2.1. In silico-predicted OBP sequences of O. aries and C. hircus
The blast searches for putative ovine and caprine OBPs allowed identification of 16 and 17 sequences, respectively. These sequences were analysed with Signal-P software (DTU Bioinformatics) to remove the signal peptide, the hallmark of secreted proteins, and aligned with Multalin software (22) (Fig. S1 in additional file 1). Among these sequences, 18 displayed OBP(stricto sensu)-like features (O. aries: W5PH68, W5PGV5, W5PZN0, W5PHA2, W5PGN0, W5PHS2, WPPHN1, W5PHM2 and W5PGW3; C. hircus: XP_017899539.1, XP_017899538.1, XP_017900101.1, XP_017899208.1, XP_017899536.1, XP_005701296.1, XP_017899515.1, XP_017899207.1, and XP_017899516.1), 8 were close to pig salivary lipocalin (SAL: O. aries: W5P8Y1, W5P8W4, W5P4T6 and W5P4W8; C. hircus: XP_017908098.1, XP_017908099.1, AHZ46504.1, and XP_017910280.1), and 7 were aligned with Von Ebner’s gland protein (VEG: O. aries: W5P559, W5NUS5, and W5NV32; C. hircus: XP_005687416.1, XP_017910286.1, XP_017911671.1, and XP_017899201.1). In addition to the typical lipocalin GxW pattern at N-terminal position (14-16 in OBP, 19-21 in SAL, 15-17 in VEG) and the YxxxYxG motif (at position 79-85 in OBP), some common patterns could be observed in some OBP, SAL or VEG-like sequences, but in not all (Fig. S1). In OBP sequences, the most conserved regions are from position 14 to 46 including the GxW motif, and at the C-terminus from residues 151 to 169. In SAL sequences, the GxW hallmark of lipocalins is also included in a well conserved region (12 to 30), whereas in VEG the predicted sequences do not share highly conserved regions. Meanwhile, there is a strong sequence conservation inside each species and between species. It is worth to notice that the number of sequences is much higher in these two ungulate species than in pig and cow (one sequence in each group of OBP, SAL, and VEG). Most of OBP, SAL, and VEG sequences start with a Q at position 1, which can be under either pyroglutamate or glutamate forms in porcine OBP, and possibly modified in ovine and caprine proteins as well. In OBP group, three predicted ovine sequences (W5PH68, W5PGV5, W5PZN0) are more closely related to bovine OBP than to porcine ones, as they have no cysteines at all, and a well-conserved GxW additional motif at position 62-64 instead of the conserved C64 (Supp. Fig. S1). These sequences are unable to form disulphide bridges, but could form dimers by domain swapping, as it was reported for bovine OBP (23). Other sequences were close to porcine OBP (W5PHA2, XP_017899539.1, XP_017899538.1, XP_017900101.1) with 2 cysteines possibly engaged in one disulphide bridge. A sub-group comprised sequences with 2 (W5PGN0, WPPHN1, XP_005701296.1, XP_017899536.1), 3 (XP_017899207.1), or 4 additional cysteines (W5PHS2, XP_017899515.1, XP_017899516.1). The predicted sequence W5PGW3 was not well-aligned with the others, both at the N-terminal end, and along the sequence, but displayed a typical OBP C-terminal end (DDCPA). The SAL group was rather homogenous, with 3-4 conserved cysteines, except sequence W5P4W8 that aligned with other SAL-like from residue D13, but is more divergent in the internal part of the sequence (26% identity with porcine SAL). In the VEG group, two sequences were well-aligned with the porcine VEG (W5NUS5, XP_005687416.1), and W5NV32 was very similar to XP_017910286.1 (96% identity between the two sequences). On the contrary, the caprine sequence XP_017911671.1 seemed truncated at the N-terminal part and did not fit well with other VEG-like proteins.
