Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

Abstract

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45-75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.

Regulatory effects of ferritin on angiogenesis

“…We have shown that ferritin retards (but does not entirely prevent) the production of HKa at sites of inflammation (17). Here, we demonstrate that the HKa produced despite this first ferritin blockade is susceptible to a second level of inhibition, in which ferritin directly blocks the activity of HKa.…”

“…Ferritin exhibits specific and saturable binding to HK, with a K d of Ϸ140 nM (17). Binding of ferritin to HK inhibits the cleavage of HK by kallikrein, thus reducing production of bradykinin (16) and its attendant proinflammatory effects.…”

“…Binding of ferritin to HK inhibits the cleavage of HK by kallikrein, thus reducing production of bradykinin (16) and its attendant proinflammatory effects. In addition, HK and ferritin colocalize at sites of inflammation, where ferritin inhibits the cleavage of HK by tryptase and elastase (17).…”

Neutrophil Elastase Promotes Interleukin-1β Secretion from Human Coronary Endothelium

“…NE Activity-NE activity was measured spectrophotometrically using a highly specific synthetic substrate (elastase substrate I, MeOSuc-APV-pNA, 100 M) as described in detail previously (14,23). Briefly, samples (supernatant and lysate) were added to assay buffer (0.45 Tris base and 2 M NaCl (pH 8.0)) containing elastase substrate I for 6 h. The rate of substrate cleavage was measured using a plate reader (Thermo Scientific) at 410 nm.…”

The many faces of the octahedral ferritin protein

“…Ferritin is involved in inflammation, cellular development, neurological development and angiogenesis (Wang et al 2010;Shi et al 2008;Alkhateeb and Connor 2010;Coffman et al 2008;Parthasarathy et al 2002). In addition, ferritin is used as a biomarker for diseases (Beard et al 2006).…”