Hagfish defend themselves from fish predators by producing large volumes of gill-clogging slime when they are attacked. The slime consists of seawater and two major components that are ejected from the slime glands: mucus and threads. The threads are produced within specialized cells and packaged into intricately coiled bundles called skeins. Skeins are kept from unraveling via a protein adhesive that dissolves when the skeins are ejected from the slime glands. Previous work revealed that hagfish slime glands have high concentrations of methylamines including trimethylamine N-oxide (TMAO), trimethylglycine (betaine) and dimethylglycine (DMG); however, the function of these compounds in the slime glands is unknown. We hypothesized that methylamines have stabilizing effects on the skeins that prevent premature unraveling in the gland. To test this hypothesis, we quantified the effect of methylamines on skein unraveling in Pacific hagfish and found that TMAO and betaine have inhibitory effects on skein unraveling in vitro. Furthermore, we found that TMAO is a more effective inhibitor of unraveling than betaine, but the presence of TMAO synergistically boosts the inhibitory action of betaine. Glycine and DMG were far less effective inhibitors of unraveling at natural concentrations. Our results support the hypothesis that high levels of trimethylamines in the slime glands may act to hold the coiled thread skeins together within gland thread cells, and they may do so by stabilizing adhesive proteins. These results advance our knowledge of skein stabilization and deployment and provide yet another example of trimethylamines functioning to stabilize proteins in a marine organism.

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