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

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.

Full text

Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis
Through Gentisic Acid Mediated Iron Withdrawal in
Hematopoietic Cell Lines
Colin Correnti
1.
, Vera Richardson
2.
, Allyson K. Sia
3.
, Ashok D. Bandaranayake
4
, Mario Ruiz
5
, Yohan
Suryo Rahmanto
2
,Z
ˇaklina Kovac
ˇevic
´
2
, Matthew C. Clifton
6,8
, Margaret A. Holmes
1
, Brett K. Kaiser
1
,
Jonathan Barasch
7
, Kenneth N. Raymond
3
, Des R. Richardson
2
*, Roland K. Strong
1
*
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 2Iron Metabolism and Chelation Program, Discipline
of Pathology and Bosch Institute, University of Sydney, NSW, Australia, 3Department of Chemistry, University of California, Berkeley, California, United States of America,
4Department of Immunology, University of Washington, Seattle, Washington, United States of America, 5Instituto de Biologı
´a y Gene
´tica Molecular, Universidad de
Valladolid, UVa-CSIC, Valladolid, Spain, 6Emerald Biostructures, Bainbridge Island, Washington, United States of America, 7College of Physicians and Surgeons of
Columbia University, New York, New York, United States of America, 8Seattle Structural Genomics Center for Infectious Diseases (SSGCID), Washington, United States of
America
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.
Citation: Correnti C, Richardson V, Sia AK, Bandaranayake AD, Ruiz M, et al. (2012) Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid
Mediated Iron Withdrawal in Hematopoietic Cell Lines. PLoS ONE 7(8): e43696. doi:10.1371/journal.pone.0043696
Editor: Dhyan Chandra, Roswell Park Cancer Institute, United States of America
Received April 30, 2012; Accepted July 24, 2012; Published August 21, 2012
Copyright: ß2012 Correnti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by National Institutes of Health grants AI117448 (Dr. Raymond), AI59432 (Dr. Strong), DK55388 and DK58872 (Dr. Barasch), and
National Institute of Allergy and Infectious Disease Federal Contract No. HHSN272200700057C (SSGCID); the Emerald Foundation and the March of Dimes(Dr.
Barasch); National Health and Medical Research Council of Australia Senior Principal Research Fellowship 571123 and Project grant 512408 (Dr. Richardson); and a
Cancer Institute New South Wales Early Career Development Fellowship (Dr. Rahmanto). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: Dr. Clifton is employed by a commercial company, ‘‘Emerald Biostructures’’, but his participation in this project is solely through Emerald
Biostructures role in the Seattle Structural Genomics Center for Infectious Diseases (SSGCID), which is funded through National Institute of Allergy and Infectious
Disease Federal Contract No. HHSN272200700057C. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: d.richardson@sydney.edu.au (DRR); rstrong@fhcrc.org (RKS)
.These authors contributed equally to this work.
Introduction
Siderophores are low molecular weight, ferric i‘on-specific
chelators that some bacteria use to acquire iron [1]. The
mammalian antibacterial protein siderocalin (Scn), also known as
lipocalin 2 (Lcn2), neutrophil gelatinase-associated lipocalin
(NGAL) or 24p3, functions by sequestering iron as bacterial
siderophore complexes [2,3]. Scn tightly binds a variety of
bacterial siderophores including many catechol-based compounds
from enteric bacteria, such as enterobactin (Ent; equilibrium
dissociation constant (K
D
) = 0.460.1 nM), but does not bind many
hydroxamate-based siderophores, such as desferrioxamine (DFO;
Figure 1A) [2–5]. The importance of Scn in antibacterial defense
was demonstrated with Scn knock-out mice, which are profoundly
susceptible to bacterial infections [2,6]. Bacterial siderophores with
modifications that ablate binding to Scn, so-called ‘stealth’
siderophores, allow pathogens to evade the Scn defense, permit-
ting acquisition of iron during infection [2,4,5,7–9].
Scn has also been implicated in cellular processes unrelated to
antibacterial activities, including apoptosis and differentiation,
reviewed in [10,11]. 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. To provide a rationale
for its pleiotropic affects on apoptosis, siderophore-free Scn (apo-
Scn) was reported to be secreted in response to cytokine
withdrawal or tumorigenesis and internalized by a receptor-
mediated process to alternately sequester and export intracellular
iron, driving apoptosis through autocrine, paracrine, or exocrine
mechanisms [14,15]. This latter hypothesis was based on
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observations that: i) Scn transcription was maximally induced in
murine hematopoietic cell lines undergoing IL-3 withdrawal-
induced apoptosis; ii) conditioned medium from apoptotic cells
containing secreted Scn, or addition of exogenous Scn, could
induce apoptosis in susceptible cells even in the presence of IL-3;
and iii) ectopic Scn expression conferred on cells the ability to
import or export iron, in the latter case driving apoptosis by
depletion of this essential nutrient [14,15]. Internalization of Scn
was shown to be mediated by a novel receptor, brain-type organic
cation transporter (BOCT; also SLC22A17 or 24p3R), enabling
access of apo-Scn to crucial intracellular iron pools vital for
metabolism and proliferation [15].
Iron transport by Scn requires the presence of a siderophore,
since Scn has no measurable affinity for iron alone [3]. Bacterial
catecholate siderophores, like Ent and its substituent, 2,3-
dihydroxybenzoic acid (2,3-DHBA; Figure 1B), are unlikely to
fulfill the requirements of an iron delivery pathway because iron is
not released from Scn/siderophore/iron complexes (holo-Scn)
until acidification below pH 4, which is not readily achieved in
most cellular compartments, such as endocytic vesicles [7]. Two
candidate endogenous siderophores have been proposed: i) simple
catechols, including catechol itself (1,2-dihydroxybenzene), medi-
ating iron delivery [13], and ii) compounds that include gentisic
acid (GA; 2,5-dihydroxybenzoic acid) substituents mediating
cellular iron efflux [16]. Bao and coworkers reported that free
catechol binds poorly to Scn (K
D
= 0.2060.06 mM), but catechol/
iron complexes bind tightly (K
D1
= 2.160.5 nM/
K
D2
= 0.460.2 nM), and that: i) catechol can mediate iron
transport in the proximal kidney through Scn complexes
potentially by the megalin receptor complex; ii) iron from Scn/
Figure 1. Steric clashes imposed by the Scn calyx preclude binding of ferric SA and GA complexes. (A) Hexadentate siderophore
structures are shown with iron liganding atoms colored blue. (B) Structures of 2,3-DHBA, GA (2,5-DHBA), 3,4-DHBA and SA (2-hydroxybenzoic acid)
are shown in the left column and complexes with iron in the center column (only two of three bidentate groups are shown for clarity). The Scn calyx
is represented at top by a gray cylinder and the size constraint imposed by the calyx diameter is represented by dashed lines, schematically showing
clashes with all iron complexes except 2,3-DHBA. (C) A section of the Scn/carboxymycobactin complex structure (PDB accession code 1X89) showing
a GA moiety superimposed on the phenolate ring of carboxymycobactin. The steric clash of the 5-OH is indicated by penetrating the molecular
surface of Scn (dashed red circle) and the short distance to neighboring atoms (green line).
