particle tracking microrheology of purified gastrointestinal mucins

Particle Tracking Microrheology of Purified

Gastrointestinal Mucins

Pantelis Georgiades1,2

, Paul D. A. Pudney3, David J. Thornton

2*, Thomas

Waigh1,4+

1Biological Physics, Department of Physics and Astronomy, University of Manchester,

Manchester, M60 1QD, United Kingdom, 2Faculty of Life Sciences, Michael Smith Building,

University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom, 3Unilever

Discover, Colworth Park, Shambrook, Bedfordshire MK44 1LQ, United Kingdom. 4Photon

Science Institute, University of Manchester, Oxford Road, Manchester, M60 1QD.

Keywords: mucin, rheology, gastric, intestinal, particle tracking, polyphenol, EGCG

The rheological characteristics of gastric and duodenal mucin solutions, the building blocks

of the mucus layer that covers the epithelia of the two organs, were investigated using particle

tracking microrheology. We used biochemically well characterised purified porcine mucins

(MUC5AC and MUC2) as models for human mucins, to probe their viscoelasticity as a

function of mucin concentration and pH. Furthermore, we used both reducing (dithiothreitol,

DTT) and chaotropic agents (guanidinium chloride and urea) to probe the mesoscopic forces

that mediate the integrity of the polymer network. At neutral pH both gastric and duodenal

mucins formed self-assembled semi-dilute networks above a certain critical mucin

concentration (c*) with the viscosity (η) scaling as �~��.��.�for MUC5AC and

�~��.��.� for MUC2, where c is the mucin concentration. Above an even higher mucin

concentration threshold (ce, the entanglement concentration) reptation occurs and there is a

Biopolymers

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/bip.22372© 2013 Wiley Periodicals, Inc.

2

dramatic increase in the viscosity scaling, �~��.��.� for MUC5AC and �~��. ��. for

MUC2. The dynamics of the self-assembled comb polymers is examined in terms of a scaling

model for flexible polyelectrolyte combs. Both duodenum and gastric mucin are found to be

pH switchable gels, gelation occurring at low pHs. There is a hundred-fold increase in the

elastic shear modulus once the pH is decreased. The addition of DTT, guanidinium chloride

and urea disassembles both the semi-dilute and gel structures causing a large increase in the

compliance (decrease in their shear moduli). Addition of the polyphenol EGCG has a reverse

effect on mucin viscoelasticity i.e. it triggers a sol-gel transition in semi-dilute mucin

solutions at neutral pH.

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1. Introduction

Mucus is a secreted viscoelastic fluid that lines the epithelium of the eye, respiratory,

gastrointestinal and urogenital tracts. The functions of mucus vary between the different

mucosal surfaces that it is found on. Mucus primarily acts as the first line of protection

against toxins, foreign particles and pathogens at the exposed mucosal surfaces, while its

other main role is mechanical, to reduce friction at the mucosal surfaces, by moving the plane

of shear away from the surface. In addition to the aforementioned general properties, mucus

also acts as a selective barrier, for example in the intestine, where it accommodates a colossal

number of bacteria and micro-organisms responsible for breaking down nutrients, while

allowing those nutrients to diffuse through it and be absorbed. In the stomach, mucus faces a

difficult challenge. During active digestion, gastric juices are formed in the stomach, which

are highly acidic to aid with protein digestion. The role of mucus is to form a pH gradient

ranging from neutral on the epithelium to pH 1-2 in the gastric lumen, to protect the stomach

walls from ingesting foreign particles, being damaged by the acidic conditions and preventing

the stomach from digesting itself.1–6

The major components of mucus are water (95%), immunoglobulins, cholesterol, lipids,

inorganic salts, proteins and the high molecular weight (Mw ranges from 2-3 MDa to tens of

MDa for secreted oligomers) glycoproteins known as mucins, which provide the structural

matrix from which the protective properties of the mucus layer are derived.7 There are two

main classes of mucins found in the human body; the secreted forms which are either

polymerising or nonpolymerising, and the cell-tethered forms1. The focus of the current work

is on polymerising secreted mucins, which are synthesized and secreted by specialised cells

found in the epithelium (goblet cells) and submucosal glands (mucous cells). These complex

O-linked glycoproteins are characterised by at least one large region of the polypeptide which

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is rich in serine, threonine and proline residues, called the STP-region or mucin domain1.

These mucin domains act as sites for covalent attachment of O-linked oligosaccharides,

resulting in extensive glycosylation and up to 70% of the molecule’s weight is accounted for

by carbohydrates. Furthermore, a common characteristic of polymerising secreted mucins is

the presence of cysteine-rich domains at their N- and C- termini that mediate disulphide

bonding between monomers, allowing the formation of disulphide linked polymeric

assemblies.1

The large size and complex chemical composition of mucins has made the characterisation of

their structure and physical properties in solution a difficult task for researchers. Previous

studies have concluded that isolated gastric and duodenal mucins share a common

mechanism for gel-formation and noncovalent interactions between the carbohydrate chains

contribute to the formation of the gel-network.8 Isolated porcine gastric mucins (PGM) have

been shown to adopt an isotropic random coil conformation in a dilute solution of neutral pH,

with most of the fibres ranging from 200-600 nm in length. When the pH of isolated

MUC5AC solutions was decreased below pH 4 a sol-gel transition is reported,3 which is

believed to be the result of the protonation of the carboxylates of the salt bridges, which

allows the unfolding and exposure of the protein’s hydrophobic regions. This leads to a

conformational transition of the molecule to an extended anisotropic random coil

configuration.9

Here we use isolated porcine gastric and duodenal mucins as biochemically well

characterised models for human mucins, to investigate the scaling of the viscoelasticity of the

materials in aqueous solutions. Viscoelasticity is a key physical property that determines the

behaviour of these materials in the stomach and intestines. For example, the microflora in the

stomach is required to swim through these materials during their life cycle.10

Viscoelasticity

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of materials is intimately related to the dynamics of the constituent molecules and its

measurement allows detailed molecular models of molecular dynamics to be created. In the

current article we use particle tracking microrheology (PTM) to probe the linear

viscoelasticity of mucin gels and solutions. This allows the viscoelasticity to be measured

over a broad range of frequencies (0.1-103

Hz) with small amounts of biological material

(~10 µL) and with the knowledge that the fragile biological specimens will not be damaged

