International Journal of
Molecular Sciences

Article
In V itro Studies on Zinc Binding and Buf fering by
Intestinal Mucins
Maria Maares 1 , Claudia Keil 1 , Jenny Koza 1 , Sophia Straubing 1 , T anja Schwerdtle 2,3
and Hajo Haase 1 , 3 , *
1 Department of Food Chemistry and T oxicology , Berlin Institute of T echnology , Gustav-Meyer -Allee 25,
D-13355 Berlin, Germany; [email protected] (M.M.); [email protected] (C.K.); jenny [email protected] (J.K.);
[email protected] (S.S.)
2 Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-A llee 114-116, 14558 Nuthetal,
Germany; [email protected]
3 T raceAge-DFG Research Unit on Interactions of essential trace elements in healthy and diseased elderly ,
Potsdam-Berlin-Jena, Germany
* Correspondence: [email protected] ; T el.: +49-(0)30-31472788; Fax: +49-(0)30-31472823
Received: 9 August 2018; Accepted: 6 September 2018; Published: 7 September 2018
     
  

Abstract:
The investigation of luminal factors influencing zinc availability and accessibility in
the intestine is of gr eat interest when analyzing parameters r egulating intestinal zinc resorption.
Of note, intestinal mucins wer e suggested to play a beneficial role in the luminal availability of zinc.
Their exact zinc binding pr operties, however , r emain unknown and the impact of these glycoproteins
on human intestinal zinc r esorption has not been investigated in detail. Thus, the aim of this
study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes
and its transfer into the blood. In the present study ,
in vitr o
zinc binding pr operties of mucins
wer e analyzed using commercially available por cine mucins and secreted mucins of the goblet cell
line HT -29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these
glycopr oteins demonstrates that mucins contain multiple zinc-binding sites with biologically r elevant
af finity within one mucin molecule. Zinc uptake into the enter ocyte cell line Caco-2 was impaired by
zinc-depleted mucins. Y et this does not repr esent their form in the intestinal lumen
in vivo
under
zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the pr esence of mucins
with dif fering degree of zinc saturation r evealed zinc buffering by these glycopr oteins, indicating
that mucin-bound zinc is still available for the cells. Finally , the impact of mucins on zinc resorption
using thr ee-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT -29-MTX
co-cultur e and conventional Caco-2 monoculture. Her e, the mucin secreting co-cultur es yielded higher
fractional zinc r esorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate
the zinc uptake into enter ocytes and act as a zinc delivery system for the intestinal epithelium.
Keywords:
intestinal zinc r esorption; zinc binding; mucus layer; intestinal mucins;
in vitr o
intestinal
model; goblet cells; Caco-2/HT -29-MTX-model
1. Introduction
The essential trace element zinc is pr edominantly resorbed in the small intestine, where it is
absorbed by enter ocytes and transported into the blood stream, primarily mediated by the apically
located Zrt-, Irt-like transporter (ZIP)-4 and the basolateral zinc exporter (ZnT)-1 [
1
]. These two
transporters ar e complemented by the basolateral transporter ZIP-5, importing zinc from the blood
into the enter ocytes, and the apical transporter ZnT -5, which exports zinc back into the intestinal
lumen [
1
]. However , despite ongoing resear ch, the molecular mechanisms regulating zinc absorption
Int. J. Mol. Sci. 2018 , 19 , 2662; doi:10.3390/ijms19092662 www .mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018 , 19 , 2662 2 of 20
ar e not yet fully elucidated. Human intestinal zinc r esorption was shown to be a regulated pr ocess,
as the fractional zinc r esorption varies between 20–60%, generally decreasing with elevated zinc
intake [
2
]. The amount of absorbed zinc is not only influenced by oral zinc intake, but particularly
depends on its accessibility in the intestine, which is strongly influenced by food components such
as phytate, impairing the intestinal zinc availability . Additionally , amino acids and several trace
elements wer e reported to impact enter ocytes’ zinc uptake [
3
]. Notably , the intestinal availability of
trace elements in general does not exclusively depend on food components, but is also influenced
by the intestinal mucus layer . Specifically , the mucus was shown to bind ions such as iron, lead,
and zinc pr eventing their hydroxypolymerisation at intestinal pH and incr easing their solubility and
availability for the intestinal epithelium [
4
–
6
]. It has been suggested that the af finity of mucins for
metals incr eases from M
+
< M
2+
< M
3+
leading to a competitive binding to the glycoproteins and
consequentially influencing their bioavailability [
5
,
7
,
8
]. In fact, while the impact of mucins for iron
r esorption was investigated in detail, there is evidence that the mucus layer might also be important
for zinc uptake by the human intestinal mucosa, as zinc binding by mucins was observed in animal
studies [
4
,
5
,
9
,
10
]. Nevertheless, the detailed r ole of the intestinal mucus on human zinc absorption has
not yet been investigated.
Mucus, synthesized and secr eted by goblet cells, covers the entire gastr ointestinal tract protecting
the underlying epithelium against the luminal content, and plays an essential role in nutrition and
health [
11
]. While a single loosely bound mucus layer supports the r esorption of nutrients in the
small intestine, this physical barrier is extended by an additional adherent mucus layer in the stomach
and colon [
11
]. The gastrointestinal mucus is mainly constituted of water , ions, lipids and 5–10%
highly glycosylated pr oteins: the mucins. These pr oteins maintain their macromolecular network-like
structur e by being largely composed of Serin, Threonin and Pr olin tandem-repeats and O-linked
oligosaccharides, as well as of fewer O -glycosylated cysteine-rich regions (r ecently r eviewed in [
12
,
13
]).
Thus, the intestinal epithelium does not only consist of enter ocytes, absorbing the nutrients from the
luminal content, but contains a variety of other cell types of which goblet cells are the most abundant,
constantly secr eting mucins into the lumen [ 14 ].
In vitr o
intestinal models pr ovide a standardized and easy platform to analyze the bioavailability
of nutrients, such as trace elements, as well as transport kinetics [
15
], offering a pr omising tool to
illuminate distinct molecular aspects of intestinal zinc resorption. Not only are changes in cellular
zinc tracked by using inductively-coupled plasma mass spectrometry (ICP-MS) and flame atomic
absorption spectr ometry (F AAS), but the application of low molecular weight sensors as an approach
to measur e zinc uptake [
16
–
18
] gained importance to determine small changes in the intracellular zinc
pool [
19
]. These models always need to r esemble the
in vivo
situation, not only concerning buffer
and medium constituents, but also cellular composition. Until now ,
in vitr o
studies on intestinal zinc
uptake wer e mainly conducted using the Caco-2 model, which was alr eady shown to express the
main intestinal zinc transporters [
17
]. This cell line is very well characterized and differ entiates into
a cell monolayer morphologically and functionally repr esenting the enterocy tes
in vivo
[
20
]. Some
disadvantages of this
in vitr o
cell model concerning over expression of the Pgp-pr otein and lack of
a mucus layer wer e improved by intr oducing the Caco-2/HT -29-MTX co-cultur e [
21
,
22
]. This well
characterized co-cultur e of Caco-2 cells and the goblet cell line HT -29-MTX [
22
,
23
] was shown to
be cover ed by mucus with a thickness of at least 2–10
µ
m after fixation [
21
] and has alr eady been
used to investigate the role of mucins on bacterial adhesion [
24
] as well as on the r esorption of
nutrients [
21
,
25
,
26
]. Furthermor e, this co-culture was r ecently applied by our group to study the
impact of a basolateral zinc acceptor on zinc r esorption [ 17 ].
The aim of this study is to examine the r ole of intestinal mucins for zinc resorption. Her ein,
zinc binding pr operties of these glycoproteins and their zinc af finity are investigated in cell-fr ee
measur ements as well as in the presence of intestinal cells to clarify the r ole of the mucus layer on zinc
uptake. Finally , zinc transport was measured comparing a conventional Caco-2 monocultur e and the
Caco-2/HT -29-MTX co-culture to investigate the impact of mucins on the actual zinc transfer .

