Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre‐elafin (trappin‐2) expressed in Pichia pastoris

Abstract

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast a-factor cDNA and expressed in the Pichia pastoris yeast expression system. Fulllength elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fastacting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k ass values of 2-4 · 10 6 M )1

EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

“…Oxidation of the Met-358 at position P1 of the reactive inhibitory loop of ␣ 1 -PI (Rosenberg et al, 1984) and of the Met-73 at P1Ј of SLPI (HeinzelWieland et al, 1991) and the Met 25 at P1Ј of elafin (Zani et al, 2004) dramatically impairs their antielastase activities, and oxidized forms of ␣ 1 -PI and SLPI are more sensitive to proteolysis than are the native inhibitors (Rapala-Kozik et al, 1999). Thus, oxidation and proteolysis may work together to promote local inflammation.…”

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

“…The molecule displays a compact structure maintained by four conserved disulfide bridges characteristic of WAP (whey acidic protein) family and shares 40% sequence identity with SLPI. In addition to its ability to inhibit porcine pancreatic elastase, elafin is a potent inhibitor of two neutrophil serine proteases, neutrophil elastase (NE) and proteinase 3 (1,2), and is therefore thought to protect tissue from degradation by these enzymes. Elafin is released by proteolytic cleavage from a larger molecule called trappin-2 or pre-elafin, which possesses at the N terminus of the whey acidic protein domain a cementoin domain containing several motifs having the consensus sequence GQDPVK that can act as transglutaminase substrate, allowing the cross-linking of the inhibitor to extracellular matrix proteins (3)(4)(5).…”

“…Elafin is a cationic 6-kDa non-glycosylated serine protease inhibitor belonging to the chelonianins, a distinct family of the canonical inhibitors also including secretory leukoprotease inhibitor (SLPI) 2 . It is also known as SKALP (skin-derived antileukoprotease) or ESI (elastase-specific inhibitor).…”

Periodontal Disease and Gingival Innate Immunity – Who Has the Upper Hand?

“…ELAFIN, also known as skin-derived antileukoproteinase (SKALP), is expressed in human epithelia of the tongue, palate, lingual tonsils, pharynx as well as gingiva (Molhuizen and Schalkwijk 1995). ELAFIN has been shown to inhibit neutrophil elastase and proteinase 3, thus has a role in protecting tissue from degradation by the neutrophil enzymes (Ying and Simon 1993;Zani, Nobar et al 2004). ELAFIN and SLPI are chelonianin family of serine protease inhibitors and share 40% sequence identity (Ying and Simon 1993;Zani, Nobar et al 2004;Guyot, Butler et al 2008).…”

“…ELAFIN has been shown to inhibit neutrophil elastase and proteinase 3, thus has a role in protecting tissue from degradation by the neutrophil enzymes (Ying and Simon 1993;Zani, Nobar et al 2004). ELAFIN and SLPI are chelonianin family of serine protease inhibitors and share 40% sequence identity (Ying and Simon 1993;Zani, Nobar et al 2004;Guyot, Butler et al 2008). Both SLPI and ELAFIN have antimicrobial activity against Gram-positive as well as Gramnegative pathogens (Sallenave, Cunningham et al 2003;McMichael, Maxwell et al 2005;King, Wheelhouse et al 2009).…”