(only thermal forces are applied). Furthermore, the effect of chaotropic and reducing agents

on the viscoelastic properties of mucin is probed, which yields information regarding the

forces that regulate the interactions in the gelled network. Moreover, the interaction of

polyphenols, a type of molecule abundant in tea and plant derived foods which is linked with

various health benefits, with mucins was examined. The polyphenol Epigallocatechin gallate

(EGCG), which is found in abundance in green tea and is believed to have a variety of health

benefits, was introduced to mucin solutions and its effect on the rheology of them was

investigated. It has been previously shown that EGCG has significant impact of the interfacial

shear rheology of human whole saliva, thus limiting the bioavailability of the molecule in the

body.11

Due to its importance in respiratory disease there have been a large number of rheology

studies on the viscoelasticity of mucus3,12–18

. Healthy animal mucus viscosities have been

measured in the range 0.0012-1000 Pas. This large variability in reported viscosities has in

part been due to the chemical heterogeneity and the variability in the mucin concentrations of

the samples. Therefore in the current study we used purified mucin samples and examined in

detail the concentration dependence of the mucin viscoelasticity.

Purified porcine mucins were chosen as model mucins, since previous studies using

commercially available Orthana mucin and partially purified mucin from Sigma, have

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concluded that the action of various proteases during their isolation has irreversibly altered

mucin structure and suppressed mucin-mucin interactions, which inhibits their ability to form

gels. Furthermore the carbohydrate side-chains are significantly deglycosylated in Orthana

samples and it is known that side chain dynamics can have a large impact on comb

polyelectrolyte viscoelasticity19

. Previous studies on these non-gelling Orthana mucins,

genetically similar to human MUC6, provided evidence for the double globular structure of

these materials and used scaling models for the structure and dynamics of flexible non-

associating polyelectrolytes to describe their behaviour.15,20,21

. In the current article we extend

on our previous studies to characterise gastrointestinal mucins whose purification process

was oriented towards maintaining their native structure and behaviour. Our findings suggest

that mucins demonstrate behaviour of large scale self-assembly of mucin oligomers into

fibrous aggregates which then cross-link to form gels at low pH.18

We expect differences in

the physical behaviour will still occur between purified mucins and naturally occurring

mucus secretions, but in terms of their linear viscoelasticity reasonably close agreement is

found between sample types i.e. mucin is the dominant contributing factor to the

viscoelasticity of mucus from healthy animals.

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2. Results

2.1 Microrheology of Porcine Gastric Mucin (PGM). Figure 1 shows the ensemble

averaged MSD curves of polystyrene spheres obtained for concentrations of PGM

(MUC5AC) in solution ranging from 1 to 50 mg/ml. The slope of the MSD curves obtained

from solutions of concentrations up to 20 mg/ml, when plotted on a loglog scale, is equal to

1. This indicates that stomach mucin solutions of up to 20 mg/ml behave as purely viscous,

Newtonian liquids and there is a monotonic increase of viscosity with mucin concentration.

The gradient of the loglog plot of the MSDs in the Newtonian regime is directly related to the

diffusion coefficient of the spheres’ Brownian motion in the mucin solutions. This allowed us

to calculate the viscosity of the samples in mPas, using Eq. (3). The scaling of the viscosities

as a function of concentration is shown in Figure 2. Viscosity increases with concentration, as

expected, but only to the relatively small value of 7 mPas at 15 mg/ml, about 7 times the

viscosity of water at the same temperature. The scaling of the viscosity of the solutions as a

function of mucin concentration (c) in the low concentration Newtonian regime follows a

power law behaviour of η ~ c0.53±0.0.08

characteristic of semi-dilute linear polyelectrolytes (the

Fuoss law)22

and also observed previously with non-associating Orthona mucin solutions.15

There is reasonable agreement between the current viscosity measurements and previous

measurements using optical tweezers and bulk rheology (diamond symbols on figure 2).

At the physiologically relevant concentration of 25 mg/ml (2-3% w/w of gastrointestinal

mucus is made up of mucin) a critical concentration is reached and a viscoelastic solution

forms. At this point the solution behaves as a complex liquid, exhibiting both viscous and

elastic behaviour, i.e. power laws with α<1 (Eq. 2) are required to describe the MSDs. The

slope of the loglog plot of the MSD at 25 and 30 mg/ml was found to be very close to 0.75, a

preliminary indication that mucins could self-assemble into semi-flexible fibres. Further

investigation of the compliance curve acquired by transforming the MSD dataset as per Eq. 4

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and fitting the Maxwell equation at long times for transition concentrations of 25 mg/ml,

enabled us to calculate the modulus of the semi-dilute network to be G = 1.4±0.1 Pa and its

viscosity, η = 70±4 mPas. Combining the two (using equation 6), the relaxation time of the

gel was found to be ��= 0.06±0.01 s, a value typical for viscoelastic polymer solutions.

At 40 mg/ml the viscosity of the solution was found to be 165±2 mPas, whereas the modulus

of the polymer network increased to 2.3±0.2 Pa. Consequently, the relaxation time of the

complex fluid was found to be 0.07±0.03 s, a clear indication of the formation of a stronger

entangled network. At even higher concentrations, namely around 50 mg/ml, the solution was

very viscous and the Brownian motion of the beads was barely detectable, thus further

investigation of higher concentrations of mucin was not possible with PTM. At this

concentration the viscosity of the solution was found to be 780±4 mPas, the elastic modulus

5.2±0.1 Pa and the relaxation time 0.15±0.03 s.

The most important physiological function of the mucus layer in the mammalian stomach is

to protect the epithelium from its hostile environment. In order to do so, a pH gradient is

maintained from near neutral on the epithelium to as low as pH 1 in the lumen. Figure 3

shows the effect of dropping the pH of a 10 mg/ml solution of stomach mucin to pH 1. It is

evident that the mobility of the embedded microspheres is severely compromised in the

acidic solution as a result of the sol-gel transition the solution undergoes due to the pH

change. The viscosity of the solution is increased by more than a 100-fold, namely to 3.0±0.2

Pas, whereas the elastic modulus was calculated to be 3.0±0.1 Pa. This results in a relaxation

time of 1.0±0.3 s, typical of a weak polymeric gel.

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2.2 Microrheology of Porcine Duodenal Mucin (PDM).

