Methods: Horse sweat was collected after exercise. Sweat was centrifuged and sterile filtered and then subjected to alcohol precipitation using 60%-75% ethanol and centrifugation. Pellets and supernatants were analyzed by SDS-PAGE and mass-spectrometry. Bactericidal activity was tested by incubating P. aeruginosa and E. coli for 2hr@37C in 10mM Na-phosphate with sweat supernatant or partially-purified latherin. Surviving bacteria (CFU) were enumerated on agar plates. Agglutination was visualized by microscopy of bacteria in the presence or absence of sweat supernatant or partially purified latherin or BSP30. The agglutinating peptide GL13NH2 was used as a positive control
Result: Latherin is partly soluble in 75% ethanol, as has been shown for related bovine and human salivary proteins. Incubation of P. aeruginosa or E. coli (2x107 and 2x105CFU/ml) in 10mM sodium phosphate with horse sweat supernatant (1.4 mg-protein/ml) or partially-purified latherin (150 µg/ml) did not reduce the number of viable bacteria. Control peptides killed 100% of both bacterial species. Bovine BSP30(100 µg/ml) caused agglutination of P. aeruginosa and E. coli. In contrast, neither sweat supernatant nor partially purified latherin showed agglutination activity.
Conclusion: Latherin exhibits similar solubility properties as other BPI-like proteins. Despite the predicted structural similarity, the biological function of latherin appears to differ from that of the human and bovine salivary proteins BSP30 and PSP. Latherin is not bactericidal and does not promote bacterial agglutination. These results suggest that members of the BPI-superfamily that are expessed in saliva of horse, cow and humans have divergent biological functions.
Keywords: Animal, Bacterial, Biochemistry, Proteins and Saliva