Mucin-Inspired Lubrication on Hydrophobic Surfaces - ACS Publications


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Mucin-inspired lubrication on hydrophobic surfaces Benjamin Tillmann Käsdorf, Florian Weber, Georgia Petrou, Vaibhav Srivastava, Thomas Crouzier, and Oliver Lieleg Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00605 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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Mucin-inspired lubrication on hydrophobic surfaces

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Benjamin T. Käsdorf1,, Florian Weber1, Georgia Petrou2, Vaibhav Srivastava2,

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Thomas Crouzier2, and Oliver Lieleg1,*

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1

Department of Mechanical Engineering and Munich School of Bioengineering, Technical University of Munich, Boltzmannstrasse 11, 85748, Garching, Germany 2

Division of Glycoscience School of Biotechnology

Royal Institute of Technology, 106 91 Stockholm, Sweden

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ABSTRACT

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In the human body, high-molecular-weight glycoproteins called mucins play a key role in

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protecting epithelial surfaces against pathogenic attack, controlling the passage of molecules

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towards the tissue and enabling boundary lubrication with very low friction coefficients.

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However,

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biomacromolecules involved in these fundamental processes are fully understood. Thus,

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identifying the key features that render biomacromolecules such as mucins outstanding boundary

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lubricants could set the stage for creating versatile artificial superlubricants. We here

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demonstrate the importance of the hydrophobic terminal peptide domains of porcine gastric

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mucin (MUC5AC) and human salivary mucin (MUC5B) in the processes of adsorbing to and

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lubricating a hydrophobic PDMS surface. Tryptic digestion of those mucins results in removal of

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those terminal domains which is accompanied by a loss of lubricity as well as surface adsorption.

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We show that this loss can in part be compensated by attaching hydrophobic phenyl groups to

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the glycosylated central part of the mucin macromolecule. Furthermore, we demonstrate that the

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simple biopolysaccharide dextran can be functionalized with hydrophobic groups which confers

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efficient surface adsorption and good lubricity on PDMS to the polysaccharide.

neither

the

molecular

mechanisms

nor

the

chemical

motifs

of

those

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Keywords: Biopolymers, Boundary lubrication, Hydrated layer, Hydration lubrication,

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Amphiphiles

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INTRODUCTION

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In the human body, all wet epithelia are covered with mucus, a transparent viscoelastic

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hydrogel which has two important functions: first, mucus shields the underlying tissue from

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pathogenic attack 2, 3, and second, it provides mechanical protection for the tissues when they are

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exposed to shear forces. The macromolecular key component of mucus is mucin, a complex

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glycoprotein which can have molecular weights up to several MDa 4. Those densely glycosylated

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proteins can be divided in three distinct groups: membrane-bound epithelial mucins, secreted

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non-gel-forming mucins and secreted gel-forming mucins with the latter being the major

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constituent of mucus

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properties of native mucus, i.e. they reduce viral activity 6, limit biofilm formation 7, and (when

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used as coatings) reduce cell

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mucus systems such as saliva

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artificial and biological surfaces 12-16: in the boundary lubrication regime, the friction coefficient

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µ measured with mucin-based lubricants can be as small as µ = 0.01 or even less.

3, 5

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. Reconstituted solutions of manually purified mucins reproduce key

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and bacterial 9 adhesion to surfaces. Moreover, similar to native 10

and tear fluid 11, mucin solutions reduce friction both on

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In this boundary regime, hydrodynamic effects are negligible and two opposing surfaces come

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into direct contact 17. The very low friction coefficients observed with mucin-based lubricants are

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therefore critically related to the ability of the highly glycosylated mucin molecules to strongly

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adsorb to a broad range of surfaces

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assemble into well-hydrated macromolecular layers 9, 22, 23, which prevent two opposing surfaces

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from getting into direct contact with each other. This process enables mucin or mucin-like

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molecules to lubricate numerous tissues in the human body: those tissues often comprise a

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mixture of hydrophilic and hydrophobic parts (e.g. the corneal epithelium, the tongue or the

18-21

. Both on hydrophilic and hydrophobic surfaces, mucins

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surface of articular cartilage) with the latter being rendered hydrophilic by adsorption of e.g.

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mucins 24-28.

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In part, this separation of two counter surfaces during shear is achieved by the macromolecule

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itself which is adsorbing to the surfaces and forms a hydrated layer. Friction is reduced by

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shearing off this polymer layer (‘sacrificial layer mechanism’) 29, 30. The second mechanism that

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is involved in reducing friction between opposing surfaces is based on surface-bound water

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molecules (‘hydration lubrication’) trapped by the various hydrophilic moieties on the mucin

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molecule

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quickly reattach to a surface after it was detached by shear forces during a friction process. The

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second mechanism involves the exchange of free water molecules from the lubricant solution

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with water molecules in the hydration layer of surface-adsorbed mucin molecules. This exchange

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takes place as a consequence of the shear occurring during the friction process and provides a

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surface-bound water layer that reduces friction.

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(Figure 1a). To be efficient, the first mechanism requires the mucin molecule to

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Figure 1: Schematic overview of the molecular mechanisms responsible for the lubricity of mucin based lubricants and mucin structure. a) Both the formation of a sacrificial surface layer and hydration lubrication contribute to the lubricity of mucin solutions (see main text for details). The amino acid sequence of human MUC5AC (b) and human MUC5B (c) is analyzed in terms of polar/nonpolar side chains and net charge (see methods for details). The red marks indicate the fragments of the mucin polypeptide which were detected by mass spectrometry after tryptic digestion (see Table S1 and S2 in the Supporting Information).

