Molecular complexity of the major urinary protein system of the Norway rat, Rattus norvegicus

Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Cluster analysis shows the existence of only 10 predicted mup genes. Further, detailed sequencing of the urinary MUP isoforms reveals a less complex pattern of primary sequence polymorphism in the rat than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications, including the phosphorylation of Ser4 in some isoforms, and exoproteolytic trimming of specific isoforms. Our results raise the possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse. Shotgun proteomics data are available via ProteomExchange with identifier PXD013986.

INTRODUCTION 13], driving assessment of genetic heterozygosity [48] and avoidance of inbreeding [12,49]. It was of 166 interest therefore to explore the heterogeneity in rat urinary MUPs. We have previously used ESI-MS to 167 profile the isoforms of the MUPs secreted in mouse urine [6,13,25]. MUPs yield strong signals on  and the intact masses can be determined to within ± 1 Da, permitting matching to predicted mature protein 169 masses from genomic or cDNA sequences [25]. The masses obtained by ESI-MS correspond to the neutral 170 average mass of the mature form of the protein, after the removal of the predicted signal peptide [50], and 171 subtraction of 2 Da for the formation of a single disulphide bond, based on homology with known MUP 172 structures [19,36]. ESI-MS also allows semi-quantitative assessment of the relative amounts of each isoform 173 [51]. less polymorphism in protein isoforms as evidenced by the ESI-MS pattern of wild rats. To explore this in 201 more detail, the same samples of wild rat urine were resolved by isoelectric focusing (IEF) ( Figure 4B) to 202 separate proteins by net charge -since MUPs were the predominant bands, they would be most prominent 203 bands after isoelectric focusing. The protein banding patterns of nine individual male wild rats were similar 204 and most of the urine samples resolved to three major and a few low intensity discrete bands. This was 205 consistent with previous IEF studies of laboratory rat MUPs [22,30,52] but with fewer bands than recorded 206 for house mice [10].

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Roberts et al. [13] showed that mice are sensitive to changes in the relative ratios of MUP isoforms in urine.

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In rats, despite the absence of qualitative polymorphism between urine samples, the relative amount of 210 each isoform differed between individuals. We quantified the relative abundance of each MUP mass from 211 the peak area of the ESI-MS deconvoluted spectra, and calculated the correlation between the amounts of 212 each protein, per individual ( Figure 5). While laboratory strains showed high correlations between the 213 relative amounts of the isoforms, this was not the case for samples derived from wild caught rats. The two 214 protein masses that correlated in intensity most strongly among the wild caught individuals was the pair at  around 11 kDa, and no mass peaks in the expected range of MUPs (18-19 kDa). We have not investigated 223 these 11 kDa proteins further, but they are likely to be RUPs (rat urinary proteins). As anticipated, ESI-MS 224 provides further confirmation of the lack of MUP expression in female rats.  Table 1.

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Supplementary Figure 9 shows a comparison of the protein sequences of the predicted rat MUP isoforms 234 highlighting unique peptides for each isoform. From this detailed analysis, we could compile the evidence 235 for each of the predicted proteins in the rat gene assembly. Rnor_6.0 (v6) (July 2014) from the Rat Genome Sequencing Consortium) using the 242 annotations compiled in the Rat Genome Database (http://rgd.mcw.edu/) and Uniprot 243 (http://www.uniprot.org/). Where possible, data relating predicted mature protein product is 244 cross-correlated with experimental data that confirm true protein products.

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In female rat samples, shotgun 'bottom-up' proteomics allowed the identification of MUPs at very low 247 levels, in good agreement with previous papers establishing the presence of trace levels of MUPs in female 248 urine [29,30]. However, the very low abundance of these proteins meant that few peptides were observed 249 and the resulting protein coverage did not allow confident assignment to any of the predicted proteins. By 250 contrast, the same approach revealed the MUP isoform composition of male samples. Below, we discuss the 251 protein-level evidence for each of the genes and include information from transcripts published to date 252 (Table 1), focusing predominantly on genome assembly v4 for these assignments. For these analyses, we   of the MUP gene cluster. Further, unlike nasal MUPs in mice, which seem to be tissue specific, it is possible 265 that the same MUP could play a dual role in odour reception and scent signalling, as it is expressed at high  There is a report of phosphorylation of MUPs in Rattus rattus, specifically from the preputial gland [60]. Analysis of the protein products of the gene cluster 438 We provide a detailed analysis of the isoforms of the major urinary protein system expressed in the urine of 439 Rattus norvegicus. We characterized the urinary MUPs from two of the most widely used laboratory strains, The relative amount of each isoform was quantified based on the peak area of the deconvoluted spectra.