Fig 1.
Analysis of the human cathepsin G preparation used in the determination of the extended cleavage specificity.
The enzyme had previously been purified from blood neutrophils and the purity of the preparation was determined by separation on SDS-PAGE and visualized with Coomassie Brilliant Blue staining. In panel A commercial preparations of two additional neutrophil proteases N-elastase (NE) and proteinase 3 (P3) were also included in the figure for comparison. Proteinase 3 and hCG were intact after short term storage at +4°C. However, N-elastase showed several degradation products indicating self-cleaving activity. In panel B three additional enzymes, the human mast cell chymase (HC), human thrombin (Th) and human granzyme B (GB) were included as they were used as reference proteases in the chromogenic and recombinant substrate assays.
Fig 2.
A panel of different chromogenic substrates was used to determine the primary specificity of hCG and HC. The panel included different chymase, elastase, tryptase and aspase substrates. The amino acid sequences of the substrates are listed at the left side of the figure. Human thrombin and human granzyme B were included as reference enzymes for tryptase and asp-ase activities, respectively.
Fig 3.
Phage displayed nonamers susceptible to cleavage by hCG after five biopannings.
After the last selection step, phages released by proteolytic cleavage were isolated and the sequences encoding the nonamers were determined. The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. The protein sequences were aligned into a P5-P4´ consensus, where cleavage occurs between positions P1 and P1´. The amino acids are colour coded according to the side chain properties as shown in a separate panel in the bottom of the figure. The sequences with one aromatic amino acid (potential cleavage site) are aligned first, followed by sequences containing two, three or four aromatic amino acids. It is not a perfect alignment but so far the best that has been obtained by any method.
Fig 4.
Distribution of amino acids in positions P4 to P4´ in phage displayed nonamers cleaved by hCG after five biopannings.
Based on the alignment (Fig 3) the percentage of each amino acid present in each position P4 to P4´ as calculated. The amino acids are ordered from left to right: aromatic, aliphatic, hydrophilic, basic (positively charged) and acidic (negatively charged).
Fig 5.
Analysis of the cleavage specificity by the use of recombinant protein substrates.
Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the hCG and HC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His6-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one Bam HI and one SalI site, which are indicated in the bottom of panel A. In panel B a hypothetical cleavage is shown to highlight possible cleavage patterns. Panels C and D shows the cleavage of 4 different substrates by hCG and HC, the HC consensus obtained from phage display, the consensus of a HC double mutant, the dog chymase consensus and the opossum chymase consensus [34, 46, 52]. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of approximately 25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa. In panel E we show the results from scanning of the gels where on e clearly can see the effect of P2´negative charge for both HC and hCG. The gel pictures in panels C and D were densitometrically scanned and the result was presented as two separate diagrams in panel E, one for HC and one for hCG.
Fig 6.
Analysis of the cleavage specificity by the use of recombinant protein substrates.
Panels A and B show the cleavage of two substrates, one containing Phe in the P1 position and one where this residue has been exchanged for a Val residue, thereby the construct does not contain any aromatic amino acids or a Leu serving as a negative control. Panels C and D show the analysis of the chymotrypsinogen-like P1 preference for the two enzymes with substrates having different aromatic amino acids or Leu in the P1 position. Panels E and F show an analysis of the P1´and P2´specificities. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. The gel pictures in panels A, B, C and D were densitometrically scanned and the result was presented as two separate diagrams in panels E and F.
Fig 7.
Analysis of the cleavage specificity by the use of recombinant protein substrates.
Panels A and B show an analysis of the P1´and P2´specificities. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels.
Fig 8.
Analysis of the potential tryptase activity by HC and hCG using recombinant protein substrates.
Using the Phe substrate (Fig 6) as a reference to determine the cleavage rate by HC and hCG on three substrates having Arg, Lys or Asp in the P1 position. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels. The gel pictures in panels A and B were densitometrically scanned and the result was presented as two separate diagrams in panel C, one for HC and one for hCG.
Fig 9.
Analysis of the cleavage specificity by the use of recombinant protein substrates.
An analysis of the effect of changing amino acids in and around the cleavage site on the cleavage by HC and hCG. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels.
Fig 10.
Analysis of the effect of particular amino acids in the P6 position for the cleavage by hCG.
The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in min is also indicated above the corresponding lanes of the different gels.
Fig 11.
Analysis of the effect of human lactoferrin on the activity of hCG.
The effect on the activity by hCG on the HC consensus substrate (VVLFSEVL) was analysed in a cleavage reaction as before (Figs 5–9). In panel A three different concentrations of lactoferrin were used: 25, 250 and 2500μg/ml. A 3–5 fold enhancement was observed at the highest lactoferrin concentration (2500μg/ml) and a 2–3 fold enhancement at the middle concentration (250μg/ml) but no enhancement at the low (250μg/ml) lactoferrin concentration. In panel B, bovine serum albumin (BSA) was used as a control to ensure that the enhancing effect was not simply an effect of higher protein concentration and thereby a stabilizing effect on hCG, but a specific lactoferrin dependent effect. As seen in panel B, no enhancement but instead a slight reduction in the cleavage of the recombinant substrate was observed by the addition of BSA.
Fig 12.
Analysis of the cleavage of a panel of consensus substrates for four human neutrophil proteases.
The cleavage of a number of consensus cleavage sites for four different human neutrophil proteases (N-elastase, hCG, hPR3 and NSP4) were studied with the 2x-Trx system. The ‘A’ consensus sites come from phage display analyses performed in our lab and the substrates B and C comes from a proteomics study by O’Donoghue et al [36]. The substrates originating from the two different methods for N-elastase and proteinase 3 both show very good cleavage, whereas for hCG the consensus sites obtained by the proteomics method shows only minor cleavage, indicating a relatively poor site. No phage display has yet been performed on hNSP4, therefore only a proteomics site was studied where minor cleavage after using a relatively high concentration of the enzyme was seen.