CONICET | Buscador de Institutos y Recursos Humanos

The freshwater snails of Pomacea genus deposit pigmented eggs above the water. Pigmentation is provided by abundant oligomeric carotenoproteins which supply nutrients and photoprotection to the growing embryo. Homologous egg carotenoproteins have evolved different defensive roles. For instance, in the invasive canaliculata clade, PcOvo and PmPV1, provide bright reddish colors, a warning coloration advertising the presence of toxins in the eggs; on the contrary, in the non-invasive bridgessi clade, PsSC with a pale color (non-warning signal), have a remarkable lectin activity not found in the canaliculata clade. The aim of our work is to begin to unveil the structure-function evolution of Pomacea carotenoproteins. We studied the structural stability and the resistance to proteolysis of PpPV1, a pale P. patula carotenoprotein member of the flagellate clade (the most basal of the genus) and compare it with the other clades of Pomacea. The stability of PpPV1 was studied against pH, temperature and guanidinium chloride (GdnHCl), using spectrophotometry and spectrofluorometry; the resistance to proteolysis was determined by in vitro simulated gastrointestinal digestion and proteinase K. PpPV1 remained stable over temperatures up to 85°C and over a wide range of pH (4.0-12.0), with slight alterations at pH 2.0. The unfolding equilibrium to GdnHCl showed that while 50% of PmPV1 and PcOVO are unfolded at 2.7 M, basal PpPV1 do so at 5.4 M. The oligomer was resistant to proteolysis, a feature that, together with the resistance to SDS denaturation, are shared by kinetically stable proteins. The resistance to thermal denaturation, to extreme pHs and digestion proteolysis of PpPV1 is similar to that of PsSC, PcOvo, and PmPV1. However, PpPV1 has a remarkable greater resistance to chemical denaturation than its homologs. The loss of stability could have contributed to the gain of new functions, such as acquiring a strong warning coloration in the most derived species.