doi:10.1371/journal.pone.0043696.g001
Siderocalin/24p3/Lcn2 Does Not Drive Apoptosis
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catechol complexes is released at pHs below 6; and iii)
Fe(catechol)
x
can be directly visualized by X-ray crystallography
bound in the Scn ligand-binding site or ‘calyx’. Devireddy and
coworkers reported that: i) GA can be isolated from conditioned
media; ii) binds to Scn tightly in the absence of iron (K
D
= 12 nM);
iii) supports iron transport by Scn in vitro; and iv) is synthesized
endogenously by a cytosolic type II R-b-hydroxybutyrate dehy-
drogenase (DHRS6) also known as BDH2.
Identification of GA alone or as a substituent of siderophores
enabling Scn-mediated iron transport was surprising as GA and
GA-based siderophores obligately interact with iron in a manner
(salicylate-mode) that, either alone, but especially in combination
with 5-OH groups, precludes binding to Scn (Figures 1B and 1C).
In agreement with this prediction, we show here that both GA and
a synthetic tris-GA analog (TRENGEN; Figures 1A and S1) bind
weakly to Scn either in the presence or absence of iron. Like
salicylic acid (SA; Figure 1B), we also show that GA on its own
does not efficiently form iron complexes at neutral pH. These
results show that GA does not meet the necessary biochemical
criteria required for an endogenous siderophore or siderophore
substituent enabling Scn-mediated iron transport.
Due to our failure to confirm the ability of GA to serve as a
siderophore or to bind to Scn, we then also re-examined the
reported roles of Scn in iron transport and apoptosis. In contrast to
previous results using identical methodologies [14,15], we
demonstrated that HeLa cells ectopically expressing BOCT did
not induce cellular iron efflux via Scn. Moreover, we showed Scn
did not drive apoptosis in hematopoietic cell lines (FL5.12 and
32D.3) reported to be susceptible to this protein, even when Scn
was added at levels exceeding those used previously by 200-fold.
We also generated stable transductants, secreting high levels of
Scn, in 32D.3 and FL5.12 cells without decreasing viability.
Finally, we were unable to detect Scn protein secreted from
FL5.12 or 32D.3 cells undergoing IL-3 withdrawal-induced
apoptosis. We conclude: i) GA cannot bind Scn or serve as a
siderophore under physiological conditions; ii) Scn does not
participate in iron efflux mediated by interactions with BOCT in
HeLa cells; and iii) does not affect apoptosis in hematopoietic cell
lines.
Results
GA Binding to Scn was Weak and not Affected by Iron
To qualitatively test binding, an ultrafiltration assay [13] with
Ent, catechol, 2,3-DHBA and GA showed greater than 50% iron
retention with Ent, catechol and 2,3-DHBA and less than 10%
iron retention with GA, comparable to background (Figure S2A).
The binding of GA, SA and 2,3-DHBA to human Scn, either
without (Figure 2A) or with (Figure 2B) iron, was then analyzed
quantitatively at neutral pH using a fluorescence quenching (FQ)
assay [13] to compare with the previous FQ analysis of desferri
GA binding [16]. The solution speciation as a function of pH was
calculated for iron complexation with 2,3-DHBA, GA, SA and
catechol ligands (L) under the conditions used in the FQ
experiments ([Fe
3+
]=20mM, [L] = 60 mM; Figures 2C, 2D, 2E
and S2B). The quenching of inherent Scn fluorescence upon
addition of either desferri or ferric ligands was monitored at the
characteristic Scn emission wavelength; K
D
values were deter-
mined with Hyperquad [17]. Various Fe(2,3-DHBA)
x
complexes
were modeled and those that generated satisfactory fits to the data
were based on predominant complex formed at pH 7.2. While
both 2,3-DHBA and catechol formed ferric complexes at
physiological pH, GA and SA only formed appreciable complexes
with iron at low pH. For mixtures of iron and 2,3-DHBA, the Fe:L
complex was predominant in solution and was successfully
modeled in an association equilibrium with Scn
(K
D
= 0.10160.002 nM); the interaction between Scn and desferri
2,3-DHBA was more than a thousand-fold weaker
(K
D
= 0.4060.01 mM). Binding models for SA were based on the
major solution species, Fe(SA)
2
. While the addition of 2,3-DHBA
to Scn resulted in a prominent change in fluorescence, neither the
addition of GA or SA, alone or in the presence of iron, showed
significant quenching that could be quantitatively analyzed,
indicating weak binding to Scn. Since hexadentate tris-catecholate
siderophores like Ent are more potent iron chelators than their
bidentate counterparts, e. g. 2,3-DHBA, and may bind more tightly
to Scn [3], the tris-GA analog TRENGEN was synthesized
(Figures 1A and S1). Like GA, TRENGEN did not show
significant quenching as desferri or ferric forms, indicating weak
binding to Scn (Figures 2A and 2B). The equivalent tris-2,3-DHBA
analog, TRENCAM, binds to Scn tightly (K
D
= 0.3260.01 nM)
[8].
Since Fe(catechol)
x
complexes were readily observed binding in
the Scn calyx by crystallography [13], Scn was co-crystallized in
the presence of Fe(2,3-DHBA)
3
and a 1:3 mixture of iron and GA
to mimic the 2,3-DHBA co-crystallization conditions (Table S1).
Initial phases were determined by molecular replacement with a
previous Scn structure as the search model (PDB accession code
1L6M). The Scn/Fe(2,3-DHBA)
3
structure showed clear electron
density for three 2,3-DHBA groups and bound iron in the calyx
(Figure 2C), while the Scn/Fe/GA structure showed only weak
electron density features in the calyx consistent with water
molecules and the unresolved side-chain of W79 (Figure 2D),
despite $millimolar concentrations of protein and GA in the
crystallization mix. While the former structure was fully refined
(final R
work
/R
free
= 25.1%/28.8%) and deposited (PDB accession
code 3U0D), no further refinement was performed on the empty
Scn/Fe/GA structure.