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to decrease friction by up to two orders of magnitude compared to lubrication with simple buffer

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12, 33

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mucin physisorption to surfaces can be mediated by two different types of physical forces:

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electrostatic interactions between charged moieties of the glycoprotein and oppositely charged

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groups on the surface of the material on the one hand, and hydrophobic interactions between the

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glycoprotein and the surface on the other hand. A mathematical analysis of the amino acid

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sequence of human MUC5B (salivary mucin) and human MUC5AC (gastric mucin) reveals that

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both types of interactions are indeed possible for either mucin variant studied here. Both mucins

Together, sacrificial layer formation and hydration lubrication enable mucin-based lubricants

. Mucins carry both charged and polar moieties as well as hydrophobic residues 4. Thus,

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exhibit a mixed distribution of charged amino acids throughout the whole sequence, and the

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terminal domains of both MUC5AC and MUC5B feature an increased density of hydrophobic

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amino acids compared to the central (glycosylated) region of the glycoprotein (Figure 1b and c).

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In this central part of the molecule, the high amount of serine and threonine (both hydrophilic

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amino acids to which glycans are typically attached to via O-glycosidic bonds) is responsible for

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the comparably strong hydrophilic character of the peptide sequence. Together, this unfolded

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central hydrophilic part and the globular hydrophobic termini result in a daisy-chain-like

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configuration with amphiphilic character 34, 35. It was already shown that the high glycosylation

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density on the mucin glycoprotein is crucial for mucin hydration, and that both the hydration

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state of mucin

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mucins . This particular molecular architecture suggests that the hydrophilic amino acids in the

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central region of the polypeptide serve as anchor points for the glycosylation of the protein

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promoting mucin hydration. In contrast, the hydrophobic terminal regions may be responsible for

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mediating the attachment of the glycoprotein to hydrophobic surfaces.

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and the detailed glycosylation pattern

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are directly related to the lubricity of

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We here show that the hydrophobic terminal peptide sequences of both human salivary mucin

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and porcine gastric mucin are crucial for the lubricating abilities of these glycoproteins on

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hydrophobic surfaces. Enzymatic removal of those peptide sequences not only eliminates mucin

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lubricity but also reduces the adsorption efficiency of the glycoproteins to PDMS. Vice versa, we

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demonstrate that the addition of hydrophobic groups to synthetic dextran molecules promotes the

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dextran adsorption to PDMS and conveys lubricity to the dextran solution if a hydrophobic

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surface is part of the tribological material pairing. Finally, we present a molecular repair

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approach that partially restores the lubricating potential of enzymatically treated mucins on

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hydrophobic surfaces by grafting artificial hydrophobic groups onto the damaged mucins.

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MATERIALS & METHODS

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Mucin purification

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The purification process of mucins was described in detail previously 33. In short, mucus was

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obtained by manual scraping pig stomachs after rinsing them gently with tap water. The mucus

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was diluted 5-fold in 10 mM sodium phosphate buffer (pH 7.0) containing 170 mM NaCl and

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stirred overnight at 4 °C. Cellular debris was removed via several centrifugation steps and a final

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ultracentrifugation step (150000 x g for 1 h at 4 °C). Afterwards, the mucins were separated by

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size exclusion chromatography using an ÄKTA purifier system (GE Healthcare) and a

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XK50/100 column packed with Sepharose 6FF. The obtained mucin fractions were pooled,

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dialyzed against ultrapure water and concentrated by cross-flow dialysis. The concentrate was

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then lyophilized and stored at -80 °C. For purification of human salivary mucin (MUC5B),

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unstimulated human whole saliva was collected from healthy, non-smoking, 20-30 year old

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donors, which refrained from consuming food or beverages other than water for 1 h prior to

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saliva donation. Saliva samples were stored on ice during collection and purified using the same

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protocol as used for MUC5AC. Monomeric MUC5AC and MUC5B has a molecular weight of

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~ 3 MDa including the glycan motifs attached to the protein backbone. As the formation of

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oligomers is possible for both mucin types, the molecular weight range for oligomeric MUC5AC

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and MUC5B is expected to be heterogeneous.

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Dextrans

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Dextrans with a MW of 150 kDa were obtained from TdB Consultancy (Uppsala, Sweden).

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The polysaccharides were either unmodified or modified with carboxymethyl (CM),

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diethylaminoethyl (DEAE), or phenyl groups - the latter of which were present on the dextrans at

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densities of either 0.15 phenyl groups/glucose or 0.40 phenyl groups/glucose, respectively. 40

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kDa dextran with a phenylation degree of 0.32 to 0.40 phenyl groups/glucose was obtained from

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Sigma Aldrich (St. Louis, MO).

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Enzymatic digestion of mucin

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Enzymatic treatment of mucins with trypsin was performed as described in Madsen et al. 37. In

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brief, mucin was dissolved at 10 mg/mL in a 50 mM ammonium bicarbonate solution, and

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disulfide bonds were reduced by adding 5 vol% 200 mM DTT (dissolved in 100 mM ammonium

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bicarbonate) at room temperature for 1 h. 4 vol% of 1 M iodoacetamide dissolved in a 100 mM

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ammonium bicarbonate solution was added to alkylate the mucin again at room temperature for

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1 h. The reaction was quenched by adding 20 vol% of DTT. For proteolytic degradation,

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40 vol% of 1 mg/mL trypsin (from bovine pancreas, Sigma Aldrich, St. Louis, MO) dissolved in

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a 100 mM ammonium bicarbonate solution was added to the mucin solution and incubated at

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30 °C for 18 h. Afterwards, the trypsinated mucin was purified and desalted via size exclusion

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chromatography and cross-flow filtration as described above for native mucin.