Figure 4 shows the scaling of the MSD curves of polystyrene spheres in porcine duodenum

mucin (MUC2) as a function of mucin concentration. A similar trend as with PGM

(MUC5AC) was observed, with the transition from a purely viscous liquid to a complex fluid

occurring at a similar, physiologically relevant concentration of 30 mg/ml. Up to this

concentration the solutions behaved as a purely viscous, Newtonian liquid with a measured

viscosity of the order of a few mPas, with no elastic component detectable using PTM.

Above this critical entanglement concentration the slope of the loglog plot of MSDs was

found to be smaller than 1, indicating the formation of a viscoelastic polymer network.

Indeed, at 30 mg/ml the viscosity of the solution was found to be 11.0±0.1 mPas and the

elastic modulus was found to be 0.27±0.01 Pa. At concentrations of 40 mg/ml a considerable

elastic modulus is measured and the viscosity is of the order of tens of mPas, as seen in

Figure 5. It was concluded that the critical concentration for porcine duodenum mucin

solutions lies slightly above that of PGM, at 30 mg/ml.

The majority of the digestion and absorption of nutrients happens in the small intestine, after

the chyme from the stomach enters the duodenum. Here the acidic pH of the gastric juices is

neutralised by the secretion of bile, pancreatic and intestinal juices, thus the pH in the small

intestine is kept close to neutral. The hypothesis that both gastric and small intestinal mucins

polymerise and form gels by the same mechanism was tested by reducing the pH of a

10 mg/ml duodenum mucin solution to 1, using HCl. This resulted in the solution undergoing

a sol-gel transition, much like PGM. The effect of dropping the pH in duodenum mucin

solution is seen in Figure 6.

The viscosity of the neutral pH 10 mg/ml duodenum mucin solution was found to be

5.0±0.1 mPas and no elastic component could be measured. Decreasing the pH of the

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solution had a large effect on its rheological characteristics, since the measured viscosity was

found to be 0.82±0.01 Pas (100-fold higher than the neutral solution’s) and its elastic

modulus was found to be 0.67±0.02 Pa. This is a somewhat unexpected finding, since there is

no physiological reason why small intestinal mucin should protect the epithelium from acidic

secretions, although it does provide a secondary protection mechanism for variations in pH

that may be caused by gastric juice leakage in the initial parts of the duodenum.

2.3 Microheology of chemically treated porcine gastric mucin (PGM)

To investigate the bonding forces between mucin monomers, a 25 mg/ml PGM solution was

treated with 10 mM dithiothreitol (DTT), a reducing agent commonly used for disrupting

disulphide bonding, followed by 2 hr incubation. In order for DTT to have an effect the pH of

the solution was regulated at pH 8, using 1M NaOH. Following incubation, the mobility of

embedded microspheres between the control and the treated sample was compared, as seen in

Figure 7a. The viscosity of the solution was considerably decreased from 87±4 mPas to

42±1 mPas, whereas the measured elastic shear modulus were 1.4±0.1 Pa and 0.54±0.02 Pa

for the untreated and treated solutions respectively.

In order to study the effect of GuHCl on the rheological properties of mucins, a 20 mg/ml

PGM solution was treated with 8 M GuHCl and incubated for 10 min. Figure 7b shows the

MSD curves obtained from the two samples. The absolute MSD values are increased 10-fold

and no elastic component was detectable in the treated solution, where the elastic shear

modulus of the untreated solution was found to be 0.29±0.01 Pa. The viscosity of the treated

solution was found to be 9.03±0.01 mPas, decreased from 53.0±0.1 mPas for the untreated

20 mg/ml solution.

To study the effect of urea, a 20 mg/ml PGM solution at pH 7 (complex liquid) and at pH 1

(gel) were treated with 2 M urea and incubated for 1 hour. The MSD curves obtained from

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tracking the embedded microspheres in the aforementioned solutions can be seen in Figure 8.

Comparing the pH neutral solutions, we observe a decrease in viscosity from 52.5±1.0 mPas

to 16.1±0.5 mPas and a decrease in the elastic modulus from 0.31±0.03 Pa to 0.03±0.01 Pa.

Furthermore, the slope of the loglog plot of the MSD increased from 0.82 to 0.89, indicating

the increased mobility of the embedded micro-spheres. The greatest change is observed when

treating the PGM solution at pH 1 with 2 M urea. The transition from a thick gel to a complex

fluid was immediately observed by visual inspection, but also seen in the transitions of power

law exponents of the MSD curves, namely from 0.3 to 0.85 as a result of the large increase in

the absolute values of the MSDs. The viscosity and the elastic shear modulus of the acidic

solution were measured to be 4.10±0.14 Pas and 0.47±0.02 Pa, whereas the same parameters

were found to be 47±1 mPas and 0.21±0.01 Pa for the 2 M urea treated sample.

The bioavailability of polyphenols in the human body and their effect on human health have

been the centre of research for the past years. The interaction of a 10 mg/ml PGM solution

and EGCG, a polyphenol molecule found in abundance in tea, was investigated by treating

the solution with 1% w/w lyophilised EGCG. . This allowed us to probe the effect of phenolic

molecule on the rheological characteristics of the mucin solution. Immediately an adverse

effect was observed, since treating the PGM solution with EGCG caused it to undergo a sol-

gel transition. The viscosity of the gelled PGM sample could not be measured, since it was

too high for PTM, but an estimate of its elastic modulus can be made from the intercept of the

plateau’s extension to the compliance axis, which was found to be ~1 Pa, shown in Figure 9.

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3. Discussion

The viscoelastic properties of mucins are of great physiological significance, especially in the

airways and gastrointestinal tract. Over production of polymerising mucins (namely MUC5B

and MUC5AC) in the airways as a result of cystic fibrosis inhibits the removal of various

pathogens by mucociliary transport, with life-threatening consequences. Furthermore,

malfunction of the mucus layer in the stomach can lead to ulcers caused by the bacterium,

Helicobacter Pylori or even by the secretions in the stomach itself. The scaling of the

viscoelasticity of both porcine gastric and duodenal mucins as a function of mucin

concentration shows that at physiologically relevant concentrations the mucins form a semi-

dilute network. This will allow the diffusion of small particles through it, like nutrients in the

small intestine, but severely reduces the diffusion coefficient of larger particles, like dust

particles and bacteria23

. At concentrations higher than the typical concentration of mucins in

mucus (~3% w/w), both types of mucins form thick entangled semi-dilute networks.

