FIBROBLAST AND NEUTROPHIL COLLAGENASES CLEAVE AT 2 SITES IN THE CARTILAGE AGGRECAN INTERGLOBULAR DOMAIN

The actions of recombinant human fibroblast collagenase (MMP1), purifi ed polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan GI-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and trypsin, both collagenases were abl e to degrade human and porcine aggrecans to a similar extent. An N-ter minal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain b etween G1 and G2. Two specific sites were found; one at an Asn341-Phe3 42 bond and another at Asp441-Leu442 (human sequence). This specificit y of the collagenases for aggrecan G1-G2 was identical with that of th e truncated metalloproteinase matrilysin (MMP7), but different from th ose of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; M MP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp -Leu site. In addition, collagenase catalytic fragments lacking C-term inal 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 inf luenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matri lysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domai n, and additionally that both fibroblast and neutrophil collagenases c leave at a second site in the interglobular domain that is not availab le to stromelysin or gelatinases.