Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-A rg(656) has been expressed in Sf21 cells using a baculovirus expression sys tem. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluron an-Sepharose affinity chromatography followed by high performance liquid ch romatography 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 express ed G1-G2 bound to both hyaluronan and link protein indicating that the immu noglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded, Further analysis of secondary structure by rotary s hadowing electron microscopy confirmed a double globe appearance, but revea led 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 fo llowing 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 m edium from cartilage explant cultures, and the products analyzed on SDS gel s by silver stain and immunoblotting, Neoepitope antibodies recognizing the N-terminal F(342)FGVG C-terminal DIPEN341 sequences were used to confirm M MP cleavage at the Asn(341) down arrow Phe bond, while neoepitope antibodie s recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) down arrow Ala bo nd. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. I ncubation 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 alpha or retinoic acid, Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sit es, 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 ke ratanase treatment was cleaved by atrolysin C at both the aggrecanase and M MP sites. The results suggest that the presence or absence per se of kerata n sulfate on native G1-G2 does not affect the activity of atrolysin C towar d the two sites.