title: Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain.
abstract: The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.
authors.affiliation: University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria, Australia.
authors.forename: A J
authors.initials: AJ
authors.lastname: Fosang
authors.forename: K
authors.initials: K
authors.lastname: Last
authors.forename: V
authors.initials: V
authors.lastname: Knäuper
authors.forename: P J
authors.initials: PJ
authors.lastname: Neame
authors.forename: G
authors.initials: G
authors.lastname: Murphy
authors.forename: T E
authors.initials: TE
authors.lastname: Hardingham
authors.forename: H
authors.initials: H
authors.lastname: Tschesche
authors.forename: J A
authors.initials: JA
authors.lastname: Hamilton
mesh_terms: D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D017364:Collagenases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology; D010446:Peptide Fragments; D011509:Proteoglycans / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine
chemical_list: D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D010446:Peptide Fragments; D011509:Proteoglycans; D011994:Recombinant Proteins; D017364:Collagenases