Andrew Smith. Keynote Speaker. “Double networks and gastropod glue: unlocking the potential of adhesive gels" I also gave a workshop at the conference, providing practical advice on the design of tough gels for use as adhesives. This was a joint networking event of the European Network of BioAdhesion expertise (ENBA) and BioSmart Trainees. Cambridge, UK. March 2019.
Andrew Smith. Keynote Speaker. "Double networks and slug glue: Integrating mechanics and sequence data to characterize an unusually tough hydrogel adhesive". Adhesion section of the Society of Experimental Biology's annual meetings in Goteborg, Sweden. July 2017.
Andrew Smith. Invited Speaker. "Terrestrial slug glue: gaining toughness through a double network mechanism". 2016 Beilstein Nanotechnology Symposium, "Biological and Bioinspired Adhesion: from Macro- to Nanoscale". Potsdam, Germany. September 2016.
The defensive glue produced from the dorsal surface of the slug Arion subfuscus is an unusually tough, adhesive gel. Despite consisting of only 3% organic matter, it can resist stresses greater than 100 kPa. Previous work has identified the cross-linking mechanisms involved in stiffening the glue and causing it to set. Nevertheless, stiffness alone is not sufficient; the glue must also be tough. It needs a mechanism to increase the energy required to achieve fracture. We show that the glue gains its toughness from a double network mechanism. The glue has two interpenetrating networks of polymers: a ductile network of sulfated polysaccharides, and a network of cross-linked proteins. These work synergistically; experiments demonstrated that neither provides measurable toughness on its own but together they require a large amount of energy to fracture. This is because the ductile network must extend greatly before ultimate failure of the material. In a double network, this extension spreads damage through a large volume of the stiff network rather than a simple fracture plane. Analysis of hysteresis loops in stress-strain data, stress relaxation experiments, and the strain rate dependence of stiffness supports this. The material behaves as a cross-linked solid, but continuously dissipates energy through rupture of cross-links as the material extends. Previous work has shown that there are two types of cross-links between proteins: coordination of metal ions such as iron and calcium between polymers, and imine bonds derived from metal-catalyzed oxidations. Sequence data for the proteins in the glue shows numerous calcium binding sites, and biochemical evidence supports this binding. Thus, we propose that calcium forms sacrificial bonds in a stiff protein network, which dissipate a large amount of energy as the glue extends. Large, heparan-sulfate like glycosaminoglycans provide an interpenetrating ductile network to allow this extension and spread the damage through the stiff network. This double network mechanism represents an important way of toughening biological materials.
Andrew Smith. Society for Integrative and Comparative Biology, San Francisco, California, January 2013: “Double network gels and biological glues: a powerful new toughening mechanism”.
Limpets, marsh periwinkles and some terrestrial slugs produce remarkable glues that are gels. A key question has been how they can achieve tenacities on the order of several hundred kilopascals using only a dilute gel that is a modified lubricating mucus. Previous work has shown that the essential change is the addition of relatively small, cross-linked proteins. Nevertheless, highly cross-linked gels are typically brittle and fail easily. Molluscs may avoid this through the use of a “double network”. Recent work in materials science has found that combining two highly dissimilar, interpenetrating gel networks can increase gel strength by a factor of 100 to 1000 over the strength of the two gels separately. A prototypical double network gel combines a deformable network of very large polymers and a highly cross-linked network of much smaller polymers. Initial fracture occurs in the stiffer, highly cross-linked network. Fracturing the soft network as well, though, requires extensive deformation. This deformation damages the rigid network in a large volume surrounding the crack. This can increase the energy required to propagate the crack by several orders of magnitude. Such a mechanism is likely at play in molluscan adhesive gels given their structure. In fact, any biological gel containing proteoglycans or similarly large polymers in combination with smaller cross-linked proteins has the potential to operate this way. This talk will outline the structural and mechanical criteria for double network gels and consider the applicability of this mechanism to different biological materials.
Andrew Smith. Keynote speaker at the 1st International Conference on Biological and Biomimetic Adhesives in Lisbon Portugal (May 9-11, 2012). His talk was titled "Multiple metal-based cross-links: protein oxidation and metal coordination in a biological glue".
