Exploring amino acids responsible for the temperature profile of glycoside hydrolase family 45 endoglucanase EGL3 from Humicola grisea

EGL3 and RCE1 are glycoside hydrolase family 45 endoglucanases isolated from Humicola grisea and Rhizopus oryzae respectively. The amino acid sequences of the two endoglucanases are homologous; on the other hand, the optimum temperature of EGL3 is higher than that of RCE1. In this study, four chimeric endoglucanases, named ER1, ER2, ER3 and ER4, in which one of four sequential amino acid regions of the EGL3 catalytic domain (CAD) was replaced by the corresponding RCE1 amino acids, were constructed to explore the region responsible for the EGL3 temperature profile. Then their temperature profiles were compared with that of the recombinant EGL3. Replacement of the N-terminal region of EGL3 with that of RCE1 caused the EGL3 temperature profile to shift to a lower temperature. These results suggest that the N-terminal amino acids of the EGL3 are responsible for the EGL3 temperature profile.     

Amino acid regions of family 45 endoglucanases involved in cotton defibrillation and in resistance to anionic surfactants and oxidizing agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.     

Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces

DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed.     

Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons

A molecular basis of survival from neuronal injury is essential for the development of therapeutic strategy to remedy neurodegenerative disorders. In this study, we demonstrate that an EF-hand Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1), one of the key proteins for various neuronal functions, also acts as an important survival factor. Overexpression of NCS-1 rendered cultured neurons more tolerant to cell death caused by several kinds of stressors, whereas the dominant-negative mutant (E120Q) accelerated it. In addition, NCS-1 proteins increased upon treatment with glial cell line-derived neurotrophic factor (GDNF) and mediated GDNF survival signal in an Akt (but not MAPK)-dependent manner. Furthermore, NCS-1 is significantly up-regulated in response to axotomy-induced injury in the dorsal motor nucleus of the vagus neurons of adult rats in vivo, and adenoviral overexpression of E120Q resulted in a significant loss of surviving neurons, suggesting that NCS-1 is involved in an antiapoptotic mechanism in adult motor neurons. We propose that NCS-1 is a novel survival-promoting factor up-regulated in injured neurons that mediates the GDNF survival signal via the phosphatidylinositol 3-kinase-Akt pathway.     

Identification of a sialate O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8-linked sialic acid

We have identified a sialate O-acetyltransferase in the lipo-oligosaccharide biosynthesis locus of Campylobacter jejuni. Strains possessing this locus are known to produce sialylated outer core structures that mimic host gangliosides, and have been implicated in triggering the onset of Guillain-Barré syndrome. The acetyltransferase, which was cloned and expressed as a fusion construct in Escherichia coli, is soluble and homologous with members of the NodL-LacA-CysE family of O-acetyltransferases. This enzyme catalyzes the transfer of O-acetyl groups onto oligosaccharide-bound sialic acid, with a high specificity for terminal alpha2,8-linked residues. The modification is directed to C-9 and not C-7 as is believed to occur more commonly in other organisms. Despite their wide prevalence and importance in both eukaryotes and prokaryotes, this is the first report to describe the characterization of a purified sialate O-acetyltransferase.     

TLR-dependent induction of IFN-beta mediates host defense against Trypanosoma cruzi

Host resistance to the intracellular protozoan parasite Trypanosoma cruzi depends on IFN-gamma production by T cells and NK cells. However, the involvement of innate immunity in host resistance to T. cruzi remains unclear. In the present study, we investigated host defense against T. cruzi by focusing on innate immunity. Macrophages and dendritic cells (DCs) from MyD88(-/-)TRIF(-/-) mice, in which TLR-dependent activation of innate immunity was abolished, were defective in the clearance of T. cruzi and showed impaired induction of IFN-beta during T. cruzi infection. Neutralization of IFN-beta in MyD88(-/-) macrophages led to enhanced T. cruzi growth. Cells from MyD88(-/-)IFNAR1(-/-) mice also showed impaired T. cruzi clearance. Furthermore, both MyD88(-/-)TRIF(-/-) and MyD88(-/-)IFNAR1(-/-) mice were highly susceptible to in vivo T. cruzi infection, highlighting the involvement of innate immune responses in T. cruzi infection. We further analyzed the molecular mechanisms for the IFN-beta-mediated antitrypanosomal innate immune responses. MyD88(-/-)TRIF(-/-) and MyD88(-/-)IFNAR1(-/-) macrophages and DCs exhibited defective induction of the GTPase IFN-inducible p47 (IRG47) after T. cruzi infection. RNA interference-mediated reduction of IRG47 expression in MyD88(-/-) macrophages resulted in increased intracellular growth of T. cruzi. These findings suggest that TLR-dependent expression of IFN-beta is involved in resistance to T. cruzi infection through the induction of IRG47.     

Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid beta-peptide cleaved from a recombinant fusion protein

The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (Abeta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Abeta(1-40) is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Abeta by pitrilysin, an overproduction system of Abeta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Abeta(1-40) derivative, Abeta(1-40)*, in which Lys16 and Lys28 of Abeta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Abeta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. Abeta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Abeta(1-40)* is conformationally similar to Abeta(1-40). However, the thioflavin T binding assay suggests that Abeta(1-40)* is more amyloidogenic than Abeta(1-40). Nevertheless, Abeta(1-40)* was cleaved by pitrilysin at the site identical to that in Abeta(1-40).