Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

Colin Correnti,Brett K Kaiser,Matthew C Clifton,Roland K Strong,Ashok D Bandaranayake,Allyson K Sia,Yohan Suryo Rahmanto,Des R Richardson,Žaklina Kovačević,Kenneth N Raymond,Mario Ruiz,Margaret A Holmes,Jonathan Barasch,Vera Richardson

doi:10.1371/journal.pone.0043696

Abstract

Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.

Highlights

Siderophores are low molecular weight, ferric i‘on-specific chelators that some bacteria use to acquire iron [1]
The solution speciation as a function of pH was calculated for iron complexation with 2,3-DHBA, GA, salicylic acid (SA) and catechol ligands (L) under the conditions used in the fluorescence quenching (FQ) experiments ([Fe3+] = 20 mM, [L] = 60 mM; Figures 2C, 2D, 2E and S2B)
The proposal that GA, or siderophores that incorporate GA substituents, would bind tightly to Scn contradicted a series of studies detailing the recognition mechanism and specificity of Scn [2,3,4,5,7,8,9,13,31,32]

Summary

Introduction

Siderophores are low molecular weight, ferric i‘on-specific chelators that some bacteria use to acquire iron [1]. Scn is observed in serum and urine in sterile kidney diseases and has been shown to be internalized by proximal tubule cells potentially after binding to the megalin receptor complex, leading to iron release from the protein [12,13] In these contexts, Scn enters endosomal compartments via the megalin receptor and passage through these low pH intracellular compartments correlates with iron release. Internalization of Scn was shown to be mediated by a novel receptor, brain-type organic cation transporter (BOCT; SLC22A17 or 24p3R), enabling access of apo-Scn to crucial intracellular iron pools vital for metabolism and proliferation [15]

Methods

Results

Conclusion