Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells

Abstract

603The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to a2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to a2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15000 units/mg (1 unit = 1 ,ug of gelatin degraded/min at 37°C) by titration with a2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.

Generation of a recombinant Sendai virus that is selectively activated and lyses human tumor cells expressing matrix metalloproteinases

“…Each of the resulting plasmids was transfected, together with a GFP-expressing plasmid, pCAGGS/GFP, into human fibrosarcoma cell line HT1080 and human stomach cancer line MKN28, which expressed and secreted MMP and tissue-type plasminogen activator (tPA), respectively, at high levels. 29,30 tPA has the same cleavage specificity as uPA. As shown in Figure 1c, the F protein with the cleavage motif compatible with MMP-subII was fully fusion competent in the relevant target cell line, HT1080, whereas the other three substrates (subI, subIII and subIV) were not.…”

Gelatinase-mediated migration and invasion of cancer cells

“…α2-macroglobulin is an abundant plasma protein and effectively inhibits the activity of most proteinases, including the MMPs (Sottrup-Jensen and Birkedal-Hansen, 1989). Binding to α2-macroglobulin is an efficient indicator of MMP activation status as only the activated enzymes bind it (Morodomi et al, 1992). The α2-macroglobulin may play an important role in the endocytic removal of proteolytic enzymes (Moestrup et al, 1993).…”

“…Indeed, some substrates show over 200-fold selectivity towards MMP-2 Kridel et al, 2001). (Morodomi et al, 1992;Okada et al, 1990) Native collagen type: I yes no (Aimes and Quigley, 1995) III yes no (Berton et al, 2000) IV yes yes (Morodomi et al, 1992) V yes yes (Morodomi et al, 1992) VII yes (Seltzer et al, 1989) X yes (Cole et al, 1993) XI yes (Smith et al, 1991) XVIII yes (Ferreras et al, 2000) Aggrecan yes yes (Fosang et al, 1992) BM-40/SPARC/ Osteonectin yes yes (Sasaki et al, 1997) Brevican yes no Decorin yes no (Imai et al, 1997) Elastin yes yes Entactin/nidogen no yes (Mayer et al, 1993;Sires et al, 1993) Fibrillin yes yes (Ashworth et al, 1999) Fibrin yes (Lelongt et al, 2001) Fibrinogen yes yes (Bini et al, 1996;Lelongt et al, 2001) Fibronectin yes no (Okada et al, 1990) Laminin yes yes (Giannelli et al, 1997;Morodomi et al, 1992) Link protein yes yes (Nguyen et al, 1993) NG2 proteoglycan yes (Larsen et al, 2003) Neurocan yes (Turk et al, 2001) Tenascin yes no (Siri et al, 1995) Vitronectin yes yes (Imai et al, 1995) α2-macroglobulin yes yes (Arbelaez et al, 1997) αB-crystallin yes (Starckx et al, 2003) Amyloid protein precursor yes (LePage et al, 1995) Big endothelin-1 yes yes (Fernandez-Patron et al, 2002) Calcitonin gene-related peptide (CGRP) yes (Fernandez-Patron et al, 2000) Complement protein C1q yes yes (Ruiz et al, 1999) Connective tissue-activating peptide-III (CTAP-III) yes (Van den Eph B1 tyrosine kinase receptor yes no Epithelial-cell derived neutrophil activating pept...…”

Digestion of C1q collagen-like domain with MMPs-1, -2, -3, and -9 further defines the sequence involved in the stimulation of neutrophil superoxide production

“…Human MMPs-1, -2, -3, and -9 were purified from human rheumatoid synovial cells and HT-1080 cells as previously described [21,22]. Preliminary experiments demonstrated that suitable conditions for collagenase digestion of C1q-CLR [2.4-4.5 mg/mL in Tris-buffered saline (TBS), pH 7.5] were achieved when incubated at 37°C for 20 h with final enzyme concentrations as follows: MMP-1 (interstitial collagenase), 1 µg/mL; MMP-2 (gelatinase A), 1.1 µg/mL; MMP-3 (stromelysin 1), 3 µg/mL; and MMP-9 (gelatinase B), 0.3 µg/mL.…”