Ulrich Bergmann

Activation of Type IV Procollagenases by Human Tumor-associated Trypsin-2

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62–65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.

Structural Insight Into the Complex Formation of Latent Matrix Metalloproteinase 2 with Tissue Inhibitor of Metalloproteinase 2

Matrix metalloproteinases (MMPs) are a family of multidomain enzymes involved in the physiological degradation of connective tissue, as well as in pathological states such as tumor invasion and arthritis. Apart from transcriptional regulation, MMPs are controlled by proenzyme activation and a class of specific tissue inhibitors of metalloproteinases (TIMPs) that bind to the catalytic site. TIMP-2 is a potent inhibitor of MMPs, but it has also been implicated in a unique cell surface activation mechanism of latent MMP-2/gelatinase A/type IV collagenase (proMMP-2), through its binding to the hemopexin domain of proMMP-2 on the one hand and to a membrane-type MMP activator on the other. The present crystal structure of the human proMMP-2/TIMP-2 complex reveals an interaction between the hemopexin domain of proMMP-2 and the C-terminal domain of TIMP-2, leaving the catalytic site of MMP-2 and the inhibitory site of TIMP-2 distant and spatially isolated. The interfacial contact of these two proteins is characterized by two distinct binding regions composed of alternating hydrophobic and hydrophilic interactions. This unique structure provides information for how specificity for noninhibitory MMP/TIMP complex formation is achieved.

Enzyme Linked Immunosorbent Assays (ELISA) for the Quantitative Determination of Human Leukocyte Collagenase and Gelatinase

A competitive and a sandwich enzyme linked immunosorbent assay (ELISA) were developed for human leukocyte collagenase and gelatinase. The competitive assay could detect 0.5 ng collagenase and 0.05 ng gelatinase. The detection limit of the sandwich ELISA was 0.05 ng for collagenase and 0.02 ng for gelatinase. No cross reactivity between human leukocyte collagenase and gelatinase was detected. The sandwich ELISA was used to determine plasma levels of these enzymes. The 90% range for collagenase was between 0 and 50 micrograms/l; the 90% range for gelatinase was between 27 and 94 micrograms/l.