It is therefore deduced that the semi-dilute networks form by self-assembly of secreted mucin

oligomers with an increase in mucin concentration. The aggregate dynamics depends on

mucin concentration, but the transition to a viscoelastic fluid occurs relatively rapidly above a

critical concentration (the entanglement concentration). At this transition point the MSD

often has a characteristic t0.75

scaling over two orders of magnitude in time (t), characteristic

of semi-flexible aggregates e.g. actin or fibrin.24

The exponent α of a power law (Eq. 2)

decreases with a further increase in concentration approaching values close to α=0, which is

characteristic of a strongly elastic solution.

The scaling of the viscosity (η) between c* and ce, �~��.��.� for MUC5AC and

�~��.��.� for MUC2, is in agreement with the Fuoss law for semi-dilute linear flexible

polyelectrolytes (1/ 2~ cη )

22. Slight modifications are predicted for the concentration

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dependence with flexible comb polyelectrolyte architectures, but the data is in broad

agreement with such predictions (the prediction with semi-flexible side chains is identical to

the Fuoss law). The prediction for the shear modulus (G) of entangled flexible comb

polyelectrolytes is 3/ 2~G c which is in good agreement with the observed result for MUC2,

1.7 0.4~G c ± and in reasonable agreement for MUC5AC �~� .��.� . The scaling of the

viscosity as a function of concentration for MUC2 above the entanglement concentration is

5.1 0.8~ cη ±and contrasts strongly with the scaling found previously for entangled Orthana

mucins that do not self-assemble and have a reduced number side-chains, 3/2~ cη 15

. The

Orthana mucins followed the scaling predictions for linear polyelectrolytes in the entangled

regime, whereas duodenal mucin follows the predictions for comb polyelectrolytes or more

specifically comb polyelectrolytes with entangled side chains (only this model can explain

the large exponent of 5.1 in the fluid phase i.e. the prediction 1/ 23~ acc eη , where a is a

constant, can only explain the high value of the exponent).25

In addition to the entanglement

of carbohydrate side-chains, there is also the possibility that the mucin aggregates create a

small number of branched nodes (the networks are not gelled, and the mucin oligomers are

known to be relatively small from scattering experiments, so the number of aggregate

branches must be relatively low, if they exist), which would result in a sparsely branched

network and a similarly large exponent is predicted for the dependence of the viscosity on the

concentration (due to the activated motion of the aggregate branches in this case). Thus above

the entanglement concentration both the backbone (protein) and the side-chains (carbohydrate

or possibly mucin aggregate branches) of MUC2 are expected to reptate. Similar behaviour is

observed with MUC5AC (Figure 2) with�~��.��.� in the entangled regime.

Based on these scaling results a schematic diagram of the mucin chains at different

concentrations can be drawn (Figure 10). Similar phase diagrams can be drawn for both

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stomach and duodenum mucin with a small rescaling of the phase boundaries. At low

concentrations the mucin molecules exist as non-interacting oligomers. As the mucin

concentration is increased the non-interacting oligomers start to interact with each and

contribute to the viscoelasticity of the solution, while also forming larger oligomers or

polymers. Further increases in concentration (� � �∗) cause the chains to overlap in the semi-

dilute unentangled regime. Above the entanglement concentration (ce) the comb solutions

begin to reptate. Dropping the pH induces permanent cross-links between neighbouring self-

assembled chains, due to protonation of salt bridges in the peptide backbone and exposure of

hydrophobic regions, and the network forms a gel. Gels can be either unentangled or

entangled depending on whether the comb polymers have reptation inducing physical

entanglements in addition to permanent cross-links, ie. whether the mucin concentration is

above or below the entanglement concentration.

Gastric and small intestinal mucins share structural homology (Supplementary material, S4).

The N- and C- termini contain von-Willebrand factor-like B, C, CK and D-domains, and the

central mucin domains, in which extensive glycosylation occurs, are interspersed with

cysteine-rich domains. Thiol groups that exist in the cysteine-rich domains at the two termini

of the glycoprotein form disulphide bonds between mucins, which eventually form fibrous

polymeric aggregates. Furthermore, recent reports have provided evidence for branched

polymerisation of MUC2.26

Treatment with DTT had a dramatic effect on both the viscous

and elastic component of the MUC5AC solution, due to effective reduction of the polymer

network. In non-chaotropic solvents DTT may not remove all of the disulphide interactions,

and a number of them are expected to reform after treatment. However it can be safely

concluded that disrupting disulphide bonding in the sample resulted in a weaker elastic

network, as a result of the reduction of a number of mucin polymers to smaller oligomers and

thus partial disassembly of the semi-dilute network.

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By its nature, biochemistry is often a reductionist science. In studying mucins, the most

common extraction method uses chaotropic agents, which break non-covalent bonds and

denature the proteins so they can be studied. A common denaturant is guanidinium

hydrochloride (GuHCl), which at concentrations of 8 M causes the proteins to lose their

structure and become randomly coiled. Treating with 8 M GuHCl effectively reduced the

viscoelastic 20 mg/ml mucin solution to a purely viscous liquid, with considerably decreased

viscosity and no measurable elastic component. Again it is deduced that the chaotropic agent

(GuHCl) has disassembled the semi-dilute network structure, this time by unfolding proteins

and breaking non-covalent bonds rather than disruption of the covalent interactions between

mucin monomers which occurred with DTT.

Another chaotropic agent commonly used in the study of complex proteins in biochemistry

laboratories is urea, an organic compound which is a powerful denaturant, since it disrupts

the noncovalent bonds in the protein. It is evident that for pH 7 and pH 1 20 mg/mL PGM

solutions (Figure 8), treatment with 2 M urea disrupts a portion of the polymer network,

resulting in weaker complex liquids. Urea is thus shown to disassemble the mucin gel at pH 1

and partially disassemble the semi-dilute network at pH 7.

The aforementioned observations imply that disrupting the non-covalent bonds reduces the

viscoelasticity of the pH neutral solution and reduces the gelled acidic solution to a

viscoelastic liquid, but steric interactions between the glycosylated regions of the mucins also

have significant contribution to the formation of the mucin network. This is in agreement

with the scaling results that imply mucin molecules above 20 mg/mL have entangled side

chains (Figure 10).

