Three Wavelength Substrate System of Neutrophil Serine Proteinases

Neutrophil serine proteases, including elastase, proteinase 3, and cathepsin G, are closely related enzymes stored in similar amounts in azurophil granules and released at the same time from triggered neutrophils at inflammatory sites. We have synthesized new fluorescence resonance energy transfer (FRET) substrates with different fluorescence donor–acceptor pairs that allow all three proteases to be quantified at the same time and in the same reaction mixture. This was made possible because the fluorescence emission spectra of the fluorescence donors do not overlap and because the values of the specificity constants were in the same range. Thus, similar activities of proteases can be measured with the same sensitivity. In addition, these substrates contain an N-terminal 2-(2-(2-aminoethoxy)­ethoxy)­acetic acid (PEG) moiety that makes them cell permeable. Using the mixture of these selected substrates, we were able to detect the neutrophil serine protease (NSP) activity on the activated neutrophil membrane and in the neutrophil lysate in a single measurement. Also, using the substrate mixture, we were in a position to efficiently determine NSP activity in human serum of healthy individuals and patients with diagnosed Wegener disease or <i>microscopic polyangiitis</i>

Selection of Effective HTRA3 Activators Using Combinatorial Chemistry

Herein, we report selection, synthesis, and enzymatic evaluation of a peptidomimetic library able to increase proteolytic activity of HtrA3 (high temperature requirement A) protease. Iterative deconvolution in solution of synthesized modified pentapeptides yielded two potent HtrA3 activators acting in the micromolar range (HCOO-CH₂O-C₆H₄-OCH₂-CO-Tyr-Asn-Phe-His-Asn-OH and HCOO-CH₂O-C₆H₄-OCH₂-CO-Tyr-Asn-Phe-His-Glu-OH). Both compounds increased proteolysis of an artificial HtrA3 substrate over 40-fold in a selective manner. On the basis of molecular modeling, the selected compounds bind strongly to the PDZ domain

The crystal structure of SplD demonstrates canonical conformation of the catalytic triad and the oxyanion hole.

<p>(Upper panel) Catalytic triad residues and the main chain fragment constituting the oxyanion hole of SplD (limon) superposed with corresponding residues of chymotrypsin (black). (Lower panel) Electron density (contoured at 1.1σ) around SplD fragment depicted in the upper panel. Red sphere represents a water molecule. Dashed lines represent hydrogen bonds.</p

Substrate specificity of the SplD protease at the P1 subsite.

<p>Substrate preference of SplD at P1 subsite was determined using a positional scanning synthetic combinatorial library (PS-SCL) of a general structure Ac-P4-P3-P2-P1-AMC as described in Materials and Methods. Vertical bars indicate the activity of the enzyme against each tested sub-library (fluorescence of released AMC) normalized to the most active sub-library. Residues fixed at P1 subsite are indicated with the single-letter amino acid code. X indicates randomized substrate position.</p

Putative binding mode of the SplD consensus substrate.

<p>(A) Residues P4 through P1’ of the consensus substrate (blue) docked to SplD (surface model). (B) Schematic representation of interactions between the consensus substrate (thick lines) and SplD (thin lines). Hydrogen bonds are shown as dotted lines.</p

Selection of an efficient fluorescence-quenched substrate of the SplD protease.

<p>Synthetic tetrapeptide substrate libraries of a general structure ABZ-X4-X3-X2-X1-ANB-NH₂ were screened for efficient fluorescence-quenched substrates of SplD as described in Materials and Methods. Vertical bars indicate the activity of the enzyme against a particular sub-library (released fluorescence) normalized to the most active sub-library in each library. Residues fixed at particular subsites (indicated at the top of each panel) are designated with the single-letter amino acid code. X indicates randomized substrate position.</p