Cross-linking by protein oxidation in the rapidly setting gel-based glues of slugs

Abstract

SUMMARY The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass (Mr) of 165×103, were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20–35% decrease in the amount of soluble protein with a Mr of 40–165 (×103) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a Mr of 15×103 were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue.

Double network gels and the toughness of terrestrial slug glue

“…Asmp-165 co-elutes more strongly with the carbohydrates, possibly indicating a closer interaction. This is not consistent with previous work that found that asmp-165 was heavily oxidized and appeared to associate with the other high molecular weight proteins in the glue (Bradshaw et al, 2011), though that study did not distinguish between asmp-165 and asmp-220. Further work will be needed to clarify the exact connections between polymers in the glue.…”

“…Second dimension SDS-PAGE showed that the region at the top of the lane contained a mixture of all the proteins from asmp-40 through to asmp-220, and occasionally some of the proteins identified as asmp-15. Note that proteins are identified as asmp (A. subfuscus mucus protein) followed by the relative molecular mass ×10 −3 (Bradshaw et al, 2011). On a 5% native gel, these proteins ran together with less mobility than a ferritin standard (450 kDa) when they were even able to enter the gel (Fig.…”

“…Previous work focused on the factors that make gastropod adhesive gels much stiffer than other forms of mucus (Smith et al, 1999a;Smith and Morin, 2002;Pawlicki et al, 2004;Werneke et al, 2007;Bradshaw et al, 2011;Braun et al, 2013). There are two main types of cross-links: direct metal crosslinks involving calcium, magnesium and possibly iron (Werneke et al, 2007;Braun et al, 2013), and cross-links that derive from metal-catalyzed oxidation (Bradshaw et al, 2011;Braun et al, 2013). There is indirect evidence that the latter are imine bonds created when amino acids, such as lysine, are oxidized to create carbonyls, which are susceptible to nucleophilic attack from primary amines (Braun et al, 2013).…”

The relative contribution of calcium, zinc and oxidation-based cross-links to the stiffness ofArion subfuscusglue

“…Protein oxidation by redox active metals like iron and copper is another way that metals can contribute to a glue (Bradshaw et al, 2011). The oxidation of the rare amino acid 3,4-dihydroxyphenylalanine (dopa), has been studied in depth as a crosslinking step in various biomaterials (Sagert et al, 2006).…”

“…This typically creates carbonyl groups, which would react readily with the primary amines of other lysine side-chains to form a Schiff base, specifically an imine bond (Tanzer, 1973). Bradshaw et al (Bradshaw et al, 2011) provide evidence that such bonds are present in A. subfuscus glue.…”