Bovine seminal plasma ASFP: Localization of disulfide bridges and detection of three different isoelectric forms

Abstract

Acidic seminal fluid protein (aSFP) is a major 13 kDa protein isolated from bull seminal plasma and characterized as a new growth factor which stimulates in vitro cell division and progesterone secretion by ovarian cells. Here, we establish that the four cysteines of oxidized aSFP form two disulfide bridges between nearest-neighbour residues. This pattern is conserved in boar spermadhesins, with which aSFP shares up to 50% amino acid sequence identity, and other proteins of the recently identified CUB domain family. Using isoelectric focusing in combination with sulfhydryl group-specific blotting, the three forms of aSFP were identified as completely oxidized (PI 4.7), partly reduced (PI 4.8) and fully reduced at pI 5.1. These results indicate that native aSFP possesses two pairs of cysteine residues of different reactivity. The observation that aSFP can protect sperm from oxidative damage might be explained by its reduction/oxidation behaviour.

Boar spermadhesin PSP‐II: Location of posttranslational modifications, heterodimer formation with PSP‐I glycoforms and effect of dimerization on the ligand‐binding capabilities of the subunits

“…2). In addition, the same cystine pattern has been reported in aSFP, a bovine seminal plasma polypeptide which is structurally related to boar spermadhesins [17]. This feature may be important for maintaining the correct three-dimensional structure of spermadhesins.…”

Two‐dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins

“…The large amount of PDC-109 detectable as different isoforms (spots 7, 8 and 9) or fragment (spot 10, for which the N-terminal sequence starts from Ala16 of the intact mature protein) in the 2-D PAGE map was expected; in fact, it is known that PDC-109 represents more than 30% of the total protein contained in bull seminal plasma and is present as a roughly equimolar mixture of a nonglycosylated polypeptide and a glycosylated one [4, 121. Another highly expressed bull seminal plasma protein, aSFP [13], has been detected in two isoelectric forms (spots 5 and 6). In this respect, we have evidenced PDC-109 and aSFP also in 2-D PAGE protein maps of ejaculated bull spermatozoa, obtained essentially as described [14], even after repetitive washing of the cells with PBS (unpublished results).…”

A cDNA cloned from pregnant mouse uterus exhibits temporo-spatial expression and predicts a novel protein

“…Bovine acidic seminal fluid protein is a member of the spermadhesin family that essentially consists of a single CUB domain [32][33][34][35]. In itro, this protein functions as a mitogen, growth factor and it stimulates progesterone secretion in cultured ovarian cells [32].…”