Foam nest components of the túngara frog: a cocktail of proteins conferring physical and biological resilience

Abstract

The foam nests of the túngara frog (Engystomops pustulosus) form a biocompatible incubation medium for eggs and sperm while resisting considerable environmental and microbiological assault. We have shown that much of this behaviour can be attributed to a cocktail of six proteins, designated ranaspumins (Rsn-1 to Rsn-6), which predominate in the foam. These fall into two discernable classes based on sequence analysis and biophysical properties. Rsn-2, with an amphiphilic amino acid sequence unlike any hitherto reported, exhibits substantial detergent-like surfactant activity necessary for production of foam, yet is harmless to the membranes of eggs and spermatozoa. A further four (Rsn-3 to Rsn-6) are lectins, three of which are similar to fucolectins found in teleosts but not previously identified in a land vertebrate, though with a carbohydrate binding specificity different from previously described fucolectins. The sixth, Rsn-1, is structurally similar to proteinase inhibitors of the cystatin class, but does not itself appear to exhibit any such activity. The nest foam itself, however, does exhibit potent cystatin activity. Rsn-encoding genes are transcribed in many tissues of the adult frogs, but the full cocktail is present only in oviduct glands. Combinations of lectins and cystatins have known roles in plants and animals for defence against microbial colonization and insect attack. Túngara nest foam displays a novel synergy of selected elements of innate defence plus a specialized surfactant protein, comprising a previously unreported strategy for protection of unattended reproductive stages of animals.

Figure 1

Lectin activity of Rsn-4. Agglutination of human red blood cells by recombinant Rsn-4 and AAA in the presence or absence of competing sugars. Positive agglutinations result in the formation of a sheet of cells over the base of the wells, negative reactions as buttons of cells in the centre. Note that, unlike the control fucolectin (AAA), agglutination by Rsn-4 was unaffected by fucose but completely inhibited by galactose or by the galactose-containing disaccharide, lactose. Other sugars showed no significant effects at this level. Similar results were obtained for cells of blood groups A, B or O.

Figure 2

Unusual hydrophobicity profile and surfactant activity of Rsn-2. (a) Kyte–Doolottle hydropathicity plot of Rsn-2 illustrating the unusual hydrophobic N-terminal region of the mature protein and notably hydrophilic C-terminus. (b) Surface tension versus concentration of recombinant Rsn-2 (filled circles) in comparison with the natural foam fluid (unfilled circles) mixture, together with a negative control (lysozyme, filled squares) and a moderately surfactant control protein (bovine serum albumin; BSA, unfilled squares).

Figure 3

Potent proteinase inhibition by túngara frog nest foam. Inhibition of papain proteolytic activity by natural foam nest fluid mixture was measured as a function of total foam fluid protein concentration. Relative proteolysis rates determined at 30°C, pH 5.0, using the chromogenic peptide substrate, Z-Phe-Arg-pNA. Note that proteinase inhibition was essentially complete at 50 μg ml⁻¹ total protein, which corresponds to more than 20-fold dilution of the natural material. Control proteins (BSA and lysozyme) showed no inhibition over the same concentration range. Error bars for each data point (from replicate measurements) are smaller than the data symbol used here.

Figure 4

Cartoon depicting the possible arrangement of ranaspumins and complex carbohydrates at the air–water interface. Surfactant proteins (predominantly Rsn-2) perform the primary role of surface tension reduction to allow initial bubble/foam production. This layer may then be further stabilized by incorporation of lectins (e.g. Rsn-3, Rsn-5 via their hydrophobic tails) to which the long-chain, branched polysaccharide components of the natural foam fluid may attach. This would create a mechanically stable, water-retaining foam matrix.