Exogenous Scn did not Affect Iron Efflux from BOCT-
expressing HeLa Cells
Apo-Scn was reported to markedly increase
59
Fe release from
human HeLa cells transfected with the putative murine Scn
receptor, BOCT (HeLa/24p3R-L), while apo-Scn had no effect on
cells transfected with an empty vector (HeLa/X7) [15]. HeLa/
24p3R-L and HeLa/X7 cells obtained from the original investi-
gators were incubated with 2.5 mM
59
FeCl
3
for 3 h at 37uCto
label intracellular iron pools, washed and then re-incubated for 5
or 24 h at 37uC in the presence or absence of 2 mM apo-Scn.
DFO (100 mM) was used as a positive control to mobilize iron
from cells [15,18]. Despite using an identical protocol [15], we did
not observe any increase of
59
Fe release after incubation of HeLa/
24p3R-L cells for 24 h with apo-Scn (16.060.6%) relative to cells
re-incubated with control medium (16.360.6%; Figure 3A). After
a re-incubation of prelabeled cells for 5 h, less
59
Fe efflux
occurred, but again there was no significant difference in
59
Fe
efflux from HeLa/24p3R-L cells in the presence or absence of
apo-Scn (Figure 3A). Apo-Scn also had no effect on increasing
59
Fe release from control HeLa/X7 cells, while a 24 h re-
incubation with DFO markedly and significantly (p,0.001)
increased
59
Fe release to 2.7-fold of that found for control medium
in HeLa/X7 cells and to 3.5-fold in HeLa/24p3R-L cells
(Figure 3A). A 5 h re-incubation of cells with DFO also increased
59
Fe release relative to control medium alone, although the extent
of release was less than that after 24 h, due to the limited
permeability of DFO [19]. The
59
FeCl
3
concentration used in
these experiments (2.5 mM) was 20-fold lower than that used
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previously [15] to minimize cytotoxicity and non-specific binding
of
59
Fe to the membrane.
Since FeCl
3
is not a physiologically relevant form of iron, as
virtually all iron in the blood of mammals is bound to transferrin
(Tf), the studies above were repeated using
59
FeTf at a
concentration ([Tf] = 0.75 mM; [Fe] = 1.5 mM) within the phys-
iological range found in extracellular fluid [20]. Cells were labeled
with
59
FeTf for 3 h at 37uC and then re-incubated with apo-Scn
or DFO for 5 or 24 h at 37uC (Figure 3B), as above. As with
59
FeCl
3
, a 5 or 24 h re-incubation with apo-Scn did not induce
Figure 2. Analysis of the binding of benzoates to iron and Scn. Normalized fluorescence is plotted against concentrations for 2,3-DHBA, SA
and GA in the absence (A) or presence (B) of iron. Comparison of the weak quenching by addition of SA, GA or TRENGEN in the presence or absence
of iron with 2,3-DHBA responses suggests that SA/Scn, GA/Scn and TRENGEN/Scn dissociation constants, while unfittable by these techniques, would
be considerably larger than the derived 2,3-DHBA K
D
(0.4060.01 mM). In order to properly model binding in quantitative fluorescence quenching
binding assays, solution speciation diagrams (left panels) of iron and 2,3-DHBA (C), GA (D) and SA (E) were calculated with HYSS [17] and confirmed
by UV/Vis spectroscopy (middle panels). Right-most panels in (C) and (D) show close-up views of the Scn calyx with Fe(2,3-DHBA)
3
bound (C) or in the
presence of iron/GA mixtures (D) in the same orientation. In these views, the calyx is represented as a molecular surface colored by electrostatic
potential; bound ligands are colored by atom-type, with the iron atom shown as an orange sphere. Difference electron density, contoured at 2s
(yellow) and 10s(red) from delete-refine F
obs
-F
calc
Fourier syntheses, is shown as nets. Note the absence of any iron peak in (D); residual density in
this view can be accounted for by tightly-bound water molecules and the unmodeled side-chain of residue W79, which adopts multiple rotamers.
doi:10.1371/journal.pone.0043696.g002
Figure 3. Scn has no effect on iron release or iron uptake from HeLa cells. Control HeLa/X7 (transfected with empty vector) or HeLa/24p3R-L
cells were labeled with either (A) 2.5 mM
59
FeCl
3
or (B) 0.75 mM
59
FeTf and re-incubated with 2 mM murine Scn or control medium for 5 h (dotted
columns) or 24 h (checked columns); 100 mM DFO was used as a positive control. Expression of BOCT in transfected HeLa/24p3R-L cells was
confirmed by RT-PCR (C). In (C), a typical result from three experiments is shown. In (D), control HeLa/X7 (white columns) and HeLa/24p3R-L cells
(black columns) were incubated for 4 h in the presence of 2 mM
59
FeCl
3
,2mM
59
FeEnt, 2 mM murine Scn with bound
59
FeEnt (
59
FeEnt+Scn) or in the
presence of 2 mM
59
FeEnt plus 2 mM human albumin (
59
FeEnt+Alb). Internalized
59
Fe was determined by c-counting. Albumin was added in (D) as an
additional control for non-specific binding. Error was calculated as the standard deviation among three experiments.
doi:10.1371/journal.pone.0043696.g003
Siderocalin/24p3/Lcn2 Does Not Drive Apoptosis
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any significant increase in
59
Fe mobilization from either cell-type.
In contrast, after either a 5 or 24 h re-incubation, addition of DFO
led to
59
Fe release from both cell-types. After a 24 h re-incubation
with DFO, we observed a significant (p,0.001) 4.1-fold (HeLa/
X7) and 5.1-fold (HeLa/24p3R-L) increase in
59
Fe release relative
to cells re-incubated with control medium alone. RT-PCR
experiments using a primer specifically designed for murine
BOCT confirmed that HeLa/24p3R-L cells expressed murine
BOCT mRNA, while Hela/X7 cells did not (Figure 3C).
Exogenous Scn did not Affect Iron uptake by BOCT-
expressing HeLa Cells
In contrast to the effect of apo-Scn on iron efflux, holo-Scn was
described to be capable of delivering iron to cells [15]. Hence, we
tested
59
Fe uptake from Scn labeled with
59
FeEnt in HeLa/
24p3R-L and HeLa/X7 cells (Figure 3D). Cells were incubated
for 4 h at 37uC in serum-free medium with 2 mM
59
Fe as
59
FeCl
3
,
2mM
59
FeEnt or 2 mM
59
FeEnt/Scn. Both cell-types internalized
similar levels of
59
Fe from FeCl
3
, although this was 6-to 7-fold
greater than
59
Fe uptake from the
59
FeEnt in both cell-types.