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Mucin sequence analysis

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The mucin peptide sequences used for analysis in this work were obtained from the UniProt

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protein database (MUC5AC, P98088; MUC5B, Q9HC84). Up to now, no peptide sequence for

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porcine gastric mucin is available yet. Thus, for our analysis, we used the sequence of human

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MUC5AC instead. Both the sequences of MUC5B and MUC5AC were first divided into sections

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of 50 amino acids each. To characterize the hydrophobic character of these sections, the number

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of amino acids with nonpolar side chains (i.e., Gly, Ala, Val, Met, Leu, Ile, Pro, Trp, Phe) and

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polar side chains (i.e., Arg, Lys, Asn, Asp, Gln, Glu, His, Tyr, Ser, Thr, Cys) was counted. To

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analyze the charge distribution along the amino acid backbone, the number of charged amino

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acids at neutral pH (negatively charged amino acids: Asp, pKa = 3.9; Glu, pKa = 4.1; positively

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charged amino acids: Arg, pKa = 12.5; Lys, pKa = 10.5) was counted. Each charged amino acid

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was assigned one elementary charge, and the net charge of each section of 50 consecutive amino

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acids is displayed as a bar in Figure 1. The glycosylation pattern was estimated based on the

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distribution of the amino acids serine and threonine. They serve as an anchor for O-linked

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glycans

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Each displayed glycan molecule in Figure 1 represents 10 monosaccharides. Cleavage sites of

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trypsin in the sequences of MUC5AC and MUC5B were analyzed with the tool PeptideCutter

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from ExPASy Bioformatics Resources Portal.

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with each O-linked oligosaccharide side chain comprising up to 20 sugar units

3, 15

.

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Adsorption measurements

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Adsorption measurements were performed with an eCell-T quartz crystal micro-balance

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(3T-analytik, Tuttlingen, Germany) and a Gamry eQCM 10M data acquisition device

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(Warminster, Pennsylvania, USA). The quartz crystals used for this study have a gold surface,

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which was spin-coated with a thin layer of polydimethylsiloxane (PDMS, Sylgard 184,

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DowCorning, Wiesbaden, Germany). Therefore, PDMS was mixed in a prepolymer/cross-linker

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ratio of 10:1 and diluted to 1 vol% in n-hexane. 100 µL of this solution was applied to the center

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of the quartz crystal distributed by operating the spin-coater at 3000 rpm for 60 seconds.

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Afterwards, the PDMS was cured at 80 °C for 4 h. A profilometric analysis of the coated crystals

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showed that the thickness of the PDMS layer was ~3 µm (see Supporting Information

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Figure S1). The concentration of biomolecules used for adsorption measurements was

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100 µg/mL, and each biomolecule type was diluted in filtered 20 mM HEPES buffer (filter

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threshold: 0.22 µm). For each measurement, a quartz crystal with a fresh PDMS coating was

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used. Prior to each measurement, the setup was equilibrated with 20 mM HEPES buffer until a

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stable frequency signal was reached. This procedure ensured that the coated PDMS layer (which

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can absorb small amounts of water 39) has reached an equilibrated state so that water uptake into

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this PDMS layer does not affect the measurement. At the beginning of each measurement,

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2 minutes of HEPES buffer signal was recorded as a baseline. Afterwards, the biomolecules were

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injected at 100 µL/min for 2 minutes, and the flow rate was set to 10 µL/min.

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Tribology

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Friction measurements were conducted on a commercial shear rheometer (MCR 302, Anton

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Paar, Graz, Austria) equipped with a tribology unit (T-PTD 200, Anton Paar). The measurements

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were performed as described previously

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geometry. As opposing friction partners, PDMS cylinders (Ø 5.5 mm) and steel spheres

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(Ø 12.7 mm, Kugel Pompel, Wien, Austria) were used. The PDMS cylinders were prepared by

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mixing PDMS prepolymer and cross-linker in a 10:1 ratio (Sylgard 184, DowCorning), exposing

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the mixture to 1 h vacuum and performing a final curing step at 80 °C for 4 h. Before a

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measurement, three pins were inserted into the sample holder and cleaned with 80 % EtOH and

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ultrapure H2O. The measurements were performed at room temperature and the PDMS cylinders

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were fully covered with lubricant. To be consistent with our previous study on the lubricity of

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mucin solutions

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~8.1 mm² (Figure S2) and thus a contact pressure of ≈ 0.35 MPa), and the friction coefficient

22

40

. In brief, the setup used was a ball-on-cylinder

, a normal force of FN = 6 N was applied (leading to a contact area of

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was recorded for sliding speeds from 1000 to 0.01 mm/s (logarithmic speed ramp, 10 measuring

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points per decade) using a measuring time of 10 s per data point. The PDMS surface topology

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was analyzed (Figure S3) after lubricating with unmodified dextrans (0.1 % in 20 mM HEPES)

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to rule out any artefacts generated by wear while sliding a steel sphere over the PDMS surface.

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All lubricants were used at a concentration of 1 mg/mL diluted in 20 mM HEPES, pH 7.4.