2.2. Identification of ewe and goat olfactory secretome
We compared the olfactory secretome of 3 females of each species, from samples collected in the same female in SA and SR. All spots obtained after 2D-E of proteins of the nasal mucus were analysed by bottom-up mass spectrometry. Nano-LC-MS/MS or MALDI-TOF MS allowed identifying specific peptides of OBPs among in silico predicted sequences above. It is worth to notice that there is no common peptide between all the OBP sequences in each species and that at least 1 unique peptide was retrieved for each predicted protein identified in analyses below. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (24) partner repository with the dataset identifier PXD014833 and 10.6019/PXD014833. Full analyses of raw data are presented in additional file 1 (Tables S1, S2, S3 for O. aries and Tables S4, S5, S6 for C. hircus).
2.2.1 Olfactory secretome of ewes
Olfactory secretome profiles obtained by 2D-electrophoresis revealed differences between SA and SR secretomes for the three ewes (Fig. 1a, 1b & Fig. S2). SR profiles were characterized by two protein strings at apparent molecular masses of 17 (4 spots) and 20 (6 spots) kDa (Fig. 1a). A third string of 5 spots appeared at 25 KDa in SA (Fig. 1b), the 20 kDa string being still composed of 6 spots, whereas the 17 kDa string was composed of 7 spots. Each spot content (numbering in Fig. 1a, 1b) was identified by nano-LC-MS/MS or MALDI-TOF MS (Table 1). According to their sequence homology with OBP, SAL and VEG already known, the identified proteins were renamed according to the insect OBPs nomenclature: first letter of the genus in capital (e. g. O. for Ovis), three first letters of the species (ari for aries) followed by the type of protein (OBP, e. g. Oari-OBP1). The correspondence between predicted sequence numbers and nomenclature is given in Table 1 for ovine proteins and in Table 2 for caprine ones. In SR the olfactory secretome was mainly composed of specific peptides of two OBP-like proteins that were named Oari-OBP2 (W5PGN0) and Oari-OBP4 (W5PHS2). The 20 kDa string was almost exclusively composed of Oari-OBP2 and the 17 kDa string was composed of a mixture of Oari-OBP2 and Oari-OBP4. Another OBP-like protein was present in only one spot of the 17 kDa string in the ewe 30094 named Oari-OBP1 (W5PHM2) and a fourth OBP sequence was identified in several spots of the ewe 30056, Oari-OBP3 (W5PGW3). In ewe 30056 a mixture of two SAL-like proteins were detected in spots 75 and 81, Oari-SAL1 (W5P8W4) and Oari-SAL2 (W5P8Y1).
SA profiles were characterized by a larger diversity, since 7 different protein sequences were identified in all females (Table 1). Oari-OBP2 and Oari-OBP4 displayed the same pattern in 17 and 20 kDa strings than in SR. The additional 25 kDa string mainly contained Oari-SAL1 and Oari-SAL2. The secretomes of ewe 30094 and 30056 slightly differed from the ewe 30118 (Fig. S2), with the two SAL-like expressed in other spots of the 20 kDa string and a mixture of Oari-OBP2 and Oari-VEG1 (W5NUS5) in spots 69 and 70 (ewe 30094, Fig.S2b). The two other OBP-like proteins (Oari-OBP3 and Oari-OBP1) were found in very few spots in ewes 30118 and 30056.
Table 1: Proteins identified in spots from SR and SA 2D-E gels of ewes
Protein name
Accession number UniProt KB
|
Ewe 30118
(Fig. 1a, 1b)
|
Ewe 30094
(Supp. Fig. S2a, S2b)
|
Ewe 30056
(Supp. Fig. S2c, S2d)
|
SR*
|
SA*
|
SR*
|
SA*
|
SR¤
|
SA¤
|
Protein found in spot n°
|
Oari-OBP1
W5PHM2
|
Not found
|
25
|
45
|
Not found
|
Not found
|
88 to 90, 92 & 93
|
Oari-OBP2
W5PGN0
|
1 to 4, 8 to 10
|
11, 15, 17, 18 & 24 to 29
|
35 to 38 & 45 to 48
|
49 to 51, 53 to 56, 58 to 68, 70 & 71
|
72, 73 & 76 to 83
|
84 to 86, 89 & 93 to 103
|
Oari-OBP3
W5PGW3
|
Not found
|
18 to 21
|
Not found
|
60
|
72, 78 & 81 to 83
|
87, 88, 95, 98, 99 & 101
|
Oari-OBP4
W5PHS2
|
1, 8 & 9
|
11, 12, 15, 17, 18 & 24 to 28
|
35, 45 & 46
|
51, 55, 56, 58, 61 to 68 & 71
|
72 to 83
|
93, 98 to 100
|
Oari-SAL1/2
W5P8W4 / W5P8Y1
|
Not found
|
11 to 15
|
Not found
|
49 to 53, 55, 56, 58 & 60
|
75 & 81
|
84 to 89, 91 & 99
|
Oari-VEG1
W5NUS5
|
Not found
|
Not found
|
Not found
|
69 & 70
|
Not found
|
Not found
|
* proteins identified by Nano-LC-MS/MS ¤ Proteins identified by MALDI-TOF MS. Full data can be found in Tables S1, S2 and S3 of additional file 1.
