Activation of the Proteolytic Activity of ADAMTS4 (Aggrecanase-1) by C-terminal Truncation

Abstract

Proteolysis of the hyalectans (aggrecan, versican, brevican) in vivo appears to result from the activity of ADAMTS4 (aggrecanase-1, herein referred to as an hyalectanase). To examine the mode of activation of AD-AMTS4, a human chondrosarcoma cell line, JJ012, has been stably transfected with the full-length c-DNA for human ADAMTS4. The cells synthesized a high molecular weight form of the enzyme (p100), which in serumfree culture was processed to three truncated forms, p75, p60, and p50. Treatment of the p100 form with recombinant furin indicated that the p75 form is generated by the removal of the prodomain by a furin-like activity. Analysis with domain-specific antisera showed that the p60 and p50 forms are generated by C-terminal truncation of the p75 form. The appearance of the p60 and p50 forms in culture medium was prevented by inclusion of a furin inhibitor, inhibitors of glycosylphosphatidylinositol synthesis, glucosamine, a hydroxamate-based matrix metalloproteinase (MMP) inhibitor, and TIMP-1, but not by AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) or E64. Only medium samples containing the p60/p50 forms exhibited aggrecanase activity, and isolation of the p75, p60, and p50 forms by preparative SDS-PAGE showed that only p60 and p50 were active in aggrecanase and versicanase assays. Pig synovium and human cartilages also contained AD-AMTS4 in the p75, p60, and p50 forms. We suggest that in vivo production of proteolytically active ADAMTS4 requires not only removal of the prodomain by a furin-like activity but also MMP-mediated removal of a portion of the C-terminal spacer domain.Aggrecan, versican, neurocan, and brevican are components of the extracellular matrix (ECM) 1 in a wide range of tissues. They are all members of the family of large aggregating proteoglycans (1), which are characterized by an N-terminal globular domain that binds to hyaluronan. They have therefore been included, along with related species such as link protein and CD44, in the molecular grouping termed hyaladherins (2). At the same time they are all synthesized with a C-terminal globular domain that is related structurally to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. Because of this structural feature they have also been given the family name of lecticans (3). In an attempt to accommodate the functionality of both the N-terminal and C-terminal globular domains, and also to indicate their proteoglycan nature, the group has also been termed the hyalectans (4).Proteolytic degradation of the hyalectans in the ECM appears to result from the activity of a subgroup of the ADAMTS family of metalloproteinases, all of which exhibit some degree of glutamyl-endopeptidase activity for specific Glu-X bonds (where X is most often Ala or Gly) in these glycosaminoglycansubstituted substrates. Thus, ADAMTS1, -4, and -5 exhibit "aggrecanase" activity (5-7), ADAMTS1 and -4 exhibit "versicanase" activity (8), and ADAMTS4 exhibits "brevicanase" activity (9). Among...

Inhibition of furin‐like enzymes blocks interleukin‐1α/oncostatin M–stimulated cartilage degradation

“…This suggests that furin-activated enzymes are involved in proteoglycan breakdown. Gao et al (34) demonstrate that Dec-RVKR-CH 2 Cl inhibits the processing of ADAM-TS4, resulting in an inactive enzyme, which is consistent with our results. The observation that Dec-RVKR-CH 2 Cl only partially blocked proteoglycan release suggests a role for enzymes that are not furin-activated or that furin inhibition was incomplete.…”

“…MT1-MMP, MT3-MMP, and MMP-11 can all be activated by furin (31)(32)(33). In addition, the aggrecanases ADAM-TS4 and ADAM-TS5 contain this furin cleavage site (9,10), and the pro-domain of ADAM-TS4 can be removed by furin (34). Furin has been detected in normal cartilage, and elevated levels are found in osteoarthritic cartilage (35).…”

Selective and non-selective metalloproteinase inhibitors reduce IL-1-induced cartilage degradation and loss of mechanical properties

“…ADAMTS-4 undergoes post-translational processing to at least 4 activation states, each with distinct substrate specificity and matrixbinding properties that could influence inhibitor potency (Gao, et al, 2002;Kashiwagi, et al, 2004;Tortorella, et al, 2000). As a result, the aggrecanase inhibitors used in this study may efficiently block aggrecanase activity in the aggrecan IGD and yet permit less destructive activity (at sites in the CS-2 domain) characteristic of lower activation states.…”

RNAi mediated MMP-1 silencing inhibits human chondrosarcoma invasion

“…55012, an established human CS cell line, was cultured as previously described [16,27]. Transfection of siRNAs was performed by using Lipofectmine 2000 siRNA transfection reagent (Invitrogen).…”