Apple snail eggs are a Pandora's box: a novel hemoprotein in the animal kingdom

MARÍA Y. PASQUEVICH; PAVÍA, IGNACIO; BAUZÁ, L.; BENITEZ, G. I. ; HERAS H

Rosario

Congreso; LIX Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2023

Sociedad Argentina de Investigaciones Bioquímicas

Apple snails are freshwater molluscs of the Pomacea genus that include Pomacea canaliculata and P. maculata two invasive snails that are agricultural pests and nematode parasite vectors. They laycalcareous and pigmented eggs out of water containing a perivitelline fluid with multifunctionalproteins (perivitelins) that nourish, defend, and protect the embryo from the surrounding Environment. Proteomic studies have shown that eggs have more than 30 proteins, >50 % of either unknown and/or uncharacterized. Some perivitellins have been characterized as the Carotenoprotein PV1, an anti-nutritive protein that protects the embryo from solar radiation and whose carotenoids provide antioxidants and a warning coloration to the eggs to deter predators. Our group is focused on classifying, identifying, and studying the structure, functions and evolution of these unknown egg proteins. We seek to understand the snail reproductive strategies from a biochemical perspective and, at the same time, to select some of these new proteins with potential as bioactive compounds to exploit their application in Biomedicine. In this work, we isolated and characterized a yellowish-colored protein found in the PV3 fraction of eggs.The protein, hereafter named PmaHP, was purified from P. maculata egg masses, combining differential centrifugation, density gradient ultracentrifugation, SEC, and anion exchange HPLC. PmaHP was then studied by UV-VIS spectrophotometry, electrophoresis, and mass spectrometry. Besides, its sequence was compared with homologs from other mollusks, and putative heme binding sites were predicted. The absorption spectrum is typical for metalloporphyrins, and ICP-MS analysis revealed the metal was iron. The metalloprotein is resolved in acidic electrophoresis under native conditions at pH 4.3, but not in basic electrophoresis, a characteristic of basic proteins. The protein was positive for heme stain with TMB. A MW 18.2 kDa was calculated and its complete amino acid sequence was identified by MS in the UniProt database. Sequence similarity with other homologous proteins of the genus is 48-52% and the sequence contains up to 5 potential binding sites to the heme group. We describe the first structural data of a new iron-containing hemoprotein of unknown function other than contributing to the coloration of P. maculata eggs. The pigment is conjugated with a protein forming an extracellular soluble hemoprotein that has no sequence homology with any other hemoprotein from the animal kingdom. More work is underway to better understand its role in the survival and reproductive success of a snail species with large economic, ecological, and health importance.