There was no significant difference in the uptake of
59
FeEnt
between HeLa/24p3R-L and HeLa/X7 cells. The greater uptake
of
59
Fe from
59
FeCl
3
than
59
FeEnt by cells can be attributed to the
presence of specific transporters on HeLa cells that are known to
transport low M
r
iron [21,22]. In contrast, the
59
FeEnt complex
did not appear to be transported into cells as effectively as
59
FeCl
3
,
which may be attributable to the larger size and charge of FeEnt
[3,23]. The addition of
59
FeEnt/Scn to HeLa/24p3R-L and
HeLa/X7 cells did not lead to significantly greater uptake than
that found for
59
FeEnt or for
59
FeEnt mixed with the non-specific
control protein albumin. Therefore, Scn did not act to enhance
the transport of
59
FeEnt into HeLa cells in the presence or absence
of exogenous BOCT expression.
Exogenous Scn did not Affect Expression of Iron-
responsive Genes
To further assess the effect of apo-Scn on cellular Fe
mobilization, the effect of Scn on genes that are sensitively
regulated by intracellular Fe levels, H-ferritin (heavy polypeptide 1;
FTH1) and N-myc downstream regulated gene-1 (NDRG1), was
monitored (Figure 4). HeLa/24p3R-L and HeLa/X7 cells were
incubated for 24 h in control media alone, with 2 mM apo-Scn, or
100 mM or 250 mM DFO. DFO has been shown to up-regulate
NDRG1 mRNA and protein expression [24,25]. Two bands for
NDRG1 were observed, likely representing different phosphory-
lation states [26,27]. While addition of DFO markedly up-
regulated NDRG1 mRNA and protein expression, apo-Scn failed
to increase NDRG1 expression (Figure 4). None of the treatments
had any significant effect on H-ferritin mRNA levels since H-ferritin
is regulated by iron at the post-transcriptional level [28]. However,
H-ferritin protein expression was decreased by addition of DFO,
consistent with previous studies [28], whereas addition of apo-Scn
did not have any effect (Figure 4).
Isolatable BOCT Subdomains do not Bind Scn
In order to attempt to confirm a functionally-relevant interac-
tion between Scn and its putative receptor BOCT, fragments of
BOCT constituting likely independently folded domains or loops
predicted to be on the cell surface by previous ([15], Figure 5A) or
our own (Figure 5B) topology analyses were synthesized as
peptides or recombinantly expressed and purified (Figures 5C
and 5D). Since BOCT is a multipass integral membrane protein,
the intact receptor is difficult to use in quantitative binding assays;
however, multipass receptors often contain identifiable minimal-
binding domains that are necessary and sufficient for interactions
with ligands. Also, Scn-interacting fragments of BOCT had been
identified in prior studies, including a minimal fragment spanning
the last predicted transmembrane domain plus the C-terminal 44-
residue domain (CTD; Figure 5A) [15], strongly suggesting that
the CTD would be sufficient to mediate Scn binding. However,
none of these peptides or domains, including a soluble form of the
CTD, displayed measurable affinities for Scn by size exclusion
chromatography (SEC; an example result is shown in Figure 5E)
or surface plasmon resonance (SPR; an example result is shown in
Figure 5F). Additional binding assays using isothermal titration
microcalorimetry or co-crystallization also failed to show measur-
able interactions (data not shown).
Exogenous Scn does not Drive Apoptosis in Murine
Hematopoietic Cell Lines
It had been reported that 32D.3 or FL5.12 cells undergo
apoptosis upon addition of apo-Scn at concentrations up to
0.5 mM [14]. However, while IL-3 withdrawal or addition of 10 or
100 mM DFO induced robust apoptotic responses in 32D.3 and
FL5.12 cells after 48 h, recombinant apo-Scn, added at concen-
trations of 10 or 100 mM (20-or 200-fold higher concentrations
than used previously), did not induce apoptosis in 32D.3 or
FL5.12 cells (Figures 6 and S3). Indeed, addition of recombinant
Scn at these concentrations had a significant (p,0.05) anti-
apoptotic effect (Figure 6). Scn used in these and prior experiments
was expressed recombinantly in E. coli, but Scn expressed in
HEK293-F cells [29], retaining native glycosylation, yielded
comparable results (data not shown).
32D.3 and FL5.12 Cells Stably Transduced with Scn are
Viable
To mimic the proposed autocrine mechanism of Scn-mediated
apoptosis [14], 32D.3 and FL5.12 cells were induced to stably
secrete murine Scn at ,2 mg/L levels with a lentivirus construct
[29] (Figures 7A, 7B and S4A). These cells show normal levels of
viability in the presence of IL-3, but undergo apoptosis as expected
in response to the addition of DFO (Figures 7C and S4B), which
induces cellular iron-depletion [30]. In order to eliminate the
possibility that the levels of endogenous siderophore available
in vitro were limiting for a hypothetical autocrine effect of Scn on
apoptosis under these conditions, iron-free Ent, 2,3-DHBA, 2,5-
DHBA and TRENGEN were added at 100 mM concentrations
(Figures 7C and S4B). Addition of Ent at this concentration
induced robust apoptosis in transduced 32D.3 and FL5.12 cells
while none of the other compounds had significant effects on
viability, together showing that Scn does not induce apoptosis
through an autocrine mechanism and supporting the hypothesis
that bidentate siderophores and TRENGEN do not chelate iron
strongly enough to affect iron metabolism in vitro.
32D.3 and FL5.12 Cells do not Secrete Detectable Scn in
Response to IL-3 Withdrawal
The initial observation underlying the Scn-apoptosis hypothesis
via iron-depletion was the up-regulation of Scn in response to
cytokine withdrawal [14]. As a control for the levels of Scn
secreted from transduced cells, the levels of Scn secreted from
32D.3 and FL5.12 cells undergoing IL-3 withdrawal-induced
apoptosis were measured by Western analyses (Figures 7B and
S4A). However, no detectable Scn was observed in concentrated
supernatants from 32D.3 and FL5.12 cells undergoing apoptosis.
