Gen-ichiro Uechi

A plant class V chitinase from a cycad (Cycas revoluta): Biochemical characterization, cDNA isolation, and posttranslational modification

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

Construction of Human Fab (Y1/K) Library and Identification of Human Monoclonal Fab Possessing Neutralizing Potency against Japanese Encephalitis Virus

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper‐immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3 × 108 Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen‐specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction‐neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 μg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21 × 10–8 M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3–23, and used a VK sequence homologous to the VκII subgroup showing 92.8% homology to germline A17.

Identification of a human monoclonal Fab with neutralizing activity against H3N2 influenza A strain from a newly constructed human Fab library

A combinatorial Fab library was constructed in pComb3H phagemid vectors, using RNA from peripheral blood lymphocytes of a healthy volunteer who had recovered from an influenza A virus infection. The library contained approximately 1.3 × 108E. coli transformants. Bio‐panning was carried out against an influenza vaccine containing components of influenza A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Shandong/7/97 for the enrichment of phages displaying human Fab specific to the viral proteins. E. coli transformed with IF1A11, 1 of 94 randomly selected clones, displayed a human Fab antibody molecule (FabIF1A11) with efficient neutralizing activity against H3N2 influenza A virus strains. The purified FabIF1A11 demonstrated neutralizing activity against A/Okayama/6/01 (H3N2) and A/Kitakyushu/159/93 (H3N2) with 50% plaque reduction neutralization titers of 0.11 μg/ml (2.2 nM) and 1.4 μg/ml (28 nM) respectively. However, FabIF1A11 did not show neutralizing activity against the influenza A virus strain A/USSR/77 (H1N1) or the influenza B virus strain B/Kanagawa/73, even at a concentration of 20 μg/ml (400 nM). The Kd of FabIF1A11 was calculated as 3.6 × 10−9 M. FabIF1A11 was estimated to recognize a conformational epitope on the hemagglutinin of A/Okayama/6/01 (H3N2). The human monoclonal Fab product FabIF1A11 may have potential as a therapeutic or short‐term prophylactic molecule for humans with influenza A H3N2 infection.