title: 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. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow 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-1alpha 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.
authors.affiliation: Department of Paediatrics, Orthopaedic Molecular Biology Research Unit, University of Melbourne, Royal Children's Hospital, Parkville 3052, Australia.
authors.forename: F A
authors.initials: FA
authors.lastname: Mercuri
authors.forename: K J
authors.initials: KJ
authors.lastname: Doege
authors.forename: E C
authors.initials: EC
authors.lastname: Arner
authors.forename: M A
authors.initials: MA
authors.lastname: Pratta
authors.forename: K
authors.initials: K
authors.lastname: Last
authors.forename: A J
authors.initials: AJ
authors.lastname: Fosang
mesh_terms: D053674:Aggrecans; D000409:Alanine / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D037181:Lectins, C-Type; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D011487:Protein Conformation; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D011994:Recombinant Proteins; D005973:Glutamine; D010649:Phenylalanine; D001216:Asparagine; D010450:Endopeptidases; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases; C089232:atrolysin C; C083641:aggrecanase; D000409:Alanine