Metal ions provide a powerful mechanism for cross-linking adhesives and other biomaterials, especially in aqueous environments. Metal ions can cross-link polymers directly through coordination, and some metals can catalyze redox reactions that drive the formation of other cross-links. It is likely that many biomaterials depend on multiple metal-based cross-links, involving variations of direct and oxidative mechanisms. The glue of gastropod mollusks demonstrates this complexity well. The terrestrial slug Arion subfuscus utilizes both types of interactions to strengthen their defensive glue. This glue contains substantial amounts of calcium, zinc, iron and copper. In addition, there are metal-binding proteins that are unique to the glue that have gel-stiffening activity. These proteins bind to both iron and zinc, and likely other metals. The function of these proteins, and the integrity of the glue overall, depends on metals. One mechanism that appears to play a central role is metal-catalyzed oxidation. Several prominent proteins in the glue are heavily oxidized, and experimental work has provided evidence that the resulting carbonyl groups link with primary amines to form imine bonds. Specific disruption of these bonds decreases glue stiffness significantly. These findings are noteworthy because common amino acids such as lysine can be readily oxidized by metals. While it has been known that oxidation of the rare amino acid 3,4-dihydroxyphenylalanine plays a role in other biomaterials, these results suggest an even broader role for protein oxidation. In addition to oxidative cross-links, metals directly cross-link slug glue, likely through coordinate covalent bonds. Evidence suggests that calcium directly cross-links the gel through interactions with sulfate on polysaccharides. Surprisingly, zinc does not strengthen the gel, though it is present in large quantities in slug glue associated with key proteins, and it is a common cross-linker in other biomaterials. Thus, it may play a different role. Overall, slug glue demonstrates interesting variations on the metal-dependent mechanisms that have been described thus far. Slug glue is also interesting because it is a dilute gel. It typically contains 97% water, and appears to be a modification of the normal lubricating slime. The ability to convert a dilute, lubricating gel into a strong glue demonstrates the power of metal-based cross-links.
Andrew Smith. "Gluing with an iron fist: the central role of metals in biological adhesives". Franklin & Marshall College, Lancaster, Pennsylvania. March 2012 as well as at the University of Scranton. Scranton, Pennsylvania. Fall 2011.
My lab studies the biochemistry and mechanics of adhesive gels. Many gastropod mollusks can form strong attachments using dilute gels. Intertidal limpets, for example, glue themselves onto wet, irregular surfaces so firmly that in some cases they cannot be detached by hand. Some terrestrial slugs can produce a similarly elastic, adhesive defensive secretion. This performance is remarkable given that the glues consist of ~97% water, and appear to be modifications of the animal’s normal lubricating mucus. Our current research focuses on the cross-linking mechanisms that govern gel mechanics. We have found specific proteins that are correlated with increased adhesive strength, and these proteins stiffen gels. We have also found that metal ions play an essential role in the glue, creating stable cross-links despite the presence of water. We are particularly interested in the different ways that metals impact gel mechanics. In the glue of terrestrial slugs, some metals cross-link polymers directly, while other redox-active metals appear to create cross-links through protein oxidation. Protein oxidation is a common post-translational modification that can significantly affect material mechanics.
“Cross-linking by protein oxidation in gastropod glues” (co-authored with A. Bradshaw*, A. Bell*, N. Litra*, M. Braun* and M. Salt*). Society for Integrative and Comparative Biology, Salt Lake City, Utah, 2011.
Protein oxidation is a common phenomenon that causes protein dysfunction in aging, but it can also be harnessed to strengthen biomaterials. Collagen and elastin in animal connective tissues are cross-linked by a metal-catalyzed oxidation system that leads to the formation of bonds between oxidized amino acids and nucleophilic amino acid side chains. Here we show that the glue produced by the terrestrial slug Arion subfuscus may use a similar mechanism but with different proteins. Immunoblotting for carbonyl groups demonstrated that several key proteins in the glue are heavily oxidized, and this oxidation appears to occur rapidly. The carbonyl groups were not easily detected unless the glue was denatured, though, suggesting that they may be unavailable due to participation in reversible cross-links. This was tested using reagents that normally modify carbonyls. The strong reducing agent sodium borohydride and the nucleophile hydroxylamine should eliminate any accessible carbonyl groups. In the glue however, borohydride had no effect on carbonyl content while hydroxylamine partially modified the carbonyls; this was consistent with the way each reagent would interact with cross-links between carbonyls and primary amines. The two treatments also impacted protein solubility in a way that was consistent with this proposed cross-linking mechanism. Thus, slugs may harness protein oxidation to strengthen their glue. Because the components involved in protein oxidation are common, it is likely that this could represent a relatively widespread but underappreciated mechanism for strengthening biomaterials.
"Cross-linking in slug glue: gelled plaster of Paris?”, co-authored with Meghan Menges (Biology ’10). Society for Integrative and Comparative Biology. Seattle, WA. January 2010.
