Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Rescue of LEKTI processing in furin-complemented furin-deficient CHO cells
  pEFDEST51-SPINK5_fl was used to transfect furin-deficient CHO cells stably re-transfected with a murine cDNA of furin. Extracellular extracts was analysed in Western blot using αD1-D6 and αD8-D11 LEKTI antibodies. The same LEKTI fragments are detected in the extracellular fraction of furin-complemented furin-deficient CHO cells as in wild type CHO cells. Moreover, using αD1-D6 antibody, in addition to the expected 10-, 13- and 20 kDa, a band at 15 kDa is also detected. A band at the same molecular weight can be detected in NHK extracellular fraction as well as human epidermis, suggesting that LEKTI processing is almost complete inside these cells, possibly due to higher amount of furin in complemented furin-deficient CHO cells.
• Supplemental Figure 2 - Presence of disulfide bridges in Origami-produced LEKTI fragments
  Disulfide status of rLEKTI fragments (D1, D5, D6, D8-D11) expressed in bacterial cells were examined using SDS-PAGE under reducing condition and non reducing condition.
  Samples in lanes 1, 3, 5, 7 were prepared under nonreducing condition; samples in lanes 2, 4, 6, and 8 were prepared under reducing conditions (5% β-mercaptoethanol, 95°C heat for 10 minutes). Proteins were visualized by Coomassie brilliant blue R-250 staining. Positions of molecular mass markers are noted to the left of the gels.
  After Coomassie blue staining, a single band was observed at the expected molecular weight with a slight difference between reducing and non reducing conditions. This is consistent with the fact that the recombinant LEKTI fragments expressed in the Origami bacteria contained intramolecular disulfide bonds. In addition, no higher molecular band representing rLEKTI intermolecular complexes was visible in the non reducing SDS-PAGE.
• Supplemental Figure 3 - Effect of pH on KLK5 and KLK7 activity and LEKTI inhibition
  KLK5 or KLK7 were incubated in absence or presence of D8-D11 LEKTI for 5 min at pH 7.5, 6.5, 5.5 or 4.5. The proteinase activity was initiated by adding the appropriate synthetic substrate, and the activity of free proteinase was determined spectrophotometrically at 405 nm by monitoring the release of p-nitrophenol. All time courses were performed at 25ºC, during 15 min, in duplicate. Reaction velocities were linear over the course of the reaction. The activities were represented as a pourcentage of the maximal activity (pH7.5 without inhibitor).
  The proteinase activities decrease following the acidification of buffer but remain detectable at pH 4.5. Inhibition by LEKTI D8-D11 is efficient until pH7.5 for KLK5 and pH 6.5 for KLK7 and is strongly diminished under.