There has been great interest in the biological effects of plant-derived polyphenols, a family

of molecules that are characterised by the presence of several phenolic groups. Polyphenols

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have been shown to have a variety of beneficial health effects in humans and animals.27

Epicatechin (EC), found in various fruits and tea, has been postulated to act as a strong

antioxidant and promote the healthy functioning of hearts. Epigallocatechin gallate (EGCG),

which has a similar structure as EC with the addition of a galloyl ring, is found in abundance

in green tea, and has been reported to prevent cancer in animal models. However, the galloyl

ring in the molecule’s structure has also been reported to be toxic at high concentrations.

Experiments performed on HT29 adenocarcinoma cell lines, used to simulate the gut

epithelium in which mucin production is inhibited, have confirmed this toxicity. However,

the mucus layer on the surface of the HT29-MTX-E12 sub-clone successfully constrained the

EGCG molecules, thus protecting the cells from the toxic environment. No toxicity was

reported when using EC on the same cell-lines.8

Furthermore, the interfacial shear rheology of human whole saliva has shown the formation

of a strong intermolecular network between salivary proteins when treated with EGCG, as a

result of the complexation of salivary proline rich proteins (PRPs) with the galloyl group

found in the EGCG molecule. It was postulated that this mechanism of protein binding of

polyphenols is the body’s first line of defence against the toxic effects of EGCG at high

concentrations, possibly related to GI tract irritation after high consumption of green tea.11

However, the effect of EGCG on mucins remains largely unknown. It is evident in

microrheology experiments that addition of EGCG to the purely viscous PGM solution

causes a strong intermolecular network to form, which forms a gel matrix. It was concluded

that EGCG causes the purely viscous PGM solution to gel, and this could act as a defence

mechanism against high concentrations of this type of polyphenol.

The linear viscoelasticity (the viscosity and shear modulus) of the microrheology results

generated here from purified MUC2 and MUC5AC are compared with results from native

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mucus rheology experiments2. Good agreement is observed with a range of bulk rheology

measurements for pig intestinal mucin. It is also clear that pH and mucin concentration both

have a strong impact on the viscoelasticity of the solutions. The viscoelasticity of the purified

mucins is close to that of native mucus secretions, which indicates that MUC5AC and MUC2

are the dominant contributing factors to the viscoelasticity of gastric and intestinal mucins

respectively. These values of gastrointestinal mucins are reasonably close to those from

respiratory mucin from healthy animals. Sputum from human cystic fibrosis patients can have

viscosities as high as ~30 Pas, an order of magnitude higher than observed in our gastric

mucin studies and bacterial DNA is thought to be a major contributor to its viscoelasticity

(DNases are used in medical treatments)28

.

Future work will investigate the structure and dynamics of gastrointestinal mucins with other

experimental techniques to test the model for self-assembled entangled flexible

polyelectrolytes combs in more detail. Some microscopy data exists in the literature to

suggest that mucin chains can bundle side-by-side into lateral aggregates29

. It may be possible

to test these predictions in small angle X-ray and neutron scattering experiments.

Furthermore the mechanistic interaction of polyphenols with mucin will be studied in more

detail with a range of experimental methods.

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4. Conclusion

Particle tracking microrheology experiments demonstrate that both MUC5AC and MUC2

mucins (from the stomach and duodenum respectively) self-assemble to form viscoelastic

solutions at high mucin concentration. Above the semi-dilute overlap concentration, but

below the entanglement concentration, unentangled polyelectrolyte comb dynamics are

observed. Above the entanglement concentration the chains begin reptating due to physical

entanglements and the scaling for MUC2 and MUC5AC is in broad agreement with a model

of polyelectrolyte combs with entangled backbones and side-chains. Reduction of the pH of

solutions of both type of mucin subsequently causes the mucin solution to gel due to the

creation of cross-links. DTT, guanidinium hydrochloride and urea cause the disassembly of

the gels and a large decrease in the shear modulus (increase in the compliance). ECGC (a key

polyphenol molecule in tea) causes PGM to gel through the introduction of cross-links.

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5. Materials and Methods

5.1 Mucin purification. Porcine gastrointestinal mucin (PGM) was used as a model for

human mucin. Porcine stomachs and small intestines were collected from a local abattoir

shortly after death, washed with PBS and brought back to the laboratory. Following a second

wash the mucus was scraped from the epithelium and stored at -20 oC. Gastrointestinal (GI)

mucins were extracted by homogenising mucus for 1 min in 0.2M NaCl containing Protease

Inhibitors and Sodium Azide, followed by two steps of CsCl density gradient centrifugation.

No chaotropic agents were used during mucin extraction, to ensure that their native structure

was unaltered. In each CsCl-gradient step, the solution was divided in 20 fractions of

decreasing density and their glycoprotein content was assessed by periodic acid/Schiff’s

staining (PAS) staining and UV light (280 nm) absorbtion (supplementary information, S1

and S2). The mucin-rich fractions were pooled, extensively dialysed against water and

lyophilized.30

The presence of MUC5AC (stomach) and MUC2 (small intestine) was

confirmed as the dominant glycoprotein present by tandem mass spectrometry

(supplementary information, S3). The main fractions identified by chromatography are

relatively polydisperse as expected for molecules with heterogeneous carbohydrate side-

chains and with different numbers of subunits in their oligomers. Around 70% of the mucins’

weight is found to be carbohydrate and 30% protein. The carbohydrate side-chains are

relatively small with 1-10 monomeric subunits (the carbohyrdrate side-chains are shown to

scale on figure 10). The side chains are highly negatively charged at neutral pH due to sialic

acid groups on the monomers. Purified mucin solutions were prepared by dissolving mucin in

10 mM Sodium Phosphate with 10 mM Sodium Succinate buffer and the pH was adjusted

using aqueous HCl. All the solutions were prepared 48 hours prior to the experiments, so that

equilibrium was reached.

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5.2 Particle Tracking Microrheology. Carboxyl-activated polystyrene microspheres

(505 nm, Polysciences) were embedded in the mucin solutions. Polyethylene glycol coated

microspheres were also used, which exhibited similar behaviour as their uncoated

counterparts (supplementary information S4). This behaviour is in sharp contrast to that

found previously with fibrin glycoprotein microrheology in which PEGylation was found to

be necessary to measure microrheology representative of fibrin networks31

. We deduce that

the interaction between the beads and the mucin solutions are in the non-associating limit. A

previous study of O.Lieleg et al32

found larger effects of surface modification, but they were

examining much lower concentration mucin solutions (at or below the semi-dilute overlap

concentration measured with our purified specimens). For consistency purposes it was

decided to use the uncoated beads throughout. Following preparation, standard flat

microscope slides with Secure-seal imaging spacers containing 8 µl of sample were loaded

onto an Olympus IX-71 inverted microscope equipped with a 100× immersion oil objective.

