Domain 5 of High Molecular Weight Kininogen Is Antibacterial

Abstract

Antimicrobial peptides are important effectors of the innate immune system. These peptides belong to a multifunctional group of molecules that apart from their antibacterial activities also interact with mammalian cells and glycosaminoglycans and control chemotaxis, apoptosis, and angiogenesis. Here we demonstrate a novel antimicrobial activity of the heparin-binding and cell-binding domain 5 of high molecular weight kininogen. Antimicrobial epitopes of domain 5 were characterized by analysis of overlapping peptides. A peptide, HKH20 (His 479 -His 498 ), efficiently killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and the Gram-positive Enterococcus faecalis. Fluorescence microscopy and electron microscopy demonstrated that HKH20 binds to and induces breaks in bacterial membranes. Furthermore, no discernible hemolysis or membrane-permeabilizing effects on eukaryotic cells were noted. Proteolytic degradation of high molecular weight kininogen by neutrophil-derived proteases as well as the metalloproteinase elastase from P. aeruginosa yielded fragments comprising HKH20 epitopes, indicating that kininogen-derived antibacterial peptides are released during proteolysis.The innate immune system, based on antimicrobial peptides (AMP), 2 provides a rapid and nonspecific response against potentially invasive pathogenic microorganisms. AMPs, first isolated from human leukocyte extracts by Zeya and Spitznagel in 1963 (1), were subsequently discovered in invertebrates (2) and cold-blooded vertebrates (3). At present, over 880 different AMPs have been identified in eukaryotes. During recent years it has become increasingly evident that many antimicrobial peptides are multifunctional (4, 5). They are found to mediate chemotaxis (defensins, LL-37) (6 -8), apoptosis (lactoferricin, LL-37) (9 -11), and angiogenesis (PR-39, LL-37) (12, 13). Conversely, molecules previously not considered as AMPs, including proinflammatory and chemotactic chemokines (14), neuropeptides (15-19), and peptide hormones (16,20), have recently been found to exert antibacterial activities. The proinflammatory, chemotactic, and anaphylatoxic peptide C3a, generated during activation of the complement system, displays potent antibacterial effects (21). Many AMPs, by virtue of their cationicity and amphipathicity, also interact with heparin (22, 23).High molecular weight kininogen (HMWK) is a multifunctional 120-kDa glycoprotein found in plasma (ϳ80 g/ml) (24) and in ␣-granules of platelets (25). The protein is composed of six domains, each having different properties and specific ligands (24). Domains D1-D3 have a cystatin-like structure, and the two latter domains serve as specific inhibitors of cysteine proteinases such as cathepsins and calpains. The D4 domain contains the bradykinin sequence, which is released by plasma kallikreins during contact activation (24, 26). Biologically active kinins can also be generated by the cooperative action of mast cell tryptase and neutrophil elastase (27). Thus, similar to complement degrad...

Proteolytic Activation Transforms Heparin Cofactor II into a Host Defense Molecule

“…2B). These results were similar to the bacterial destruction caused by membrane-acting antimicrobial peptides (12,14,15,22). Correspondingly, studies using the impermeant fluorescent probe FITC demonstrated that only HCIIa caused membrane permeabilization of bacterial cells (Fig.…”

“…For instance, although ATIII levels are reduced in almost all conditions associated with disseminated intravascular coagulation, reduced plasma levels of HCII are primarily detected during infection (8)(9)(10)(11). Increasing evidence pointing toward a close link between hemostasis, proteolysis, and host defense (12)(13)(14)(15)(16)(17) raised the hypothesis that HCII could play a role in host defense against infections.…”

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

“…Microbicidal Potency and Proteolytic Stability-Focusing on two kininogen-derived peptides, GKHKNKGKKNGKHN-GWK (GKH17) and HKHGHGHGKHKNKGKKN (HKH17) (14), hydrophobic end tags were investigated concerning effects of the chemical nature, length, and position of the tags on bactericidal and cytotoxic properties of these peptides. As can be seen in Fig.…”

“…Because cholesterol should preclude membrane tag insertion (25), selectivity between bacteria and eukaryotic cells could also be expected. In the present investigation, this concept was investigated with a focus on previously identified highly charged AMPs derived from kininogen, interesting from a therapeutic perspective because of their ability to withstand infection-related proteolysis (14), another key defense and resistance mechanism of bacteria against AMPs (6).…”

“…From a therapeutic perspective, AMPs should ideally rupture bacterial walls specifically, leaving (human) eukaryotic cells unaffected. Approaches for reaching this include quantitative structure-activity relationship in combination with directed amino acid modifications (9 -12) and identification of selective AMPs generated through infection-related proteolysis of endogenous proteins (13)(14)(15).…”