Reversible Supramolecular Assembly of Velvet Worm Adhesive Fibers

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Reversible supramolecular assembly of velvet worm adhesive fibers via electrostatic interactions of charged phosphoproteins Alexander Baer, Sebastian Hänsch, Georg Mayer, Matthew J. Harrington, and Stephan Schmidt Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01017 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 18, 2018

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Reversible supramolecular assembly of velvet worm adhesive fibers via electrostatic interactions of charged phosphoproteins Alexander Baer1, Sebastian Hänsch2, Georg Mayer1, Matthew J. Harrington3,4* and Stephan Schmidt5*

1

Department of Zoology, Institute of Biology, University of Kassel, Heinrich-Plett-Str. 40,

34132 Kassel, Germany, 2Center for Advanced Imaging (CAi), Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany, 3Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam 14424, Germany, 4Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada, 5Institute of Organic and Macromolecular Chemistry, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße Universitätsstr. 1, 40225 Düsseldorf, Germany

Email: Stephan Schmidt – [email protected] Email: Matthew J. Harrington – [email protected] * Corresponding authors

Keywords responsive nanoparticles, coacervate, polyelectrolytes, mechano-responsive, self-assembly, Onychophora 1

ACS Paragon Plus Environment

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Abstract Velvet worms secrete a fluid hunting slime comprised of a dispersion of nanoglobules that form micro-fibers under small mechanical shear forces, facilitating the rapid formation of stiff biopolymeric fibers. Here, we demonstrate that the nanoglobules are held together and stabilized as a dispersion by electrostatic interactions reminiscent of coacervate-based natural adhesives. Variation of ionic strength and pH affect the stability of nanoglobules and their ability to form fibers. Fibers mainly consist of large (~300 kDa), highly charged proteins, and current biochemical analysis reveals a high degree of protein phosphorylation and presence of divalent cations. Taken together, we surmise that polyampholytic protein sequences, phosphorylated sites and ions give rise to transient ionic crosslinking, enabling reversible curing of ejected slime into high-stiffness fibers following dehydration. These results provide a deeper understanding of velvet worm adhesive fibers, which may stimulate new routes towards mechano-responsive and sustainable materials.

1. Introduction Supramolecular polymers are an important class of advanced materials with dynamic and tunable properties in which macromolecules are able to reversibly assemble and disassemble via non-covalent interactions.1 Whereas focused development of synthetic supramolecular polymers dates back less than thirty years, nature has evolved remarkable biopolymer supramolecular assembly mechanisms over eons of evolution, which have emerged as important role models for sustainable fabrication of high performance polymers.2-5 In particular, recent investigations of the adhesive slime ejected by the velvet worms (Onychophora) revealed that biopolymeric fibers with material stiffness comparable to polyamide thermoplastics can be formed rapidly and reversibly under environmentally friendly processing conditions by simple mechanical drawing and subsequent dissolution in 2


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distilled water for extended periods.2 In the present study, we investigate the role of noncovalent electrostatic interactions in mediating this remarkable supramolecular assembly behavior in the prey capture slime of the onychophoran species Euperipatoides rowelli. Velvet worms are invertebrate animals living in the underbrush of temperate and tropical forests of the southern hemisphere and around the equator. Their hunting strategy involves a projectile adhesive slime, which is ejected from nozzle-like extensions on either side of the head, known as slime papillae.6-8 During ejection, the fluid slime transitions into viscoelastic adhesive fibers that can adhere to and trap prey, such as crickets, spiders, wood lice and other small arthropods (Figure 1a).6, 7 Shortly thereafter, the slime adhesive rapidly dries, forming a very stiff fibrous material with an elastic modulus of around 4 GPa, comparable to high performance polymers such as Nylon®.2 More remarkable, perhaps, is the fact that fiber formation is completely reversible and circular – dry fibers from E. rowelli dissolve in water after several hours and new fibers can be regenerated from the dissolved fiber solutions.2, 9 The fact that velvet worm fiber formation occurs outside the body sets this fabrication process apart from other biopolymer assembly processes that involve bio-mediated control of processing conditions (e.g., silks10 and mussel byssus5), making it highly relevant for biomimetic transfer into synthetic polymer systems. However, this first requires a more indepth understanding of the molecular level driving forces controlling reversible supramolecular assembly – a response that must be programmed in the fluid itself. Biochemical analysis of the slime in two closely related species of the Australian genus Euperipatoides has revealed that it is comprised of several protein-building blocks, as well as some fatty acids.11,

