Recent Presentations

“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.

Abstract:

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.

Abstract:
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.