Int. J. Mol. Sci. 2018 , 19 , 2662 3 of 20
2. Results
2.1. Zinc Binding by Intestinal Mucins
First, the pr operty of mucins to bind and release zinc was investigated using the zinc-chelating
chr omophore 4-(2-pyridylazo)r esorcinol (P AR) (additional spectr ophotometric titration of the
zinc-(P AR)
2
-complex in Supplementary Figur e S1). Figur e 1 shows a significant decrease of the
fr ee zinc concentration after applying 2.5–10 mg/mL zinc-depleted mucins (one-way analysis of
variance (ANOV A) with Dunnett’s multiple comparison test; p < 0.001), indicating zinc binding by the
gastr ointestinal glycoproteins.
Figure 1.
Effect of mucins on zinc availability for 4-(2-pyridylazo)r esorcinol (P AR). Effect of mucins on
zinc availability for P AR is shown as free zinc r elative to the initially added zinc concentration. Dif ferent
concentrations of zinc-depleted porcine mucin wer e incubated with 8
µ
M zinc and fr ee zinc was
analyzed using the colorimetric zinc chelator P AR. Data are pr esented as means + standard deviation
(SD) of at least thr ee independent experiments. Significant differ ences to the control ar e indicated
(*** p < 0.001; one-way analysis of variance (ANOV A) with Dunnett’s multiple comparison test).
Subsequently , the binding capacity of these mucins was further investigated after dialysis of
por cine mucins with differ ent zinc concentrations for 12 h against T ris(hydr oxymethyl)aminomethane
(T ris)-buffer ed saline (TBS) (selection of the appropriate dialysis time in Supplementary Figur e S2).
Her e, the zinc content of zinc-loaded mucins increased significantly compar ed to mucins without added
zinc (Figur e 2 ). The addition of 10,000
µ
M zinc befor e dialysis resulted in a beginning zinc-saturation
of the glycopr oteins (Figure 2 A). These samples wer e defined as high zinc-loaded mucins. In total,
thr ee differ ent degrees of zinc-loaded mucins wer e selected for further analysis: in addition to the
high zinc-loaded mucins, also medium and low zinc-loaded mucins wer e dialyzed in the presence of
5000
µ
M or 2500
µ
M zinc, r espectively . Next, zinc values wer e normalized to the total glycopr otein
and pr otein content of the mucin samples after dialysis. These also depended on the amount of zinc
pr esent during dialysis, and maximum amounts of 5.7 mg zinc per g glycoprotein (Figur e 2 B) and of
94.2 mg per g pr otein (Figure 2 C) wer e determined for the high zinc-loaded mucins.
Finally , the zinc binding affinity of zinc-depleted por cine mucin was analyzed using the
colorimetric r eagent 2-carboxy-2
0
-hydr oxy-5
0
-sulfoformazylbenzene monosodium salt (zincon)
(additional spectr ophotometric titration in Supplementary Figure S3) and compar ed to the affinity
of zinc-depleted mucins harvested fr om the goblet cell line HT -29-MTX (Figur e 3 A,B). This analysis
yielded similar dissociation constants for the zinc-mucin complexes with 6.8
µ
M for por cine and 5
µ
M
for HT -29-MTX mucin.

Int. J. Mol. Sci. 2018 , 19 , 2662 4 of 20
Figure 2.
Zinc binding properties of gastr ointestinal mucins. Differ ent zinc concentrations were
added to 25 mg/mL zinc-depleted por cine mucins and dialyzed against (T ris)-buffered saline (TBS)
for 12 h. Subsequently the amount of zinc retained by binding to mucins was measur ed using flame
atomic absorption spectrometry (F AAS) (
A
). Mor eover , the zinc content of mucins after zinc loading is
shown relative to the glycoprotein content measured by quantitative periodic acid Schif f (P AS)-assay
( B ), and relative
to pr otein content of mucins measured by bicinchoninic acid (BCA)-assay (
C
). Data are
shown as means
±
SD of at least thr ee independent experiments. Significant differ ences to the control
are indicated (** p < 0.01; *** p < 0.001; one-way ANOV A with Dunnett’s multiple comparison test).
Figure 3.
Zinc binding affi nity of gastrointestinal mucins. Shown ar e zinc binding affinities of two
differ ent zinc-depleted mucins analyzed with the chromophor e Zincon. For this, 1 mg/mL porcine
mucins (
A
) and mucins harvested from HT -29-MTX (
B
) were used. Data wer e analyzed with GraphPad
Prism softwar e version 5.01 (GraphPad Software Inc., San Diego, CA, USA) and a non-linear regr ession
assuming a one site-specific binding with Hill slope as a function of the zinc concentration was applied
to calculate the dissociation constants of the mucin-zinc-complex as indicated. Data are pr esented as
means ± SD of three independent experiments.
2.2. Role of Zinc Buffering by Mucins on Zinc Uptake into Goblet Cells
Next, the impact of mucins on zinc uptake by goblet cells was investigated. Zinc absorption of
mucin-pr oducing HT -29-MTX cells with or without mucin r emoval as well as the enterocyte cell line
HT -29 was analyzed with the fluorescent low molecular weight zinc pr obe Zinpyr-1 and is depicted
as the incr ease of intracellular free zinc (Figur e 4 A–C). Of note, the term “fr ee” zinc is frequently
used to describe the zinc pool that is complexed by small molecule ligands [
27
], which was alr eady
employed to investigate short-term zinc uptake in intestinal cells [
16
,
17
]. In detail, HT -29 showed
a concentration-dependent zinc absorption (Figur e 4 A), while zinc uptake of the mucin-producing
HT -29-MTX cells was very slight and concentration-independent (Figure 4 B). When extracellular
mucins wer e depleted with N -acetylcysteine (NAC), zinc uptake of HT -29-MTX cells incr eased
(Figur e 4 C). Notably , analysis of the intracellular distribution of the fluorescent zinc sensor r evealed

