Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex

Abstract

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal-catechol-Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal-siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

Figure 1

Determination of the affinity of catechol:iron in complex with Scn-Ngal. (a) Fluorescence quenching analysis of Scn-Ngal with free catechol ligands (L; Top) (b) or with iron^III catechol ligands (FeL₃, Bottom). Symbols show the fluorescence output at 340 nm; lines are fitted using a model constructed with two dissociation constants. Note that iron^III dramatically enhanced the affinity of Scn-Ngal for different catechols. (c). Calculated binding constants for free catechol (L) and iron^III catechol (FeL₃) ligands.

Figure 2

UV-visible spectra of complexes of Scn-Ngal, siderophores, and iron. (a) apo-Scn-Ngal, Ent:iron^III and Scn-Ngal:Ent:iron^III (left) and apo-Scn-Ngal, catechol:iron^III, and Scn-Ngal:catechol:iron^III (right). While ligand-metal charge-transfers between Ent and ironIII (λ_max = 498 nm) were not modified by the addition of Scn-Ngal protein (note red coloration in B, 2 left tubes), catechol:iron^III converted from a FeL complex (blue, λ_max = 575 nm) to a FeL₃ complex (red, λ_max = 498 nm) when bound to Scn-Ngal (b, right tubes). (c) Speciation diagram of catechol:iron^III (10:1) in solution. FeL₂ was the predominant species at pH 7.4. FeL₃ may be observed in more basic conditions. The speciation diagram was calculated in HySS (Hyperquad Simulation and Speciation) based on catechol thermodynamic values.

Figure 3

Scn-Ngal binds to catechol:iron^III as well as to 4-methylcatechol:iron^III. (a, b) The images show close up views of the electrostatic surface of pocket#1 within the calyx of the protein. Positive (blue), neutral (white) and negative (red) charges are shown. Pair-wise alignment on all Cα’s were used to compare the structures. The ligand in molecule A (gray) and in molecule C (yellow) are bound in pocket #1 of the calyx. (c,d) These panels show a side view of each ligand comparing molecule A (grey) and molecule C (yellow). Catechol (c) has rotated 55° towards the outside of the protein. 4-methylcatechol (d) has rotated 10°. Hydroxyls groups facing out of the calyx are potentially protonated or oxidized to form a semi-quinone species. Iron is shown in orange for molecule A and yellow for molecule C in panels a–d. (e, f) 2Fo-Fc electron density map of molecule A for catechol (e) and 4-methylcatechol (f) contoured at 1 sigma. Waters are shown in red, chloride in green, iron in orange, and the molecule in gray.

Figure 4

The formation and distribution of the Scn-Ngal:catechol:Fe complex in vivo. (a) The Scn-Ngal:catechol complex can form in serum and be detected five minutes after i.p. injection of each component. The complex was identified by gel filtration of serum followed by immunoblots and scintillation counting (dark blue line), which revealed Ngal:¹⁴C-catechol:Fe centered at fraction 20 (n = 3 independent fractionations). Also shown is the authentic Scn-Ngal:¹⁴C-catechol:Fe complex formed in vitro (grey line) and serum taken after the injection of free ¹⁴C-catechol (light blue line). (b) The distribution of the Scn-Ngal:¹⁴C-catechol:Fe complex vs free ¹⁴C-catechol was reported as a percentage of the injected ¹⁴C-catechol (n = 4 independent experiments; data represent mean ± s.d.). At 20 and 180 min, ns signifies non-significant differences, *P < 0.05; **P < 0.005; ***P < 10⁻⁴ as assessed by two tailed Students t-test. (c) The complexes, Scn-Ngal:catechol:⁵⁵Fe, citrate:⁵⁵Fe, and transferrin:⁵⁵Fe were recovered at 3 hrs. Whereas Scn-Ngal:catechol:⁵⁵Fe located predominately to the kidney, citrate:⁵⁵Fe and transferrin:⁵⁵Fe located predominantly to the liver or bone marrow (n = 5–7 independent experiments, data represent mean ± s.d., *P < 0.05; **P < 0.005). (d, e) Trafficking of ⁵⁵Fe to the kidney was visualized by radioautography using Ilford K5D emulsion. Note the black silver grains in proximal tubules (Pt) after introduction of (d) Scn-Ngal:catechol:⁵⁵Fe but not after the introduction of (e) citrate:⁵⁵Fe. Glomeruli (G), proximal tubules (Pt), examples of nuclei (arrows) and tubular lumen (L) are indicated (n = 2 independent experiments).

Figure 5

Release of ligands from Scn-Ngal as a result of acidification. (a) Fluorescence titration of Scn-Ngal by catechol:Fe^III, 2,3 DHBA:Fe^III, 3-methycatechol:Fe^III and pyrogallol:Fe^III. Subsequently, upon acidification, the ligands were released and fluorescence returned to baseline. Basification, where relevant, caused rebinding. Note that Scn-Ngal:pyrogallol and 2,3 DHBA complexes required lower pH for dissociation. (b) Low pH released ⁵⁵Fe from Scn-Ngal:catecholate:Fe^III complexes. In this comparison, iron^III loading at pH 7.0 was defined as 100% of the assay. Catechol differed significantly from Ent (P = 0.00012) and pyrogallol (P = 0.0017) at pH 5.5 (n = 4 independent preparations of Scn-Ngal, data represents mean ± s.d., two tailed t-test). (c) The capture of ⁵⁵Fe from the Scn-Ngal:catechol:⁵⁵Fe complex by kidney stromal cells and kidney proximal tubule LLCPK cells in vitro. ⁵⁵Fe uptake was inhibited at 4°C or by bafilomycin, an inhibitor of the vacuolar H⁺ATPase. Data represent mean ± s.d., n =5 independent preparations of Scn-Ngal, and statistical significance (**P < 0.005; ***P < 10⁻⁴) was assessed by two tailed Students t-test comparing uptake at 37°C and 4°C.