Molecular and Functional Characterization of the Rabbit Epididymal Secretory Protein 52, REP521

Abstract

As part of a systematic study of rabbit epididymal proteins involved in sperm maturation, we have identified and characterized a novel glycoprotein (rabbit epididymal secretory protein 52 [REP52]) of 52 kDa. REP52 is synthesized and secreted in a tissue-specific manner by the mid (region 6) and distal (region 7) corpus epididymidis and associates weakly with the sperm surface overlying the principal piece of the tail. Sequencing of cloned REP52 cDNA demonstrated that this protein represents a novel member of the highly conserved fibronectin type II (FN2) module protein family. The protein appears related but not homologous to ungulate seminal plasma proteins and is the first known example to be identified as a rabbit epididymal secretory protein. Consistent with other members of this protein family, REP52 possessed a high level of sequence identity within the FN2 module-encoding domains, but a highly variable N-terminal sequence that failed to show significant homology with published sequences. By analogy with evidence from studies of the ungulate seminal plasma proteins it is hypothesized that the tandemly arranged FN2 modules could facilitate the association of REP52 with sperm phosphatidylcholine residues on the outer leaflet of the sperm tail. It is also considered likely that these domains represent key elements for the function of this novel protein, a conclusion supported by the fact that antisera raised against the REP52 protein blocked in vitro fertilization in a concentration-dependent fashion.

Epididymal Binder of SPerm genes and proteins: what do we know a decade later?

“…With the exception of bull BSP4 , all other newly identified genes are most strongly expressed in the epididymis. Around the same time, a 52‐kDa glycoprotein related to BSP proteins was isolated from the rabbit epididymides (Nixon et al ., ). This protein previously called REP52 was renamed BSP1 according to new nomenclature for BSP genes and proteins (Manjunath et al ., ).…”

“…For example, proteins of the BSP subfamily expressed by seminal vesicles can represent between 1 and 50% of total seminal plasma proteins. In line with this, BSP1 from rabbit, also part of the BSP subfamily, represents 2–4% of the proteins from the fluid in the region 8 of the epididymis (Nixon et al ., ). On the other hand, only traces of BSP protein homologs from human and mouse (part of the BSPH1 and BSPH2 subfamilies) have been found in seminal plasma.…”

“…Analyses of the expression pattern of the epididymal BSPs revealed that rabbit BSP1 is different from the others and is found only in mid‐ (region 6) and distal (region 7) corpus epididymis (Table ) (Nixon et al ., ). In the other species, proteins are mostly expressed in early segments of the epididymis.…”

“…Analyses of BSP sequences from mouse, human and rabbit showed they all have putative signal peptide for secretory proteins at the N‐terminal end composed of 15 to 22 amino acids (Lefebvre et al ., ; Nixon et al ., ). However, little is known about post‐translational modifications of epididymal BSP proteins or their regulation.…”

“…No information on any other modifications is available. Rabbit BSP1 is again different; potential post‐translational modifications identified include one N‐ and 15 O‐glycosylation sites, one amidation site, three myristylation sites and several phosphorylation sites modifying the protein's molecular weight from 49.6 kDa to 52 kDa (Nixon et al ., ). Deglycosylation experiments with rabbit BSP1 confirmed the presence of glycosylation similar to those found in proteins from the BSP subfamily.…”