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Hydration measurements

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The hydration of the mucin and dextran coatings was measured by combining two

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complementary techniques. First, the hydrated mass of mucin and dextran coatings was assessed

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using quartz crystal microbalance with dissipation monitoring (QCM-D, E4 system, Q-Sense)

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using gold coated crystals (QSX 301, Q-sense) that were cleaned prior to use with a mixture of

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20 % hydrogen peroxide and 80 % ammonium heated at 80 °C for 10 minutes. The mucin and

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dextran solutions were prepared at 1 mg/mL in 20 mM HEPES pH 7 buffer and injected into the

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instrument at a rate of 200 µL/min. In QCM-D, the changes in dissipation reflect the viscoelastic

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properties of the adsorbed coating. The frequency and dissipation shifts were fed into a

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Voigt-based model which was used to accurately estimate the hydrated mass (Q-tools

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software) 41. The density of the mucin coating was fixed at 1050 kg/m3 which is between that of

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pure water (1000 kg/m3) and pure protein (1350 kg/m3) 42.

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Second, the dry mass was measured using a surface plasmon resonance technique (SPR,

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Biacore 2000, GE Healthcare). Mucins or dextrans were injected at a concentration of 1 mg/mL,

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dissolved in 20 mM HEPES, pH 7. The adsorbed mass density was estimated assuming one

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converting response units corresponded to 0.1 ng/mm2 43. The hydration level of the coatings was

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deduced from the dry and hydrated mass using the relationship described in Equation 1.

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ℎ  −    100   ℎ 

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Repair of trypsinated mucin

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Since chemical modification of the termini of trypsin treated mucins is very challenging,

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phenylation of trypsin treated MUC5B was performed by carbodiimide coupling of

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phenylethylamine (Sigma-Aldrich) to the carboxylic groups of sialic acid motifs along the

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central part of the trypsin-treated mucin molecules. Trypsin treated MUC5B was dissolved in a

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20 mM MES buffer solution (pH 5.5) together with 58 mM EDC (1-ethyl-3-(3-

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dimethylaminopropyl)carbodiimide hydrochloride, ThermoFisher) and 27 mM NHS (N-

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hydroxysulfosuccinimide,

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phenylethylamine dissolved in 20 mM MES buffer solution (pH 5.5) were added to the mixture,

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and the pH was adjusted to pH 5.5 by addition of HCl. The solution was left to react at room

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temperature for 2 hours. The mucin was then desalted and purified by chromatography (PD-10

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column, GE Healthcare) and lyophilized before further use. The addition of the phenyl

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functionalities was verified by UV spectra measurement using 1 mg/mL solutions of the mucin

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variants.

Simga-Aldrich).

After

complete

dissolution,

173 mM

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RESULTS AND DISCUSSION

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Previous studies revealed that commercially available mucins (e.g. porcine gastric mucin from

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Sigma Aldrich) lack key properties observed for native mucin systems 6, 44. Prominent examples

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are that solutions of industrial gastric mucins lack the ability to form oligomers, they do not

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exhibit a sol-gel transition at acidic pH, and they are not able to reduce friction in the boundary

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lubrication regime at neutral pH

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gastric mucin following a previously described protocol 33, which preserves its native functional

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properties. To investigate the contribution of the terminal hydrophobic peptide domains of

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mucins to the lubrication potential of mucin solutions, we performed an enzymatic digestion of

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both MUC5AC and MUC5B using trypsin. This enzyme usually cleaves a broad range of peptide

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sequences. For this enzyme, an analysis of the sequences of human MUC5AC and MUC5B

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predicts ~300 cutting sites for either mucin (see Methods), and those cutting sites are distributed

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equally throughout the peptide sequence. However, both mucins are densely glycosylated, and

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protein glycosylation is generally known to protect the polypeptide against proteolytic activity 38,

265

45, 46

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peptidases 47, and accordingly, we expect only partial degradation of MUC5AC and MUC5B by

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trypsin.

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(Figure 2a and b) indicated that the treated mucins in fact retained a very large molecular

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weight and that the cleaved groups were much smaller than the remaining glycoprotein.

13, 19, 33

. As a consequence, we here manually purify porcine

. Indeed, it has been demonstrated that porcine gastric mucin is partially resistant to

A

chromatographic separation

after enzymatic treatment

of

either mucin

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Figure 2: Lubricity of native and enzymatically treated mucins. Chromatograms of size exclusion chromatography of MUC5AC and MUC5B after tryptic digestion are shown in (a) and (b), respectively. Tribological measurements were performed with a steel/PDMS pairing using 0.1 mg/mL mucin dissolved in HEPES buffer. The measurements show the lubricating abilities of either native or tryptic digested MUC5AC (c) or MUC5B (d), respectively. HEPES buffer is included as a reference. The error bars denote the standard deviation as obtained from three independent measurements.

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Mass spectrometry confirmed that the peptide fragments obtained after trypsin treatment of

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human MUC5B indeed originate from the terminal region of the glycoprotein (Figure 1c and

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Table S2). We interpret this result such that the central part of human MUC5B, which is densely

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glycosylated, is shielded against proteolytic degradation. The unprotected terminal parts,

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however, are accessible for the enzyme and seem to be broken down into small fragments of

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similar size. For porcine MUC5AC, a detailed peptide sequence is not available yet in the

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literature. However, the chromatographic profiles obtained for both trypsin treated mucins are

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very similar and only exhibit one second peak at later fractions in addition to the mucin main

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peak, which occurs at similar fractions as untreated mucin. Additionally the analysis of the

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peptide fragments of trypsinated porcine MUC5AC via mass spectroscopy show a coverage with

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the human MUC5AC sequence (Table S2). The overall coverage is lower than for MUC5B but

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the pattern is similar, as fragments only of the terminal mucin domains can be identified. This

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suggests that, also for the porcine MUC5AC used here, trypsin treatment did mostly generate

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small peptide fragments which are likely to originate from the terminal, unglycosilated region of

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the glycoprotein. After separating them from the proteolytic fragments, the remaining mucin

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glycoproteins were tested for their lubricity. When the treated mucins were used as a 0.1 % (w/v)

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lubricant in a steel/PDMS tribology setup, we observed an almost complete loss of their

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lubricating abilities, especially in the boundary lubrication regime (Figure 2c and d).