Int. J. Mol. Sci. 2018 , 19 , 2662 5 of 20
a vesicular accumulation in both cell lines (Figur e 4 D). Extracellular mucins were visualized with the
high molecular fluor escein isothiocyanate (FITC)-dextran 20 kDa (FD-20). It intercalates in the mucus
layer due to its high molecular weight [
28
], and was analyzed using confocal laser scanning microscopy
(CLSM) together with staining of the cell membrane (Figur e 4 E). Z-scans showed differ ences in the
mucin thickness of HT -29 and HT -29-MTX, with HT -29 showing only slight FD-20-staining, wher eas
the HT -29-MTX goblet cells produced a thick extracellular mucin layer , which decreased visibly after
mucin depletion. Moreover , mucin secr etion of HT -29-MTX cells and its successful depletion with NAC
was further investigated by immunofluorescent staining of the MUC5AC-apopr otein together with
nuclear staining using Hoechst (Figure 4 F). It shows a dif fuse distribution of the MUC5AC-apoprotein
over the cell layer of HT -29-MTX, whereas no MUC5AC-staining was observed for mucin-depleted
HT -29-MTX and HT -29 cells.
Figure 4.
Effect of mucin depletion on zinc-r esorption in HT -29-MTX. (
A
–
C
) Zinc uptake of the
enterocytes HT -29 was analyzed with the fluorescent pr obe Zinpyr-1 (
A
) and compared to the
zinc-uptake of the goblet cell line HT -29-MTX (
B
). Additionally , the effect of mucin depletion on
zinc absorption of HT -29-MTX was analyzed after removing extracellular mucins using 10 mM
N -acetylcysteine (
C
). Data are pr esented as means
±
standard err or of the mean (SEM) of thr ee
independent experiments. (
D
) Cellular distribution of Zinpyr -1 in HT -29 and HT -29-MTX cells together
with nuclear staining using Hoechst was analyzed by fluor escence microscopy . Scale bar 20
µ
m.
(
E
) V isualization of extracellular mucins with fluorescein isothiocyanate (FITC)-dextran was conducted
using confocal laser scanning micr oscopy . Shown ar e z-stacks of HT -29, HT -29-MTX and HT -29-MTX
without mucins after incubation with FITC dextran (green). The cell layer is stained with a cell
membrane-tracker (red). Scale bar 50
µ
m. (
F
) Immunochemical detection of MUC5AC and nuclear
staining using Hoechst using fluorescence micr oscopy in HT -29, regular HT -29-MTX and HT -29-MTX
after removal of mucins. Scale bar 20 µ m.

Int. J. Mol. Sci. 2018 , 19 , 2662 6 of 20
2.3. Impact of Extracellular Mucins on Zinc Uptake into Enterocytes
The impact of dif ferent mucin concentrations on short-term zinc uptake into the intestinal cell
line Caco-2 was investigated with Zinpyr -1 (Figure 5 A). Cellular fr ee zinc is increasing after adding
25 and 50
µ
M zinc, r espectively , but decreases significantly with added zinc-depleted por cine mucin
(one-way ANOV A with the Bonferroni post hoc test: 1.25 mg/mL (25
µ
M): p < 0.05; 2.5 mg/mL (25
µ
M
and 50
µ
M): p < 0.05; 5 mg/mL (25
µ
M and 50
µ
M): p < 0.05). Mor eover , Figure 5 B pr esents imaging of
the cellular localization of Zinpyr -1 fluorescence in Caco-2 cells unveiling a pr edominantly vesicular
distribution of the fluor escence.
Figure 5.
Impact of zinc-depleted mucins on zinc uptake by enterocytes. (
A
) Zinc uptake in Caco-2
cells after zinc incubation for 40 min in the presence of dif ferent concentrations of zinc-depleted por cine
mucin is shown as the incr ease of free zinc using the fluor escent zinc probe Zinpyr -1. Data ar e shown
as means + SD of at least three independent experiments. Significant dif ferences fr om 0 mg/mL
zinc-depleted porcine gastric mucin within one zinc concentration ar e indicated (*
, #,
p < 0.05;
** p < 0.01
; *** p < 0.001, one-way ANOV A with Dunnett’s multiple comparison test). (
B
) Fluorescence
microscopy showing the intracellular distribution of the zinc-dependent signal of the fluorescent pr obe
Zinpyr-1 in Caco-2 cells together with nuclear staining using Hoechst. Scale bar 20 µ m.
Short-term zinc uptake in the pr esence of 5 mg/mL zinc-depleted porcine mucins was also
investigated by F AAS, yielding no significant change of the cellular zinc content either with or without
zinc-depleted mucins (Figur e 6 A). In comparison, long-term zinc absorption in the presence of 0 and
5 mg/mL zinc-depleted mucins, also conducted with F AAS, resulted in a significant incr ease of the
cellular zinc content of Caco-2 cells after applying zinc without mucins (Figur e 6 B; one-way ANOV A
with Dunnett’s multiple comparison test; 25
µ
M: p < 0.001; 50
µ
M: p < 0.01). The incubation of zinc
together with 5 mg/mL mucins did not significantly change the cellular zinc content.

Int. J. Mol. Sci. 2018 , 19 , 2662 7 of 20
Figure 6.
Impact of extracellular mucins on zinc uptake in Caco-2 cells measured with F AAS.
The influence of extracellular addition of zinc-depleted mucins on the uptake of dif ferent zinc
concentrations in enter ocytes was investigated after incubation for 30 min (
A
) and 24 h (
B
). Cellular zinc
content was analyzed using F AAS and is shown relative to cellular pr otein. Data ar e shown as means
+ SD of three independent experiments. Means significantly differ ent from the untr eated controls ar e
indicated (* p < 0.05; ** p < 0.01; one-way ANOV A with Dunnett’s multiple comparison test).
Next, the zinc buffering capacity of mucins and their zinc r elease into enterocytes was investigated
in an experimental setting closer to the
in vivo
situation by using the afor ementioned low , medium
and high zinc-loaded por cine mucins. First, cellular zinc uptake fr om zinc-loaded mucins diluted to
a final zinc concentration of 25
µ
M was compar ed to the absorption of 25
µ
M zinc without mucins
(Figur e 7 A). While low and medium zinc-loaded mucins resulted in a comparable rise of intracellular
fr ee zinc, high zinc-loaded mucins caused a significant increase. However , they did not r each the levels
of cellular zinc that wer e observed without mucins (Figure 7 A).

