Production and Purification of Prostromelysin and Procollagenase from IL-1 Beta-Stimulated Human Gingival Fibroblasts

Abstract

Conditions were established to stimulate human gingival fibroblast explant cultures to synthesize milligram quantities of the metalloproteinase proenzymes, prostromelysin and procollagenase. To stimulate enzyme production, cells were treated with 1 nM recombinant human IL-1 beta for approximately 7 days under serum free conditions. Using a combination of rapid column chromatography steps, approximately 10 milligrams of prostromelysin and 5 milligrams of procollagenase were purified from 1 liter of conditioned media. Prostromelysin electrophoresed as a doublet with molecular weights of 55,57 kD, whereas, procollagenase migrated with slightly lower molecular weights of 52, 54 kD. Both proenzymes were treated with trypsin or aminophenylmercuric acetate to generate active species. The molecular weights of the active enzymes were approximately 10 kD smaller than the proenzymes. Active enzymes were inhibited by metal chelators and the natural metalloproteinase inhibitor, tissue inhibitor of metalloproteinase (TIMP), but not by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). Activated stromelysin degraded a number of substrates including transferrin, proteoglycan monomer, proteoglycan aggregated with hyaluronic acid, and substance P. By contrast, collagenase degraded interstitial type I collagen and the peptide thioester, Ac-Pro-Leu-Gly-SCH(iBu)Co-Leu-GlyOEt. Identity of both enzymes were confirmed by amino-terminal protein sequence analysis as well as by immunoblot analysis using monoclonal antibodies.

Changes in cartilage composition and physical properties due to stromelysin degradation

“…The femoropatellar groove was isolated and 3-mm diameter by 1-mm thick cartilage disks were harvested using a dermal punch (Miltex Instruments, Lake Success, NY) and a sledge microtome (model 860; American Optical, Buffalo, NY), as described previously (38). Recombinant human prostromelysin (200 pg/ml) in 25 mM Tris HC1, 10 mM CaCl,, 0.05% Bnj 35, and 0.01% NaN, (39) was activated with trypsin, as described previously (40). The enzyme had specific activity as native human gingival fibroblast SLN using 3H-transferrin as a substrate (40).…”

Cytokine Expression in Periodontal Health and Disease

“…Another important activity of IL-I in the pathological process of periodontitis is to induce the production of matrix metalloproteinases (MMPs) (HavemosePoulsen and Holmstrup, 1997). IL-1 gives rise to an elevated level of procollagenase in both gingival fibroblasts and periodontal ligament (PDL) cells (Meikle et al, 1989;Lark et al, 1990;Richards and Rutherford, 1990;Tewari et al, 1994). No significant change in tissue inhibitors of metalloproteinase (TIMP) synthesis was observed after treatment of gingival fibroblasts and PDL cells with IL-I D (Meikle et al, 1989) as well as no change in TIMP mRNA level in PDL cells (Alvares et al, 1995).…”

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

“…In some cases, as a positive control, unfixed cryosections were digested with trypsin-activated recombinant human stromelysin-1 (100 g/ml in 25 mM Tris- HCl, 150 mM NaCl, 10 mM CaCl 2 , 0.02% NaN 3 ) for 30 min at 37 Њ C. The tissue sections were then stained for the neoepitopes described above. Trypsin was removed from all activated stromelysin samples using soybean trypsin inhibitor-agarose (35). The only proteolytic activity remaining in the sample was due to stromelysin.…”