Construction of yeast Pichia pastoris to produce Man5GlcNAc2 mammalian mannose-type glycoprotein / 生物工程学报

Glycosylation is vital for activity, higher structure and function of protein. Glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated, while those from mammalian cells contain N-glycan of hybrid or complex type. We introduced the alpha-1,2-mannosidase I (MDSI) into yeast cells, which catalyzed an essential proceeding of N-glycan structures from Man8GlcNAc2 to Man5GlcNAc2. The plasmids contained MDSI genes from Homo sapiens [HMDSI(delta185)] or Arabidopsis thaliana [ATMDSI(delta48)], and three ER-signals were used to be transformed a mutant Pichia pastoris GJK01, respectively. The reporter protein HSA/GM-CSF (human serum albumin and granulocyte-macrophage colony stimulating factor fusion protein) was expressed and its N-glycans were analyzed by DSA-FACE (DNA sequencer assisted fluorophore-assisted carbohydrate electrophoresis). The plasmid contained ER-ScMnsI-ATMDSI(delta48) was expressed in Pichia pastoris, the Man5GlcNAc2 N-glycan on secreted glycoprotein HSA/GM-CSF was observed. The research reported here provided basic substrate to obtain the hybrid- and complex-type glycans in mammalian cell.

The Enzymatic Approach of Zygomycosis - Causing Mucorales / 대한의진균학회지

Various oxidases and hydrolytic enzymes were analyzed to investigate the relationship between these enzymes and the skin pathogenicity of 18 Mucorales strains. Each strain was cultured in a nutrient medium containing starch as a carbon source. The cells grew quickly and were at a good state of growth after incubation for three days. Oxidase activity was not detected in any strain, whereas Mucor spp. including Mucor racemosus IFM47053 typically had high alcohol dehydrogenase (ADH) activity and all the strains had catalase activity. The culture filtrate and the cell free extract of each strain were applied to APIZYM test system, which revealed that all the strains examined produced many hydrolytic enzymes both inside and outside their mycelia. In the case of Absidia corymbifera strains, lipase activity was comparatively high, and polysaccharide hydrolytic enzymes such as alpha-glucosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and alpha-fucosidase were produced.

Differential gene expression in nasopharyngeal carcinoma cell with reduced and normal expression of 6A8 alpha-mannosidase / 中国医学科学院学报

<p><b>OBJECTIVE</b>To detect the differential display of mRNA expression between human nasopharyngeal carcinoma cell CNE-2L2 with reduced malignancy caused by transduction of a DNA antisense to 6A8 alpha-mannosidase cDNA (AS cell) and the wild type cell (W cell).</p><p><b>METHODS</b>Differential display of mRNA expression was analyzed using DNA microarray analysis. The datasets were confirmed by Northern blotting and RT-PCR.</p><p><b>RESULTS</b>Out of the 1069 genes analyzed, 34 genes were up-regulated in AS cells relative to W cells. Conversely, 42 genes were down-regulated. The genes, up-regulation of which might have suppressive effect on tumor malignant behaviors, were P130 mRNA for 130K protein, TGF-betaIIR alpha, GABBR1, TGFBR1, TNFAIP1, STANIN, E-CADHERIN, CTNNA1 and 2, RFX2, TMPO, etc. The genes, down-regulation of which might have suppressive effect on tumor malignant behaviors, were CD44, NDRG1, TGFB1, RPS5, LEGUMAIIN, CBS, CD59, SNRPA1, etc. The microarray datasets were confirmed by Northern blot and RT-PCR analysis.</p><p><b>CONCLUSIONS</b>In comparison to the W cell, AS cell has up-regulation of 34 genes and down-regulation of 42 genes. Changes of the gene expression may play a role in the malignancy reduction of AS cell.</p>

Inhibition of 6A8 alpha-manosidase expression induces decrease of adhesion to laminin and reduction of lamellipodia of human nasopharyngeal carcinoma cell CNE-2L2 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of 6A8 alpha-manosidase expression on the adhesiveness of CNE-2L2 cells to laminin and the lamellipodia on cell surface.</p><p><b>METHODS</b>6A8 alpha-manosidase expression was detected by Western blotting. For assaying the adhesion of cells to laminin, cells were incubated in laminin-coated plate at 37 degrees C for 1 h, the adhered cells were stained with crystal purple dissolved in 0.1 mol/L Sodium Citrate/50% ethanol. Absorbance 540 nm was measured. Adhesion rate (R) was calculated according to formula R = AT/A100 x 100%. Here A100 represents 100% adhesion. lamellipodia on cell surface was observed upon a scanning electron microscopy.</p><p><b>RESULTS</b>The adhesion rate of two clones (AS1 and AS2) with inhibition of 6A8 alpha-manosidase expression to laminin was 0.447 +/- 0.096 and 0.533 +/- 0.065 respectively. The adhesion rate of three controls with normal expression of 6A8 alpha-manosidase to laminin was 0.78 +/- 0.035, 0.7 +/- 0.05 and 0.80 +/- 0.04 respectively. The difference was significant (P < 0.01). CNE-2L2 cells with normal expression of 6A8 alpha-manosidase was rich in lamellipodia on their surface. Lamellipodia nearly disappeared on the cells with inhibition of 6A8 alpha-manosidase expression.</p><p><b>CONCLUSIONS</b>Inhibition of 6A8 alpha-manosidase expression results in decrease of adhesion to laminin and reduction of lamellipodia of human nasopharyngeal carcinoma cell CNE-2L2.</p>