Int. J. Mol. Sci. 2018 , 19 , 2662 8 of 20
Figure 7.
Effect of mucin zinc saturation on zinc uptake by enter ocytes. The impact of the degr ee of
zinc saturation of extracellular added mucins on zinc uptake in Caco-2 cells is shown by measuring the
increase of fr ee zinc using the fluorescent zinc pr obe Zinpyr-1. Porcine mucins wer e incubated with
2500
µ
M, 5000
µ
M and 10,000
µ
M zinc, resulting in mucins with dif fering zinc content (low , medium,
high) and degree of zinc saturation. (
A
) Zinc absorption of Caco-2 cells after 40 min of treatment
with 25 µ M zinc alone or with zinc-loaded mucins, diluted to a final zinc concentration of 25 µ M zinc
(final mucin concentration: 1 mg/mL low zinc mucin, 0.46 mg/mL medium zinc mucin, 0.31 mg/mL
high zinc mucin). (
B
) Zinc uptake after 40 min incubation with 0
µ
M or 25
µ
M zinc in the presence
of 0 mg/mL or 0.25 mg/mL zinc-loaded or zinc-depleted mucins, respectively . Data are pr esented
as means + SEM of at least thr ee independent experiments. Significant dif ferences wer e analyzed by
repeated-measur es ANOV A with the Bonferr oni post hoc test. Bars sharing a letter (a, b, c, d) ar e not
significantly differ ent.
T o elaborate the influence of the zinc buffering capacity of mucins on cellular zinc uptake,
the absorption of 25
µ
M zinc in the pr esence of 0.25 mg/mL zinc-loaded or zinc-depleted mucins was
analyzed (Figur e 7 B). This was then compared to the uptake of 25
µ
M zinc without mucins. Here,
intracellular fr ee zinc increased significantly , whereas the pr esence of zinc-depleted mucins diminished
the zinc uptake comparable to the results shown in Figur e 5 . Incubating the cells with 0.25 mg/mL
zinc-loaded mucins (w/o additional zinc), a similar slight increase of fr ee zinc regar dless of the mucins’
r emaining zinc-binding capacity and zinc content was detected. Adding zinc-loaded mucins and
25
µ
M zinc simultaneously to the cells yielded a significant rise of free zinc in the pr esence of high
zinc-loaded mucins (r epeated-measures ANOV A; high zinc-loaded mucins with 25
µ
M zinc compar ed
to high zinc mucins with 0
µ
M: p < 0.05). This increase was similar to the absorption of 25
µ
M zinc in
the absence of mucins. In contrast, low and medium zinc mucins, still not completely saturated with
zinc, showed no additional zinc absorption in the pr esence of 25 µ M zinc.
2.4. Comparison of Zinc Resorption in Different Intestinal Cell Cultur e Models: The Role of Mucins
Finally , the role of intestinal mucins on zinc r esorption was analyzed comparing zinc transport
by a Caco-2/HT -29-MTX co-culture with a Caco-2 monocultur e. The integrity of the cell monolayers
was monitor ed during the experiments by measuring the paracellular permeability for FD-20 and
by detecting transepithelial electrical resistance (TEER) at the beginning and end of the experiments,
r evealing no impairment of both parameters during the resorption study (
Supplementary Figur e S4
).
The pr esence of goblet cells in the co-culture did not influence the permeability of the cell

Int. J. Mol. Sci. 2018 , 19 , 2662 9 of 20
monolayer , as the transepithelial resistance of the co-cultur es and Caco-2 monocultures did not
dif fer significantly (co-cultures: 1146.7
±
25.5
Ω ·
cm
2
; monocultur es: 1288.9
±
239.2
Ω ·
cm
2
) and
the paracellular permeability was comparable to those measur ed in the absence of goblet cells
(Supplementary Figur e S4).
Apical zinc uptake by the monoculture, relative to the initially applied zinc concentrations,
was declining with incr easing amounts of zinc. In contrast, the Caco-2/HT -29-MTX co-culture
absorbed comparable amounts between 12.8% and 14.2% of all added zinc concentrations (Figur e 8 A,B).
Regar dless of the differ ences in the apical zinc uptake, the fractional zinc resorption into the basolateral
compartment of both intestinal models declined inversely related to the initially added zinc. Y et,
the fractional r esorption was significantly higher in Caco-2/HT -29-MTX co-cultur es (Figure 8 C,D;
two-way ANOV A with the Bonferroni post hoc test comparing the mono- and co-cultur es: 25
µ
M:
p < 0.001
; 50
µ
M: p < 0.05). More pr ecisely , fractional r esorption by monocultures dr opped from 1.6%
to 0.9%, showing only slight concentration dependence, wher eas resorption by co-cultur es declined
fr om 4.2% to 1.9% of the initially added zinc. Additionally , in both intestinal models the cellular zinc
uptake incr eased with added zinc, yielding a significantly higher uptake after addition of 50
µ
M zinc
by the co-cultur e (Figure 8 E,F; two-way ANOV A with the Bonferroni post hoc test, p < 0.05).
Figure 8.
Comparison of zinc resorption in Caco-2 monocultur es and Caco-2/HT -29-MTX co-cultures.
Zinc transport studies wer e conducted using the enterocytes Caco-2 (
A
,
C
,
E
) and a co-culture of Caco-2
with the mucus-producing goblet cell line HT -29-MTX (
B
,
D
,
F
). Shown are the decr ease of apical zinc
(apical zinc uptake) (
A
,
B
) and fractional zinc resorption after 4 h incubation r elative to the initially
added amount of zinc (
C
,
D
). Moreover , cellular zinc uptake is shown relative to cellular pr otein
content (
E
,
F
). Data are shown as means + SD of thr ee independent experiments and means significantly
differ ent from the untr eated controls ar e indicated (* p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOV A
with Dunnett’s multiple comparison test). Accor ding to a two-way ANOV A with the Bonferroni post
hoc test comparing the results within one added zinc concentration of the mono- and co-cultur es there
are significant di fferences r egarding the fractional zinc r esorption (25
µ
M: p < 0.001; 50
µ
M: p < 0.05),
and zinc uptake (50 µ M: p < 0.05).

Int. J. Mol. Sci. 2018 , 19 , 2662 10 of 20
Supplementary T able S1 summarizes detailed quantitative data of zinc uptake into the cells,
cellular zinc content, and the amount of zinc transported to the basolateral compartment. Over all,
the zinc transport study r esulted in higher zinc transport rates for the mucin-producing co-cultur e
(Figur e 9 A,B; monoculture: 0.3–1.29 nmol zinc/cm
2
; co-culture: 1.1–2.3 nmol zinc/cm
2
). In detail,
accor ding to a two-way ANOV A with the Bonferroni post hoc test comparing the r esults of the two
intestinal models within one added zinc concentration, the co-culture r esulted in a significantly higher
zinc transport rate of initially added 100 µ M ( p < 0.05).
Figure 9.
Zinc transport rates in Caco-2 monocultures and Caco-2/HT -29-MTX co-cultur es. Zinc
transport rates in nmol zinc per cm
2
resorption ar ea in mono- and co-cultures ar e displayed. Data ar e
presented as means + SD o f three independent experiments. Significant differ ences to control cells
(0
µ
M zinc) are indicated (** p < 0.01; one-way ANOV A with Dunnett’s multiple comparison test).
According to a two-way ANOV A with the Bonferroni post hoc test comparing the r esults within one
added zinc concentration of the mono- and co-cultures, ther e is a significant differ ence between the
zinc transport rate at 100 µ M ( p < 0.001).
3. Discussion
Zinc binding by the mucus layer was alr eady observed in animal studies [
4
,
5
,
9
,
10
], suggesting
that mucins could play a r ole in human intestinal zinc absorption. However , little was known about
the distinct zinc binding properties of these glycopr oteins. The pr esent study demonstrated that
zinc binding is not only dependent on the amount of the available, or rather , free zinc concentration,
but also on the zinc:mucin ratio. Assuming an appr oximate molecular mass of intestinal mucins of
2.5 MDa [
29
,
30
], the employed mucin concentrations between 2.5–10 mg/mL correspond to 1
µ
M–4
µ
M mucin, resulting in a 2–8 fold molar excess of zinc in the assay . When applying an 8-fold molar
zinc excess, 50% of zinc was retained by the glycopr oteins, declining to almost no free zinc at a molar
zinc:mucin ratio of 2 (Figur e 1 ). This suggests multiple zinc-binding sites within one mucin molecule
with biologically r elevant affinity .
Notably , gastrointestinal mucins wer e able to decrease the amount of available zinc for
the high af finity colorimetric reagent P AR (dissociation constant of the zinc-(P AR)
2
-complex
7.08 × 10 − 13 M 2 [ 31 ]
), indicating that at least some of the binding sites have high affinity for zinc.
The coor dination of metals by proteins str ongly depends on the metal ion prefer ence for particular
electr on donors [
32
]. Zinc tends to form stronger covalent binding to nitr ogen and sulfur and weak
complexes with oxygen [
32
]. The latter are highly pr esent in mucins by carboxylate gr oups of the
O -glycans [
13
]. Nitr ogen and Sulfur ar e part of both human and porcine intestinal mucins containing
N -acetyl gr oups ( N -acetyl-galactosamine (GalNac), N -acetylneuraminic acid (NeuAc)) [
33
,
34
] and on
average about 8–14% fr ee thiols [
35
,
36
], possibly playing an important r ole in the zinc binding affinity
of mucins.
The zinc binding capacity of mucins was further investigated by dialysis of pig gastric mucins
against dif ferent zinc concentrations, r esulting in low , medium and high zinc-loaded mucins (Figure 2 ).
Accor ding to the present study , mucins have an average molar zinc binding capacity of about
200, indicating a multiplicity of zinc-binding sites within one mucin molecule, possibly providing