The setup was placed on an active optical table to reduce the effect of vibrations. Videos of

the spheres’ Brownian motion were recorded using a FastCam 1024-PCI fast camera and a

bright CoolLED light source. This allowed the motion of the spheres to be followed at up to

27 000 frames per second and their two-dimensional tracks were extracted using a parallel

version of the MatLab based Polyparticletracker software.33

This is based on a polynomial fit

of the intensity around each feature point, weighted by a Gaussian function of the distance

from the centre.34

Special care was taken to only track spheres away from the surfaces of the

microscope slides to avoid detecting the hydrodynamic interactions between them (the

Faxen’s law corrections are negligible). These trajectories were subsequently converted to

mean-square displacements (MSDs) and the ensemble averaged MSD was plotted as a

function of lag time, τ. No substantial cage hopping events were observed in the tracks of the

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probe spheres, which have been shown to lead to artefacts with microrheology experiments

on actin solutions35

.

For Brownian motion of a probe in a Newtonian fluid the mean square displacement,

⟨��⟩, is linearly dependent on the lag time (τ) with proportionality constant D (the

diffusion coefficient):

⟨∆��⟩ � 2� � (1)

where n is the number of dimensions (equal to 2 for microscopy experiments). More

generally a power law time dependence of the MSDs is observed for viscoelastic fluids

⟨∆��⟩∼�! (2)

α=1 corresponds to spheres diffusing in a purely viscous material and combination of

equation (1) with the Stokes-Einstein relationship gives:

� "#/6&�' (3)

where η is viscosity, b is the sphere’s radius and kT is the thermal energy. This allows the

viscosity of the material to be calculated from the fluctuations of a probe sphere in the

solution. In the case where 0<α<1 (Eq. 2), the material’s response is dependent on the

timescale of the measurement relative to the characteristic time of the material, that is the

relaxation time ��.36

The MSD curve can be converted to the time dependent compliance

(J(τ)), a standard measure of viscoelasticity with units of Pa-1

(which assumes the generalised

Stokes Einstein relationship for its derivation) using:37

(�� )!

*+⟨∆��⟩ (4)

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22

This can be further investigated using the Maxwell model for linear viscoelasticity, which

relates J(τ) to both viscosity and its elastic modulus, G, as:

(��

,-

.

/ (5)

Viscosity is related to the relaxation time (τrel) and the modulus (G) by the expression:

� � �� (6)

This expression is exactly true when a material is well described by a Maxwell model, but it

is also a good approximation with a wide range of other viscoelastic fluids. This relation

allows us to further characterise the viscoelastic properties of the material since τrel, the

characteristic relaxation time of the material, can be calculated e.g. the slowest characteristic

time scale of the molecules’ motions.38

The semi-dilute overlap concentration for a mucin chain can be calculated using

�∗ �012

3)45

3 (7)

where Mw is the molecular weight of a mucin monomer and Rg is the radius of gyration.

From separate light scattering, neutron scattering and X-ray scattering measurements (to be

presented separately) we have measured the radius of gyration of a MUC5AC mucin

monomer to be 43±5 nm and the molecular weight is approximately ~2.5 MDa. Thus, from

equation (7) the semi-dilute overlap concentration of monomers is 12 mg/mL. However the

semi-dilute overlap concentration of self-assembled aggregates is expected to be much

smaller than this value due to the much larger effective radius of gyration.

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6. Acknowledgements

We would like to acknowledge Unilever Discover for funding this project. Furthermore, we

would like to thank Heather Davies, Karine Rousseau, Caroline Ridley, Bill Frith and Gleb

Yakubov for useful conversations.

Table 1

Comparison of the viscosity and shear moduli for purified MUC5AC and MUC2 compared to

bulk rheology measurements in the literature2.

Porcine mucin type Viscosity (Pas) Shear modulus (Pa)

Previous studies Gastric 0.13-63 3.3-34

Small intestine 0.063-5.0 0.19-12

Colonic 0.16-1000 64-160

Current study Purified pH7

MUC5AC

0.001-0.6 2-5 (30-50 mg/mL)

Purified pH7 MUC 2 0.002-5.0 0.67-5 (50-100

mg/mL)

Purified pH1

MUC5AC

3 (10 mg/mL) 3 (10 mg/mL)

Purified pH1 MUC2 0.82 (10 mg/mL) 0.67 (10 mg/mL)

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7. References

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70, 459–86.

(2) Lai, S. K.; Wang, Y.-Y.; Wirtz, D.; Hanes, J. Advanced drug delivery reviews 2009,

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Erramilli, S. Rheology of gastric mucin exhibits a pH-dependent sol-gel transition.

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(7) Bansil, R. Annual Review of Physiology 1995, 57, 635–657.

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H.; McKinley, G. H.; So, P.; Erramilli, S.; Bansil, R. Proceedings of the National

Academy of Sciences of the United States of America 2009, 106, 14321–6.

(11) D’Agostino, E. M.; Rossetti, D.; Atkins, D.; Ferdinando, D.; Yakubov, G. E. Journal

of agricultural and food chemistry 2012, 60, 3318–28.

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(13) Haward, S. J.; Odell, J. a.; Berry, M.; Hall, T. Rheologica Acta 2010, 50, 869–879.

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(19) Papagiannopoulos, A; Fernyhough, C. M.; Waigh, T. A. The Journal of chemical

physics 2005, 123, 214904.

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(21) Yakubov, G. E.; Papagiannopoulos, A.; Rat, E.; Waigh, T. A. Biomacromolecules

2007, 8, 3791–9.

(22) Dobrynin, A. V; Colby, R. H.; Rubinstein, M.; York, N. Macromolecules 1995, 28,

1859–1871.

(23) Lai, S. K.; Wang, Y.-Y.; Cone, R.; Wirtz, D.; Hanes, J. PloS one 2009, 4, e4294.

(24) Xu, J.; Palmer, A.; Wirtz, D. Macromolecules 1998, 31, 6486–6492.

(25) Papagiannopoulos, a.; Waigh, T. A.; Hardingham, T. E. Faraday Discussions 2008,

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(26) Ambort, D.; Johansson, M. E. V; Gustafsson, J. K.; Nilsson, H. E.; Ermund, A.;

Johansson, B. R.; Koeck, P. J. B.; Hebert, H.; Hansson, G. C. Proceedings of the

National Academy of Sciences of the United States of America 2012, 109, 5645–50.

