Submitted on August 23, 2007
Accepted on January 10, 2008
Cleavage of High Molecular Weight Kininogen by Elastase and Tryptase is Inhibited by Ferritin
Lan G. Coffman1, Julie C. Brown2, David A. Johnson3, Narayanan Parthasarathy2, Ralph B. D'Agostino Jr.4, Mark O. Lively5, Xiaoyang Hua6, Stephen L. Tilley7, Werner P. Muller-Esterl8, Mark C. Willingham9, Frank M. Torti10, and Suzy V Torti11*
1 Program in Molecular Medicine, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States
2 Biochemistry, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States
3 Biochemistry and Molecular Biology, East Tennessee State University, Johnson City, Tennessee, United States
4 Public Health Sciences, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States; Cancer Center, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States
5 Biochemistry, Wake Forest University School of Medicine, Winston Salem, North Carolina, United States
6 Medicine, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, United States
7 Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
8 Inst Biochem, Johann Wolfgang Goethe U, Frankfurt, Germany
9 Pathology, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States; Cancer Center, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States
10 Cancer Biology, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States; Cancer Center, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States
11 Biochemistry, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States; Cancer Center, Wake Forest University Health Sciences, Winston Salem, North Carolina, United States
* To whom correspondence should be addressed. E-mail: storti{at}wfubmc.edu.
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 pro-inflammatory peptide bradykinin (BK) and other biologically active products, such as 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. Immunopreciptiation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134nM. 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. Further, 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.