Int. J. Mol. Sci. 2018 , 19 , 2662 11 of 20
a br oad spectrum of differ ent binding af finities. Mucins ar e highly glycosylated proteins, consisting
of appr oximately 80% carbohydrates [
11
,
13
]. Thus, the total amount of zinc bound per g pr otein is
one or der of magnitude higher than per g glycoprotein (Figur e 2 B,C). These results ar e comparable to
those obtained by Quarterman et al. analyzing zinc binding of porcine mucins at dif fer ent pH levels,
which led to a zinc content of 10 mg zinc per g mucin at pH 7.5 [
4
]. Furthermor e, the binding constants
of por cine gastric mucin and mucins obtained from the cell line HT -29-MTX are in good agr eement
with each other (Figur e 3 ), and additionally are of the same magnitude as luminal zinc levels [
37
,
38
].
Given the high number of binding sites, they pr obably repr esent an average dissociation constant of
the zinc/mucin complex, constituting a mixtur e of several binding sites with varying affinities.
Diet-derived luminal factors influence intestinal zinc bioavailability [
39
]. T ogether with
physiological factors such as luminal fluid and mucus layer , these components repr esent the luminal
matrix, which influences zinc speciation, consequently af fecting its availability for enterocytes [
4
,
18
].
T o illuminate the impact of apically present zinc-binding pr oteins on zinc uptake into enterocytes,
a r ecent study of our group investigated the ef fect of albumin on zinc absorption. Albumin significantly
r educed short-term zinc uptake measured with the low molecular weight sensor Zinpyr -1 [
17
]. By the
same technique, the r ole of mucins in short-term zinc absorption was analyzed in the present study .
The goblet cell line HT -29-MTX produces and secr etes mucins covering the cell surface, as shown by
qualitative analysis using immunochemical staining of the MUC5AC-apopr otein (Figure 4 E), which is
comparable to pr evious MUC5AC-stainings of HT -29-MTX mucins [
40
]. Y et, the impact of the mucus
layer on their zinc uptake has not been investigated before. Zinc uptake of HT -29-MTX before and after
mucin depletion was analyzed and compar ed to the intestinal absorptive cells HT -29. Overall, mucin
depletion caused a FD-20- and MUC5AC staining almost similar to that of HT -29 cells, confirming
a successful r emoval of extracellular mucins by N-acetylcysteine. Notably , short-term zinc uptake
of HT -29-MTX was impaired by extracellular mucins, which indicates zinc binding and buf fering by
the glycopr oteins produced by these cells. In Caco-2 cells, cellular zinc absorption also decr eased
significantly with elevated concentrations of zinc-depleted por cine mucins (Figure 5 A), comparable
to the impairment by albumin [
17
]. In addition to the analysis with the low molecular weight sensor
Zinpyr -1, short-term zinc uptake by Caco-2 cells was also measured with F AAS resulting in no
significant change of cellular zinc, either with or without mucins (Figure 6 A). Only long-term zinc
incubation of Caco-2 cells r esulted in a significant increase of cellular zinc in the absence of mucins
(Figur e 6 B). Consistent with previous findings, changes in intracellular zinc after short-term incubation
of Caco-2 cells ar e probably too small compar ed to the cellular zinc content to be detected by F AAS [
17
].
Fr om the uptake studies shown in Figures 5 A and 6 it could be concluded that mucins impair
intestinal zinc availability . However , these por cine mucins were zinc-depleted, which is compulsory
for investigating zinc-binding capacity and af finity , but does not r epresent the
in vivo
situation in
the intestinal lumen under zinc adequate conditions [
38
,
41
]. This is corroborated by the fact that the
commer cially available porcine mucins contained considerable amounts of zinc befor e zinc-depletion
with Chelex
®
100 Resin. Accordingly , the impact of zinc-loaded mucins on zinc uptake into enterocytes
was examined using zinc-containing mucins (low , medium and high zinc-loaded mucins; Figure 7 ).
Her e, all three types of mucins seemed to r elease part of their zinc, resulting in an incr ease of cellular
fr ee zinc compared to contr ol cells (Figure 7 A). Inter estingly , this release appear ed to be largely
independent of the mucins’ zinc content and their r emaining zinc binding capacity , adding the same
concentration of dif ferentially loaded mucins to the cells r esulted in similar zinc uptake (Figure 7 B).
These observations suggest that mucins buf fer the cellular available zinc concentration in the lumen,
still keeping it available for enter ocytes. In animal studies, intestinal mucins wer e discussed to absorb
zinc fr om the luminal content transferring it to the mucosa [
9
,
10
]. Zinc transfer to the intestinal cells
occurr ed slower than the initial zinc binding by mucins [
9
]. In this manner , the intestinal mucus
layer might lead to r etention of luminal available zinc, possibly providing intestinal cells with zinc
for extended periods of time after food intake. This implies that mucins act as a zinc delivery system
fr om the lumen to the intestinal epithelium, which was already postulated for ir on [
5
], and led