2.2.2 Olfactory secretome of goats
The olfactory secretome profiles of goats in SR (Fig. 1c, Figs. S3a, S3c) revealed a major protein string of zip shape at an apparent molecular mass of 17 kDa and a smaller string at 15 kDa. The SA profiles were similar to the SR ones for the three goats (Fig. 1d, Figs. S3b, S3d), even if the profiles of goat 30363 (Fig. 1c, 1d) differed slightly from those of the two other animals (Fig. S3), by the lack of the 15 kDa string.
In SR samples, the strings at 17 kDa and at 15 kDa were mainly composed (numbering in Fig. 1c, 1d) of Chir-OBP2 (XP_017899208.1) and Chir-OBP4 (XP_017899515.1) specific peptides for the three goats (Table 2). But contrary to ewes SR profiles, several other proteins could be identified: Chir-VEG2 (XP_017911671.1) is present in few spots in the three females, whereas Chir-OBP3 (XP_005701296.2), Chir-OBP5 (XP_017899538.1) and Chir-VEG1 (XP_005687416.1) are present only in goat 30422. Chir-SAL1 (XP_01708099.1) and Chir-SAL2 (XP_01708098.1) were detected in mixture in some spots of this string in this female (Table 2). The spot in the basic part of the gel in goat 40322 profile (spot n°73, Fig. S3a) contained specific peptides of Chir-OBP2, Chir-OBP4 and Chir-VEG2 isoforms.
Chir-OBP2 and Chir-OBP4 were the major components of the 17 kDa string in SA, with some isoforms of Chir-OBP3 and Chir-VEG2 for the three females. For the goat 40322 the Chir-OBP4 was not as present as in the other females. Specific peptides of Chir-OBP5, Chir-OBP6, Chir-SAL1, Chir-SAL2 were identified in this string, except in goat 30432. In addition, peptides of two new proteins were identified in the 17 kDa string: Chir-OBP1 (XP_017899536.1) in goat 30432, Chir-OBP6 (XP_017900101.1) and Chir-VEG3 (XP_017910286.1) in goats 30363 and 30422. As well as the majority of spots in these profiles, the 15 kDa spots in SA (spot 139 to 143 of goat 30432) contained specific peptides of Chir-OBP2 and Chir-OBP4, and basic spots of SA profiles (spots 144 & 145 of goat 30432), except for spot n°144 of the goat 30432 that contained peptides of Chir-OBP1 (Table 2, Fig. S3d).
Spots of the 25 kDa string, specific to goat 30432, contained mainly Chir-OBP2 and Chir-OBP4 in both SR and SA, plus some peptides of Chir-SAL1 in SR, and Chir-OBP5 in SA.