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Siderocalin/24p3/Lcn2 Does Not Drive Apoptosis
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An Anti-Scn Antibody does not Block Apoptosis
In order to begin mapping interactions between Scn and cell-
surface receptors mediating endocytosis and subsequent apoptosis,
an anti-Scn antibody (R&D Systems MAB1857) was tested for the
ability to affect IL-3 withdrawal-induced apoptosis in 32D.3 and
FL5.12 cells (Figure 8A) and co-crystallized with murine Scn as an
Figure 4. Added Scn does not affect the expression of iron responsive genes. Expression of H-ferritin (FTH-1) and NDRG1 in HeLa/X7 and
HeLa/24p3R-L cells was assayed by RT-PCR (A) and Western blot (B). Cells were untreated or treated with 2 mM murine Scn or DFO (100 mMor
250 mM) for 24 h. Densitometry results (right) were calculated relative to b-actin; error was calculated from the standard deviation among three
experiments; a typical result from three experiments is shown in (A) and (B).
doi:10.1371/journal.pone.0043696.g004
Figure 5. BOCT N-and C-terminal domains do not bind Scn. Predicted BOCT membrane topologies are shown, either as determined in [15] (A)
or calculated here (B), with transmembrane-spanning helices shown as blue cylinders. The sequence lengths of the NTD (green), CTD (red) and
connecting loops are indicated; loops synthesized as peptides for binding analyses are indicated with numbered black circles, corresponding to the
numbering in the Materials & Methods section. The amino termini of fragments used to originally identify BOCT as a Scn receptor [15] are
indicated with orange arrows in (A). PAGE analyses of bacterially-expressed soluble, purified NTD (C) and CTD (D) are shown. SEC analysis of NTD/Scn
is shown in (E). Complex formation would have been indicated by a shift in the Scn+NTD peak to lower elution volumes; in this case, the Scn/NTD
mixture runs as the simple summation of the Scn and NTD alone peaks, indicating no binding under these conditions. (F) SPR analysis of Scn/CTD
binding, with Scn analyte concentrations indicated. The bar indicates the sample injection period (association phase); gaps in the sensorgrams cover
transients associated with injections.
doi:10.1371/journal.pone.0043696.g005
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Fab fragment to determine its binding footprint on Scn (Figure 8B
and Table S1). However, since Scn did not drive apoptosis in these
cells in the above experiments, this analysis was not informative,
though characterizing this interaction is useful for future studies of
interactions between Scn and bona fide receptors.
Figure 6. Scn does induce apoptosis in murine 32D.3 or FL5.12 cells. FL5.12 (A) and 32D.3 (C) cells were incubated with 10 mM Scn and DFO
for 48 h (NT: no treatment; -IL-3: in the absence of added IL-3). Apoptosis was assayed by annexin V-FITC staining and DAPI was used as a vital stain;
percentages of cells positive for annexin staining are indicated. Average annexin V-positivity from three independent experiments are shown for
FL5.12 (B) and 32D.3 (D) cells; error was calculated as the standard deviation of three replicates. Statistical significance is indicated as *p,0.05;
**p,0.01; ***p,0.001. Note that while the effect of adding Scn was significant, the effect was anti-apoptotic.
doi:10.1371/journal.pone.0043696.g006
Siderocalin/24p3/Lcn2 Does Not Drive Apoptosis
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Figure 7. Stably-induced expression of Scn does not drive apoptosis in FL5.12 cells. (A) FL5.12 cells were transduced with the pCVL-SFFV-
muScn-IRES-GFP lentivirus and GFP mean fluorescence intensity was determined one-week post-transduction by cytometry, confirming transgene
functionality. (B) A Western blot of supernatants, concentrated from 32 mL, from FL5.12 cells shows that the transduced cells constitutively express
Scn, while parental cells in the presence or absence of IL-3 do not secrete detectable amounts of Scn after 72 h in culture. (C) Transduced FL5.12 were
incubated with a variety of siderophores in order to assess the role of exogenous siderophores on cell viability (NT: no treatment). The hexadentate
chelators DFO and Ent at 100 mM produce robust apoptosis, while the bidentate chelators at 300 mM do not affect viability.
doi:10.1371/journal.pone.0043696.g007
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Discussion
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
[2–5,7–9,13,31,32]. Modifications to the catechol functional group
that widen the complex with iron, such as 3,4-DHBA (proto-
catechuic acid) substituents in the anthrax siderophore petrobactin
[9], glucose modifications of the salmochelins [4] or adducts in
synthetic siderophore analogs [9], do not bind to Scn as iron
complexes because of steric clashes with the Scn calyx. At neutral
pH, Ent and 2,3-DHBA chelate ferric ions in solution through the
adjacent catechol 2-and 3-OH groups, generating complete,
hexadentate FeEnt complexes or incomplete Fe(2,3-DHBA)
x
complexes. As the pH is lowered, iron binding shifts from
catecholate-mode to salicylate-mode, engaging the 2-OH and
carbonyl oxygens, and then to iron release [33,34]. The shift from
catecholate-to salicylate-mode binding is accompanied by an
outward swing of the catechol groups, widening the ferric complex
to a diameter that is also sterically incompatible with two of three
pockets in the rigid calyx of Scn. Ferric complexes of SA and
obligate salicylate-mode analogs of Ent [7] do not bind to Scn for
this reason.
This transition is illustrated in the Fe(2,3-DHBA)
3
structure
reported here (Figure 2C); crystallized at low pH, one of the three
2,3-DHBA moieties shifted to salicylate-mode binding in the only
pocket where this transition is tolerated. GA, because it lacks
neighboring (ortho) hydroxyl groups, must chelate iron in salicylate-
mode. The position of the 5-OH group of GA maximizes collisions
with the Scn calyx, accentuating the steric clash, though the weak
binding of ferric SA complexes to Scn showed that salicylate-mode
binding is sufficient on its own to ablate binding. Therefore, ferric
complexes with GA or siderophores with GA substituents should
Figure 8. An anti-Scn antibody does not block IL-3 withdrawal-induced apoptosis. (A) 32D.3 and FL5.12 cells in the presence (NT) or
absence (-IL3) of IL-3 were incubated for 48 h with 10 mM of the anti-Scn antibody MAB1857; percent annexin-V positivity is indicated. (B) The
structure of Fab MAB1857 with Scn, shown in a ribbon representation (Fab in gray and Scn in orange), reveals the interface that is occluded in the
complex. Had Scn had an effect on apoptosis through receptor-mediated uptake, the effect of the antibody on the process would have identified a
potential receptor-interacting surface on Scn, the rationale for this approach. However, since Scn does not affect apoptosis, an anti-Scn antibody
cannot reveal a receptor-interacting surface by blocking a non-existent effect.
doi:10.1371/journal.pone.0043696.g008
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not bind to Scn with appreciable affinity, confirmed here in
qualitative, quantitative and crystallographic binding studies,
demonstrating that GA alone cannot facilitate iron binding by
Scn. The synthetic tris-GA compound TRENGEN also failed to
show measurable interactions with Scn in contrast to the
analogous synthetic tris-2,3-DHBA analog TRENCAM which
binds tightly [8].