Int. J. Mol. Sci. 2018 , 19 , 2662 12 of 20
to our hypothesis that zinc-saturated mucins might facilitate zinc delivery to enter ocytes. In the
pr esent study , however , cellular zinc uptake in the presence of zinc-saturated mucins was similar , but
not augmented, compared to the absence of mucins (Figur e 7 B). This discrepancy might be due to
insuf ficient equivalence to the
in vivo
situation: first, the basolateral compartment, wher e the absorbed
zinc can be exported, is lacking. Second, the mucins applied in this study wer e commercially available
isolated por cine mucins, which might not be entirely comparable to the native mucins pr oduced by
goblet cells [
42
]. Although these mucins are often used as a standar d model for the mucus layer to
investigate characteristics of gastr ointestinal mucins [
43
] and their r ole in intestinal metal uptake [
28
],
they ar e also known to have weaker gel-forming abilities [
44
], possibly due to pr otease treatment
during the isolation and purification pr ocess [ 42 ].
T o overcome both issues, the impact of chemically unprocessed mucins on intestinal zinc
r esorption in a three-dimensional cultur e was investigated by transport studies in the mucin-producing
Caco-2/HT -29-MTX co-culture, which wer e then compar ed to zinc transport in the absence of a mucus
layer using conventional Caco-2 monocultur es. Her ein, the mucin-producing co-cultur e clearly yielded
higher zinc absorption than the mucus-lacking monoculture (Figur e 8 ). The Caco-2/HT -29-MTX
model r esulted in a stronger decr ease of the apical zinc concentration, varying ar ound 12.7–14.1%
independent of the initially added zinc, as well as an elevated cellular zinc uptake (Figure 8 E,F),
indicating that the cellular zinc uptake is facilitated by the mucus layer . Indeed, this supports the
afor ementioned hypothesis that the mucus layer might act as a zinc delivery system for the intestinal
epithelium. Moreover , the glycopr oteins seemed to assist the zinc transfer across the intestinal
epithelium as the fractional zinc resorption was significantly higher (1.8–2.4 fold higher) in the pr esence
of mucin-pr oducing goblet cells. Likewise, the zinc transport rate was elevated using co-cultures.
Her e, the basal zinc transport rate without the addition of exogenous zinc was already 3.8 fold higher
(1.13 ng zinc/cm
2
r esorption area) than that of monocultur es (0.3 ng zinc/cm
2
) (Figur e 9 ), possibly
due to r esorption of the basal zinc levels of mucins. These basal zinc levels might originate from
the cell cultur e medium (containing 3
µ
M zinc), and were incorporated by the mucins during the
21 days of cultivation of the cell model. Thus, the mucus-secreting co-cultur e not only absorbed
mor e zinc from the apical compartment, but showed augmented zinc export to the basolateral side,
supporting pr evious observations that intestinal mucins repr esent an important factor for intestinal
zinc r esorption [ 4 , 9 , 10 ].
The beneficial r ole of mucins for the resorption of other trace elements, such as ir on, is already
well documented [
21
,
28
,
45
]. Concerning the essential nutrient ir on, the mucus layer not only mediates
its availability for the intestinal epithelium by maintaining its solubility [
5
], but was also proposed
to pr ovide its delivery to the absorptive cells by the mucin-integrin mobilferrin pathway [
45
,
46
].
A similar mechanism might also be ef fective for zinc, as the present study indicates that mucins not
only influence zinc uptake by incr easing its luminal solubility , as discussed befor e [
4
], but rather
pr omote zinc absorption by additionally acting as a zinc delivery system for the mucosa, which would
be in good agr eement with observations from animal studies [ 9 , 10 ].
Metal ion binding by mucins might have further implications for metal ion homeostasis.
The impact of other trace elements on zinc r esorption was previously investigated and is still a topic
of ongoing r esearch [
3
,
47
]. Mucins ar e not only involved in the mucosal uptake of single trace
elements, but wer e also suggested to influence their bioavailability by competitively binding differ ent
metals [
5
]. Thus, in addition to competing for transport pr oteins, competition for binding sites in
mucins could be another factor for the mutual interferences observed in intestinal trace element
absorption. Furthermor e, mucins not only support intestinal zinc absorption, but might also be
involved in fecal zinc loss. A considerable amount of endogenous zinc is excreted with feces [
2
,
48
].
Even during extr eme zinc deficiency , the fecal excretion of zinc can be observed, which is defined as
the “obligatory fecal loss” [
2
]. The intestinal epithelium and the overlying mucus layer under go cycles
of r enewal [
49
,
50
] r esulting in a complete turnover of the mucins, which is said to occur much faster
than that of the underlying mucosa [
51
]. Considering the amount of zinc bound to mucins, the fast

Int. J. Mol. Sci. 2018 , 19 , 2662 13 of 20
r enewal of the intestinal mucus layer might play an important role in the fecal zinc loss as the mucins
ar e possibly excreted together with a r emainder of tightly bound zinc.
On the one hand, gastr ointestinal mucins are important for zinc absorption in the intestinal
tract. On the other hand, zinc was also reported to be important for mucin synthesis, as the gene
expr ession profile of dif ferent mucin pr oteins was shown to depend on zinc supply [
52
], and abdominal
pr oduction as well as secretion of mucins wer e also decreased in zinc-deficient animals [
53
,
54
]. Thus,
further investigations of the interplay of zinc with intestinal mucins and distinct molecular mechanisms
of the mucosal delivery system ar e needed for a better understanding of their role in intestinal zinc
uptake as well as gastr ointestinal disorders.
4. Materials and Methods
4.1. Materials
CellMask
™
DeepRed (ThermoFisher Scientific, W altham, MA, USA); Cy3
®
goat anti rabbit IgG
(Jackson ImmunoResear ch, Dianova, Hamburg, Germany); Chelex
®
100 Resin (Bio-Rad, Hercules,
CA, USA); Dulbecco’s modified Eagles medium (DMEM) (P AN-Biotech, Aidenbach, Germany);
Hoechst 33258 (Sigma Aldrich, Munich, Germany); fluorescein isothiocyanate (FITC)-dextran 20
(TdB, Uppsala, Sweden); fetal calf serum (FCS) (CCPro, Ober dorla, Germany); mucin from por cine
stomach T ype II (Sigma Aldrich, Munich, Germany); P AR (Sigma Aldrich, Munich, Germany);
T ranswell inserts (Corning, New Y ork, NY , USA); N , N , N
0
, N
0
-T etrakis(2-pyridylmethyl)ethylenediamine
(TPEN) (Sigma Aldrich, Munich, Germany); W illCo-dish
®
glass bottom dish (W illCo, Amsterdam,
The Netherlands); Zincon (Sigma Aldrich, Munich, Germany); ZnSO
4 ·
7H
2
O (Sigma Aldrich, Munich,
Germany). All other chemicals were pur chased from standar d sources.
4.2. Cell Culture
Caco-2, HT -29-MTX and HT -29 cells wer e cultured at 37
◦
C, 5% CO
2
and in a humidified
atmospher e in DMEM, containing 10% FCS, 100 U/mL penicillin and 100
µ
g/mL str eptomycin.
Additionally , medium for HT -29-MTX contained 1% non-essential amino acids (NEAA). Media wer e
changed every other day . Analysis of pr oper dif ferentiation, morphology and barrier integrity of
Caco-2 cells cultur ed in our lab into an enterocyte-like phenotype after 21 days, as shown befor e [
20
,
55
],
was r eported previously [
56
]. Caco-2, HT -29-MTX and HT -29 cells wer e obtained from Eur opean
Collection of Cell Cultur es (ECACC, Porton Down, UK).
4.3. 4-(2-Pyridylazo)Resorcinol (P AR) Assay
Zinc binding by mucins was investigated with the colorimetric r eagent P AR. The assay was
performed in TBS, containing 50 mM T ris(hydroxylmethyl)aminomethan and 150 mM NaCl, at pH 7.5.
Stock solutions of 25 mg/mL por cine mucins were pr epared in TBS and zinc-depleted using Chelex
®
100 Resin accor ding to the manufacturer ’s pr otocol (zinc content before depletion: 436
µ
g zinc/g mucin;
zinc content after depletion: 16.2
µ
g zinc/g mucin). Differ ent mucin concentrations were incubated
with 8
µ
M ZnSO
4 ·
7H
2
O and 20
µ
M P AR (stock solution 25 mM in H
2
O) was added. Absorption of the
zinc-(P AR)
2
-complex was measur ed at 485 nm using a well plate reader (M200, T ecan, Switzerland)
and the ef fect of mucins on the availability of zinc for P AR was analysed using an external calibration
of 0–10 µ M zinc obtained by spectr ophotometric titrations followed by linear regr ession analysis.
4.4. Zinc-Binding Capacity
The zinc-binding capacity of gastr ointestinal mucins was investigated by dialysis. 25 mg/mL
mucins wer e incubated with 0–100 mM ZnSO
4 ·
7H
2
O overnight and dialyzed against 1.5 L TBS
for 6, 12 and 24 h. Finally , zinc concentrations wer e determined with F AAS using a Perkin Elmer
AAnalyst800 (Perkin Elmer , Rodgau, Germany) and zinc-binding capacity was calculated relative to
the zinc concentrations befor e dialysis. Furthermore, the amount of pr otein in the mucin samples after

