Time-lapse monitoring of TLR2 ligand internalization with newly developed fluorescent probes.

As a mammalian toll-like receptor family member protein, TLR2 recognizes lipoproteins from bacteria and modulates the immune response by inducing the expression of various cytokines. We have developed fluorescence-labeled TLR2 ligands with either hydrophilic or hydrophobic fluorescence groups. The labeled ligands maintained the inflammatory IL-6 induction activity and enabled us to observe the internalization and colocalization of the TLR2 ligands using live-cell imaging. The time-lapse monitoring in the live-cell imaging of the fluorescence-labeled TLR2 ligand showed that TLR2/CD14 expression in the host cells enhanced the internalization of TLR2 ligand molecules.

A Comprehensive Study of the Interaction between Peptidoglycan Fragments and the Extracellular Domain of Mycobacterium tuberculosis Ser/Thr Kinase PknB.

The Mycobacterium tuberculosis Ser/Thr kinase PknB is implicated in the regulation of bacterial cell growth and cell division. The intracellular kinase function of PknB is thought to be triggered by peptidoglycan (PGN) fragments that are recognized by the extracytoplasmic domain of PknB. The PGN in the cell wall of M. tuberculosis has several unusual modifications, including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues and amidation of d-Glu in the peptide chains. Using synthetic PGN fragments incorporating these diverse PGN structures, we analyzed their binding characters through biolayer interferometry (BLI), NMR spectroscopy, and native mass spectrometry (nMS) techniques. The results of BLI showed that muropeptides containing 1,6-anhydro-MurNAc and longer glycan chains exhibited higher binding potency and that the fourth amino acid of the peptide stem, d-Ala, was crucial for protein recognition. Saturation transfer difference (STD) NMR spectroscopy indicated the major involvement of the stem peptide region in the PASTA-PGN fragment binding. nMS suggested that the binding stoichiometry was 1:1. The data provide the first molecular basis for the specific interaction of PGN with PknB and firmly establish PGNs as the effective ligands of PknB.

Total Synthesis of Cardiolipins Containing Chiral Cyclopropane Fatty Acids.

Cardiolipin (CL) is a phospholipid located in both the eukaryotic mitochondrial inner membrane and the bacterial cell membrane. Some bacterial CLs are known to contain cyclopropane moieties in their acyl chains. Although the CLs are thought to be involved in the innate immune response, there have been few attempts at chemical synthesis of the CLs, and detailed studies of their biological activities are scarce. Thus, we have developed a synthetic route to CLs containing chiral cyclopropane moieties.

Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis.

A screening assay for inhibitory activity against trypsin in skin mucus from 29 species of fishes reveals a wide distribution of trypsin inhibitors in skin mucus and relatively high antitryptic activity in pufferfish of the family Tetraodontidae. Two trypsin inhibitors termed TPTI 1 and 2 were purified to homogeneity from the skin mucus of Takifugu pardalis by salting out, lectin affinity, anion exchange FPLC and gel filtration HPLC. Both inhibitors are acidic glycoproteins, with an apparent molecular mass of 57 kDa in SDS-PAGE, pI below 4 and 1.9% reducing sugar for TPTI 1 and with an apparent molecular mass of 47 kDa in SDS-PAGE, pI 5.2 and 0.8% reducing sugar for TPTI 2. The inhibitors effectively repress the catalytic activity of trypsin and alpha-chymotrypsin, and therefore can be classified as serine protease inhibitors. The inhibitory constants against trypsin were 4.9x10(-8) M for TPTI 1 and 3.9x10(-8) M for TPTI 2. Both inhibitors react with trypsin at a molar ratio of 1:1, although TPTI 1 reversibly inactivates the proteolytic activity of trypsin non-competitively and TPTI 2, competitively. The trypsin inhibitors in the skin mucus of T. pardalis may function as defense substances to neutralize serine proteases released by invasive pathogens.