Influence of Charge Distribution at the Active Site Surface on the Substrate Specificity of Human Neutrophil Protease 3 and Elastase

Abstract

The biological functions of human neutrophil protease 3 (Pr3) differ from those of neutrophil elastase despite their close structural and functional resemblance. Although both proteases are strongly cationic, their sequences differ mainly in the distribution of charged residues. We have used these differences in electrostatic surface potential in the vicinity of their active site to produce fluorescence resonance energy transfer (FRET) peptide substrates for investigating individual Pr3 subsites. The specificities of subsites S5 to S3 were investigated both kinetically and by molecular dynamic simulations. Subsites S2, S1, and S2 were the main definers of Pr3 specificity. Combinations of results for each subsite were used to deduce a consensus sequence that was complementary to the extended Pr3 active site and was not recognized by elastase. Similar sequences were identified in natural protein substrates such as NFB and p21 that are specifically cleaved by Pr3. FRET peptides derived from these natural sequences were specifically hydrolyzed by Pr3 with specificity constants k cat /K m in the 10 6 M ؊1 s ؊1 range. The consensus Pr3 sequence may also be used to predict cleavage sites within putative protein targets like the proform of interleukin-18, or to develop specific Pr3 peptide-derived inhibitors, because none is available for further studies on the physiopathological function of this protease.Protease 3 (Pr3) 3 was initially described as an elastin-degrading protease whose structural and functional properties are similar to those of human neutrophil elastase (HNE) (1, 2). Pr3 is stored as an active enzyme within the primary granules of human neutrophils, together with HNE and cathepsin G, and is released from activated cells as a free or membrane-bound protease (3, 4). Its three-dimensional structure is very similar to that of HNE, with which its sequence is 57% identical (5). Pr3 and HNE also have extended interaction sites that greatly influence substrate binding and specificity (5). The active sites of the two proteases are also very similar, and both preferentially accommodate small aliphatic residues in their S1 subsite 4 (6, 7). This is why there was no substrate that discriminated between the two proteases until fluorescence resonance energy transfer (FRET) peptides became available. These can be used to study the specificity on both sides of the cleavage site (8, 9). However, kinetic and structural studies have shown that the substrate binding site in Pr3 is more polar, and some subsites are more restrictive than those in HNE (5, 10). This partly explains why Pr3, but not HNE, is not inhibited by the low molecular weight inhibitor SLPI present in the upper airways, even though both proteases are inhibited by the ␣1-protease inhibitor (␣1-Pi) in lung secretions (6,11).Although the primary function of Pr3 and HNE is commonly thought to be the intralysosomal degradation of phagocytized microorganisms, both also act extracellularly to break down matrix proteins (6, 12, 13), release cytokines from their ...

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

“…4B). In agreement with structural data, HNE and PR3 preferentially accommodate small hydrophobic residues (Val, Cys, Ala, Met, Ile) (Brubaker et al, 1992;Koehl et al, 2003;Korkmaz et al, 2007;Wysocka et al, 2007) in the S1 pocket, whereas CG has both a chymotrypsin-like and a trypsin-like specificity, permitting both large, hydrophobic P1 residues (Phe, Leu, Met) and positive Lys or Arg residues (Tanaka et al, 1985). The S2 subsites of HNE and CG are bordered by Phe215, Leu99, and the flat side of the imidazole ring of the catalytic triad His57, which is quite hydrophobic.…”

“…Indeed, most commercially available chromogenic and/or fluorogenic substrates used to measure HNE and CG display a proline at the P2 position. Because of the presence of Lys99, PR3 preferentially accommodates a negatively charged residue at P2 (Hajjar et al, 2006;Korkmaz et al, 2007). The S3 subsite of CG is formed by a Lys at position 192, which favors interaction with an acidic P3 residue (Tanaka et al, 1985).…”

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

“…Substrate hydrolysis was followed by measuring the fluorescence at ex ϭ 320 nm and em ϭ 420 nm, as reported earlier (18,21).…”

Mapping of Conformational Epitopes on Human Proteinase 3, the Autoantigen of Wegener’s Granulomatosis

“…The only exception is the very conservative Ile-208 substitution on the back side of the hPR3 molecule by a Val, which is one methyl group shorter. Active site residues K99, D61, and R143 implicated in the substrate binding of hPR3 and conferring its specificity are not substituted on gPR3 (24)(25)(26). Fig.…”