Int. J. Mol. Sci. 2018 , 19 , 2662 14 of 20
dialysis was analyzed using BCA-assay as described [
57
] and the glycopr otein content in the mucin
samples was detected using the quantitative P AS-assay from Schömig et al. [ 44 ].
4.5. Zinc-Binding Affinity of Mucins
The binding af finity of commercially available por cine gastric mucins and mucins produced by the
cell line HT -29-MTX were investigated with the chelating chr omophore zincon [
31
]. Secr eted mucins
fr om HT -29-MTX wer e collected after culturing the cells for 13 days, washing with phosphate buffer ed
saline (PBS) and additional incubation in DMEM without phenol red (with 100 U/mL penicillin
and 100
µ
g/mL str eptomycin, w/o FCS) for 24 h. Subsequently , the cell supernatant was collected
and secr eted mucins were concentrated to an 8-fold incr ease after desalting and washing with TBS
using ultrafiltration (molecular weight cut-of f: 50 kDA) followed by zinc depletion using Chelex
®
100 Resin (zinc content befor e depletion: 59.2
µ
g/g pr otein; zinc content after depletion: 4.1
µ
g/g
pr otein). Pr otein content was analyzed using BCA assay as described [
57
]. Finally , zincon in TBS pH 7.5
(final concentration 50
µ
M) was added to either 1 mg/mL commer cially available porcine mucin or
the equivalent pr otein amount of secreted mucins fr om HT -29-MTX, r espectively , and titrated with
0–60
µ
M ZnSO
4 ·
7H
2
O. Subsequently , the absorption was determined on a well plate reader (M200,
T ecan, Switzerland) at 620 nm. The amount of mucin-bound zinc was calculated using an external
calibration. Data wer e analyzed with GraphPad Prism softwar e version 5.01 (GraphPad Software Inc.,
San Diego, CA, USA) and a non-linear regr ession assuming a one site-specific binding with Hill slope
as a function of the zinc concentration was applied.
4.6. Cellular Zinc Uptake Measured by Zinpyr-1
Short-term zinc uptake in Caco-2, HT -29 and HT -29-MTX was quantified as the incr ease of free
zinc [nM] using the low molecular zinc pr obe Zinpyr-1. The concentration of fr ee zinc was determined
using the following equation of Grynkiewicz et al.
[Zinc] = Kd × [(F − F min )/(F max − F)] ([58])
and a dissociation constant for the zinc-Zinpyr-1-complex of 0.7 nM [
59
]. Cells wer e transferred to
96-well plate and cultur ed for 21days (Caco-2; initial cell number per well: 5000) or 7 days (HT -29 and
HT -29-MTX; initial cell number per well: 10,000), respectively . On the day of the experiment, cells
wer e incubated with 2.5
µ
M Zinpyr -1 and the uptake of zinc, in the presence or absence of mucins as
indicated in the r espective figure legends, was measur ed as the increase of fr ee zinc on a fluorescence
well plate r eader (Spark, T ecan, Switzerland; Zinpyr-1 fluor escence:
λ ex
= 508 nm and
λ em
= 527 nm)
as described [ 17 ].
4.7. Removal of Extracellular Mucins by N-Acetylcysteine
Extracellular mucins of the goblet cell line HT -29-MTX were r emoved by r educing the intra- and
intermolecular disulfide bounds in the glycopr oteins using NAC [
21
,
60
]. Befor e analyzing zinc uptake
of HT -29-MTX, cells were tr eated twice with 10 mM NAC in PBS for 10 min. Between treatments,
cells wer e incubated in DMEM for 1 h. Successful removal of extracellular mucins was examined
using immunochemical staining of the main glycopr otein of HT -29-MTX, MUC5AC, and fluor escence
micr oscopic visualization with FD-20.
4.8. Immunochemical Staining of the MUC5AC Glycoprotein
Immunochemical staining of MUC5AC was performed using the mucin-specific antiserum
MAN-5ACI for the polypeptides of MUC5AC [
61
,
62
]. Ther efore, HT -29-MTX and HT -29 cells were
seeded on glass slides and cultivated for 7 days. Depletion of extracellular mucins of HT -29-MTX prior
to immunochemical staining was performed as described above. Cells were fixed on ice with a final
concentration of 3.7% formaldehyde dir ectly added to the cell medium, washed with cold PBS and

