Functional characterization of the domains of the bovine binder of SPerm 5 (BSP5) protein

Abstract

Background: Bovine BSP5 is a multifunctional protein primarily involved in sperm capacitation. BSP5 consists of long N-terminal part followed by two similar and highly conserved fibronectin type II domains designated A and B.

Methods: In order to assess the role of these domains in the sperm binding and capacitation processes, we created recombinant individual domains (N, A, B), series of overlapping domains (NA and AB) and full-length BSP5 in an Escherichia coli expression system. The recombinant constructs were also tested for their ability to interact with ligands such as gelatine, heparin, chondroitin sulphate B and phosphatidylcholine liposomes by affinity chromatography and co-sedimentation studies.

Results: With the exception of the N domain, all recombinant constructs retained gelatine, phosphatidylcholine, heparin and chondroitin sulphate B binding activities. Domain-wise studies showed clearly that AB domain is capable of performing its biological functions as well as the full-length protein, as it was able to potentiate heparin-mediated sperm capacitation.

Conclusions: These results indicate that the C-terminal domain composed of two Fn2 domains is sufficient and crucial to maintain the biological functions of BSP proteins. The N-terminal part of the protein did not bind to any of known BSP5-ligands including epididymal sperm and did not seem to be required for either sperm binding or sperm capacitation. This study also confirmed that glycosylation is not required for BSP-mediated sperm capacitation or any of the binding characteristics displayed by BSP5.

Fig. 1

Schematic representation of bovine BSP5 and the recombinant constructs. a Native full-length bovine BSP5 is a 158 amino acid-protein composed of an N-terminal part and two Fn2 domains (a and b) joined by a short linker. BSP5 is glycosylated on six residues of its N-terminal domain. b Recombinant constructs of the BSP5 proteins used in this study. c The tag encoded by the pET32a(+) expression vector consists of a thioredoxin, followed by a (His)₆-tag and a S-tag

Fig. 2

SDS-PAGE pattern of purified BSP5 recombinant constructs. pET32a(+) constructs of full length (FL), A domain (A); B domain (B); NA domain (NA) and (AB) domain were expressed in E. coli origami(DE3) cells and purified by Ni²⁺-NTA affinity chromatography. The pET30a(+) N-terminal (N) construct was expressed in E. coli BL21(DE3) cells. Five micrograms of purified proteins were analyzed by 15 % SDS-PAGE followed by Coomassie Blue staining

Fig. 3

Effect of recombinant BSP5 constructs on lyso-PC-induced acrosome reaction of epididymal sperm incubated with heparin. Sperm collected from cauda epididymis were incubated for 20 min alone or in media containing increasing concentrations of the different BSP5 constructs, 60 μg/ml Trx-His-S or 30 μg/ml native BSP5. Sperm were then washed, incubated with 12 μg/ml of heparin for 5 h and incubated an additional 15 min in presence (white bars) or absence (black bars) of 100 μg/ml lyso-PC. Acrosome reaction was assessed by naphtol yellow S-erythrosin B staining. Data are presented as the mean ± SD of three independent experiments. Differences compared to control (no proteins added) were analyzed by one-way covariance analysis followed by LSD multiple comparison tests (* p < 0.05). Panels (a-f) correspond respectively to Domain N, Domain A, Domain B, Domain NA, Domain AB and Protein FL (Full length)

Fig. 4

Effect of recombinant BSP5 constructs on the viability of epididymal sperm incubated with heparin. Sperm collected from epididymis were incubated for 20 min alone or in media containing 40 μg/ml of constructs N, A and B, 60 μg/ml of constructs NA, AB and FL, 60 μg/ml Trx-His-S or 30 μg/ml native BSP5. Sperm were then washed, incubated with 12 μg/ml of heparin for 5 h. Viability was assessed by staining with eosin/nigrosin solutions. Data are presented as the mean ± SD of three independent experiments

Fig. 5

Immunostaining of recombinant BSP5 constructs on heparin-capacitated sperm. Sperm from cauda epididymis were incubated 20 min alone (Ctrl) or in media containing 60 μg/ml of each construct, 60 μg/ml Trx-His-S or 30 μg/ml native BSP5. Sperm were then washed and incubated for 5 h with 12 μg/ml of heparin. Aliquots of the samples were dried on poly-L-lysine slides. Slides were then incubated with anti-BSP5 or His-probe (Trx-His-S slide) antibodies at dilution of 1:100 and treated with FITC-conjugated IgG. (Original magnification × 630; DIC, Differential Interference Contrast)