12

The most prominent component is a large proline-rich protein (~300

kDa) localized in the fiber core and previously proposed to be largely unstructured.12 Recent studies indicate that the fluid slime is a dense dispersion of spherical nanoglobules (diameter ~70 nm) in which both lipids and proteins were observed to co-localize (Figure 1b).2 3



Moreover, nanoglobules exhibited a strong tendency to self-organize into nano- and microfibrils under mechanical stress, suggesting that they are the active agent during fiber selfassembly.2 Thus, the key to understanding fiber assembly from fluid slime is elucidating the forces and interactions that guide reversible supramolecular assembly of slime nanoglobules – a process which has been previously proposed to involve both electrostatic and hydrophobic interactions.12 Towards achieving a deeper molecular comprehension of this fascinating self-assembly process, we aim here to generate a physical-chemical understanding of the nanoscopic and molecular nature of the globules and their peculiar ability to reversibly form polymeric fibers. Along these lines, we investigated the effect of pH and ionic strength on the colloidal stability of nanoglobules and on fiber formation. Additionally, we examined the ionic composition of the slime and potential role of post-translational protein modification as a route to supramolecular assembly. Our results confirm that electrostatic interactions, likely mediated in part via phosphate groups on the major constituent protein, play a crucial role in both nanoglobule stability and fiber formation. In addition to furthering the understanding of onychophoran physiology and biochemistry, these findings have potential relevance for inspiring sustainable polymer fabrication processes via biomimetic supramolecular chemistry.

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Figure 1 Hunting slime of Euperipatoides rowelli. a) Ejection of capture slime by the animal after provoking defensive behavior. Image courtesy of Alexander Baer and Ivo de Sena Oliveira. b) Native slime droplets contain nanoglobules (left) and form stiff fibers after mechanical shearing and drying (right). Scale bar: 500 nm.

2. Experimental Section Animals and sample collection Experiments were performed on the peripatopsid species Euperipatoides rowelli Reid, 1996.13 Specimens of E. rowelli were obtained from decaying wood at the corresponding localities and maintained in the laboratory as described previously.14 The animals were collected and exported under the following permit numbers: SL101720/2016, issued by NSW National Parks & Wildlife Service (Australia) and PWS2016-AU-001023, issued by Department of Sustainability, Environment, Water, Population and Communities (Australia). All animal treatments complied with the principles of laboratory animal care and the German law on the protection of animals. Slime samples for each experiment were obtained from several specimens by stimulating them to eject the slime into 500 µl Eppendorf tubes. Collected slime was stored in the fridge no longer than for 3–4 days at 4°C to avoid bacterial growth or potential protein degradation.

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Dynamic light scattering Dilutions (1:100) of crude slime were prepared using NaCl solutions from 10 mM to 1 M in water. For pH variation, the dilutions were prepared in three different buffer systems, all at 10 mM concentration. For pH 4.8 acetate buffer, for pH 5.2 and pH 5.7 MES buffer (2-(N-Morpholino)ethanesulfonic acid), for pH 6.3 and pH 7.2 and 8.2 phosphate buffer. The DLS measurement procedure is thoroughly described in Supplementary Information S4. In brief, measurements were conducted at 20 °C in disposable cuvettes on a Malvern HTTPS Particle Sizer in backscattering configuration with fixed scattering angle of 173 degrees. DLS autocorrelation functions were evaluated by regularization to obtain the hydrodynamic radius distributions. Zeta potential measurements The same slime samples used in DLS were used for Zeta potential measurements. 1 ml of slime dilutions were transferred to disposable capillary cells (Malvern instruments) and then the zeta potential was measured on a Malvern Zetaziser Nano Z at an electric field strength of 10 mV/cm. AFM imaging 1:10 dilutions of crude slime were prepared in the different buffer systems (50 mM). Then a droplet of the samples was placed on a silicon surface (cleaned by RCA procedure: water, ammonia, hydrogen peroxide 30% at 70°C for 20 min) and allowed to rest for 10 minutes. Next, the droplet was rinsed from the surface using the respective buffer (5 mM) followed by drying the silicon surface under a stream of nitrogen. The surfaces were imaged on a Nanowizard II AFM (JPK instruments, Germany) using tapping mode cantilevers XSC11/no Al (Micromasch Bulgaria) with a nominal spring constant of 40 N/m. Fiber imaging with optical microscope 1:10 dilutions of crude slime were prepared in 1.5 ml Eppendorf tubes using the different buffer systems (50 mM) or with NaCl solutions to obtain 10, 100 and 1000 mM final NaCl concentrations. Then samples were agitated with a pipette tip. Immediately after agitation, samples were transferred to microwells for optical microscopy. Images were collected using in phase contrast mode on an Olympus IX81 6


