Biol. Chem., Vol. 389, pp. 955–962, July 2008 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.101 2008/318 Article in press - uncorrected proof Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis Mariusz Olczak*, Katarzyna Indyk and Teresa Olczak Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland * Corresponding author e-mail: Mariusz.Olczak@biotech.uni.wroc.pl Abstract Azurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed in Sf9 insect cells possessing the reconstituted His41-Asp89- Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8–9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C- terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity. Keywords: azurocidin; catalytic triad; insect cell expression system; proteolytic activity; site-directed mutagenesis. Introduction Neutrophils provide a primary form of defense against various infectious agents by releasing oxidants and proteases. The azurophil granules of human neutrophils contain a family of proteins with structural homology to serine proteases termed serprocidins (Campanelli et al., 1990; Gabay, 1994). These proteases are thought to be implicated in the killing and digestion of microbes and the destruction of extracellular matrix proteins (Chertov et al., 2000). Apart from targeting components of extra- cellular matrix, a variety of cell surface ligands, soluble proteins, and a number of important adhesion molecules are also susceptible to their activity (Wiedow and Meyer- Hoffert, 2005; Chua and Laurent, 2006). There is a grow- ing body of evidence implicating neutrophilic proteases to play important roles in the pathophysiology of different diseases, such as rheumatoid arthritis, respiratory dis- tress syndromes, inflammatory bowel disease, Papillon- Lefe `vre syndrome, Wegener’s granulomatosis, congenital neutropenia (Henson and Johnston, 1987; Weiss, 1989; Wiedow and Meyer-Hoffert, 2005). Human azurocidin, also termed heparin-binding pro- tein (HBP) or cationic antimicrobial protein (CAP37), is homologous to human neutrophil elastase (45%), pro- teinase 3 (42%), and cathepsin G (35%), but despite these similarities, it is devoid of proteolytic activity due to a substitution of His and Ser residues in the active site. Azurocidin is a multifunctional protein originally iso- lated from human neutrophils (Shafer et al., 1984), but now known to be also induced in endothelial cells (Lee et al., 2002), corneal epithelial cells (Ruan et al., 2002), and smooth muscle cells (Gonzalez et al., 2004) in response to inflammatory mediators, such as lipopoly- saccharide and cytokines or growth factors. Originally identified for its antimicrobial activity, the protein is known to exert a variety of immunoregulatory effects on host cells (reviewed in Watorek, 2003). Azurocidin stim- ulates contraction of fibroblasts and endothelial cells (Ostergaard and Flodgaard, 1992), activates endothelial protein kinase C (Pereira et al., 1996b), upregulates adhe- sion molecules on endothelial cells (Lee et al., 2003), increases macromolecular efflux in microvessels (Gautam et al., 2001), serves as a chemoattractant for monocytes (Pereira et al., 1990; Chertov et al., 1997) and microglia (Pereira et al., 2003), and regulates the secretion of cyto- kines from monocytes and microglia (Rasmussen et al., 1996; Pereira et al., 2003). Several reports also suggest that azurocidin may be associated with diseases, such as atherosclerosis (Lee et al., 2002) and Alzheimer’s disease (Pereira et al., 1996a). Serprocidins are synthesized as inactive precursors, but after processing, they are stored in azurophil granules as active enzymes (Lindmark et al., 1990; Salvesen and Enghild, 1990, 1991; Gullberg et al., 1995; Rao et al., 1996). Neutrophil elastase, cathepsin G, and proteinase 3 are synthesized as preproproteins with a signal sequence which is cleaved off immediately after trans- lation/insertion into endoplasmic reticulum (Lindmark et al., 1990, 1999; McGuire et al., 1993; Rao et al., 1996; Gullberg et al., 1997). Further removal of the dipropeptide allows folding of the N-terminal isoleucine into the hydro- phobic interior, the substrate-binding pocket becomes available, and a mature enzymatically active protein is generated (Lindmark et al., 1990; Murakami et al., 1995; Fujinaga et al., 1996; Rao et al., 1996; Gullberg et al., 1997). Native proforms of cathepsin G, neutrophil elas- tase, and proteinase 3 also show cleavage of C-terminal propeptides concurrently with enzymatic activation. Neu- trophil elastase shows processing of a 20-amino acid C-terminal propeptide (Salvesen and Enghild, 1990; Takahashi et al., 1988), whereas procathepsin G has a Brought to you by | Universität Osnabrück Authenticated Download Date | 5/26/15 2:01 PM