title: Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14.
abstract: In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76-85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality.
authors: Departamento de Bioquímica y Biología Molecular y Celular, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA), Facultad de Veterinaria, Spain.
authors: Edith
authors: E
authors: Serrano
authors: Rosaura
authors: R
authors: Pérez-Pé
authors: Lucía
authors: L
authors: Calleja
authors: Natalia
authors: N
authors: Guillén
authors: Adriana
authors: A
authors: Casao
authors: Ramón
authors: R
authors: Hurtado-Guerrero
authors: Teresa
authors: T
authors: Muiño-Blanco
authors: José A
authors: JA
authors: Cebrián-Pérez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002457:Cell Extracts; D002474:Cell-Free System; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D007313:Insecta; D008297:Male; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012661:Semen / Q000737:chemistry; D029589:Seminal Plasma Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012756:Sheep / Q000235:genetics*; D013094:Spermatozoa / Q000737:chemistry
chemical_list: D002457:Cell Extracts; D018076:DNA, Complementary; D011994:Recombinant Proteins; D029589:Seminal Plasma Proteins; D012313:RNA