Functions and structural stability of PmPV1 carotenoprotein, the major egg perivitelline from Pomacea maculata

PASQUEVICH M. Y.; DREON M. S.; HERAS H.

La Serena

Congreso; Physiomar-14; 2014

Freshwater snails of the genus Pomacea have among Ampullarids, amphibious habits. Thus, Pomacea lay calcareous and pigmented eggclutches above water bodies on vegetation or other surfaces, exposing the eggs to fluctuant temperatures, air desiccation and terrestrialpredators. The striking pigmentation of these eggs is provided by highly glycosilated oligomeric carotenoproteins with astaxanthins astheir major carotenoid prosthetic group. Previous data from our laboratory suggest that carotenoproteins are playing a particular role inegg protection against both environmental factors and predation. The carotenoprotein PmPVI, the main source of color in P. maculataeggs, has recently been biochemically characterized. A great similarity with carotenoproteins from other members of the genus (P.canaliculata and P. scalaris) was found.The aim of the present study is to determine the structural stability of PmPVl as a function of pH and temperature. In addition, theresistance to digestion and capacity to inhibit trypsin were performed, which complements previous functional studies related withcarotenoid protection.Structural stability analysis by fluorescence and absorption spectroscopy showed that PmPVl was structurally stablebetween pH 2.0 to 12.0, although a minor alteration of fine structure in the visible absorption spectrum wasobserved at pH 2.0. The thermal stability was recorded up to 85°C and no denaturalization throughout thetemperature range, as shown by its absorption and fluorescent spectra, was observed. Moreover, electrophoreticbehavior of PmPVl was not altered after boiling for 10 min. Using 2D electrophoresis we determined that althoughall subunits are glycosilated, this was not the only postraductional modification. Capacity to inhibit proteases wasevaluated using N-benzoil-L-arginine ethyl ester (BAEE) as substrate. While P. maculata perivitelline fluid inhibitsthe BAEE hidrolization by trypsin, no inhibiting activity against trypsin was found when purified PmPVl wasincubated with this protease. Capacity to withstand gastrointestinal digestion was assayed by in vitro simulatedgastrointestinal digestion. PmPVl resist digestion at enzyme concentrations that completely digest other proteinssuch as bovine serum albumin.In conclusion PmPVl retained its native structure after exposure to physical and biological factors. Taken togetherthese results indicate that PmPVl is highly resistant to pH and temperature, and a difficult to digest protein. Theseproperties could be part of the P. maculata eggs defensive strategy against predation and environmental factors. Thisstudy increases the knowledge on the reproductive strategies of these mollusks and further highlights the importanceof these perivitellins in the reproductive strategy of Pomacea.