The urine of bank voles (Myodes glareolus) contains substantial quantities of a small protein that is expressed at much higher levels in males than females, and at higher levels in males in the breeding season. This protein was purified and completely sequenced at the protein level by mass spectrometry. Leucine/isoleucine ambiguity was completely resolved by metabolic labelling, monitoring the incorporation of dietary deuterated leucine into specific sites in the protein. The predicted mass of the sequenced protein was exactly consonant with the mass of the protein measured in bank vole urine samples, correcting for the formation of two disulfide bonds. The sequence of the protein revealed that it was a lipocalin related to aphrodisin and other odorant-binding proteins (OBPs), but differed from all OBPs previously described. The pattern of secretion in urine used for scent marking by male bank voles, and the similarity to other lipocalins used as chemical signals in rodents, suggest that this protein plays a role in male sexual and/or competitive communication. We propose the name glareosin for this novel protein to reflect the origin of the protein and to emphasize the distinction from known OBPs.

Mouse lemurs are basal primates that rely on chemo- and acoustic signalling for social interactions in their dispersed social systems. We examined the urinary protein content of two mouse lemurs species, within and outside the breeding season, to assess candidates used in species discrimination, reproductive or competitive communication. Urine from Microcebus murinus and Microcebus lehilahytsara contain a predominant 10 kDa protein, expressed in both species by some, but not all, males during the breeding season, but at very low levels by females. Mass spectrometry of the intact proteins confirmed the protein mass and revealed a 30 Da mass difference between proteins from the two species. Tandem mass spectrometry after digestion with three proteases and sequencing de novo defined the complete protein sequence and located an Ala/Thr difference between the two species that explained the 30 Da mass difference. The protein (mature form: 87 amino acids) is an atypical member of the whey acidic protein family (WFDC12). Seasonal excretion of this protein, species difference and male-specific expression during the breeding season suggest that it may have a function in intra- and/or intersexual chemical signalling in the context of reproduction, and could be a cue for sexual selection and species recognition.

Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum ( Trichosurus vulpecula ) contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling.

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