Positive Selection Shaped the Convergent Evolution of Independently Expanded Kallikrein Subfamilies Expressed in Mouse and Rat Saliva Proteomes

We performed proteomics studies of salivas from the genome mouse (C57BL/6 strain) and the genome rat (BN/SsNHsd/Mcwi strain). Our goal was to identify salivary proteins with one or more of three characteristics that may indicate that they have been involved in adaptation: 1) rapid expansion of their gene families; 2) footprints of positive selection; and/or 3) sex-limited expression. The results of our proteomics studies allow direct comparison of the proteins expressed and their levels between the sexes of the two rodent species. Twelve members of the Mus musculus species-specific kallikrein subfamily Klk1b showed sex-limited expression in the mouse saliva proteomes. By contrast, we did not find any of the Rattus norvegicus species-specific kallikrein subfamily Klk1c proteins in male or female genome rat, nor transcripts in their submandibular glands. On the other hand, we detected expression of this family as transcripts in the submandibular glands of both sexes of Sprague-Dawley rats. Using the CODEML program in the PAML package, we demonstrate that the two rodent kallikrein subfamilies have apparently evolved rapidly under the influence of positive selection that continually remodeled the amino acid sites on the same face in the members of the subfamilies. Thus, although their kallikrein subfamily expansions were independent, this evolutionary pattern has occurred in parallel in the two rodent species, suggesting a form of convergent evolution at the molecular level. On the basis of this new data, we suggest that the previous speculative function of the species-specific rodent kallikreins as important solely in wound healing in males be investigated further. In addition to or instead of that function, we propose that their sex-limited expression, coupled with their rapid evolution may be clues to an as-yet-undetermined interaction between the sexes

Lipopolysaccharide-induced elevation and secretion of interleukin-1β in the submandibular gland of male mice*

The intraperitoneal injection of lipopolysaccharide (LPS) (400 µg/kg body weight) induced the expression of mRNAs of inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α in the submandibular gland (SMG) of C3H/HeN mice but not that of C3H/HeJ mice, a mutant strain for Toll-like receptor-4 (TLR-4(–) mutant). The mRNA levels of these cytokines in the SMG of the wild-type mice increased as early as 3 hr after injection, peaked at 3–6 hr, and had decreased again by 24 hr. In this study, we particularly focused on IL-1β, and induction by this endotoxin was investigated in detail. Denervation of the superior cervical trunk and chorda tympani nerve did not diminish the LPS-induced elevation of IL-1β mRNA in the SMG, indicating the irrelevance of the central nervous system in this induction. TLR-4 mRNA and protein were shown to be strongly expressed in the SMG, suggesting the direct action of LPS on this gland. IL-1β proteins were localized in the secretory granules of granular convoluted tubular (GCT) cells, and their molecular weights in the gland were 17·5 and 20 kDa. IL-1β of the same size appeared in the saliva 6 hr after LPS injection in C3H/HeN but not in C3H/HeJ mice. The present study thus suggests that IL-1β, an inflammation cytokine, is induced and secreted into the saliva in response to endotoxin injected intraperitoneally