Molecular characterization of a Bufo arenarum oviductal protease

BARRERA, DANIEL; LLANOS, RICARDO; VALDECANTOS, PABLO; MICELI, DORA CRISTINA

Pinamar, Buenos Aires (Argentina)

Congreso; XLI Reunión anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2005

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

In amphibians, the envelope surrounding the egg exhibits differences according to the functional state of the female gamete. The eggs obtained from the coelomic cavity are not fertilized, but they are after their passage through the Pars Recta (PR) portion of oviduct. This acquisition of egg fertilizability is due to an oviduct-induced alteration of egg envelope. The biochemical and ultrastructural vitelline envelope modifications are caused by the action of a trypsin-like protease named oviductin. The aim of this work was to clone the oviductin complete cDNA and to analyze their functional domains. Total RNA was isolated from oviductal PR of hormonally stimulated animal. Sets of primers were designed based on homology sequences. We first amplified, cloned and sequenced an internal 530 pb partial cDNA. The 5' cDNA end was amplified using a new group of specific designed primers. To complete the mRNA sequence, a 3' rapid amplification of cDNA ends (3' RACE) was performed. The overlapping sequence showed a 3,203 bp-long oviductin cDNA with one open reading frame coding for a 980 aminoacids protein. The molecular structure comprise two protease domains (alpha and beta) and three CUB domains. The alpha domain has three important aminoacids for catalytic activity (His, Asn, Ser), while in the beta domain a His residue was replaced by Asp. Thus, this domain is not likely to be proteolytically active. These results would indicate that the Bufo arenarum oviductin alpha domain produce the partial hydrolysis of the envelope glycoproteins. At this moment, the exact function of the CUB domains and beta domain is still unknown.