297

To efficiently reduce friction by enabling hydration lubrication, macromolecular lubricants

298

such as mucins have to form well-hydrated surface coatings. Thus, the inability of trypsin-

299

digested mucins to lubricate in a hydrophobic PDMS/steel pairing could be due to a reduced

300

hydration of such glycoprotein coatings compared to untreated mucins. Since the strong

301

hydration of mucins is established by the high density of hydrophilic glycans in the central

302

region of the protein, the amount of mucin-bound water should not be strongly influenced by our

303

proteolytic degradation procedure. Indeed, hydration measurements of trypsin-digested

304

MUC5AC and MUC5B showed no significant reduction in the amount of mucin-bound water

305

(Figure 3, for detailed information see Table S3 in the Supporting Information).

306 307 308

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309 310 311 312 313 314 315 316

Figure 3: Hydration measurements of surface adsorbed layers of different macromolecules. The hydration is calculated for surface coatings of native MUC5AC and MUC5B, trypsin treated MUC5AC and MUC5B, unmodified dextrans and phenylated dextrans (phenylation degree 0.15 and 0.40, respectively), as well as trypsin treated MUC5B with attached phenyl groups. The error bars denote the standard deviation as obtained from three independent measurements.

317

An alternative explanation for the almost complete loss of mucin lubricity in the boundary

318

lubrication regime could be that the adsorption efficiency of enzymatically treated mucins to

319

surfaces is weakened compared to native mucins. It has been put forward that the terminal

320

(hydrophobic) regions of mucins are involved in the adsorption process of the glycoproteins to

321

hydrophobic surfaces whereas the glycosylated (hydrophilic) central region of mucins are

322

relevant for mucin adsorption to hydrophilic surfaces

323

hydrophobic moieties of the mucin glycoprotein might also be necessary for conveying lubricity

324

on apolar surfaces. Thus, in a next step, we analyzed the adsorption behavior of native and

325

trypsin treated mucins to a hydrophobic PDMS surface. Indeed, in contrast to native MUC5AC

326

and MUC5B, the trypsin treated mucins showed a strongly reduced adsorption to the

327

hydrophobic PDMS as indicated by the drastically reduced shift in resonance frequency reported

328

by QCM (Figure 4a and b).

18

. This model suggests that the

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Figure 4: Adsorption kinetics of different mucin and dextran variants onto PDMS. The adsorption of native and trypsin treated mucins (MUC5AC (a) and MUC5B (b), respectively) and different dextrans variants (phenylated (c) and charged (d)) to PDMS-coated QCM sensors is shown. The error bars denote the standard deviation as obtained from three independent measurements.

335

This result motivates that, whereas mucin hydration is still high after trypsin treatment, the

336

efficiency of hydration lubrication will be drastically reduced as this mechanism requires surface

337

adsorbed mucin molecules to take effect. Also, the formation of a sacrificial layer, i.e. a dynamic

338

shear-off and readsorption cycle, will be hampered if mucin adsorption is reduced. Together,

339

these findings explain the observed loss in mucin lubricity very well and suggest that the

340

hydrophobic terminal domains of MUC5AC and MUC5B are crucial for promoting mucin

341

adsorption to PDMS as required for a good boundary lubricant on hydrophobic surfaces.

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342

To verify our hypothesis that hydrophobic groups are required for the adsorption and

343

subsequent lubricity of well-hydrated macromolecules on hydrophobic surfaces, we make use of

344

a bottom up approach. The idea is to test whether a macromolecule that is not an efficient

345

lubricant on hydrophobic PDMS can be turned into a good lubricant when its adsorption to

346

PDMS is improved. Of course, the trypsin treatment performed on the two mucin variants did

347

not only remove hydrophobic amino acids from the terminal region of the polypeptide but also

348

cleaved positively and negatively charged amino acids. As a molecular platform to

349

systematically probe the influence of charged and hydrophobic modifications on the adsorption

350

efficiency and lubricity of the molecule, we chose dextrans. Dextrans are strongly hydrated

351

molecules 48, and their good hydration is due to the high density of hydrophilic hydroxyl groups

352

along the polysaccharide 49. However, solutions containing unmodified dextrans hardly adsorb to

353

PDMS surfaces at all (Figure 4c). Consistently, the Stribeck curves obtained with solutions

354

containing such dextrans are very similar to those obtained with buffer lacking any

355

macromolecules (Figure 5a).

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Figure 5: Rotational tribology with dextran solutions as lubricants. Tribological measurements between a steel/PDMS pairing with 0.1 mg/mL dextrans solution. Measured were the lubricating abilities of either hydrophobic (phenyl) dextrans (a) or charged dextrans (b). The dotted line represent the Stribeck curve for HEPES buffer as a reference, the dashed line the Stribeck curve of native MUC5AC. The error bars denote the standard deviation as obtained from three independent measurements.

368

In a next step, we test a dextran variant which was modified to carry hydrophobic moieties.

