Gene Cloning, Characterization, and Molecular Simulations of a Novel Recombinant Chitinase from Chitinibacter Tainanensis CT01 Appropriate for Chitin Enzymatic Hydrolysis.

Chitin, a polymer of N-acetyl-d-glucosamine (GlcNAc), can be degraded by chitinase, which is produced by higher plants, vertebrates, and bacteria. Chitinases are characterized by the ability to hydrolyze the beta-1,4-linkages in the chitin chain by either an endolytic or an exolytic mechanism. Chitinase 1198 is a novel endochitinase from the genome sequence of CT01.

Neuroprotective effect of tempeh against lipopolysaccharide-induced damage in BV-2 microglial cells.

This study evaluated the bioactive composition of tempeh products and examined the effects of tempeh on BV-2 microglial cell cytotoxicity, neurotrophic effects, and expression of inflammatory genes. Tempeh products included soybean fermented by , soybean fermented through cocultivation with and , and red bean fermented through cocultivation with and (RT-C). We analyzed the bioactive contents of tempeh extracts and evaluated the effects of tempeh water extract on lipopolysaccharide (LPS)-treated BV-2 cells.

Distal M domain of cobra ADAM-like metalloproteinase mediates the binding of positively charged cysteine-rich domain to αvβ3 integrin in the suppression of cell migration.

We have previously identified two new P-III type ADAM-like snake venom metalloproteinases (SVMPs), i.e., atragin and kaouthiagin-like, from Taiwan cobra venom and determined their 3D structures with a distinct C- and I-shaped metalloproteinase/disintegrin-like/cysteine-rich (MDC) modular architecture.

Identification of a new androgen receptor (AR) co-regulator BUD31 and related peptides to suppress wild-type and mutated AR-mediated prostate cancer growth via peptide screening and X-ray structure analysis.

Treatment with individual anti-androgens is associated with the development of hot-spot mutations in the androgen receptor (AR). Here, we found that anti-androgens-mt-ARs have similar binary structure to the 5α-dihydrotestosterone-wt-AR. Phage display revealed that these ARs bound to similar peptides, including BUD31, containing an Fxx(F/H/L/W/Y)Y motif cluster with Tyr in the +5 position.

Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes.

The role of sulfatide lipid domains in the membrane pore-forming activity of cobra cardiotoxin.

Cobra CTX A3, the major cardiotoxin (CTX) from Naja atra, is a cytotoxic, basic β-sheet polypeptide that is known to induce a transient membrane leakage of cardiomyocytes through a sulfatide-dependent CTX membrane pore formation and internalization mechanism. The molecular specificity of CTX A3-sulfatide interaction at atomic levels has also been shown by both nuclear magnetic resonance (NMR) and X-ray diffraction techniques to reveal a role of CTX-induced sulfatide conformational changes for CTX A3 binding and dimer formation. In this study, we investigate the role of sulfatide lipid domains in CTX pore formation by various biophysical methods, including fluorescence imaging and atomic force microscopy, and suggest an important role of liquid-disordered (ld) and solid-ordered (so) phase boundary in lipid domains to facilitate the process.

Cell surface heparan sulfates mediate internalization of the PWWP/HATH domain of HDGF via macropinocytosis to fine-tune cell signalling processes involved in fibroblast cell migration.

HDGF (hepatoma-derived growth factor) stimulates cell proliferation by functioning on both sides of the plasma membrane as a ligand for membrane receptor binding to trigger cell signalling and as a stimulator for DNA synthesis in the nucleus. Although HDGF was initially identified as a secretory heparin-binding protein, the biological significance of its heparin-binding ability remains to be determined. In the present study we demonstrate that cells devoid of surface HS (heparan sulfate) were unable to internalize HDGF, HATH (N-terminal domain of HDGF consisting of amino acid residues 1-100, including the PWWP motif) and HATH(K96A) (single-site mutant form of HATH devoid of receptor binding activity), suggesting that the binding of HATH to surface HS is important for HDGF internalization.