Table 2: Proteins identified in spots from SR and SA 2D-E gels of goats
Protein name
Accession number GenPept
|
Goat 30363
(Fig. 1c, 1d)
|
Goat 30422
(Fig. S3a, S3b)
|
Goat 30432
(Fig. S3c, S3d)
|
SR*
|
SA¤
|
SR¤
|
SA¤
|
SR*
|
SA*
|
Protein found in spot n°
|
Chir-OBP1
XP_017899536.1
|
Not found
|
Not found
|
Not found
|
Not found
|
85
|
124, 129 & 144
|
Chir-OBP2
XP_017899208.1
|
1, 2, 5 to 15, 17, 20, 22, 24 & 25
|
27 to 29, 31 to 43 & 45
|
46 to 64, 66 to 70 & 73
|
74 to 82
|
83 to 105
|
108, 109, 121, 122, 124, 126 to 134, 136, 137, 139, 140 & 142 to 145
|
Chir-OBP3
XP_005701296.1
|
Not found
|
30 to 32
|
47 to 49, 51 to 54, 57, 59, 62, 66 & 67
|
75, 76 & 80
|
Not found
|
127
|
Chir-OBP4
XP_017899515.1
|
1 to 3, 5 to 15 & 17 to 19
|
31 to 35, 38, 40, 41 & 45
|
46 to 48, 51 to 63, 65 & 73
|
76 & 78
|
83 to 95 & 97 to 104
|
108 to 114, 117 to 119, 122 to 124, 127 to 134, 136 to 138, 140, 142, 144 & 145
|
Chir-OBP5
XP_017899538.1
|
Not found
|
28, 30 & 32
|
46, 47, 50, 52 to 56, 59, 60, 64, 68, 70 & 71
|
76, 78 & 79
|
86
|
121 & 122
|
Chir-OBP6
XP_017900101.1
|
1
|
28 to 31, 36, 37 & 45
|
47, 49 & 72
|
74 & 76 to 78
|
86
|
Not found
|
Chir-SAL1/2
XP_017908099/8.1
|
2
|
31, 32, 35, 41, 43 & 44
|
46 to 48, 50 to 57, 59 to 67, 69, 71 & 72
|
74, 77, 78 & 80 to 82
|
87 & 90
|
Not found
|
Chir-VEG1
XP_005687416.1
|
10
|
30 & 34
|
54, 55 & 63
|
Not found
|
Not found
|
Not found
|
Chir-VEG2
XP_017911671.1
|
11
|
32, 35 & 39 to 43
|
46 to 67, 69, 71 & 73
|
78 & 80 to 82
|
97 to 100
|
134
|
Chir-VEG3
XP_017910286.1
|
Not found
|
32 & 38
|
Not found
|
74 & 77
|
Not found
|
Not found
|
* proteins identified by nano-LC-MS/MS ¤ Proteins identified by MALDI-TOF MS. Full data can be found in Tables S4, S5 and S6 of additional file 1.
2.2.3 Comparison between the 2 species
The olfactory secretome profiles of ewe and goat differed in the number of detected spots and in the number of protein strings, but share a common main string at apparent molecular mass of 17 kDa. The interindividual variability in goats participated to this difference between the two species. The composition of theses profiles was also different, as more proteins are present in goat than in ewe in both SR and SA. In goats, six OBP-like proteins were identified, instead of four in ewes, 3 VEG-like in goat and only 1 in ewe, but two SAL-like proteins were identified in both species. The olfactory secretome profile of ewe is remarkably similar to the pig’s one (21), where SAL-like proteins are dispatched in a protein string with a decreasing molecular weight gradient from acid to basic spots, and with VEG proteins in the basic part of the main spots (17 kDa string in ewe, Fig. S2b). Even if the distribution of these protein families is not as clear in goat as in ewe, the olfactory secretomes are mainly composed of isoforms of two OBP-like sequences: Oari-OBP2 and Oari-OBP4 in ewe, and Chir-OBP2 and Chir-OBP4 in goat. These proteins were identified in almost all spots whenever the season in the 2 species, and remarkably close to each other in the previous alignment (Fig. S1). To confirm this phylogenetic proximity between the two OBP2 and the two OBP4, the nucleotide sequences were amplified from ewe and goat olfactory epithelium.
