A variety of antimicrobial peptides against infection have been identified from the skin of amphibians. However, knowledge on amphibian defensins is very limited. A novel anionic defensin designated PopuDef was purified from the skin of tree frog Polypedates puerensis, and the cDNA encoding PopuDef precursor was cloned from the skin cDNA library. The amino acid sequence of PopuDef (net charge: -2, pI: 4.75) shared the highest identity of 57 % (25/44) with the salamander defensin CFBD-1 (net charge: 0, pI: 6.14) from urodela amphibians. PopuDef showed moderate antimicrobial activities against P. aeruginosa and S. aureus (MICs are 19.41 and 17.25 μM, respectively), and relatively weak activities against E. coli and B. subtilis (MICs are 38.82 and 43.14 μM, respectively). Tissue distribution analysis indicated that relatively high expression level of PopuDef mRNA was observed in immune-related tissues including skin, gut, lung and spleen. Furthermore, the expression level of PopuDef was significantly upregulated in these tissues after tree frogs were infected with different bacteria strains mentioned above. Interestingly, the induction of PopuDef challenged with E. coli or B. subtilis, which was less sensitive to PopuDef, was much higher than that did with P. aeruginosa or S. aureus. These findings highlight the key role of PopuDef in innate immunity against infection. To our knowledge, PopuDef is the first anionic defensin characterized from amphibians.

The N-glycome of human serum exosome is reported and its perspective as a source of biomarker discovery is revealed.

BackgroundBlack flies (Diptera: Simuliidae) are haematophagous insects that can cause allergic reactions and act as vectors of pathogens. Although their saliva has been thought to contain a diverse array of physiologically active molecules, little information is available on antimicrobial factors in black fly salivary glands, especially no defensins have been reported so far.MethodsA novel cationic defensin designated SibaDef was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense. The amino acid sequence of SibaDef was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-positive bacteria Staphylococcus aureus or Bacillus subtilis treated with SibaDef were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the expression of SibaDef mRNA in whole bodies of insects after oral infection with the bacteria S. aureus or B. subtilis.ResultsSurprisingly, the phylogenetic analysis of defensin-related amino acid sequences demonstrated that SibaDef is most closely related to defensins from the human body louse Pediculus humanus corporis (Anoplura: Pediculidae), rather than to other dipteran defensins. SibaDef showed potent antimicrobial activities against Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.83 μM to 2.29 μM. SEM analysis indicated that SibaDef killed microorganisms through the disruption of cell membrane integrity. The transcript levels of SibaDef in the bacteria-immunized flies increased with the time course, reaching maximum at 36 h and then slowly decreased from that time point.ConclusionsOur results indicate that SibaDef is involved in the innate humoral response of the black fly S. bannaense, and it might play a significant role in the defence against microorganisms in both sugar and blood meals.

No abstract

The identification and quantification of low-abundant proteins are always impeded by high-abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. Also, the quantification was achieved by introducing isotopically labeled threonine in the motif LPXTG. In this strategy, both the enrichment of N-terminal glycine peptides and the stable isotope labeling were achieved in a single step. We applied this approach for the proteome analysis of MCF-7 cell line. It was demonstrated a significant reduction in sample complexity via highly selective and efficient enrichment of N-terminal glycine peptides, thereby detecting lots of less abundant proteins and enhancing proteome coverage. In comparison to the untreated sample, an increase of 34% of proteins was additionally identified. Furthermore, 97% of proteins were successfully quantified with high accuracy. In summary, this quantitative N-terminal glycine peptides enrichment strategy is expected for high-throughput qualitative and quantitative proteomic analysis as a complementary approach to conventional shotgun proteomics.