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microscope equipped with an UPlan FL N 20x/0.5 Ph1 objective and CMOS camera (DMK 23UX174, The Imaging Source, Germany). STED Microscopy Coverslip coating procedure to obtain polycationic, and PEGylated glass surfaces are described in the Supplementary Information S5. Slime was labeled by first gently diluting (1:100) in 10 mM phosphate buffer (pH 5.2) to a final volume of 100 µl. Nanoglobules were labeled by addition of mCling (Atto647N-labeled, Synaptic Systems, Germany) at a final concentration of 0.4 µM. The diluted slime was incubated for 10 min and transferred to glass slides for microscopy. Settling and spreading of the slime was allowed for another 15 min before microscopic and nanoscopic measurements were applied. STED measurements were performed using a TCS SP8 STED 3X (Leica) equipped with an HC PL APO CS2 100x objective (NA 1.4) at a scan speed of 1000 Hz. As excitation and depletion LASER wavelength 640 nm and 775 nm were chosen respectively. The detection range for the emitted fluorescent signals was set from 660 to 700 nm. SDS-PAGE and phosphoprotein staining Different dilutions of crude slime (1:5 ≙ approx. 55 µg slime protein) were reduced (5% β-mercaptoethanol) and separated using denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The polyacrylamide concentration in gels varied between 5% and 15% (total concentration of both acrylamide and the crosslinker bisacrylamide) with 3% C (concentration of the crosslinker) for separating small and large proteins, respectively. Protein samples were run in a Mini-PROTEAN II electrophoresis cell (Bio-Rad, Hercules, CA, USA) and performed either according to Laemmli15, with a TrisGlycin SDS running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.3) at 40 mA for approximately 1.5 h (SDS-PAGE), or according to Schägger16 using Tris anode buffer and Tris-Tricine cathode buffer at constant 120 V for approximately 3 h (Tricine SDS-PAGE). As protein standards, the prestained protein ladders PageRuler (Fermentas, St. Leon-Rot, Germany; for proteins in the molecular range of 15–120 kD) and Protein Standard HiMark 7



(Invitrogen, Darmstadt, Germany; for larger proteins between 70–300 kD) were used. Phosphoprotein stains were performed according to the Pro-Q® Diamond Phosphoprotein Gel Stain protocol (Thermo Fisher Scientific, Waltham, MA, USA). Total protein was performed using Coomassie Blue (0.1% w/v Coomassie Blue R350 for 20 min and destained in destaining solution (50% v/v methanol and 10% v/v glacial acetic acid) for 3 h. Gels were imaged using the documentation system Syngene G-Box supported by the software GeneSnap v7.05 (Synoptics, Cambridge, England). Final image editing and panel design were performed using Adobe (San Jose, CA, USA) Photoshop CS5 and Illustrator CS5. Prediction of posttranslational modifications Theoretical phosphorylations of previously identified slime proteins by Haritos et al.12 were predicted using NetPhos3.1.17 Atom absorption spectroscopy Fluid slime (1:10) was dissolved in 500µl aqua regia and heated for 1h at 96°C. 3 samples of 20µl and element standards (Ca, Cu, Fe, K, Mg, Na, P, Zn) were measured using ICP-OES (Optima 2100 DV, Perkin-Elmer) at a back pressure of 169 kPa and a flow of 0.55 L/min.