2.3 Amplification and molecular cloning of nucleotide sequences coding for OBP2 and OBP4
2.3.1 cDNA analysis of ovine and caprine main sequences
The cDNA encoding the 2 most abundant OBPs, OBP2 and OBP4, identified in ewe and goat secretome were amplified by RACE-PCR. Indeed, the predicted sequences available from databases are unverified, often automatically deduced from high-throughput sequencing data (e. g. contigs assembly) and should be amplified from tissues to ascertain the sequence. The full-length sequences (GenBank accession numbers: MK908982, MK908983, MK908984 and MK908985), when translated, showed high identity with the predicted Oari-OBP2 and Oari-OBP4 (Fig. 2), Chir-OBP2 and Chir-OBP4 (Fig. S4). Oari-OBP2 was 98 % identical to the predicted sequence, the main differences were located in the middle of the sequence with two frameshifts modifying the sequence (deleting C51 and C56 in the predicted), and at the 3’ end modifying the C-terminal sequence (GDCSLA - predicted and GCQAQ - cloned). For the three other sequences the identity between predicted and cloned sequences were higher (Oari-OBP4: 99 %, Chir-OBP2: 99.4 % and Chir-OBP4: 100 %). These four sequences were added to the predicted olfactory-binding proteins and reference sequences to perform a Blast search in order to build a phylogenetic tree (Fig. 3). As expected from the predicted sequences, ovine and caprine sequences segregated into the three OBP, VEG and SAL groups. The tree revealed the high proximity between sheep and goat sequences inside protein families, e. g. between Chir-VEG1 and Oari-VEG1, Oari-SAL1 & SAL2 and Chir-SAL1 & SAL2.
2.3.2 Comparison of sheep and goat sequences
Chir-OBP2 and Oari-OBP2 shared 97.72 % identity of nucleotide sequence (98.3 % of homology and 96 % of identity between protein sequences), whilst Chir-OBP4 and Oari-OBP4 shared 98.1 % identity at the nucleotide level (96.5 % of homology and 95.3 % of identity at the protein sequence level). Such identities are unusual for OBPs of different species, generally 20% of similarities are observed between species (17). This suggests that OBP2 and OBP4 could have a common role in odours reception of the two species. The two OBP2 sequences are close to porcine OBP in the phylogenetic tree (Fig. 3) and share 58% of similarity, and could display common binding properties for steroids and fatty acids (18). This is supported by the molecular modelling experiments performed to predict the 3D-structure of these sequences. The predicted model by I-TASSER software (25) showed that the four sequences have a 3D-structure typical of lipocalins, with eight b-strands forming a b-barrel in addition to an a-helix, and a disulphide bridge (Cys63-Cys155) conserved in all Mammalian OBPs except in the bovine one (17). If the two OBP2 have a 3D-structure very similar to porcine OBP (26), a second disulphide bridge is formed in the OBP4 between Cys44 and Cys48 (Fig. 4). This bridge is only found in rodents OBP, more specifically in Cricetidae, in proteins expressed in fluids involved in chemical communication, such as hamster aphrodisin (27) and bank vole glareosin (28). In aphrodisin, the disulphide bridge forms a loop where an asparagine, not conserved in OBP4, is N-glycosylated (29). Hence, the two OBP4 seem rather specific to the small ungulates, and could have a role in the binding of specific odours of these species. It could be the same for the Chir-SAL1 & SAL2 and Oari-SAL1 & SAL2 which are very close in the tree, and known to be Pheromone-Binding Proteins (30). Their specific expression in SA in ewe could reflect an adaptation of female secretome to odours emitted by a sexually active ram. In goat, SAL are expressed in SR and not systematically present in SA, in this case the modification of the secretome could result on the increasing number of isoforms of OBP sequences in SA. In particular, new isoforms could be generated by post-translational modifications (PTM), as it was evidenced in pig (21).
2.4 Sheep and goat olfactory-binding proteins are modified by Post-Translational Modifications
2.4.1 N-Glycosylation
Samples were treated with PNGase F, an enzyme that specifically removes N-glycan chains from proteins (Fig. 5). A clear shift in molecular mass was observed only in treated sample of ewe in SA, characterized by the emergence of an additional band compared to the non-treated sample (arrow, Fig. 5a). This decrease in molecular mass suggests that one or several glycan chains were removed by PNGase F from the band migrating at 25 kDa (spots 11-14 in Fig. 1b) and/or from the band migrating at 20 kDa (spots 15-21, Fig. 1b) in SA, from proteins of the 17 kDa string in SR (spots 7-10, Fig. 1a). To confirm that one or more proteins bear glycan-chain, a lectin-blot with Con A lectin was performed on the non-treated samples (Fig. 5c). For the goat samples and the sample of ewe secretome in SR, no signal was obtained. The reactivity to Con A lectin indicated the presence of poly a-mannoses chain(s) on ewe proteins. The strong signal in SA could be due to Oari-SAL1 and Oari-SAL2 that are not expressed in SR (Table 1). Indeed, pig SAL, which is phylogenetically close to Oari-SALs (Fig. 3) is N-glycosylated (31). Four sites of N-glycosylation were indeed predicted by the NetNGlyc software (http://www.cbs.dtu.dk/services/NetNGlyc/) for Oari- and Chir-SAL. These results suggest the existence of two groups of SAL-like proteins in ewe, one group of isoforms in the 25 kDa string in SA, N-glycosylated, and one group of isoforms in the 20 kDa string (Table 2), which are not N-glycosylated. As one site of N-glycosylation was predicted by NetNGlyc for Oari-OBP2, its isoforms in the 20 kDa string could be N-glycosylated as well.
