Cellular Localization of Membrane-type Serine Protease 1 and Identification of Protease-activated Receptor-2 and Single-chain Urokinase-type Plasminogen Activator as Substrates

Abstract

Membrane-type serine protease 1 (MT-SP1) was recently cloned, and we now report its biochemical characterization. MT-SP1 is predicted to be a type II transmembrane protein with an extracellular protease domain. This localization was experimentally verified using immunofluorescent microscopy and a cell-surface biotinylation technique. The substrate specificity of MT-SP1 was determined using a positional scanning-synthetic combinatorial library and substrate phage techniques. The preferred cleavage sequences were found to be (P4-(Arg/Lys)P3-(X)P2-(Ser)P1-(Arg)P1-(Ala)) and (P4-(X)P3-(Arg/Lys)P2-(Ser)P1(Arg) P1(Ala)), where X is a non-basic amino acid. Protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator are proteins that are localized to the extracellular surface and contain the preferred MT-SP1 cleavage sequence. The ability of MT-SP1 to activate PARs was assessed by exposing PAR-expressing Xenopus oocytes to the soluble MT-SP1 protease domain. The latter triggered calcium signaling in PAR2-expressing oocytes at 10 nM but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 nM. Singlechain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 nM), but plasminogen was not cleaved under similar conditions. The membrane localization of MT-SP1 and its affinity for these key extracellular substrates suggests a role of the proteolytic activity in regulatory events.

3-Bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate inhibits cancer cell invasion in vitro and tumour growth in vivo

“…Consequently, further experimental investigations are required to determine the exact mechanism of action of the bromo coumarin derivative and its biological target. For example, the recently described MT-SPS which are membrane-bound serine proteases implicated in tumorigenesis are possible targets for protease inhibitors like coumarins (Takeuchi et al, 2000;Hooper et al, 2001). In the light of our in vivo and in vitro results, the bromo coumarin derivative appears to be very promising as potential antitumoral agent.…”

Emerging roles of serine proteinases in tissue turnover in arthritis

“…Thus, increased expression of serine proteinases capable of activating PARs (Figure 1) has the potential to exacerbate inflammation. For example, PAR-2 can be activated by trypsin, mast cell tryptase (163), tissue factor-factor VIIa and factor Xa (15), some kallikreins (164), PR3 (165), and matriptase (166).…”

In vitro characterization of TMPRSS2 inhibition in IPEC-J2 cells

“…All TTSPs contain six conserved cysteine residues within their catalytic domain, which form three intradomain disulfide bonds as known for all serine proteases of the S1 fold. TTSPs have high affinity towards substrates containing an Arg residue in the P1 position compared to Lys containing sequences and they may be activated by other members of the family or via intermolecular autoactivation, as shown in vitro for HAT, matriptase, matriptase-2 and TMPRSS2 [3][4][5][6][7] . After activation, the C-terminal protease domain remains covalently linked to N-terminal located domains by a conserved disulfide bond and can develop their activity in cellular compartments or on the cell surface 8 .…”