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(28) Shak, S.; Capon, D. J.; Hellmiss, R.; Marsters, S. a; Baker, C. L. Proceedings of the

National Academy of Sciences of the United States of America 1990, 87, 9188–92.

(29) Cone, R. A. In Mucosal Immunology (3rd Edition); Jiri Mestecky, Lamm Michael,

Jerry R. McGhee, John Bienenstock, L. M. and W. S., Ed.; Elsevier Inc., 2002; pp. 49–

72.

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H. Biophysical journal 1999, 76, 1250–8.

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T. J.; Weitz, D. A. Biophysical journal 2004, 86, 4004–14.

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(33) Kenwright, D.; Harrison, A.; Waigh, T.; Woodman, P.; Allan, V. Physical Review E

2012, 86, 031910.

(34) Rogers, S. S.; Waigh, T. A; Zhao, X.; Lu, J. R. Physical biology 2007, 4, 220–7.

(35) He, J.; Tang, J. X. Physical Review E 2011, 83, 041902.

(36) Waigh, T. A. Reports on Progress in Physics 2005, 68, 685–742.

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(38) Goodwin, J W and Hughes, R. W. Rheology for Chemists: An Introduction; The Royal

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Caption list:

Figure 1: The mean square displacement of probe spheres in PGM solutions at pH 7 as a

function of mucin concentration. The scaling of the MSD curves from increasing

concentration of stomach mucin reveals that mucins undergo a network self-assembly

transition at a physiological relevant concentration of 25 mg/ml. The trend lines of τ1 and τ

0.75

are a guide for viscous and semi-flexible behaviours respectively.

Figure 2: The scaling of the viscosity (η) of MUC5AC (stomach) mucin solutions as a

function of mucin concentration(c) followed a power law dependence �~��.��.� for the

dilute regime and �~��.��.� in the semi-dilute regime (○). The scaling of the elastic

modulus with concentration is seen in the inset and was found to follow a power law

dependence of �~� .��.� . The triangles show micro-DLS and bulk rheology data for

purified MUC5AC from the literature.39

Figure 3: Mean Square displacements of polystyrene spheres in MUC5AC solutions at

10 mg/ml at pH 7 (�) and pH 1 (�). Decreasing the pH of the solution causes a dramatic

effect on its viscoelastic properties. The MSD values obtained from the acidic solution are

one order of magnitude smaller than those obtained from the neutral sample. Power law fits

to the MSDs give ( )2 1.03 0.01~r τ τ ±∆ and ( )2 0.28 0.01~r τ τ ±∆ for the ungelled and gelled

samples respectively.

Figure 4: The ensemble averaged MSD curves of polystyrene spheres as a function of lag

time obtained from various concentrations of small intestinal mucin (MUC2) exhibit a similar

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28

behaviour to MUC5AC. The transition from a Newtonian liquid to a viscoelastic fluid occurs

at a slightly higher concentration when compared to MUC5AC, that is 30 mg/ml. The dashed

trend lines of τ1 and τ

0.75 are a guide for viscous and semi-flexible behaviours respectively.

Figure 5: Scaling of the viscosity (η) and the elastic shear modulus (G) of the porcine

duodenum mucin solutions as a function of mucin concentration (c), obtained with particle

tracking microrheology. c* is the semi-dilute overlap concentration and ce is the

entanglement concentration. The viscosity follows a scaling law of �~��.��.� between c*

and ce. Above ce the viscosity has a scaling law of 5.1 0.8~ cη ±

. The shear modulus follows a

scaling relation, 1.7 0.4~G c ± .

Figure 6: The mean square displacement of probe spheres in duodenum mucin solution at

10 mg/ml, at pH 1 (□) and pH 7 (○). The mobility of the embedded microspheres is severely

compromised upon the reduction of the solution’s pH. The duodenum mucin solution

undergoes a sol-gel transition when the pH is dropped to 1. At high pH the scaling of the

mean square displacement is ( )2 1.02 0.01~r τ τ ±∆ , whereas at low pH ( )2 0.22 0.03~r τ τ ±∆ .

Figure 7: The mean square displacement of probe spheres in PGM solutions at pH 7 with and

without the addition of DTT (a) and 6M GuHCl (b). The mobility of embedded microspheres

in the DTT treated mucin solution is considerably larger than the untreated solution.

Disruption of a number of disulphide bonds in the solution results in a weaker elastic

component, whereas treatment with 6M GuHCl (○) results in a purely viscous Newtonian

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Biopolymers

29

liquid, with no detectable elastic component (⟨��⟩~� .� ��.��) (□). The MSD scaling of

the 25 mg/ml PGM solution (○) was found to be ⟨��⟩~��.6��.�� and treatment with 10

mM DTT (□) did not induce a significant change in the power law (⟨��⟩~��.6��.��).

Figure 8: The mean square displacement of probe spheres in 20 mg/mL PGM solutions at pH

7 and pH 1 with and without the addition of 2M urea. Treatment of the mucin solution with

2 M urea significantly increases the mobility of the embedded microspheres in the PGM

solutions. The most dramatic effect is observed with the acidic solution, since it irreversibly

disrupts the mucin network that forms the gel, resulting in a significantly weaker viscoelastic

liquid. The scaling exponents for the MSD curves obtained are ⟨��⟩~��.��,�� and

⟨��⟩~��.��,�� for the untreated and treated neutral PGM solutions respectively and

⟨��⟩~��.��,�� and ⟨��⟩~��.��,�� for the acidic solutions.

Figure 9: The ensemble averaged mean square displacement of probe spheres in 10 mg/ml

PGM solution with and without the addition of 1% w/w EGCG, a plant polyphenol abundant

in green tea. The plateau of the MSD curve of the EGCG treated solution at long times

implies gelation of the purely viscous untreated mucin solution. An estimation for the elastic

shear modulus could be made using the plateau value of the compliance curve (inset). For

untreated PGM ( )2 1.03 0.01~r τ τ ±∆ whereas with EGCG ( )2 0.04 0.01~r τ τ ±∆ .

