New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs)

<div><p>Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1) and 2 (BSPH2). The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria) belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2), which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, <i>Ovis aries</i> BSP5b).</p></div

Genome context analysis for Binder of Sperm Proteins.

<p>The three different patterns (BSP, BSPH1 and BSPH2)were found in Genomicus (G) or in NCBI (N) databases. Abbreviations on the right hand of each box represent chromosome/scaffold number (i.e. 14), species (i.e. Ov for <i>Ovis aries</i>) and database (i.e. G, Genomicus), respectively. In <i>Ovis aries</i>, letters bellow arrows mean the corresponding UniProt Code and letters in red correspond to trivial name. Letters in blue bellow arrows represent the correct name of missanotated proteins. Names with? at the end mean new BSP proteins not previously described. See text for gene and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137008#pone.0137008.t001" target="_blank">Table 1</a> for species abbreviations, respectively.</p

<i>In silico</i> analysis of Binder of Sperm Proteins.

<p>(A) Ribbon representation of the modelled <i>Ovis aries</i> RSVP20 protein. Different colors represent different conserved blocks: Cyan (Block I and VI), red (Block II and VII), green (Block III and VIII), magenta (block IV and IX) and orange (Block V). Amino acids forming both phosphorylcholine (PC) binding sites are shown in sticks and labeled. A PC molecule in the proposed binding site is shown in ball and stick representation. (B) Surface representation of 1FN2 PC binding site. (C) Surface representation of 2FN2 PC binding site. Amino acids involved in both binding sites are labeled according RSPV20 numbering and colours are the same as blocks described in (A) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137008#pone.0137008.s002" target="_blank">S2 Fig</a>).</p

Western blot membrane indicating the presence of recombinant and native RSVP20 in ram sperm lysates.

<p>Supernatant fraction (SN) and pellet fraction (P) of sperm samples incubated with (100 ug) or without (0 ug) recombinant RSVP20.</p

Updated nomenclature for Binder of Sperm Proteins.

<p>^§Accession numbers in parenthesis mean duplication of the same protein in UniProt</p><p>Updated nomenclature for Binder of Sperm Proteins.</p

Flow cytometry analysis of the sperm binding capacity of Alexa Fluor 488-conjugated RSVP20.

<p>Cytometry analysis of the effect of sperm incubation with increasing concentration of Alexa-conjugated RSVP20: A) 3 μg; B) 7.5 μg; C) 15 μg per 4 x 10⁷ cells. D) Plots showing the number of cells linked with protein (events) and the different fluorescence intensity (FL1 Log) for increasing concentrations of Alexa-conjugated RSVP20.</p

Phylogenetic analysis of Binder of Sperm Proteins.

<p>BSP proteins are divided into the main subfamilies: BSP (green), BSPH1 (purple) and BSPH2 (hot pink). In addition, the BSP subfamily is composed of four different clades corresponding to BSP1 (olive green), BSP2 (yellow green), BSP3 (camouflage green) and BSP5 (light green). The structures behind each protein code represent domain composition: signal peptide (cyan), 1FN2 (red) and 2FN2 (fluorescent green). C-terminal section of the protein is the outer part of the domain representation. The neighbor-joining (NJ) tree was obtained from 1000 replicates. Bootstrap values are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137008#pone.0137008.s001" target="_blank">S1 Fig</a>.</p

<i>In silico</i> analysis of 2FN2 phosphorylcholine binding site.

<p>The size of PC binding site 2 is reduced from (A) RSVP20 (BSP5b) to (B) murine BSPH1 and from the latter to (C) murine BSPH2. The first decrease in size is due to a change from a serine (S127 in RSVP20) to a glutamic acid (E109 in mBSPH1). The second decrease in size is due to the change of the latter E109 to a tyrosine (Y104 in mBSPH2). Cartoon representation of the backbone is in tan and surfaces are displayed in CPK (carbon in grey, oxygen in red and nitrogen in blue).</p

Patient Characteristics.

<p><b>Abbreviations:</b> +8, trisomy 8; PB, peripheral blood; PHA, phytohemagglutinin; RA, refractory anemia; RCUD, refractory cytopenia with unilineage dysplasia; RCMD, refractory cytopenia with multilineage dysplasia; RAEB, RA with excess of blasts; MDS-U, myelodysplastic syndrome unclassified; RARS-T, RA with ringed sideroblasts and thrombocytosis; CMML, chronic myelomonocytic leukemia; AML, acute myeloid leukemia; AML-MDRC, AML with myelodysplasia-related changes; AML NOS, AML not otherwise specified; APL, acute promyelocytic leukemia. In bold patient with constitutional trisomy 8 mosaicism.</p><p>Patient Characteristics.</p