Basic Residues in Azurocidin/HBP Contribute to Both Heparin Binding and Antimicrobial Activity

Abstract

Azurocidin/CAP37/HBP is an antimicrobial and chemotactic protein that is part of the innate defenses of human neutrophils. In addition, azurocidin is an inactive serine protease homolog with binding sites for diverse ligands including heparin and the bovine pancreatic trypsin inhibitor (BPTI). The structure of the protein reveals a highly cationic domain concentrated on one side of the molecule and responsible for its strong polarity. To investigate the role of this highly basic region, we produced three recombinant azurocidin mutant proteins that were altered in either one or both of two clusters of 4 basic residues located symmetrically on each side of a central cleft in the cationic domain. Two of the mutant proteins (Loop 3: R5Q, K6Q, R8Q, and R10Q; Loop 4: R61Q, R62Q, R63Q, and R65Q) exhibited little or no change in heparin and BPTI binding or in antimicrobial function. In contrast, the Loop 3/Loop 4 mutant (R5Q, K6Q, R8Q, R10Q, R61Q, R62Q, R63Q, and R65Q) in which all 8 basic residues were replaced showed greatly decreased ability to bind heparin and to kill Escherichia coli and Candida albicans. Thus, we report that the 8 basic residues that were altered in the Loop 3/Loop 4 mutant contribute to the ability of the wild-type azurocidin molecule to bind heparin and to kill E. coli and C. albicans. Because BPTI binding was comparable in wild-type and Loop 3/Loop 4 mutant protein, we conclude that the same 8 basic residues are not involved in the binding of BPTI to azurocidin, supporting the notion that the binding site for BPTI is distinct from the site involved in heparin binding and antimicrobial activity. Finally, we show that removal of all 4 positively charged amino acids in the 20 -44 azurocidin sequence (DMC1: R23Q,H24S,H32S,R34Q), a region previously thought to contain an antimicrobial domain, does not affect the activity of the protein against E. coli, Streptococcus faecalis, and C. albicans.Azurocidin/CAP37/HBP is an antimicrobial and chemotactic protein (1-7) that forms part of the innate defenses of human neutrophils (8 -11). It has also been reported to play a role in neutrophil-induced increase in vascular permeability (12). The mechanism for these functions is largely unknown. Structurally, azurocidin belongs to the serprocidin family, a group of serine proteases with antimicrobial function but lacking enzymatic activity (13-16). Its three-dimensional structure has been elucidated and shows a highly cationic region made of 16 basic amino acid residues and located at one pole of the molecule (17,18). In addition to its high density of positive charges, the cationic region also contains potential heparin binding consensus sequences that are located in loop areas of azurocidin tertiary structure (4, 17).Several ligands have been shown to bind to azurocidin, including heparin and the serine protease inhibitor BPTI 1 (4,19). In this report, we show that heparin and BPTI bind to distinct regions of the molecule and that these two ligands differentially affect antimicrobial activity. Indeed, binding ...

Proteomic analysis of the peritrophic matrix from the gut of the caterpillar, Helicoverpa armigera

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