Int. J. Mol. Sci. 2018 , 19 , 2662 15 of 20
permeabilized using 0.5% T riton-X-100 in PBS for 20 min on ice. After washing with PBS, cells wer e
blocked with 10% FCS in TBS for 1 h, incubated with MAN-5ACI antiserum (1:500 in TBS with 20%
T ween (TBST)) overnight, followed by additional washing and blocking. Subsequently , Cy3
®
goat anti
rabbit IgG (indocarbocyanin goat-anti rabbit immunoglobulin G) (1:500 in TBST) was incubated for
1 h at 37
◦
C. Additionally cellular nuclei wer e stained with Hoechst 33258. Finally , cells were washed
with TBST and evaluated by fluor escence microscopy (Axio Imager M1, Zeiss, Germany) at excitation
wavelengths of 546 nm (Cy-3) and 358 nm (Hoechst).
4.9. V isualizing Extracellular Mucins with Fluorescein Isothiocyanate (FITC)-Dextran
Extracellular mucins pr oduced by HT -29-MTX wer e visualized using high molecular dextran
labeled with fluor escein isothiocyanate (FD-20). Due to its size, FD-20 is trapped in the high molecular
glycopr oteins, possibly due to steric hindrance [
63
]. T o this end, HT -29 and HT -29-MTX cells wer e
cultur ed in glass bottom dishes for 14 d. Prior to the experiment, medium was car efully removed and
100
µ
M FD-20 together with cell membrane tracker CellMask
™
DeepRed (3.3 ng/mL) in assay buf fer
(120 mM NaCl, 5.4 mM KCl; 5 mM Glucose; 1 mM CaCl
2
, 1 mM MgCl
2
, 1 mM NaH
2
PO
4
, 10 mM
HEPES, pH 7.35) was incubated for 15 min at 37
◦
C. Subsequently , cells were washed with assay
buf fer and live cell imaging of fluorescently labeled extracellular mucins was performed with a CLSM
(Leica TCS SP8;
λ ex
(FITC) = 488 nm,
λ em
(FITC) = 510 nm;
λ ex
(CellMask
™
DeepRed) = 552 nm,
λ em
(CellMask™DeepRed) = 695 nm).
4.10. T otal Cellular Zinc Content Measured by Flame Atomic Absorption Spectr ometry (F AAS)
For the determination of long-term zinc uptake with F AAS, 1.2
×
10
5
Caco-2 cells wer e seeded
in 6 well plates and cultur ed for 21 days. Fully dif ferentiated cells wer e incubated with differ ent
zinc concentrations with 0 or 5 mg/mL por cine mucin in DMEM w/o phenol red and incubated for
24 h. Short-term uptake was conducted after 30 min incubation using 0 or 5 mg/mL mucin in assay
buf fer . Finally , cells wer e harvested on ice with a cell scraper , and an aliquot was collected for protein
quantification as described [
57
]. Subsequently , cells were dissolved in a mixtur e of 67% ultrapure
HNO
3
and 30% H
2
O
2
(50/50; v / v ) and dried at 92
◦
C overnight using a thermoshaker . Residues wer e
dissolved in 0.67% HNO 3 and samples wer e analyzed by F AAS.
4.11. Zinc T ransport Assay
Zinc transport studies wer e performed using monocultures of Caco-2 and Caco-2/HT -29-MTX
co-cultur es. Co-cultures wer e r ealized with an initial cell ratio of 75% Caco-2 and 25% HT -29-MTX
cells and alternated cell seeding, modified after Nollevaux et al. [
64
]. Her ein, the co-cultur e of Caco-2
and HT -29-MTX cells in our lab was characterized concerning the proper cellular ratio by investigating
the mucin secr etion and adequate differ entiation of the enterocytes as r eported before [ 17 ].
Eighty thousand cells wer e transferred onto polycarbonate transwell membranes (por e size
0.4
µ
m, cultur e area 1.12 cm
2
) and cultur ed for 21 days in DMEM with 10% FCS, 100 U/mL penicillin,
100
µ
g/mL str eptomycin and 1% NEAA. For the co-cultures, 25% 20,000 HT -29-MTX cells wer e added
2 days after seeding of Caco-2. After 21 days, the cells were incubated with 0
µ
M, 25
µ
M, 50
µ
M and
100
µ
M ZnSO
4 ·
7H
2
O in 0.5 mL transport buf fer [
17
] on the apical side of the transport chamber for 4 h.
The basolateral compartment constituted 1.5 mL cell cultur e medium with 30 mg/mL BSA. Prior and
after the experiment, barrier integrity was monitor ed by measuring TEER with the epithelial volt-ohm
meter Millicell
®
ERS-2 (Millipor e, Burlington, MA, USA). Additionally , permeability of cell monolayers
during the experiment was determined using FD-20 [
64
] as r eported before [
17
]. At the end of the
experiment, the media of the apical and basolateral compartments were collected, and cells were
harvested on ice in PBS, homogenized and centrifuged (800
×
g ). An aliquot of cell homogenates was
collected for pr otein quantification using BCA [
57
]. Subsequently , cells wer e dried at 92
◦
C overnight
as described above and dissolved in 0.67% HNO
3
. Zinc quantification in apical, basolateral and cellular
compartment was conducted by ICP-MS after dilution (1:10 and 1:200) in 2% HNO
3
containing 5
µ
g/L

Int. J. Mol. Sci. 2018 , 19 , 2662 16 of 20
r hodium, using an Agilent 8800 ICP-QQQ (Agilent T echnologies Deutschland GmbH, Böblingen,
Germany) in the single quad-mode [ 17 ].
4.12. Statistical Analysis
Statistical significance was analyzed by one- or two-way ANOV A (for multiple comparisons),
followed by Bonferr oni or Dunnett’s multiple comparison post hoc tests, as indicated in the respective
figur e legends, using GraphPad Prism software version 5.01 (GraphPad Softwar e Inc., San Diego, CA,
USA). Err or bars repr esent standard deviation or standar d error of the mean, as indicated, of at least
thr ee independent biological replicates.
5. Conclusions
This study pr ovides the first comprehensive assessment of the zinc binding pr operties of mucins
and their impact on
in vitr o
intestinal zinc r esorption. By clarifying the molecular zinc binding
capacity of these glycopr oteins and their average affinity for the bivalent cation, we could demonstrate
that mucins bind multiple zinc ions with physiologically relevant af finity . Her eby , zinc-fr ee mucins
impair zinc uptake, but this is not the form in which they are pr esent in the gastrointestinal tract.
2D-experiments with isolated por cine mucins show that mucin-bound zinc is still available for cellular
uptake, but not superior to fr ee zinc. In contrast, the 3D co-culture of enter ocytes and mucin-secreting
goblet cells suggests that mucins even facilitate zinc uptake by enterocytes, making them an integral
part of intestinal zinc r esorption.
Supplementary Materials:
Supplementary materials can be found at http://www .mdpi.com/1422- 0067/19/9/
2662/s1 .
Author Contributions:
Conceptualization, M.M., C.K. and H.H.; Data curation, M.M.; Formal analysis, M.M.
and H.H.; Funding acquisition, T .S. and H.H.; Investigation, M.M., J.K. and S.S.; Methodology , T .S.; Pr oject
administration, H.H.; Resources, T .S. and H.H.; Supervision, M.M., C.K. and H.H.; W riting—original draft, M.M.;
W riting—review and editing, C.K., T .S. and H.H.
Funding:
The work of H.H. and T .S. is funded by the Deutsche Forschungsgemeinschaft (T raceAge–DFG Research
Unit on Interactions of essential trace elements in healthy and diseased elderly , Potsdam-Berlin-Jena, FOR 2558/1,
HA 4318/4-1, SCHW903/16-1).
Acknowledgments:
The authors would like to thank V era Meyer from the Department of Applied and
Molecular Microbiology (Berlin Institute of T echnology) for their kind support with the CLSM measur ements,
David J. Thornton
(University of Manchester) for generously pr oviding the mucin-specific antiserum MAN-5ACI,
and A y ¸ se Duman for her excellent technical work.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ANOV A analysis of variance
BCA bicinchoninic acid
BSA bovine serum albumin
CLSM confocal laser scanning microscopy
DMEM Dulbecco’s modified Eagles medium
ECACC European Collection of Cell Cultur es
HBSS Hanks’ balanced salt solution
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
F AAS flame atomic absorption spectrometry
FCS fetal calf serum
FD (FITC)-Dextran
FITC fluorescein isothiocyanate
ICP-MS inductively-coupled plasma mass spectrometry
NAC N -acetylcysteine
NEAA non-essential amino acids
P AR 4-(2-pyridylazo)resor cinol
P AS periodic acid Schif f

Int. J. Mol. Sci. 2018 , 19 , 2662 17 of 20
Papp apparent permeability
PBS phosphate buffer ed saline
TBS tris(hydr oxymethyl)aminomethane (T ris)-buffer ed saline
TBST tris-buffer ed saline with T ween 20
TEER transepithelial electrical r esistance
TPEN N , N , N 0 , N 0 -T etrakis(2-pyridylmethyl)ethylenediamine
ZnT zinc transporter
ZIP Zrt Irt-like transporter
Zincon 2-carboxy-2 0 -hydroxy-5 0 -sulfoformazylbenzene monosodium salt
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