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.
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.
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