µg/ml (0.2 µM; Fig. 8a). Saturability of the decorin-C1q interaction was investigated by adding increasing amounts of radiolabeled decorin (specific activity 200,000 cpm/µg) to wells coated with C1q or 1% BSA. The inset in Fig. 8, b and c, demonstrates that specific binding of decorin proteoglycan and core to C1q reaches a plateau at 0.6 µg of proteoglycan and 0.4 µg of core protein added. Assuming a single binding site on the C1q molecule, Scatchard analysis (31) yielded a dissociation constant for the decorin-C1q interaction of 50 and 26 nM for the binding of the recombinant decorin proteoglycan and core proteins, respectively.

DISCUSSION

In this study, differentially glycosylated forms of decorin have been overexpressed in a eukaryotic cell line with a connective tissue origin (HT-1080). These decorin glycoforms were separated from other secreted molecules to greater than 95% purity, and further partially resolved by metal chelating affinity chromatography into two distinct populations: proteoglycan forms and core protein forms. The proteoglycan population was further partially resolved into two species distinguished by different sized chondroitin sulfate chains with distinct disaccharide composition; the core protein subpopulation was also represented by two distinct forms, distinguished by the extent of N-linked oligosaccharide substitution. The core protein preparations generated by chondroitinase ABC digestion of the proteoglycan pool comprised both the 2 and 3 N-linked oligosaccharide forms; we cannot exclude the possibility that the different sized GAG chains are on different core proteins (i.e. the 2 and 3 N-linked oligosaccharide forms, respectively), however, this would seem unlikely given a similar bi-modal population of GAG chains was observed for recombinant biglycan (34) where there is no evidence for expression of two core proteins.

The two non-proteoglycan forms of recombinant decorin had an apparent mobility of ~49 and ~53 kDa on SDS-PAGE. Differential N-glycosylation of decorin synthesized by human skin fibroblasts in vitro has been demonstrated previously (32), whereby chondroitinase ABC-digested decorin migrated as a doublet core protein preparation which further reduced to a single band when synthesized in the presence of tunicamycin. Although these investigators were able to induce N-glycan-free decorin proteoglycan synthesis through exogenous addition of tunicamycin, we saw no evidence for expression of similarly N-glycan-free decorin core protein expression, nor is there any evidence that this is a biosynthetic product in vivo. In fact, it is likely that core protein devoid of both GAG and N-linked oligosaccharides may be insoluble, as evidenced by the difficulties associated with bacterial expression of these molecules (33).

The different glycoforms of decorin expressed in this system are similar to the variety of decorin glycanated and non-glycanated species already identified in tissues. The proteoglycan form of the recombinant decorin is characteristic of material extracted from tissues in terms of chondroitin sulfate chain length and pattern of sulfation, as well as differential N-glycosylation. The recombinant decorin secreted devoid of GAG chains is likely a product of overexpression of core protein exceeding the capacity of one or more glycosyltransferases. Purification and quantitation of the recombinant decorin indicates that the HT-1080 cells secreted up to 30-40 mg of decorin/10⁹ cells per 24 h, about 75% as a proteoglycan form and the remainder as core glycoprotein. Under native conditions we experienced significant losses of material, but are nevertheless able to purify chemical amounts that are correctly folded and biologically active.

Circular dichroism spectra and secondary structure prediction clearly demonstrate that about 70% of the native recombinant intact decorin assumes a secondary structure with a high proportion of -sheet content. This is not surprising given that about 70% of the core protein is comprised of the LRR domain which is predicted to assume a complex structure in solution. However, both recombinant decorin subjected to denaturing conditions and decorin extracted from cartilage by standard techniques (29, 30) demonstrated a loss of secondary structure in their CD spectra. This strongly argues that techniques commonly employed for isolation of decorin and biglycan from tissues (23, 24, 29) have a permanent effect on the conformation of the core protein. These changes in secondary structure may have implications on the interpretation of data reported by others related to interactions between decorin and biglycan with other extracellular matrix molecules. A recent study (23) reported that decorin extracted from cartilage with GdnHCl was not an effective competitor of native decorin binding to type I collagen. Given many previous reports of GdnHCl-extracted decorin interacting with type I collagen in a variety of assays, it is possible that there are both secondary structure-dependent and -independent interactions. It is likely that interactions requiring correctly folded decorin are important in vivo so it is important to investigate these interactions using native preparations.

Decorin bound very effectively to the complement component C1q, showing an 18-fold increase in binding with core protein compared to BSA, and a 9-fold increase in binding with the proteoglycan form. Since the presence of a GAG chain appears to have attenuated the extent of binding it is likely that this interaction is mediated via the core protein, and the GAG chain may interfere with the ability of C1q to access the core protein domain. C1q is a subcomponent of the C1 complex, the first component of the classical pathway of complement activation (21, 22). The C1q molecule consists of two domains, a collagen-like triple helical domain at the N terminus and a globular configuration at the C terminus and circulates in the plasma as a Ca²⁺-dependent complex with C1r and C1s (22). Although the biological significance of C1q-decorin interaction is not known, decorin may modulate complement-mediated inflammation at sites where extracellular matrices containing decorin are exposed to complement components (21). The C1q-decorin interaction was competitively inhibited by both core protein and proteoglycan preparations, however, an intact proteoglycan (i.e. substituted with a GAG chain) is required for inhibition of the complement cascade (21). Although both native and denatured decorin bind to C1q, it remains to be determined if the two forms interact via the same domain or whether the interaction with denatured decorin is simply nonspecific binding. The specificity and affinities of native decorin glycoforms for collagen-like molecules is the subject of further study.

This study, and the accompanying paper (34), have demonstrated the high level expression and purification under native conditions of the small interstitial proteoglycans, decorin and biglycan. These glycoconjugates possess a high degree of secondary structure not apparent in similar molecules extracted from hard tissues by standard techniques, in addition to maintaining biological activity in several binding assays. Furthermore, several glycoforms have been isolated that are similar to subpopulations present in a variety of tissues in vivo. These recombinant decorin proteoglycan and core protein forms isolated in chemical amounts will allow further study on the biological role and molecular interactions of this class of LRR-containing molecules.