X-ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase.

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10-5  s-1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.

Male parental assistance in embryo hatching of barred-chin blenny Rhabdoblennius nitidus.

Most teleostean embryos develop and hatch without parental assistance, though some receive parental care. We focused on a paternal brood-care species, the barred-chin blenny (Rhabdoblennius nitidus [Günther, 1861]). As hatching approached, fanning behavior by the male parent drastically increased and then embryos hatch. In the absence of the male parent, most embryos failed to hatch. However, the hatching rate was greatly assisted by introducing an artificial water current, suggesting that paternal assistance other than for aeration is required for successful embryo hatching. Next, we analyzed genes for the hatching enzyme and egg-envelope protein, which were successfully cloned from barred-chin blenny, and found the expression patterns differed from those of other euteleosts. Generally, high choriolytic enzyme swells the intact egg envelope, and then low choriolytic enzyme solubilizes the swollen envelope. The expression levels of both the enzymes, but especially the latter, were much lower in barred-chin blenny that is known in most other oviparous species. In addition, the main component of the egg envelope was changed into ChgHm and choriogenin L (ChgL) in barred-chin blenny, whereas ChgH and ChgL for other euteleosts. These in barred-chin blenny would result in ineffective egg-envelope digestion because the posthatching egg envelopes were observed to be swollen but not solubilized. Male parental assistance by fanning until hatching may compensate for this insufficiency. Our study illustrates an example of the evolution of parent-embryo interaction built on a novel relationship: Degradation of the hatching enzyme/egg-envelope digestion system, accompanied by male parental hatching assistance.

Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

Highly sensitive detection of influenza virus in saliva by real-time PCR method using sugar chain-immobilized gold nanoparticles; application to clinical studies.

A highly sensitive and convenient method for detecting influenza virus was developed using modified end-point melt curve analysis of a RT-qPCR SYBR Green method and influenza virus-binding sugar chain-immobilized gold-nanoparticles (SGNP). Because SGNPs capture influenza viruses, the virus-SGNP complex was separated easily by centrifugation. Viral RNA was detected at very low concentrations, suggesting that SGNP increased sensitivity compared with standard methods. This method was applied to clinical studies. Influenza viruses were detected in saliva of patients or inpatients who had been considered influenza-free by a rapid diagnostic assay of nasal swabs. Furthermore, the method was applied to a human trial of prophylactic anti-influenza properties of yogurt containing Lactobacillus acidophilus L-92. The incidence of influenza viruses in saliva of the L-92 group was found to be significantly lower compared to the control group. Thus, this method was useful for monitoring the course of anti-influenza treatment or preventive measures against nosocomial infection.