Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin.
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.
Coffman
Lan G
LG
Program in Molecular Medicine, Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
Brown
Julie C
JC
Johnson
David A
DA
Parthasarathy
Narayanan
N
D'Agostino
Ralph B
RB
Lively
Mark O
MO
Hua
Xiaoyang
X
Tilley
Stephen L
SL
Muller-Esterl
Werner
W
Willingham
Mark C
MC
Torti
Frank M
FM
Torti
Suzy V
SV
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D005293:Ferritins / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D047468:Immunoprecipitation; D007249:Inflammation / Q000503:physiopathology; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism*; D016676:Macrophages, Alveolar / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism; D011485:Protein Binding; D013388:Succinimides / Q000737:chemistry; D053802:Tryptases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D019679:Kininogen, High-Molecular-Weight; D013388:Succinimides; C015752:N-chlorosuccinimide; D005293:Ferritins; D009195:Peroxidase; D010196:Pancreatic Elastase; D053802:Tryptases; D015032:Zinc