Recombinant Human Aggrecan G1-G2 Exhibits Native Binding Properties and Substrate Specificity for Matrix Metalloproteinases and Aggrecanase

Abstract

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val 1 -Arg 656 has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M r 90,000 -95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. sequences were used to confirm aggrecanase cleavage at the Glu 373 2 Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1␣ or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.Aggrecan is a chondroitin sulfate and keratan sulfate-bearing proteoglycan. It is present in cartilage as large multimolecular aggregates bound noncovalently to hyaluronan (HA). 1Aggregate formation is mediated via the proteoglycan tandem repeat loops in the N-terminal G1 domain which bind to decasaccharide units within the polymeric HA. This binding is further stabilized by link protein, which binds to form stable trimeric complexes. The formation of large aggregate structures provides a mechanism for trapping aggrecan in the tissue and preventing its loss by diffusion. A major feature of the pathology of arthritis is the gradual loss of aggrecan from the cartilage matrix. This loss involves proteolysis of the core protein and release into culture medium (1, 2) or synovial fluid (3) of large fragments lacking G1 domains that are t...

ADAMTS4 Cleaves at the Aggrecanase Site (Glu373-Ala374) and Secondarily at the Matrix Metalloproteinase Site (Asn341-Phe342) in the Aggrecan Interglobular Domain

“…Although it has been widely accepted (22,23,30,(37)(38)(39)(40)(41)(42) that ADAMTS4 (aggrecanase-1) cleaves only at the Glu 373 -Ala 374 bond within the IGD, the results described here suggest that both of the major cleavages that are known to occur in vivo, at Glu 373 -Ala 374 and Asn 341 -Phe 342 , can be catalyzed by ADAMTS4. Previously, cleavage at the Asn 341 -Phe 342 site in the aggrecan IGD was considered a specific property of the MMP family, with the exception of a snake venom enzyme (30,34) 341 -Phe 342 bond in the experiments described here was due to contamination of the ADAMTS4 preparation with an MMP-like activity was eliminated by the observation that although 10 mM EDTA and TIMP-3 totally blocked the cleavage, neither TIMP-1 nor TIMP-2 was inhibitory, even at 750 nM. Collectively, these results suggest that the distinction between MMP, cathepsin B, and ADAMTS activities based on neoepitope immunoreactivity of the products can no longer be considered definitive (see Table I for summary).…”

“…The ability of ADAMTS4 to cleave the Asn 341 -Phe 342 bond does not, however, appear to be sensitive to the presence or absence of keratan sulfate substitution because DIPEN 341 fragments were generated from both native aggrecan and non-glycosylated rG1-G2 substrate. This is in contrast to the snake venom reprolysin atrolysin C, which failed to cleave non-glycosylated rG1-G2 at the Asn 341 -Phe 342 site (30). Substrate specificity studies have suggested that ADAMTS enzymes associate with a region of aggrecan at or near the MMP cleavage site; however, this association was presumed to be more in the nature of a substrate-docking site for the enzyme (31,35 (48).…”

“…conditioned medium (30,31) have also failed to detect this dual cleavage specificity. The most likely explanation for this is that, in general, studies with aggrecanase in this area have been optimized in terms of enzyme activity, sample loadings, and gel exposures to detect the products expected from cleavage at the Glu 373 -Ala 374 bond.…”

“…Digests of Recombinant G1-G2-Recombinant G1 (rG1)-G2 substrates (wild-type, an aggrecanase site mutant (A 374 RGSV to N 374 VYSV), or an MMP site mutant (deletion of residues EN 341 2F 342 F)) were expressed and purified as described previously (30,31). For digests, ADAMTS4 (381 nM) was incubated with the rG1-G2 substrate (1.4 M) in digestion buffer at 37°C for 16 h. The reactions were stopped by the addition of 20 mM EDTA.…”

N-Linked Keratan Sulfate in the Aggrecan Interglobular Domain Potentiates Aggrecanase Activity

“…Other studies comparing the presence or absence of endogenous keratan sulfate chains have suggested that aggrecanase cleavage in the IGD may be increased in the presence of keratan sulfate and reduced when keratan sulfate is enzymatically removed (36,37). However, recombinant substrates that lack keratan sulfate are not totally resistant to cleavage by aggrecanases (38,39). Collectively, these studies suggest that keratan sulfate in the IGD may influence aggrecanolysis.…”