2.4.2 Phosphorylation
The OBP phosphorylation was investigated on ewe and goat proteins of olfactory secretome, as previous work has shown that porcine olfactory-binding proteins are phosphorylated (18,32). Anti-phosphoserine antibody labelled all samples from ewe and goat, whenever the season (Fig. 6). In ewes, two bands were labelled in SA and only one in SR (Fig. 6a). In goat, labelling of SR samples seemed stronger than in the SA samples (Fig. 6b). The western-blot after 2D-E showed that, in ewe in SR, 4 spots of the main string at c. a. 17 kDa were labelled (Fig. 6c), whereas 5 spots of the same protein string are labelled in SA (Fig. 6d). In goat, the same number of spots were labelled in SR and SA samples (9 spots of the 17 kDa string, Fig. 6e, 6f). Western-blot and Coomassie blue stained gels of 2D-E were merged, which allowed identification of the labelled spots. The map of labelled spots can be found in Fig. S5 (a-d) in additional file 1. The same samples were digested with alkaline phosphatase to assess the specificity of Q5 antibodies (Fig. S6a). Indeed, dephosphorylated samples were not labelled by this antibody after western-blot. Comparing the proteins identified in these spots and the antibody labelling, we propose that OBP2 and OBP4 could carry this modification in both species. The physiological status of the female could have an impact on the number of phosphorylated isoforms expressed in the secretome, leading to more phosphorylation in SA in ewe and more phosphorylation in SR in goat. Conversely, antibody Q7, raised against phosphothreonine labelled all samples (Fig. S7).
To confirm and if possible, localize the phosphorylation sites in ewe and goat OBPs, nano-LC-MS/MS data were analysed with Scaffold software. Ion scores, identity scores and retention times were compared between phosphorylated and naked spectra for each phosphorylated peptide (spectra of peptide 18-40 of Oari-OBP2 are compared in Fig. S8 of additional file 1). Despite the immunodetection of potential phosphorylated serine in both species (Fig. 6) no corresponding sites were identified by mass spectrometry, contrary to phosphothreonine sites identified in accordance with the labelling in western-blot (Fig. S7). In ewe, phosphorylation sites were identified on Oari-OBP2, Thr18, Thr29 and Th65, only in SA. Thr18 site was confirmed in spot 67 of ewe 30094 (Fig. S2b) by 2 spectra, Thr29 site by one spectrum in spot 27 of ewe 30118 (Fig. 1b), and Thr65 site in ewe 30094 (spot 68, Fig. 1b) and 30118 (spot 32, Fig. S2b) by 2 spectra for each spot. Except for spot 32 of ewe 30118, all spots were located in the 17 kDa string.
In goat, 3 sites were identified on Chir-OBP2, Thr29, Tyr38 and Thr64, with some interindividual differences. In goat 30432 in SA (Fig. S3d), Tyr38 site was identified by 2 spectra on spots 138 and 140. In SR, Thr29 site was identified by 6 spectra in goat 30363 (spots 6 to 9, Fig. 1c) and by 3 spectra in goat 30432 (in spots 100, 101, and 105, Fig S3c). Tyr38 site was phosphorylated on peptides found in many spots of goat 30363 (SR, Fig. 1c) by eleven spectra in spots 5 to 9, and 11. In goat 30432 (SR, Fig. S3c), Tyr38 site was identified by 9 spectra of two different peptides in spots 99, 100, 105, and 106. Thr64 was identified in spot 8 of goat 30363 in SR by three spectra.