369

The idea is that the addition of hydrophobic groups to dextrans should improve the adsorption of

370

the molecules to hydrophobic PDMS and thus provide lubricity to the dextran solution. Indeed,

371

phenylated dextrans with a phenyl content of 0.15 phenyl substituents per glucose molecule

372

adsorb to a hydrophobic PDMS surface, although the recorded shift in crystal resonance

373

frequency is smaller than that observed for adsorption of either MUC5AC or MUC5B (Figure

374

4c). A possible explanation for the comparably lower adsorption efficiency of phenyl-dextran

375

might be that the amount of hydrophobic groups present on the dextran molecule is smaller than

376

the corresponding number of hydrophobic moieties on the mucin glycoprotein. This idea would

377

be consistent with the biochemical structure of the mucins which comprises large areas with

378

numerous hydrophobic amino acids. To test this hypothesis, we repeated the adsorption

379

measurements with dextrans carrying an increased density of phenyl groups (0.40 phenyl groups

380

per glucose molecule), i.e. a higher number of hydrophobic groups per dextran molecule. Indeed,

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381

for this dextran variant, we observe a stronger shift in the crystal resonance frequency

382

corresponding to more efficient adsorption than for the 0.15 phenyl-dextran variant. For the 0.40

383

phenyl-dextran, this frequency shift is now also similar in magnitude as that observed for the

384

adsorption of native mucins (Figure 4c). These findings suggest that the increased degree of

385

dextran phenylation leads to stronger interactions with the hydrophobic PDMS surface and

386

therefore should aid in maintaining a hydrated polymer film on the surface – provided that the

387

phenylation procedure did not interfere with dextran hydration. QCM measurements, however,

388

show that the high degree of hydration observed for native dextrans is maintained after

389

introducing phenyl groups to these polymers (Figure 3). As a consequence, we expect that both

390

phenylated dextrans should show improved lubricity on PDMS compared to unmodified

391

dextrans, but the dextran variant with the higher phenylation degree should exhibit better

392

lubricating abilities than the 0.15 phenyl-dextran.

393

Tribological measurements with the two phenylated dextran variants on hydrophobic PDMS

394

indeed agree with this expectation: both phenyl-dextran variants significantly reduce the friction

395

coefficient by up to two decades, especially in the mixed lubrication regime, i.e. for sliding

396

speeds between 1 mm/s and 100 mm/s (Figure 5a). In the boundary lubrication regime, i.e. at

397

low sliding speeds below 1 mm/s, the 0.40 phenyl-dextran is more efficient: we here measure

398

constantly low friction coefficients on the order of µ ~ 0.03 which is a bit higher than the value

399

obtained for mucins but more than an order of magnitude lower than the friction coefficient

400

measured for the 0.15 phenyl-dextran in this regime. This observation is consistent with our

401

notion that a stronger adsorption of hydrated molecules, i.e. a more stable hydrated polymer film

402

on the PDMS surface, will lead to better lubrication.

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403

At this point, it is important to recall that the enzymatic treatment of the mucin glycoproteins

404

has not only removed hydrophobic groups from the macromolecule, but also positively and

405

negatively charge amino acids. However, we do not expect that those charged groups contribute

406

significantly to the adsorption of mucins to hydrophobic PDMS surfaces. To verify this, we next

407

test two dextran variants which were modified with anionic carboxymethyl (CM) and cationic

408

diethylaminoethyl (DEAE) groups, respectively. For those two charged dextran variants, we

409

observe similarly low adsorption to PDMS surfaces as for the unmodified dextrans (Figure 4d).

410

This underscores our notion that there are no strong binding interactions between the charged

411

CM or DEAE groups and hydrophobic PDMS, and it suggests that solutions containing those

412

macromolecules should be poor boundary lubricants. Indeed, the lubricity of those

413

macromolecular solutions on PDMS is low: the Stribeck curves measured in lubrication tests

414

performed with these charged dextran variants resemble the results obtained with either simple

415

buffer or unmodified dextrans (Figure 5b). Together, these findings are in agreement with our

416

hypothesis that it is the hydrophobic character of the terminal peptide sequences of mucins and

417

not the charged groups in this region of the glycoproteins that confers adsorption and lubricity on

418

hydrophobic surfaces such as PDMS to mucins.

419

The results presented so far highlight the importance of hydrophobic moieties for the

420

lubrication process on hydrophobic surfaces. Furthermore the extent of these hydrophobic

421

modifications on dextran molecules seems to be linked to the lubricating potential of those

422

polymers. However, a higher phenylation degree not only increases the number of phenyl groups

423

on the dextran molecule but also the density of those hydrophobic groups. Thus, we now ask,

424

whether the overall number of hydrophobic groups on dextrans is important for conveying

425

lubricity or if a certain density of hydrophobic motifs, i.e. spatial proximity of phenyl groups, is

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426

required for dextran molecules to act as an effective boundary lubricant. To tackle this question,

427

we probe the lubricating abilities of another dextran variant having a lower molecular weight (i.e.

428

40 kDa) than the dextrans tested so far (150 kDa) but a similar phenylation degree of ~0.40

429

phenyl groups/glucose. Interestingly, for the small dextran molecules, we obtain Stribeck curves

430

which are virtually identical to those obtained with the larger dextrans (Figure 5a). Since the

431

phenylation degree is 0.40 in both cases, this may indicate, that it is indeed the density of

432

hydrophobic groups on a hydrated macromolecule rather than the total number of hydrophobic

433

motifs which is relevant for providing low friction in the boundary lubrication regime.

434

Together, these results demonstrate that the presence of hydrophobic moieties is crucial for

435

biopolymers such as mucin or dextran to act as a boundary lubricant on hydrophobic surfaces.