The defensive glue of the slug Arion subfuscus sets rapidly into a sticky, elastic mass. There appear to be several cross-linking mechanisms, but the initial gelation may occur through complex coacervation involving sulfate- and calcium-binding polymers. In this mechanism, electrostatic forces bring together charged polymers creating locally high concentrations. These may cross-link to create a reticular network. Calcium and sulfate are particularly interesting, as their interaction causes setting in plaster of Paris. Slug glue was shown to contain a strikingly high concentration of calcium (40 mM) as measured by atomic absorption spectrometry and energy dispersive SEM. It also contains a comparable amount of sulfate (40-50 mM) as measured by a colorimetric assay and SEM. The sulfate is likely bound to polysaccharides, while several of the proteins bind strongly to metals. The sulfate would create a high negative charge density, which would be neutralized by the calcium leading to coacervation, drawing metal-binding proteins and sulfated polysaccharides together. Several assays were developed to determine if any proteins in the glue bound to sulfate in this way. These assays identified a 15 kDa protein that was known to be unique to the glue and has been shown to stiffen gels. This protein precipitated with sulfated sugars, but only in the presence of metals. Furthermore, it bound to sulfate groups in column chromatography when metals were present. Chelating the metals often blocked this binding. Thus, there is a specific metal-based interaction between the primary cross-linking protein and sulfate groups. The glue is not soluble in acid, however, and the calcium is not tightly bound to the glue, suggesting that a strong solubility-based interaction, as seen in plaster, does not occur.
“Multiple cross-linking mechanisms in molluscan adhesive gels”, and was co-authored by two IC students, Sarah Garcia and Aaron Bloom (Biology '09 and '08, respectively). Annual conference of the Society for Integrative and Comparative Biology, "Biomaterials: properties, variation and evolution". Boston, MA. January 2009.
Some terrestrial slugs produce remarkably sticky and elastic gels as defensive secretions. Previous work on these gels has shown that metals play a central role in their cross-linking. The transition metals iron and zinc are common in these gels, as are calcium and magnesium. A major question is how these metals cross-link the gel, and whether there is more than one mechanism by which they do so. Chelation of metals with EDTA for an extended time breaks down the mechanical integrity of the gel, thus demonstrating a direct effect of the metals on gel mechanics. Furthermore, metals, particularly calcium, were shown to have a general stiffening effect on commercial gels at the concentrations seen in the glue. Metal removal does not completely break down the gel, however, as size exclusion chromatography experiments show that the major cross-links involve a 40 kDa protein and these are unaffected by metal chelation after the glue sets. If chelation occurs before the glue sets, however, this cross-link does not form either. Measurements of the stiffness of commercial gels with metals and glue proteins added separately and together show that both stiffen gels on their own, but the effect is merely additive; they are not necessarily interdependent. The findings suggest that the mechanical strength of the gel depends in part on metals such as calcium and zinc forming direct cross-links and also on other cross-links involving the 40 kDa protein, which are catalyzed by metals before the glue sets.
"Metals, molluscan glues and gel mechanics", annual meeting of the Society for Integrative and Comparative Biology. San Antonio, Texas. January 6, 2008.
Metals, molluscan glues and gel mechanics. SMITH. A. M. Ithaca College. Molluscan adhesive gels possess many useful properties, most notably their remarkable combination of strength and deformability, as well as their ability to adhere to wet, irregular surfaces. Recent work has found that the glue of the terrestrial slug Arion subfuscus contains substantial amounts of iron, manganese, zinc and some copper. Furthermore, the presence of transition metals was critical for the glue to set. This study addresses the relative roles of the different metals. Do they all function similarly, with similar effectiveness? Are they incorporated into the glue in a similar way? We used atomic absorption spectroscopy to characterize the metal content of several different gastropod glues. We also tested the effect of these metals on the mechanics of several commercial gels. The metal content of the glue from the terrestrial snail Helix aspersa and the terrestrial slugs A. subfuscus and Ariolimax columbianus was markedly similar. When hydrated, all three had over 40 mM calcium and 0.07-0.08 mM iron. A. subfuscus also had 0.9 mM zinc while A. columbianus had 0.5 mM manganese. For comparison, sodium and chloride concentrations were roughly 10 mM. Soaking A. subfuscus glue in EDTA caused all the metal concentrations to drop to 1-5% of their original value, except iron, which was not significantly different (t-test, P = 0.18). All the metals stiffened agar and pectin gels. Notably, despite its poor solubility iron was 20-40x as effective as calcium. Zinc was roughly 10x as effective as calcium. These results suggest that iron is more effective in controlling the gel mechanics than other metals, and it is more tightly incorporated into the glue. The other metals are present in higher concentrations, so they would still contribute substantially, but likely in different ways.