Figure 2
Activity of KLK7 and mKLK7. (a) Coomassie-stained 1% casein zymography: cleared area at approximately 25 kDa shows digestion of casein by active KLK7 while mKLK7 has not digested casein, suggesting no activity. (b) Activity with a KLK7-specific peptide substrate MeO-Suc-Arg-Pro-Tyr-MCA: time versus absorbance (corrected mean ± SD) plot showing activity of KLK7 compared to no activity with mKLK7 and substrate only control (n = 6; mean ± SD). (c) Activity against a KLK7 protein substrate fibronectin: KLK7 and mKLK7 incubated with fibronectin (KLK7/FN: 1/10 molar ratio) visualised by silver-stained SDS-PAGE. Black arrow heads indicate KLK7-generated cleavage fragments. No cleavage was observed for mKLK7. (d) For KLK7 and (f) mKLK7 the percentage amino acid occurrences in P6-P6′ derived from both libraries were calculated and are shown in the form of a two-dimensional heat map. Both libraries confirm the predominant KLK7 specificity for Y, L and F in P1. The mKLK7-treated (f) tryptic and GluC libraries confirm the mKLK7 specificity for Y in P1. White line depicts the scissile peptide bond between P1 and P1′. (e) KLK7 and (g) mKLK7 cleavage sites derived from both libraries are displayed as icelogos by analysing their multiple sequence alignments. The height of the single amino acid residue (in bits) reflects its occurrence rate for each position in P10-P10′. Amino acids are coloured according to their physico-chemical properties.
