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.

Polysaccharide microarray technology for the detection of Burkholderia pseudomallei and Burkholderia mallei antibodies.

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray technology over the current serodiagnosis of the above bacterial infections were discussed.

Monoclonal antibodies passively protect BALB/c mice against Burkholderia mallei aerosol challenge.

Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against Burkholderia mallei, the etiologic agent of glanders, and were shown to be effective in passively protecting mice against a lethal aerosol challenge. The antibodies appeared to target lipopolysaccharide. Humoral antibodies may be important for immune protection against B. mallei infection.

Construction of a rhamnose mutation in Bacillus anthracis affects adherence to macrophages but not virulence in guinea pigs.

Carbohydrate analyses of whole-spore extracts have confirmed the presence of rhamnose in the spore of the fully virulent Ames strain of Bacillus anthracis. A gene cluster containing loci with high homology to the rhamnose biosynthetic genes, rmlACBD, was identified within the B. anthracis chromosome. The first gene of this cluster, rmlA, was inactivated by forming a merodiploid cointegrate using an internal fragment of the gene within the Ames strain of B. anthracis to construct the mutant strain Ames-JAB1. Carbohydrate analysis of spores from this mutant demonstrated the loss of rhamnose. When assaying for spore infection of macrophages, we detected a significant decrease in the recovery with the Ames-JAB1 strain compared to the recovery with the Ames wild-type strain. When pre-treating macrophages with cytochalasin-D, spores of the mutant were further hindered in recovery, indicating that the spores were not able to bind as well to the macrophages. However, in guinea pigs challenge experiments, no difference in virulence was observed between the mutant and wild-type strains. These results suggest that the incorporation of rhamnose into the spore coat of B. anthracis is required for optimal interaction with macrophages but is not required for full virulence in this animal model.

Ferritin binds to light chain of human H-kininogen and inhibits kallikrein-mediated bradykinin release.

Ferritin is an iron-storage protein that exists in both intracellular and extracellular compartments. We have previously identified H-kininogen (high-molecular-weight kininogen) as a ferritin-binding protein [Torti and Torti (1998) J. Biol. Chem. 273, 13630-13635]. H-Kininogen is a precursor of the potent pro-inflammatory peptide bradykinin, which is released from H-kininogen following cleavage of H-kininogen by the serine protease kallikrein. In this report, we demonstrate that binding of ferritin to H-kininogen occurs via the modified light chain of H-kininogen, and that ferritin binds preferentially to activated H-kininogen. We further demonstrate that binding of ferritin to H-kininogen retards the proteolytic cleavage of H-kininogen by kallikrein and its subsequent release of bradykinin from H-kininogen. Ferritin does not interfere with the ability of kallikrein to digest a synthetic substrate, suggesting that ferritin specifically impedes the ability of kallikrein to digest H-kininogen, perhaps by steric hindrance. Based on these results, we propose a model of sequential H-kininogen cleavage and ferritin binding. These results are consistent with the hypothesis that the binding of ferritin to H-kininogen may serve to modulate bradykinin release.