Scn generally crystallizes at pHs in the 4 to 4.5 range. While this
is below the release pH for catechol complexes, a partially-released
complex was observed because of the high concentration of
components and the very non-physiological conditions in the
crystallization trials [13]. Low pH release of iron from Scn
complexes is a necessary component of the iron delivery
hypothesis. However, the apoptosis through iron withdrawal
hypothesis requires tight binding of iron in Scn complexes at both
neutral and low pHs, to both efficiently sequester iron intracel-
lularly and outcompete Tf extracellularly, otherwise iron would
simply be returned to cells through normal trafficking. However,
speciation analyses showed little SA/iron and essentially no GA/
iron complex formation at neutral pH, while the binding studies
showed no GA/Scn ligation at neutral or low pHs.
The inability to confirm the iron-or Scn-binding properties of
GA led to a reconsideration of the remaining elements of the Scn-
induces-apoptosis hypothesis laid out in three seminal papers [14–
16]. Despite using the same experimental models, exogenously-
added Scn did not affect iron efflux or uptake, or affect the
expression levels of iron-responsive genes, in BOCT-expressing
HeLa cells. While failing to confirm the role of Scn in iron
mobilization, these results also failed to demonstrate a functional
association between Scn and BOCT, consistent with recent results
that failed to demonstrate binding between the rat orthologs of Scn
and BOCT [35] and our own inability to identify a murine BOCT
subdomain sufficient to mediate murine Scn binding. Further-
more, exogenously-added Scn did not drive apoptosis in IL-3
dependent murine hematopoietic cell lines, even at levels 200-fold
higher than reported by Devireddy and coworkers to induce
robust responses. In fact, stable, Scn-secreting transductants of
these cell lines were viable in culture even in the presence of added
GA or 2,3-DHBA, but readily apoptose in the presence of added
DFO or Ent. Hexadentate chelators, like DFO and Ent, are able
to effectively compete with Tf for iron, but bidentate chelators, like
2,3-DHBA, while effective at solubilizing iron in solution, do not
display affinities sufficient to outcompete Tf for iron [36]. GA and
TRENGEN, which cannot bind iron at the neutral pH used in cell
culture studies, did not significantly affect iron metabolism in these
experiments. Finally, FL5.12 and 32D.3 cells undergoing apopto-
sis in response to IL-3 withdrawal did not secrete detectable levels
of Scn.
The failure of GA to function as a siderophore under
physiological conditions suggested a reexamination of the logic
behind the identification of DHRS6, as DHRS6 was not directly
shown to catalyze the synthesis of GA from any hypothetical
precursor [16]. DHRS6 was identified on the basis of sequence
homology to the enterobacterial enzyme trans-2,3-dihydro-2,3-
dihydroxybenzoate dehydrogenase (EntA), which catalyzes the
conversion of 2,3-diDHBA to 2,3-DHBA as part of Ent
biosynthesis: DHRS6 is the closest mammalian homolog of EntA
[16]. However, the converse is not true; the closest bacterial
homologs of DHRS6 are a family of specific hydroxybutyrate
dehydrogenases, with structural features associated with hydroxy-
butyrate binding in DHRS6 (three arginine residues) conserved
across vertebrate DHRS6 orthologs and at least one bacterial
DHRS6 ortholog (from Bordetella bronchiseptica) [37]. B. bronchiseptica
produces the hydroxamate-type siderophore alcaligin, not cate-
cholate siderophores [38], so does not require an EntA-like activity
for iron acquisition, consistent with annotating the B. bronchiseptica
protein as a hydroxybutyrate dehydrogenase and not as an EntA
analog. DHRS6 is a highly specific enzyme, showing considerable
activity against (R)-OH butyrate, but no measurable activity
against the closely related compounds (S)-OH butyrate, 3-OH-R-
2-methylbutyrate or 3-OH-S-2-methylbutyrate, consistent with the
tight constraints imposed by a highly specific substrate binding site
[37]. EntA is also highly selective, efficiently converting 2,3-
diDHBA but poorly tolerating substituents on the 4 and 5
positions [39], as on diGA. While EntA is unlikely to efficiently
catalyze conversion of both diGA and 2,3-diDHBA substrates, it is
possible that a vertebrate EntA analog could, though this should
be formally demonstrated since indiscriminate conversion of
dihydroxybenzoate isomers would be unusual for this class of
enzymes. However, the expectation would be that DHRS6 is
simply a highly stereospecific hydroxybutyrate dehydrogenase with
insufficient reactivity towards unrelated substrates, like diGA, to
generate GA.
The logical framework of the hypothesis that Scn drives
apoptosis of hematopoietic cells through iron depletion, mediated
by interactions with GA as endogenous siderophore and BOCT as
cell-surface receptor, constitutes an interdependent chain predi-
cated on the integrity of each experimental link. We have shown
that multiple links in this chain are questionable on the basis of
first principles in the absence of direct experimental support (i.e.,
DHRS6 catalyzes the production of GA) or cannot be reproduced
(i.e., GA binds to Scn, GA is a siderophore, BOCT mediates Scn
iron export, Scn drives apoptosis, Scn is secreted in response to
cytokine withdrawal). In light of these results, where any single
break in the logical chain invalidates the overall hypothesis, the
endogenous role of Scn in apoptosis needs to be fully reevaluated.
Materials and Methods
Filter Retention Binding Assay
Apo-Scn (10 mM),
55
Fe
III
(1 mM), cold Fe
III
Cl
3
(9 mM) and a
candidate siderophore (10 mM) were incubated in 150 mM NaCl,
20 mM Tris (pH 7.4) and incubated at ambient temperature for
60 min as described in [13]. The mixture was then washed four
times with the Tris buffer on YM-10 ultrafilters (Millipore) and the
retained
55
Fe measured with a scintillation counter.