The spots where Thr29 site was identified belonged, in the two goats in SR, to the 17 and 15 kDa strings. In SA of goat 30432, Tyr38 was phosphorylated only in spots at 15 kDa or lower. In SR, Tyr38 site was found in spots of the 17 kDa string in goat 30363, and in goat 30432 except for spot 106 (Fig. S3c) which is located in a more basic part of the 2D-E gel.
The fact that no site of phosphoserine was identified by mass spectrometry, despite the strong labelling of specific antibody is surprising regardless the literature, since phosphorylation of serine is described as the most abundant phosphorylation in Mammals (ratio Ser/Thr/Tyr of 1800/200/1) (33). This could come from the lability of this modification submitted to the high energy of HCD ionization, and the possibility of serine modification cannot be excluded. As porcine OBP is phosphorylated (32), the identity between the primary sequences of porcine OBP, Oari-OBP2 and Chir-OBP2 led us to compare the phosphorylation pattern of these sequences. Recently, phosphorylation sites on porcine OBP were identified by CID-nano-LC-MS/MS (18). Several sites are common between the 3 species (Ser13, Ser23, Ser24, Ser41, and Ser67) and are predicted as phosphorylated by the NetPhos server (http://www.cbs.dtu.dk/services/NetPhos/). Thr112, which is phosphorylated in pig, was not detected as a phosphorylation site in sheep and goat sequences (Fig. S1).
2.4.3 O-GlcNAcylation
A major difference between the two species appeared with the detection of O-GlcNAcylation on OBP. In goat (Fig. 7b), the samples of the three females in SA and SR were labelled by CTD110.6 antibody, suggesting an equal level of O-GlcNAcylation. For the three ewes, only the SA samples were labelled, no O-GlcNAcylation was detected in SR (Fig. 7a). The labelling at a higher molecular mass could correspond to oligomers frequently observed for OBPs (21), which could be more reactive to antibodies than monomers, as a result of their tendency to form oligomers in solution. Western-blot performed after 2D-E brought more information on the number of O-GlcNAcylated spots: in ewe (in SA) four spots of the 25 kDa string were labelled and five spots of the 17 kDa string (Fig. 7c). In the SR sample of goat, 8 spots of the 17 kDa string were labelled (Fig. 7d), and 6 spots of this string in SA (Fig. 7e). The map of labelled spots can be found in Fig. S5 (e-g) in additional file 1. The competition assay between CTD110.6 antibody and free GlcNAc assessed the specificity of the antibody (Fig. S6b) and O-GlcNAcylation of OBPs in ewe and goat olfactory secretome. The HCD-nano-LC-MS/MS is not a suitable method to identify O-GlcNAcylation sites, as GlcNAc moieties are removed during ionization. However, spots were specifically labelled by CTD110.6 antibody (Fig. 6, Fig. S5f, S5g) suggesting in goat that Chir-OBP2 and Chir-OBP4 could be O-GlcNAcylated as their isoforms were identified in all labelled spots. In ewe, isoforms of Oari-OBP2 and Oari-OBP4 of the 17 kDa string could carry this modification as well, in addition to Oari-SAL1 and SAL2 which mainly composed the 25 kDa string (Fig. 6e). The sequence similarity between Oari-OBP2, Chir-OBP2 and porcine OBP reinforces this hypothesis, as porcine OBP is O-GlcNAcylated (21) on Ser13 and Thr18 (18) that are conserved in sheep and goat OBP2 sequences. Even if Oari-OBP4 and Chir-OBP4 are phylogenetically more distant to the porcine OBP (Fig. 3), the number of their isoforms in the labelled spots does not totally exclude the O-GlcNAcylation of these proteins. The potential O-GlcNAcylation of SAL-like proteins (both in sheep and goat) is supported by the similarity between these sequences and the porcine SAL (64% of similarity), which is O-GlcNAcylated (21). VEG-like sequences found in the spots labelled by CTD110.6 could also be modified by GlcNAc (34), but it has to be confirmed by ETD-nano-LC-MS/MS, a technique more suitable to localize O-GlcNAcylation sites (35).