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

Abstract

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.

Keywords: C-lectin; adhesive; hydrogel; iron; protein oxidation; transition metals.

Figure 1.

Identification of iron-binding proteins by immobilized metal affinity chromatography. The lanes are Ext (original dissolved extract), FT (flow through, unbound material), W1 and W2 (first two washes), then two washes each at pH 8 and 9. The numbers on the left identify the molecular mass of two prominent bands.

Figure 2.

Identification of zinc-binding proteins by immobilized metal affinity chromatography. The left-most lane shows the original extract that was loaded. The two lanes on the right show the proteins that bound to the zinc in regular loading buffer, and loading buffer with 8 M urea.

Figure 3.

The effect of pH on zinc binding in the glue. The amount of zinc dissociating from the glue relative to the original amount was measured using atomic absorption spectroscopy.

Figure 4.

The effect of pH on the stiffness of the glue. Stiffness was measured on intact strips soaked in buffer of different pH values using a uniaxial tensometer. The iron-binding proteins dissociate from iron at pH 9.

Figure 5.

Native gel electrophoresis of samples collected and rapidly dispersed directly into Tris-acetate NaCl buffer with different treatments. All samples were collected into ice-cold buffer except as noted. The relative mobilities of bands that consistently appeared are shown on the left. Samples in lanes 1, 3 and 5 were treated with 8 M urea before loading on the gel. Lane 1 is the negative control (with urea), lane 2 is the negative control in warm buffer, lane 3 also had 15 mM NH₂OH (with urea), lane 4 had 10 mM deferoxamine, lane 5 had 10 mM EDTA (with urea), lane 6 had glue that was allowed to set into a solid, then dissolved in the buffer, lane 7 is BSA.

Figure 6.

Second dimension SDS–PAGE of bands that appeared on native gels after glue was collected directly into cold buffer. The numbers across the top show the relative mobilities of bands on the native gel that were analysed by 2D PAGE. The left-most lane (Top) is taken from a strip less than 1 mm thick (0.01 relative mobility) shaved from the top of the gel (bottom of the loading well). The numbers down the left side show the mass in kDa of selected proteins on SDS–PAGE.