FQ Binding Assay
GA was obtained from TCI America (min. 98% purity) and
TRENGEN was synthesized as described in Experimental
Procedures S1; FeCl
3
stock solutions in 1 M HCl were standard-
ized by EDTA titration [40]. Quenching of human Scn was
measured on a Cary Eclipse fluorescence spectrophotometer
(20 nm slit band pass for excitation; 2.5 nm slit band pass for
emission) using characteristic Scn excitation and emission wave-
lengths, 281 nm and 320–340 nm, respectively. Measurements
were made at a protein concentration of 100 nM in Tris-buffered
saline (TBS; pH 7.2), 5% DMSO, plus 32 mg/mL ubiquitin.
Fluorescence intensities were corrected for dilution due to addition
of ligand. An aliquot of a DMSO stock solution of the free ligand
(12 mM; 25 mL) and FeCl
3
salt (27 mM, 3.7 mL, 0.33 equivalents)
were combined and diluted with TBS (pH 7.2) to form the metal
complexes at a concentration of 0.1 mM (no metal added for apo-
ligands). The solutions were equilibrated for 1.5 h and diluted to a
final concentration of 20 mM in 5% DMSO/TBS buffer.
Fluorescence data were analyzed by a non-linear regression
analysis (Figures 2A and 2B) of the normalized fluorescence
response versus ligand concentration using Hyperquad [17]. The
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model for determination of the species stoichiometry and K
D
took
into account ferric ion hydrolysis constants [41], 2,5-DHBA
protonation constants [42] and Fe-2,5-DHBA formation constants
[42]. Dissociation constants were determined from at least three
independent titrations.
UV/Vis Spectroscopy
Stock solutions (22.5 mM) of catechol, 2,3-DHBA, GA and SA
(Sigma-Aldrich) were prepared in ultrapure water; a FeCl
3
stock
was prepared at 500 mM in 1 M HCl. Iron complex solutions
were prepared at 4.5 mM siderophore and 1.5 mM FeCl
3
in
100 mM sodium acetate (pH 4.0), Tris (pH 7.5) or Tris (pH 9.0).
Scn (6 mg/mL) in 100 mM Tris (pH 7.5) was mixed with each of
the ferric siderophores and extensively washed with Tris (pH 7.5)
through multiple rounds of ultrafiltration to remove any unbound
ligand. Absorbance data (Figures 2C, 2D and 2E) were collected
immediately at ambient temperature using a Nanodrop ND-1000
spectrometer (Thermo Scientific).
Crystallography
Crystals of Scn, loaded with molar excesses of stoichiometric
iron/GA or iron/2,3-DHBA mixtures and subsequently washed
and concentrated by ultrafiltration, were grown as previously
described [13]. Scn/Fab complexes were prepared by cleaving
monoclonal anti-Scn rat IgG
2A
(R&D Systems MAB1857) with
papain, adding Scn and purifying the complex by size exclusion
chromatography. Crystals were grown by vapor diffusion at 25uC:
protein at 10 mg/mL was mixed 1:1 with a reservoir solution of
0.1 M sodium citrate (pH 4.2), 0.2 M sodium chloride and 20%
w/w PEG 8000. Crystals were cryopreserved in reservoir solution
plus 15% v/v glycerol. Diffraction data were collected at the
Advanced Light Source, beamline 5.0.1.
PCR Primers
For murine BOCT (GenBank entry NM_021551), the sense
primer used was 59-AAGCGGCAGATTGAGGAA-39and anti-
sense primer was 59-CTTCAGAAGCAAGGAGGGTAC-39. For
human NDRG1, the sense primer used was 59-TCACCCAG-
CACTTTGCCGTCT-39and the anti-sense primer was 59-
GCCACAGTCCGCCATCTT-39. For human H-ferritin (FTH1),
the sense primer used was 59-CCTCCTACGTTTACCTGTC-39
and anti-sense primer was 59-TTTCATTATCACTGTCTCCC-
39. For human b-actin, the sense primer used was 59-
CCCGCCGCCAGCTCACCATGG-39and the anti-sense prim-
er was 59-AAGGTCTCAAACATGATCTGGGTC-39.
HeLa Iron Transport Assays
Human apo-Tf (Sigma-Aldrich) was labeled with
59
Fe (Perki-
nElmer) to produce diferric
59
FeTf using the ferric nitriloacetate
complex at a iron:nitriloacetate molar ratio of 1:10 as previously
described [43]. The iron saturation of Tf was monitored by UV-
Vis spectrophotometry comparing the absorbance at 280 nm
(protein) with that at 465 nm (iron-bound complex). HeLa/
24p3R-L and HeLa/X7 cells [15] were kindly provided by M. R.
Green (University of Massachusetts Medical School). 24p3R-L
refers to a widely expressed, longer splice variant of BOCT as
compared to a short splice variant lacking the N-terminal 154
amino acids [15]. Cells were cultured as described [15] using
DMEM (Invitrogen) supplemented with 10% fetal calf serum
(Invitrogen) and 2.5 mg/mL blasticidin (Sigma-Aldrich). To
confirm expression of BOCT, total RNA was isolated using
TRIzolH(Invitrogen) and RT-PCR was performed using Super-
Script III RT/PlatinumHTaq Mix as previously described [44]
using primers detailed as above. Western blot analysis was
performed using established protocols [45] and primary antibodies
against NDRG1 (Abcam 37897), H-ferritin (Cell Signaling
Technology 3998) and b-actin (Sigma-Aldrich A5441). For
59
Fe
release experiments (Figure 3), cells growing as a monolayer were
pre-labeled with 0.75 mM
59
FeTf for 3 h at 37uC in DMEM
(Invitrogen) plus 10% fetal calf serum (Invitrogen). Cultures were
then washed four times with PBS on ice and then re-incubated in
fresh culture media with or without 2 mM apo-Scn for 5 or 24 h at
37uC; 100 mM DFO (Novartis or Sigma-Aldrich) was used as
positive control. Scn was obtained from R&D Systems, the kind
gift of L. Devireddy (Case Western Reserve University) or was
produced as previously described [46]. After this incubation, the
supernatant was collected and the cells harvested to estimate
radioactivity using a 2480 Wizard
2
c-counter (PerkinElmer). In
additional experiments, cells were pre-labeled with 2.5 mM
59
FeCl
3
(PerkinElmer) instead of
59
FeTf as in [15]. For
59
Fe
uptake experiments (Figure 3),
59
FeEnt was produced by
incubating iron-free Ent (EMC Microcollections) with
59
FeCl
3
(PerkinElmer) in a molar ratio of 1:1 for 30 min at 37uC in the
dark. Scn was incubated with
59
FeEnt in a 1:1 molar ratio at 37uC
for 30 min in the dark to generate radiolabeled holo-Scn, albumin
was similarly pre-incubated with
59
FeEnt. To measure iron
uptake, cells were incubated in serum-free DMEM (Invitrogen)
with 2 mM
59
FeCl
3
,2mM
59
FeEnt or 2 mM holo-Scn for 4 h at
37uC. Human albumin (2 mM) was added with
59
FeEnt as a
control for non-specific protein-binding and transport. After this
incubation, cells were washed on ice four times with PBS and
harvested for c-counting. Experiments were performed in
triplicate and data were compared using Student’s t-test; results
were considered statistically significant when p,0.05.