436

Since the trypsin treatment of mucins entailed a nearly complete loss of mucin lubricity on

437

hydrophobic surfaces, we now, in a last step, try to “repair” the enzymatically treated mucin by

438

grafting artificial hydrophobic groups to the “damaged” mucins. For this approach, we choose to

439

covalently add phenyl groups to the carboxylic groups of trypsin treated mucin (Figure 6a).

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441 442 443 444 445 446 447 448 449 450

Figure 6: Adsorption properties and lubricity of phenylated trypsin-treated MUC5B. A schematic representation of the “repair approach” (= phenylation) of trypsin treated MUC5B is depicted in (a). (b) Spectroscopic comparison of MUC5B-Trypsin and MUC5B-Trypsin with covalently attached phenyl groups. (c) Adsorption kinetics of native, trypsin treated and “repaired” MUC5B onto PDMS. (d) Lubricity of solutions of phenylated MUC5B as probed on a steel/PDMS pairing. The Stribeck curves obtained for native MUC5B (solid line) and trypsin treated MUC5B (dashed line) are shown for comparison. The error bars denote the standard deviation as obtained from three independent measurements.

451

Since the terminal peptide domains of the degraded mucin are no longer present and the central

452

part of the polypeptide sequence is shielded by glycans, we aim to graft those phenyl groups to

453

the carboxyl group of sialic acid, a charged monosaccharide, present among the mucin glycans 50,

454

51

455

which has a higher sialic acid content than MUC5AC

456

indicated a successful phenylation of “damaged” (= trypsin treated) MUC5B (Figure 6b), we

. For optimal efficiency of this grafting process, we perform these experiments with MUC5B 52-54

. Since UV-absorbance measurements

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457

next analyze if the addition of those hydrophobic groups would promote the adsorption of the

458

“repaired” mucin onto a hydrophobic PDMS surface. Indeed, we observe a significant increase

459

of adsorbed mucin molecules on a PDMS-coated quartz crystal (Figure 6c). However, the

460

amount of adsorbed molecules seems not to be as high as for native MUC5B. Thus, in a next

461

step, we ask if the “repaired” molecule can achieve similarly good lubricity as native mucin.

462

When performing friction tests with the “repaired” mucins (Figure 6d), we observe a strong

463

decrease of friction coefficient in the mixed lubrication regime (i.e. for sliding speeds between

464

1 mm/s and 100 mm/s) compared to trypsin treated MUC5B. This decrease in the friction

465

coefficient is, however, less pronounced in the boundary lubrication regime where we measure a

466

friction coefficient of ~0.2 - 0.3. This value is still ~3fold lower than the corresponding value

467

obtained for “damaged” mucin but more than an order of magnitude larger than the friction

468

coefficient obtained for native mucins in this regime. It is likely that the incomplete recovery of

469

both the adsorption kinetics as well as the lubricating abilities of this phenylated mucin

470

compared to native mucin can be attributed to the limited amount of phenyl groups grafted onto

471

the damaged mucin molecules. As discussed before (Figure 5a), the density of phenyl groups on

472

dextrans seems to be directly linked to both the adsorption kinetics of the macromolecule and its

473

lubricating ability. Therefore, the amount of sialic acid groups which can be targeted with our

474

phenylation procedure might be too low to achieve a phenylation density that is high enough to

475

achieve adsorption kinetics and lubricity on a level comparable to that of native mucin. This

476

assumption is in good agreement with previous work where it was shown that the adsorption

477

strength and lubricating potential of boundary lubricants are linked 55, 56. However, those findings

478

indicate that MUC5B digested with trypsin can indeed be ‘repaired’: covalently attaching phenyl

479

groups seems to compensate for the lost hydrophobic peptide termini, at least to a certain degree.

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480

CONCLUSION

481 482

We here demonstrated, that the hydrophobic domains of mucins are crucial to adsorb and

483

further lubricate hydrophobic surfaces such as PDMS. Since several tissue surfaces in the human

484

body exhibit a hydrophobic character

485

means of hydrophobic interactions is essential to provide boundary lubrication in vivo. When the

486

mucin molecules are deprived of their hydrophobic domains, this lubrication on hydrophobic

487

surfaces cannot take place anymore. However, we were able to compensate for the loss of these

488

hydrophobic domains by grafting phenyl groups onto the damaged mucins, which in part

489

recovered their ability to adsorb and lubricate hydrophobic surfaces. This approach can also be

490

transferred to other biomolecules: Dextrans, highly hydrophilic molecules, can function as good

491

boundary lubricants on hydrophobic PDMS when equipped with phenyl groups. The density of

492

attached hydrophobic groups during this bottom-up approach determines the adsorption kinetics

493

and lubricity of this mucin-inspired macromolecule. Of course, we here study macromolecular

494

lubricity on simple PDMS surfaces which are hydrophobic but uncharged. On more complex

495

surfaces which combine hydrophobic and charged characteristics, a phenyl-modified

496

polyelectrolytic dextran variant might provide even better lubricity than the uncharged

497

phenylated dextran molecules presented here.

25-28

, the interaction of mucins with these surfaces by

498

The results presented here may pave the way towards the rational design of macromolecular

499

superlubricants which provide ultra-low friction on a broad range of biological and technical

500

surfaces. A suitable polymer for such an artificial lubricant could be polyethylene glycol (PEG)

501

which is non-toxic, water soluble, and available with different molecular weights. PEG is already

502

used in numerous fields ranging from industrial to pharmaceutical applications. PEG is highly

503

hydrated and a good lubricant when utilized as surface attached polymer brushes 57. Moreover, it

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504

has been demonstrated that linking PEG polymer chains to deglycosylated mucin can serve as a

505

replacement for the hydrated glycans and restore lubricity 22. Combining those existing strategies

506

with the results shown here could lead to an artificial brush-like macromolecule with great

507

potential for providing ultra-low friction on various surfaces.