Role of heparan sulfates and glycosphingolipids in the pore formation of basic polypeptides of cobra cardiotoxin.

Cobra venom contains cardiotoxins (CTXs) that induce tissue necrosis and systolic heart arrest in bitten victims. CTX-induced membrane pore formation is one of the major mechanisms responsible for the venom's designated cytotoxicity. This chapter examines how glycoconjugates such as heparan sulfates (HS) and glycosphingolipids, located respectively in the extracellular matrix and lipid bilayers of the cell membranes, facilitate CTX pore formation.

Structures of two elapid snake venom metalloproteases with distinct activities highlight the disulfide patterns in the D domain of ADAMalysin family proteins.

The structures of snake venom metalloproteases (SVMPs) are proposed to be useful models to understand the structural and functional relationship of ADAM (a disintegrin and metalloprotease) which are membrane-anchored proteins involved in multiple human diseases. We have purified, sequenced and determined the structures of two new P-III SVMPs - atragin and kaouthiagin-like (K-like) from Naja atra. Atragin exhibits a known C-shaped topology, whereas K-like adopts an I-shaped conformation because of the distinct disulfide pattern in the disintegrin-like (D) domain.

Role of glycosphingolipid conformational change in membrane pore forming activity of cobra cardiotoxin.

The major cardiotoxin from Taiwan cobra (CTX A3) is a pore forming beta-sheet polypeptide that requires sulfatide (sulfogalactosylceramide, SGC) on the plasma membrane of cardiomyocytes for CTX-induced membrane leakage and cell internalization. Herein, we demonstrate by fluorescence spectroscopic studies that sulfatides induce CTX A3 oligomerization in sulfatide containing phosphatidylcholine (PC) vesicles to form transient pores with pore size and lifetime in the range of about 30 A and 10(-2) s, respectively. These values are consistent with the CTX A3-induced conductance and mean lifetime determined previously by using patch-clamp electrophysiological experiments on the plasma membrane of H9C2 cells.

Non-cytotoxic cobra cardiotoxin A5 binds to alpha(v)beta3 integrin and inhibits bone resorption. Identification of cardiotoxins as non-RGD integrin-binding proteins of the Ly-6 family.

Severe tissue necrosis with a retarded wound healing process is a major symptom of a cobra snakebite. Cardiotoxins (CTXs) are major components of cobra venoms that belong to the Ly-6 protein family and are implicated in tissue damage. The interaction of the major CTX from Taiwan cobra, i.

Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, and CD analysis of the effect of cis-proline conformation on three-fingered toxins.

Natural homologues of cobra cardiotoxins (CTXs) were classified into two structural subclasses of group I and II based on the amino acid sequence and circular dichroism analysis, but the exact differences in their three-dimensional structures and biological significance remain elusive. We show by circular dichroism, NMR spectroscopic, and X-ray crystallographic analyses of a newly purified group I CTX A6 from eastern Taiwan cobra (Naja atra) venoms that its loop I conformation adopts a type VIa turn with a cis peptide bond located between two proline residues of PPxY. A similar "banana-twisted" conformation can be observed in other group I CTXs and also in cyclolinopeptide A and its analogues.

Peripheral binding mode and penetration depth of cobra cardiotoxin on phospholipid membranes as studied by a combined FTIR and computer simulation approach.

Cobra cardiotoxin, a cytotoxic beta-sheet basic polypeptide, is known to cause membrane leakage in many cells including human erythrocytes. Herein, we demonstrate that the major cobra cardiotoxin from Naja atra, CTX A3, can cause leakage of vesicle contents in phosphatidylglycerol (PG) and phosphatidylserine containing, but not in pure phosphatidylcholine (PC), membrane bilayers. By the combined polarized attenuated total reflection infrared spectroscopy and computer simulation studies, CTX A3 is shown to peripherally bind to both zwitterionic and anionic monolayers in a similar edgewise manner with a tilted angle of approximately 48 +/- 20 degrees between the beta-sheet plane of the CTX molecule and the normal of the membrane surface.