Expression and Characterization of BOCT Subdomains
Predicted membrane topologies of BOCT, determined by
Devireddy and coworkers (Figure 5A) [15] or the union of results
from several computational algorithms (TMHMM [47,48],
TMpred [49], SOSUI [50]), suggest that N-and C-terminal
BOCT sequences both comprise domains large enough to form
independent folding units (NTD: in its longer form, residues 1
through 102; CTD: residues 477 through 520). NTD
1–102
and
CTD
477–520
were expressed recombinantly in E. coli, the former as
a His-tagged, periplasmically-targeted construct and the latter as a
cytoplasmically-targeted, cleavable GST-fusion (Figures 5C and
5D). Binding of NTD or CTD to Scn was assayed by SEC (e. g.,
Figure 5E) and SPR (e. g., Figure 5D). In Figure 5D, 2086 SPR
response units (RUs) of Scn were amine-coupled to CM5 sensor
chips (Biacore) following the manufacturer’s protocol. CTD
analytes, at concentrations from 3.125 to 50 mM, were injected
in duplicate, in random order, for one minute at a flow rate of
20 ml/min on a Biacore 3000 system. Sensorgrams were blank-
corrected by the double-subtraction method [51], using a capped
channel as blank. In this experiment, a saturating response on a
fully-active surface would correspond to .100 RUs; therefore, the
very weak responses observed, even at very high analyte
concentrations, show that the CTD/Scn interaction has an
equilibrium dissociation constant considerably weaker than
50 mM. Comparable results were obtained for NTD. Peptides
corresponding to predicted cell-surface loops of significant length
(.6 residues) in either topology were synthesized commercially
(Genscript) with N-terminal biotin groups:
1. b-SKDWRFLQR (residues 210 through 218)
Siderocalin/24p3/Lcn2 Does Not Drive Apoptosis
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2. b-ESARWLIVKRQIEEAQSVLRILAERNRPHGQML-
GEEAQEALQELENTSPLPATSTFS (residues 238 through
294)
3. b-FTNFIAHAIRHSYQPVGGGGSPSD (residues 313
through 336)
4. b-WDYLNDAAITT (residues 386 through 396)
5. b-QRLHMGHGAFLQ (residues 446 through 457)
BOCT loop peptides were coupled to strepavidin-coated sensor
chips and analyzed with Scn analytes using analogous methodol-
ogy; as above, no quantifiable responses were detected (data not
shown).
Apoptosis Assays
32D.3 (ATCC CRL-11346) and FL5.12 (the kind gift of L.
Devireddy (Case Western Reserve University)) cells were cultured
in modified RPMI-1640 (ATCC) containing 5% fetal calf serum
and 5 ng/mL murine IL-3 (BD). FL5.12 and 32D.3 cells were
maintained in culture at 5610
5
cells/mL and 24 h later seeded at
1610
5
cells/mL in 24 well plates. 10 mM DFO or Scn was added
to the cells and incubated for 48 h; Scn was produced as
previously described [46]. Annexin V-FITC/DAPI staining was
carried out as described by the manufacturer (BD) and each
sample was analyzed by flow cytometry. Transduction of 32D.3
and FL5.12 cells was carried out at 1610
6
cells/mL in media
supplemented with 4 mg/mL hexadimethrine bromide. The
lentiviral construct used for the transductions was described
previously [29]. Experiments were performed in triplicate and
data were compared using Student’s t-test; results were considered
statistically significant when p,0.05.
Supporting Information
Figure S1 Related to Figure 1: Synthesis of TRENGEN.
(TIF)
Figure S2 Related to Figure 2: Catechol solubilizes iron
at neutral pH and mediates iron retention by Scn. (A)
Iron retention by Scn in an ultrafiltration assay in the presence of
various candidate siderophores is shown; error was calculated from
the standard deviation of triplicate experiments. (B) HYSS
speciation analysis (left panel) and UV/Vis spectroscopic analysis
of iron/catechol/Scn interactions.
(TIF)
Figure S3 Related to Figure 6: Scn does not induce
apoptosis at high concentrations. FL5.12 (A) and 32D.3 (C)
cells were incubated with 100 mM Scn and DFO for 48 h.
Apoptosis was assayed by annexin V-FITC staining and DAPI was
used as a vital stain; percentages of cells positive for annexin
staining are indicated.
(TIF)
Figure S4 Related to Figure 7: Stably-induced expres-
sion of Scn does not drive apoptosis in 32D.3 cells. (A) A
Western blot of 32D.3 cells shows that the transduced cells
constitutively express Scn, while parental cells in the presence or
absence of IL-3 do not secrete detectable amounts of Scn after
72 h in culture; 32 mL of culture supernatants was concentrated
and loaded in the first three lanes. (B) Transduced 32D.3 were
incubated with a variety of siderophores in order to assess the role
of exogenous siderophores on cell viability. The hexadentate
chelators DFO and Ent at 100 mM produce robust apoptosis,
while the bidentate chelators at 300 mM do not affect viability.
(TIF)
Table S1 Related to Figure 8: Crystallographic statis-
tics.
(DOCX)
Experimental Procedures S1 TRENGEN synthesis.
(DOCX)
Acknowledgments
The authors thank Carmelo Sgarlata, Trisha Hoette, Della Friend and the
SSGCID for technical support and helpful discussions. The authors declare
no competing financial interests.
Author Contributions
Conceived and designed the experiments: CC VR AKS ADB YSR BKK
JB KNR DRR RKS. Performed the experiments: CC VR AKS ADB MR
YSR ZK MCC MAH BKK JB DRR. Analyzed the data: CC VR AKS
ADB MR YSR ZK MCC MAH BKK JB KNR DRR RKS. Wrote the
paper: CC VR AKS ADB MR YSR MCC JB KNR DRR RKS.
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