508 509 510

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Biomacromolecules

AUTHOR INFORMATION

512

Corresponding author

513

*Prof. Dr. Oliver Lieleg, e-mail: [email protected], phone: +49 89 289 10952, fax: + 49 89

514

289 10801

515

Present Address

516

Department of Mechanical Engineering and Munich School of Bioengineering

517

Boltzmannstraße 11, 85748 Garching, Germany

518 519

AUTHOR CONTRIBUTIONS

520

BK, OL and TC proposed the experiments. BK, FW, GP and VS performed the experiments

521

and analyzed the data. The manuscript was written by BK and OL.

522 523

ACKNOWLEDGEMENTS

524

The authors thank Christine Braig for assistance with the mucin preparation.

525 526

SUPPORTING INFORMATION

527



Analysis of PDMS-layer thickness on QCM-chips

528 529



Detailed information on the mass spectrometry analysis of trypsin digested MUC5AC and MUC5B.

530



Detailed information on the hydration measurements

531



Surface topology analysis of the PDMS pin surface

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37. Madsen, J. B.; Svensson, B.; Abou Hachem, M.; Lee, S., Proteolytic Degradation of Bovine Submaxillary Mucin (BSM) and Its Impact on Adsorption and Lubrication at a Hydrophobic Surface. Langmuir 2015, 31, (30), 8303-8309. 38. Jentoft, N., Why Are Proteins O-Glycosylated. Trends Biochem. Sci. 1990, 15, (8), 291294. 39. Verneuil, E.; Buguin, A.; Silberzan, P., Permeation-induced flows: Consequences for silicone-based microfluidics. Europhys. Lett. 2004, 68, (3), 412-418. 40. Boettcher, K.; Grumbein, S.; Winkler, U.; Nachtsheim, J.; Lieleg, O., Adapting a commercial shear rheometer for applications in cartilage research. Rev. Sci. Instrum. 2014, 85, (9), 093903. 41. Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B., Viscoelastic Acoustic Response of Layered Polymer Films at Fluid-Solid Interfaces: Continuum Mechanics Approach. Phys. Scr. 1999, 59, (5), 391. 42. Weber, N.; Wendel, H. P.; Kohn, J., Formation of viscoelastic protein layers on polymeric surfaces relevant to platelet adhesion. J. Biomed. Mater. Res., Part A 2005, 72A, (4), 420-427. 43. Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C., Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J. Colloid Interface Sci. 1991, 143, (2), 513-526. 44. Kočevar-Nared, J.; Kristl, J.; Šmid-Korbar, J., Comparative rheological investigation of crude gastric mucin and natural gastric mucus. Biomaterials 1997, 18, (9), 677-681. 45. Jensen, P. H.; Kolarich, D.; Packer, N. H., Mucin-type O-glycosylation – putting the pieces together. FEBS J. 2010, 277, (1), 81-94. 46. Russell, D.; Oldham, N. J.; Davis, B. G., Site-selective chemical protein glycosylation protects from autolysis and proteolytic degradation. Carbohydr. Res. 2009, 344, (12), 1508-1514. 47. Scawen, M.; Allen, A., Action of Proteolytic-Enzymes on Glycoprotein from Pig Gastric Mucus. Biochem. J. 1977, 163, (2), 363-368. 48. Balasubramanian, D.; Raman, B.; Sundari, C. S., Polysaccharides as amphiphiles. J. Am. Chem. Soc. 1993, 115, (1), 74-77. 49. Hunger, J.; Bernecker, A.; Bakker, Huib J.; Bonn, M.; Richter, Ralf P., Hydration Dynamics of Hyaluronan and Dextran. Biophys. J. 2012, 103, (1), L10-L12. 50. Perez-Vilar, J.; Hill, R. L., The structure and assembly of secreted mucins. J Biol Chem 1999, 274, (45), 31751-31754. 51. Authimoolam, S.; Dziubla, T., Biopolymeric Mucin and Synthetic Polymer Analogs: Their Structure, Function and Role in Biomedical Applications. Polymers 2016, 8, (3), 71. 52. Bhaskar, K. R.; Gong, D.; Bansil, R.; Pajevic, S.; Hamilton, J. A.; Turner, B. S.; Lamont, J. T., Profound Increase in Viscosity and Aggregation of Pig Gastric Mucin at Low Ph. Am J Physiol 1991, 261, (5), G827-G833. 53. Snary, D.; Allen, A., Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa. Biochem. J. 1971, 123, (5), 845-53. 54. Thomsson, K. A.; Prakobphol, A.; Leffler, H.; Reddy, M. S.; Levine, M. J.; Fisher, S. J.; Hansson, G. C., The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse. Glycobiology 2002, 12, (1), 1-14.

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55. Zappone, B.; Ruths, M.; Greene, G. W.; Jay, G. D.; Israelachvili, J. N., Adsorption, Lubrication, and Wear of Lubricin on Model Surfaces: Polymer Brush-Like Behavior of a Glycoprotein. Biophys. J. 2007, 92, (5), 1693-1708. 56. Jahanmir, S.; Beltzer, M., An Adsorption Model for Friction in Boundary Lubrication. ASLE Trans. 1986, 29, (3), 423-430. 57. Spencer, N. D., Aqueous Lubrication with Poly(Ethylene Glycol) Brushes. Tribology Online 2014, 9, (4), 143-153.

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TABLE OF CONTENTS GRAPHIC

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