Three-dimensional model of rabOBP3 as built by using the crystallographic structure of pig SAL (Boar salivary lipocalin, PDB ID: 1 GM6) as a template[10].

<p>Molecular model of rabOBP3 and pig SAL are shown in the left- and right-top panel, respectively. The corresponding sequence alignment is shown in the bottom panel, where conserved amino acids are highlighted in yellow. The conserved N-glycosylation site (Asn44), and oxidized (Cys59 and Cys152) and reduced (Cys66 and Cys133) residues are indicated by specific labelling (top) or asterisks (bottom).</p

Binding of 1-NPN (left) and selected ligands (right) to rabOBP3 purified from seminal fluid and delipidated with dichloromethane.

<p>The protein binds the fluorescent probe 1-NPN with a dissociation constant of 3.8 µM (SD 0.9, n = 3). None of the ligands tested exhibited strong affinity to the protein, except quercetin, for which a physiological role does not seem plausible. Calculated dissociation constants are 2.2, 7.8 and 11.2 µM for quercetin, 2-nonenal and geraniol, respectively.</p

MALDI-TOF-MS analysis of the purified tryptic glycopeptides from rabOBP3 as obtained after HILIC enrichment and nanoLC separation.

<p>Spectra acquired in linear mode of the fractions eluting at 15 and 16 min are reported in panel A and B, respectively; shown are the mono-, bi- and tri-antennary complex-type glycan structures N-linked to Asn44 in peptide (44–50). ▪, N-acetyl-glucosamine; •, mannose; ○, galactose; ◂, fucose; ♦, N-acetyl-neuraminic acid.</p

Expression of rabOBP3 in different tissues of male (m) and female (f) rabbit individuals.

<p>SDS-PAGE analysis of different rabbit tissues and corresponding Western blotting are shown. M: molecular weight markers; m1: nasal respiratory tissue; m2: epidydimis; m3: testis; m4: prostate; f1: nasal respiratory tissue; f2: uterine tubes; f3: ovaries; f4: uterus; P: purified rabOBP3.</p

SDS-PAGE analysis of rabbit sperm and corresponding Western blotting.

<p>SN, soluble fraction; P, sperm cells; WP, sperm cells after washing three times with buffer. A strong cross-reactivity with a polyclonal antiserum raised against pig SAL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111932#pone.0111932-Marchese1" target="_blank">[19]</a> was observed for a protein migrating at about 23 kDa. Staining was much stronger in the soluble fraction; the weak reactivity observed for the sperm cells disappeared after washing the cells, thus indicating the absence of the protein in this sample.</p

MALDI-TOF-MS analysis of the tryptic digest of rabOBP3 alkylated with iodoacetamide under denaturing, non-reducing conditions before (top) and following (bottom) treatment with dithiothreitol.

<p>Constant and variable signals are labelled in the spectra acquired in reflectron mode to highlight reduced and oxidized residues present under native conditions. Trypsin-derived peptides are indicated with an asterisk.</p

Purification of the rabbit seminal fluid OBP.

<p>A sample of crude seminal fluid, as obtained after sperm centrifugation, was resolved at first by gel filtration chromatography on a Superose-12 column and then by anion-exchange chromatography on a DE-52 column (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111932#s2" target="_blank">Materials and Methods</a> section for details). The protein was eluted as a pure component, as verified by SDS-PAGE.</p