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Articles by D Tsuji
Total Records ( 2 ) for D Tsuji
  H Akeboshi , Y Kasahara , D Tsuji , K Itoh , H Sakuraba , Y Chiba and Y. Jigami

Effective enzyme replacement therapy for lysosomal storage diseases requires a recombinant enzyme with highly phosphorylated N-glycans. Recombinant human β-hexosaminidase A is a potentially therapeutic enzyme for GM2-gangliosidosis. Recombinant HexA has been produced by using the methylotrophic yeast Ogataea minuta as a host, and the purified enzyme was tested for its replacement effect on cultured fibroblasts derived from GM2-gangliosidosis patients. Although the therapeutic effect was observed, in order to obtain the higher therapeutic effect with a little dose as possible, increased phosphorylation of recombinant β-hexosaminidase A N-glycans is suggested to be prerequisite. In the budding yeast Saccharomyces cerevisiae, the overexpression of MNN4, which encodes a positive regulator of mannosylphosphate transferase, led to increased mannosylphosphate contents. In the present study, we cloned OmMNN4, a homologous gene to ScMNN4, based on the genomic sequence of O. minuta. We overexpressed the cloned gene under the control of the alcohol oxidase promoter in a β-hexosaminidase A-producing yeast strain. Structural analysis of pyridylamine-labeled N-glycans by high-performance liquid chromatography revealed that the overexpression of MNN4 caused a 3-fold increase in phosphorylated N-glycans of recombinant β-hexosaminidase A. The recombinant enzyme prepared from strains overexpressing OmMNN4 was more effectively incorporated into cultured fibroblasts and neural cells, and it more rapidly degraded the accumulated GM2-ganglioside as compared to the control enzyme. These results suggest that β-hexosaminidase A produced in a strain that overexpresses OmMNN4 will act as an effective enzyme for use in replacement therapy of GM2-gangliosidosis.

  M Ohshima , K Inoue , H Hayashi , D Tsuji , M Mizugaki and K. Itoh

DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m5dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were contructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of light chains. The scFv library was selected against m5Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m5Cyd-BSA. Two scFvs with high reactivity for m5Cyd-BSA termed 1–2 and 1–12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1–2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1–2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1–2 was similar to scFv 1–2, and thus, AcGFP-scFv 1–2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.

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