CONICET | Buscador de Institutos y Recursos Humanos

The evolution of structure-function relationship of hyperstable proteins is poorly understood. Studying Pomacea snail carotenoproteins as a model, we observed that those involved in embryo nutrition and defense have changed during genus evolution: while brightly colored ones of the most derived clade (P. canaliculata and P. maculata) act as antinutritive defenses against predators; those of P. scalaris, pale, additionally possess a strong lectin activity. The aim of this work was to analyze the evolutionary trends of Pomacea carotenoproteins, studying PpPV1, the carotenoprotein of P. patula, a basal member of the genus. PpPV1 absorption spectrum shows a 380 nm maximum indicating a blue-shift from the basal clade compared with the derived species. Electrophoretic mobility also shifted from a mobility equivalent to a 450 kDa protein in native PpPV1 towards lower MWs in the most derived carotenoproteins (293 -380 KDa). Despite these differences, PpPV1 have similar architecture and subunits have similar MW to those of its derived species homologues. Derived clades are kinetically stable (a high energetic barrier that not allow passing from native to denaturized state). This characteristic was evaluated in PpPV1 hypothesizing that was acquired in derivative clades in species with notable reproductive success. However, PpPV1 oligomer withstands boiling and is resistant to SDS-induced denaturation, highlighting that carotenoproteins of Pomacea eggs are already thermal and kinetically stable in the most basal species. Functional analysis of hemagluttinating activity revealed that P. patula periviteline fluid surrounding the embryo has a moderate lectin activity that is not mainly ascribed to PpPV1. In conclusion, the comparative analysis shows that, while carotenoprotein color and size changed, subunits and structural stability was conserved. The later may have increased tolerance to mutations that accelerated the evolution of new functions.