Molecular mechanism of metastasis inhibition of nasopharyngeal carcinoma cell CNE-2L2 induced by reduction of 6A8 alpha-mannosidase expression / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of inhibition of 6A8 alpha-mannosidase expression on adhesiveness among and E-cadherin expression on CNE-2L2 cells, and on metastasis of the tumors from the cells inoculated in nude mice.</p><p><b>METHODS</b>Anchorage-independent adhesion among cells was examined in soft agar culture. E-cadherin expression was studied by immunofluorescence staining, immunohistological staining and RT-PCR. CNE-2L2 cells were subcutaneously inoculated into nude mice. Eight weeks later tumor metastasis was demonstrated by means of histological examination of lung sections.</p><p><b>RESULTS</b>CNE-2L2 cells with suppression of 6A8 alpha-mannosidase expression (AS) became aggregated. E-cadherin expression on wild type cells was very weak. In contrast, it was greatly enhanced on AS cells. The enhancement was detected on both protein and mRNA levels. Lung metastasis of the tumor from inoculated AS cells were heavily inhibited in nude mice.</p><p><b>CONCLUSION</b>Inhibition of 6A8 alpha-mannosidase expression results in enhancement of cell-cell adhesion and of E-cadherin expression on CNE-2L2 cells. Lung metastasis of the tumor grown from AS cell inoculate in nude mice is heavily suppressed.</p>

Inhibition of ER alpha-mannosidase expression causes reduction and shortening of microvilli on rat liver epithelial cell WB-F344 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of N-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell WB-F344 and the growth of the cells in culture.</p><p><b>METHODS</b>Recombinant adeno-associated virus (rAAV) expression vector pAGX (+) containing an antisense or a sense fragment of 6A8 cDNA encoding a human alpha-mannosidase was constructed. The recombinant vectors or the mock were transfected into WB-F344 cells by means of lipofectAmine. The transfected cells were selected in G418 medium and cloned by means of limiting dilution. Integration of the transfected DNA into host DNA was detected by neo PCR. Rat liver ER alpha-mannosidase activity in cell supernatant was measured by using P-nitrophenyl-alpha-D-mannopyranoside as a substrate. Microvilli on cell surface were observed upon a scan electron microscope. The growth curves of the cells in culture were drawn.</p><p><b>RESULTS</b>The cell clones transfected with antisense 6A8 showed reduction of ER alpha-mannosidase activity with various degrees. Clone AS1 and AS2 cell showed a pronounced reduction of the enzymatic activity. In the study on AS1 cells, Con A binding to the cells was found to be enhanced, cell growth in culture became slow from day 5. The microvilli on the cells were reduced and blunted.</p><p><b>CONCLUSIONS</b>Transfection with antisense 6A8 resulted in reduction and blunting of microvilli on the surface of growing WB-F344 cells, which might be related to N-glycosylation modification.</p>

A comparative study on hydrolase activities in Acanthamoeba culbertsoni and A. royreba

Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp. and Naegleria sp. has also been reported since the latter half of 1970's. However, no information on hydrolase activities in Acanthamoeba sp. is available. Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A. royreba. Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice. Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively. The results are summarized as follows: The mice infected with A. culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days. Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A. royreba did not. Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta- N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A. royreba. A. culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A. royreba did not show any specific cytotoxicity. About 80 % of the target cells mixed with A. culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation. Hydrolase activities in A. culbertsoni cultured with the target cell line were assayed according to the culture time. The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba extracts than in culture media up to 120 hours after cultivation, but after 120 hours of cultivation those activities were detected higher in culture media than in the amoeba lysates. Neutral proteinase activity in A. culbertsoni increased more in EBSS medium than in the lysate specimens although the activity in the extracts was generally steady according to the cultivation time. Summarizing the above results, it is concluded that there were differences in hydrolase activities between pathogenic A. culbertsoni and non-pathogenic A. royreba, and that some hydrolase activities were detected remarkably higher in A. culbertsoni which revealed strong cytotoxicity to the target CHO cell line.