On the saliva proteome of the Eastern European house mouse (Mus musculus musculus) focusing on sexual signalling and immunity

Abstract

Chemical communication is mediated by sex-biased signals abundantly present in the urine, saliva and tears. Because most studies concentrated on the urinary signals, we aimed to determine the saliva proteome in wild Mus musculus musculus, to extend the knowledge on potential roles of saliva in chemical communication. We performed the gel-free quantitative LC-MS/MS analyses of saliva and identified 633 proteins with 134 (21%) of them being sexually dimorphic. They include proteins that protect and transport volatile organic compounds in their beta barrel including LCN lipocalins, major urinary proteins (MUPs), and odorant binding proteins (OBPs). To our surprise, the saliva proteome contains one MUP that is female biased (MUP8) and the two protein pheromones MUP20 (or 'Darcin') and ESP1 in individuals of both sex. Thus, contrary to previous assumptions, our findings reveal that these proteins cannot function as male-unique signals. Our study also demonstrates that many olfactory proteins (e.g. LCNs, and OBPs) are not expressed by submandibular glands but are produced elsewhere-in nasal and lacrimal tissues, and potentially also in other oro-facial glands. We have also detected abundant proteins that are involved in wound healing, immune and non-immune responses to pathogens, thus corroborating that saliva has important protective roles.

Figure 1

Details of the Power Law Global Error Model: (a) histograms of the sequence coverage and the unique sequence coverage, (b) the model fitting on a female experimental condition, (c) MA plot with the differentially abundant proteins where significant points are colored from green (p < 0.05) to blue (p < 0.01).

Figure 2. Graphical representation of the protein abundance values in heat maps shows sexually dimorphic proteins (labelled with stars: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) with notable variation between individuals.

We provide the phylogeny dendrogram for kallikreins and a partial dendrogram for the detected lipocalins. The Maximum likelihood trees are showing the protein phylogeny based on the number of substitutions per site and with the bootstrap values. We consider a group of proteins monophyletic when bootstrap values are above 75.

Figure 3. Graphical representation of structural and biochemical properties of murine salivary protein homologs - Cathelin-like domain of human Cathelicidin L-77, human Cystatin D and murine ESP1.

Upper row: Cartoon representation of selected proteins where α-helices are in red, and beta sheets in blue color. Below are provided 3D representations of surface charge distribution of respective proteins, the charge scale is shown under each structure.