3. Results and Discussion 3.1. Nanoglobule dispersion and fiber formation at varying ionic strength and pH 3.1.1. Nanoglobules in solutions of varying ionic strength The colloidal stability of nanoglobular dispersions is typically linked to their mutual electrostatic repulsion. Conversely, electrostatic attraction of specific ions and amino acids can drive colloidal instability and lead to the formation of fibrous protein aggregates (e.g., amyloid aggregates18 and spider silk assembly19). Therefore, it stands to reason that the reversible, non-covalent fiber formation2 in the nanoglobular velvet worm secretion may be influenced by electrostatic interactions given the prominence of both positively and negatively 8


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charged amino acids in the primary protein sequence.12 To further investigate this hypothesis, we examined the effect of ionic strength and pH, which function to disrupt the charge-charge interactions, on nanoglobule stability and fiber formation. Native slime has a slightly cloudy texture and exhibits a distinct Tyndall light scattering effect resulting in a yellowish/brownish appearance when observing light that is transmitted through the slime (Figure 2a). Typically, this effect is observed when light is scattered from particle dispersions with average particle sizes a bit below or near the wavelength of visible light. However, when adding NaCl in the concentration range of 0.1 M to 1 M overall light scattering is decreased and the transparency of the fluid is increased, suggesting that light scattering globules are no longer present (Figure 2c). To gain a nano-scale understanding of this behavior, we performed dynamic light scattering (DLS) to monitor globule size as a function of salt content. Previous DLS investigation of native slime showed a large particle population with hydrodynamic radii in the range of ~70 nm and a much less prominent population at smaller length scales (5–10 nm), which were previously assigned to protein nanoglobules and free proteins, respectively.2 DLS investigation of slime in which NaCl was added indicate an increase in the intensity of the smaller radius peak relative to the peak at larger hydrodynamic radius, suggesting that nanoglobules are dissociating into free biomolecules, reducing the number of larger particles (Figure 2b, c), This, in turn, suggests that increased electrostatic screening by addition of NaCl promotes dissociation of nanoglobules. Taking into account that fiber formation is reversible (i.e. functional nanoglobules are spontaneously formed by dissolving slime fibers in water2), it seems plausible that nanoglobules are non-covalently stabilized by oppositely charged molecular species. Cationic or anionic proteins, fatty acids or bound counterions are likely chemical moieties present in the slime capable of forming such charge-charge stabilized complexes. Increasing the ionic strength in presence of such complexes would increase Debye 9



screening length, leading to destabilization and dissolution of the nanoglobules, as observed.20, 21

Figure 2 Light scattering properties of the slime as a function of NaCl concentration. a) Droplet of the slime (about 0.5 cm in diameter) in reflection (left) and transmission (right) show Tyndall light scattering effect. b) Hydrodynamic radii distribution of nanoglobules in 1:100 dilutions at varying NaCl concentration. All curves show two peaks at around 10 and 100 nm. c) At increasing NaCl concentration the peak ratio indicates that the number of the larger particles decreases (black line). Light scattering intensity also indicates decrease in strong light scatterers (large particles) at increased NaCl concentration.

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3.1.2. Nanoglobules in solutions of varying pH In addition to screening effects based on ionic strength, charge-stabilized colloids are also highly sensitive to pH changes. Native slime has an acidic pH of 5.2, which, based on the high prevalence of oppositely charged amino acids in slime proteins,12 may play an important role in stabilizing nanoglobules. To further test the role of electrostatic interactions on nanoglobule stability, slime pH was varied within the range of pH 4–8, and nanoglobule size and surface charge were assessed with DLS and zeta potential measurements, respectively. Notably, DLS indicates that globule size is smallest at the natural pH (~70 nm), but more than doubles in size at pH 4. Above pH 5, globule size steadily increases with increasing pH up to a maximum of ~1 µm at pH 7.2, before sharply dropping at higher pH. Notably, the solution also becomes extremely turbid at pH 7.2, suggesting precipitation of protein aggregates (Figure 3a, b). Results from zeta potential measurements under pH variation match the trend seen in DLS. Under native conditions (pH 5.2), the nanoglobules have a zeta potential of +13 mV. A zeta potential of 10-20 mV provides rather low colloidal stability, in line with that fact that nanoglobule aggregation can be readily induced by mechanical agitation. When reducing the pH to 4.0, the zeta potential increased, whereas a continuous increase in pH above 5.2 resulted in a steady decrease of the zeta potential. The apparent isoelectric point of the entire slime mixture was around pH ~7.1, where the zeta potential was zero (Figure 3c). Upon further pH increase, the zeta potential became increasingly negative. This fits well with DLS observations, which revealed that at neutral pH large aggregates likely formed due to the absence of surface charge and electrostatic stabilization. Further increase in pH appears to lead to weak electrostatic stabilization and nanoscopic structures.