Figure 10: a) Mucin oligomers form as the asymmetric dumbbell comb molecules self-

assemble end on end. b) A schematic diagram of the concentrations regimes that determine

the scaling of the viscoelasticity as a function of concentration and pH. Mucins are

synthesized and secreted as oligomers, which are assumed to be non-interacting below a

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Biopolymers

30

certain concentration. Above a critical concentration (c*), mucins overlap to form a semi-

dilute solution. Above a second critical concentration (ce) the molecules begin to reptate. If

the pH is dropped mucins undergo a conformational change and aggregate, forming

hydrophobic bonds between the fibres (red circles), eventually forming a gel. c* is the semi-

dilute overlap concentration and ce is the entanglement concentration.

Figure S1: Analysis of the fractions obtained from a CsCl density gradient centrifugation of

a small intestinal mucus sample (top – starting density = 1.4 g/ml). The glycoprotein rich

fractions, assessed by PAS staining (blue line and diamonds), also have a low absorption of

UV light (green line and diamonds). The density of each fraction (red line and squares) was

measured by weighing 1 ml aliquots and confirms the density gradient was formed by

centrifugation. In this case fractions 1-16 were pooled and a 2nd

density gradient (bottom -

starting density = 1.5 g/ml) was done to further isolate the mucin rich fractions. Fractions 3-

16 were pooled from the second CsCl density gradient and were subsequently dialysed

against dd H2O and then lyophilised.

Figure S2: Analysis of the 20 fractions obtained from the two steps of CsCl density gradient

centrifugation obtained during the extraction of MUC5AC from stomach mucus. The

fractions of the first density gradient (top- starting density = 1.4 g/ml) were analysed as

described and fractions 2-17 were pooled. These were further expanded with a 2nd

CsCl

density gradient (bottom – starting density = 1.5 g/ml), were fractions 2-15 were pooled,

extensively dialysed against deionised water and lyophilised.

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Biopolymers

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Figure S3: MUC5AC (a) and MUC2 (b) peptide matches from MS/MS analysis of purified

pig stomach and duodenal mucins. Since the pig genome is not yet available, we relied on

predictions of the N-terminal of pig MUC2 and MUC5AC. Matched peptides are highlighted

in red. Predictions for the N-terminal of MUC6 also exist in our database, but tandem mass-

spec experiments have not identified any peptides corresponding to MUC6.

Figure S4: The ensemble averaged MSD curves obtained from PEG coated and uncoated

polystyrene microspheres embedded in a 20 mg/ml small intestinal mucin solution. The

exponent of the power laws fitted to all of them was found to be close to ⟨��⟩~��.. The

small difference in the absolute value of MSD was attributed to small differences in size

between the coated and uncoated probe spheres.

Figure S5: MUC2 and MUC5AC share common C- and N- terminal protein domains, which

are the sites for disulphide bonding between mucins, leading to polymer formation. In

contrast, the central domain of the two mucins differs, MUC2, has only 2 cysteine-rich (cys)

domains that interrupt the central mucin domain, whereas MUC5AC has at least 8. The

tandem repeated sequences rich in serine, threonine and proline comprise the central mucin

domain. In MUC2 the number of tandem repeats can differ between individuals, a property

referred to as polymorphism.1 The different number of cys-domains could provide larger

flexibility to MUC5AC, thus explaining the slower scaling of viscosity with respect to

concentration compared to MUC2. [Illustration adapted from Ref. 1]

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Figure list:

Figure 1

10-2

10-1

100

10-15

10-14

10-13

10-12

τ0.75

⟨∆r2(τ)⟩

(m2)

Lag time (s)

1 mg/ml

2 mg/ml

3 mg/ml

4 mg/ml

5 mg/ml

10 mg/ml

15 mg/ml

20 mg/ml

25 mg/ml

30 mg/ml

35 mg/ml

40 mg/ml

45 mg/ml

50 mg/ml

τ1

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Biopolymers

33

Figure 2

1 10

1

10

100

Concentration (mg/ml)

Viscosity (mPas)

c3.92±0.38

c0.53±0.08

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Biopolymers

34

Figure 3

0.1 1

10-14

10-13

10-12

τ1.03±0.01

<∆r2(τ)>(m

2)

Lag time (s)

τ0.28±0.01

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Biopolymers

35

Figure 4

0.01 0.1 11E-16

1E-15

1E-14

1E-13

1E-12

τ0.75

5 mg/ml

10 mg/ml

30 mg/ml

40 mg/ml

60 mg/ml

50 mg/ml

60 mg/ml

70 mg/ml

80 mg/ml

90 mg/ml

100 mg/ml

<∆r2(τ)> (m2)

Lag time (s)

τ1

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Biopolymers

36

Figure 5

1 10 1001

10

100

1000

c*

Concentration (mg/ml)

Viscosity (mPas)

c5.1±0.8

c0.53±0.06

ce

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Biopolymers

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Figure 6

0.01 0.1 1

10-14

10-13

10-12

τ0.22±0.03⟨∆

r2(τ

)⟩ (m2)

Lag time (s)

τ1.02±0.01

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Biopolymers

38

Figure 7

a) b)

Figure 8

0.01 0.1 1

10-15

10-14

10-13

τ0.76±0.03

⟨∆r2(τ)⟩ (m2)

Lag time (s)

τ0.73±0.02

0.01 0.1 1

10-15

10-14

10-13

10-12

τ1.01±0.02

⟨∆r2(τ)⟩

(m2)

Lag time (s)

τ0.72±0.01

0.01 0.1 1

10-14

10-13

10-12

τ0.85±0.02

2M Urea

Untreated

pH1 untreated

pH1 2 M Urea

τ0.82±0.02

⟨∆r2

(τ)⟩

(m2

)

Lag time (s)

τ0.36±0.05

τ0.89±0.02

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Biopolymers

39

Figure 9

0.01 0.1 1

10-14

10-13

10-12

1% w/w EGCG

Untreated 10 mg/ml PGM

τ1.03±0.01

⟨∆r2(τ)⟩

(m2)

Lag time (s)

τ0.04±0.01

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Figure 10

Peptide

backbone

Carbohydrate

side-chain

(O-and N– linked

oligosaccharides)

Disulphide bonds

Mucin oligomer

a)

N Terminal

C Terminal

Mucin Concentration

pH

low

pH

hig

h p

H

Oligomers Semi-dilute

unentangled

Semi-dilute

entangled

Entangled gelUnentangled gel

c* ce

b)

Crosslink

Small aggregates

Crosslink

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particle tracking microrheology of purified gastrointestinal mucins

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