Metal-binding proteins and cross-linking in the defensive glue of the slug Arion subfuscus

The role of metals in forming the primary cross-links in slug glue was investigated. Several metal-binding proteins were identified in the defensive glue produced by the slug Arion subfuscus. Notably, the C-lectins that are unique to the glue are iron-binding proteins. This is unusual for C-lectins. Dissociating these proteins from iron does not affect the glue's stiffness. Similarly, several proteins that can bind to zinc were identified, but dissociating the proteins from zinc did not weaken the glue. These results suggest that metal coordination is not involved in the primary cross-links of this hydrogel glue. The stable cross-links that provide stiffness are more likely to be created by a catalytic event involving protein oxidation. Cross-linking was unexpectedly difficult to prevent. Collecting the glue into a large volume of ice-cold buffer with reagents aimed at inhibiting oxidative cross-linking caused a slight loss of cross-linking, as demonstrated by the appearance of uncross-linked proteins in native gel electrophoresis. Notable among these was a protein that is normally heavily oxidized (asmp165). Nevertheless, this effect was not large, suggesting that the primary cross-links form before secretion.