Recombinant expression and affinity purification of a novel epididymal human sperm-binding protein, BSPH1.

Mammalian sperm undergo a series of maturation steps before acquiring fertilization competence. Our previous work demonstrated the importance of binder of sperm (BSP) proteins in bovine sperm capacitation. Recent studies identified a BSP-homologous DNA sequence in the human genome (BSPH1) and mRNA expression in the epididymis. The aim of this study was to develop an efficient method to express and purify recombinant human BSPH1. BSPH1 accumulates in inclusion bodies when expressed with an N-terminal hexahistidine tag in BL21 (DE3) Escherichia coli cells. Similar to other BSP proteins, BSPH1 contains two fibronectin type-II (Fn2) domains, each consisting of two disulfide bonds. Therefore, when expressed in Origami B (DE3)pLysS cells, a strain favouring disulfide bond formation, an improvement in soluble protein yield was observed. However, protein was aggregated, which complicated subsequent purification steps. Expression of glutathione-S-transferase-tagged BSPH1 in both cell types also led to accumulation in inclusion bodies. Finally, successful production of soluble and active protein was achieved when BSPH1 was expressed as a His(6)-thioredoxin-tagged protein. Recombinant protein bound phosphatidylcholine liposomes, low-density lipoproteins and human sperm, therefore displayed binding activities common to all BSP-family proteins, which may indicate similar biological function(s). This approach was also successful in producing the murine orthologue of BSPH1 in the soluble and active form. Thus, fusion to thioredoxin and expression in Origami B (DE3)pLysS cells may constitute a strategy applicable to all BSP-family proteins, and possibly to other proteins containing Fn2 domains. This work is important to elucidate the role of BSPH1 in human sperm functions and fertility.

New nomenclature for mammalian BSP genes.

BSP proteins and their homologs are a family of structurally related proteins characterized by the presence of tandem fibronectin type II domains. In the bovine species, BSP proteins were shown to be involved in sperm capacitation, a posttesticular maturation event necessary for sperm to acquire the ability to fertilize an oocyte. Recently, many new genes from this family have been discovered in numerous mammalian species. However, inconsistency in the nomenclature is creating much confusion. In light of the rapid growth of the BSP superfamily of proteins, we propose a new nomenclature in collaboration with the HUGO Gene Nomenclature Committee.

Seminal plasma proteins: functions and interaction with protective agents during semen preservation.

Seminal plasma contains factors that are beneficial and/or detrimental to sperm function and/or storage. However, the nature and characteristics of these factors are not well understood. The major protein fraction (50-70%) of bovine seminal plasma is represented by a family of phospholipid-binding proteins collectively called BSP proteins. The BSP protein signature is characterised by two tandemly repeated fibronectin type 2 (Fn2) domains. It is now well established that BSP proteins and their relatives represent a new emerging superfamily of proteins in mammals. They bind to sperm membrane choline phospholipids at ejaculation. They also bind to capacitation factors, namely, high-density lipoproteins and glycosaminoglycans and promote sperm capacitation induced by these molecules, indicating their beneficial role in sperm function and fertility. In contrast, BSP proteins also induce changes in the sperm plasma membrane by stimulating cholesterol and phospholipid efflux. Thus, the continuous exposure of sperm to seminal plasma that contains BSP proteins is detrimental to the sperm membrane, which may render the membrane very sensitive to sperm storage in the liquid or frozen states. Interestingly, BSP proteins specifically bind low-density lipoproteins present in egg yolk, a compound commonly used in semen extenders. This interaction appears to abolish the detrimental effect of BSP proteins on the sperm membrane. Therefore, BSP proteins in seminal plasma act like a double-edged sword, being both beneficial and detrimental to sperm.

Bovine seminal plasma proteins and their relatives: A new expanding superfamily in mammals.

BSP proteins represent three major proteins of bovine seminal plasma: BSP-A1/-A2, -A3 and -30 kDa. The BSP protein signature is characterized by two tandemly repeated fibronectin type 2 (Fn2) domains. Although classical affinity chromatography and protein sequencing have proven that the BSP protein homologs may be ubiquitous in mammals and functionally related to sperm capacitation, only the three bovine genes have been reported thus far. In this study, we report three new BSP protein-related genes from bovine, as well as other BSP protein-related DNA sequences from human, chimpanzee, mouse, rat, dog, horse and rabbit. Analysis of the relationships between all Fn2 domain-containing proteins revealed that the Fn2 domains found in BSP-related proteins have special features that distinguish them from non-BSP-related proteins. These features can be used to identify new BSP protein-related sequences. Further molecular evolutionary analysis of the BSP protein lineage revealed that all BSP proteins and their related sequences can be grouped into three subfamilies: BSPH4, BSPH5 and BSPH6, which indicates that the BSP protein family is much bigger than previously envisioned. More interestingly, the three BSP proteins in bovine within the BSPH4-subfamily were shown to evolve rapidly. The ratio of nonsynonymous to synonymous substitutions was higher than 1. The analysis also indicated that the rate of evolution was heterogeneous between the first and second Fn2 domains of the genes. These data may reflect that some amino acids in BSP proteins are under a strong positive selection after gene duplication and that each BSP protein evolves rapidly, possibly to acquire new functions.