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Figure 3 DLS and zeta potential of nanoglobules at varying pH in 1:100 dilutions. Hydrodynamic radii distribution (a) and average values (b) measured by DLS; zeta potential distributions (c) and average values (d). Changes in particle size as a function of pH were further confirmed by AFM imaging of nanoglobules adsorbed on glass surfaces. Ten-fold diluted slime dispersions were incubated in the respective buffer systems and allowed to adsorb on glass surfaces. For slime incubated at pH 4.0–6.3, we observed distinct nanoparticles of various sizes, consistent with DLS measurements. At pH 5.7 and 6.3 morphology of the nanoglobules films becomes less distinct which is due to the combined effects of reduced nanoglobule surface charge and increased ionic strength upon drying the film on the AFM substrate. At pH values around 7.2, the dried slime layer loses its globular structure and forms unstructured films. Upon further increasing pH to 8.2, elongated hollow objects were observed on the glass surface (Figure 4).

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Figure 4 AFM images of adsorbed and dried velvet worm secrete in different pH conditions (1:100 buffer dilutions, “native pH” signifies 1:100 dilution in ultrapure water). In acidic pH (4.0 to 6.3) nanoglobules are present. At pH 7.2 non-structured films are formed. Slime at pH 8.2 forms large ellipsoidal capsules reminiscent of lamella-like structures. Scale bar: 500 nm. 3.1.3. Fiber formation from solutions of varying pH and ionic strength Fiber formation via mechanical agitation is also expected to respond strongly to different pH or ionic strength, considering that the unperturbed slime dispersion shows a strong response of nanoglobule stability due to changes in surface charge. Fiber formation was therefore tested at varying NaCl concentration, as well as varying pH. Dilutions of the slime were prepared in NaCl (10–1000 mM) and buffer (pH 4.0–8.2) solutions. In order to induce fiber formation, the diluted slime samples were mechanically agitated with a pipette tip. Immediately after, samples were imaged by optical microscopy (Figure 5). Increasing the ionic strength resulted in reduced fiber formation at 100 mM NaCl and completely clear solutions at 1000 mM NaCl. Variations in pH showed an even stronger effect on fiber formation. Not surprisingly, fiber formation was most prominent at the natural pH 5.2. Changing the pH by one unit above or below the natural pH rendered the slime almost 13



completely unable to form fibers. Under mild basic conditions (pH 8.2), the slime transitioned into an opaque gel that shows no fibers and little mechanical strength. These findings indicate that the mechanism of nanoglobule stabilization, which appears to be correlated with electrostatic interactions, also strongly influences the dynamic transition from globules to fiber. This is likely to be related to the high abundance of charged amino acids that can significantly alter protonation state outside of the native pH range (Asp, Glu, His comprise a quarter of all amino acids, see Supplementary Information S1). Thus, it is conceivable that the organism retains a high degree of control over pH conditions in the slime to optimize conditions for fiber formation.

Figure 5 Formation of fibers on 1:10 dilution at different NaCl concentrations (top) and different pH values (below). Scale bar: 20 µm. 3.2. Interaction of nanoglobules with materials of different surface charge In addition to its fiber-forming capacity, immediately after ejection the slime functions as an effective adhesive, entrapping insect prey. To investigate the role of the charged nanoglobules in initiating the adhesive response of the slime, we utilized stimulated emission depletion (STED) microscopy to study the adsorption and wetting behavior of the nanoglobules on 14


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surfaces with varying surface charge. Consistent with the finding that nanoglobules exhibit a positive surface charge, which is rather unusual for a non-synthetic colloidal dispersion22, 23, slime adhered strongly to negatively charged glass surfaces. The surfaces appear homogeneously covered indicating that nanoglobules rupture and the contents spread over oppositely charged glass surfaces (Figure 6 left). On glass surfaces treated with polyethyleneimine, a cationic polyelectrolyte, nanoglobules stay intact, likely due to electrostatic repulsion (Figure 6 right). On PEGylated neutral glass surfaces, intermediate adhesion was observed. Here, nanoglobules form clusters of 1–4 µm in diameter with single nanoglobules observed in the spaces between the clusters (Figure 6 middle, inset). Many natural material surfaces composed of carbohydrates or proteins, including hair or skin show affinity to polycationic macromolecules or colloids, which is exploited by pharmaceutical or cosmetic products, for example.24, 25 While it is tempting to speculate that initial contact between slime and prey animal is similarly enhanced due to electrostatic interactions that trigger aggregation and adhesion of the cationic nanoglobules, further studies need to be initiated to confirm this hypothesis, especially considering that insects typically possess a waxy epicuticle.26 In any case, the strong wetting behavior observed by the nanoglobules likely functions to increase the contact with the material surfaces of captured prey, which is a prerequisite for generating mechanical stress on the slime, fiber formation and mechanical entrapment. This key function of the globular particles may explain their unusual positive surface charge when compared to other natural adhesive particles like vegetable latex or adhesive proteins like fibrinogen or fibronectin showing negative surface charge.27, 28

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Figure 6 Nanoglobule adhesion on glass slides with different surface charge via STEDmicroscopy (mCling labeling). Left: glass surface with anionic silanol groups, middle: PEGylated glass bearing no charge, right: cationic polyethyleneimine coating on glass. Scale bar: 10 µm; Scale bar inset: 1 µm. 3.3. Biochemical and compositional analysis The primary protein component proposed to comprise the fiber are high molecular weight (~300 kDa), proline-rich proteins containing a high amount of both positive and negatively charged residues.11 The presence of highly charged zwitterionic macromolecules is consistent with the current hypothesis of ion-dependent formation of nanoglobular and fibrous structures. To further investigate this possibility, we performed inductively coupled plasma optical emission spectroscopy (ICP-OES) on slime samples to determine the elemental composition. ICP-OES of crude velvet worm slime indicates the presence of elevated content of Mg, Ca and P, as well as lower content of transition metals such as Zn and Fe (Table 1). Notably, many biological adhesives found in nature, such as mussel byssus4, caddisfly larvae silk glue29, sandcastle worm cement30 and snail slime31 are stabilized by electrostatic interactions 16


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between charged amino acids and divalent ions. In particular, sand castle worm cement and caddisfly silk are comprised of phosphorylated proteins that interact strongly with calcium ions to provide mechanical stability to the adhesive.29, 32 To clarify whether the high phosphorous content revealed in ICP-OES elemental analysis might be due to protein phosphorylation, we ran slime proteins on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and specifically labeled phosphoproteins. The results indicate that the high molecular weight protein believed to be the primary component of the slime fibers exhibits a strong positive staining for phosphorylated amino acids (Figure 7). Furthermore, analysis of the protein sequence with NetPhos17 algorithms indicates that the degree of phosphorylation for the high molecular weight proteins is around 50% (Supplementary Information S3). Comparison to other organisms like sea cucumbers33, sandcastle worms30 or caddisfly larvae34 shows that the degree of serine, threonine and tyrosine phosphorylation in the high molecular weight sequences of the slime is quite high. While we cannot absolutely exclude that some of the detected phosphorous might arise from phospholipids, positive protein staining suggests a significant portion of the phosphorous is associated with the large proline-rich protein. Furthermore, previous reports of lipids in the slime of E. rowelli reported only non-phosphorylated fatty acids.11 Overall, the presence of phosphorylated proteins and high abundance of divalent metal ions calcium and magnesium make a strong case for specific ionic bridging between the high molecular weight proteins to form globular and fibrous structures. Table 1 Composition of velvet worm slime atom / compound

concentration (mg/ml)

sodiuma)

0.3±0.1

potassiuma)

0.65±0.06

a)

calcium

0.66±0.07

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magnesiuma)

0.26±0.2

coppera)

0.08±0.01

iron

a)

Reversible Supramolecular Assembly of Velvet Worm Adhesive Fibers

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