Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Hepatitis B virus upregulates host expression of α-1,2-mannosidases via the PPARα pathway

AIM To assess the effects of hepatitis B virus(HBV) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms.METHODS We measured the expression levels of MAN1A1, MAN1A2, MAN1B1, and MAN1C1 in cell lines HepG 2.2.15, HepN 10, HepA D38 and Hep G2 by Western blot. Viral antigens(HBs Ag and HBe Ag) in the culture medium were measured using the chemiluminescence method. HBV DNA quantification assays were performed using a commercial real-time PCR kit. Protein levels of human liver tissue α-1,2-mannosidases were also evaluated by Western blot. Plasmids containing seven individual viral genes of HBV(PTT22-HBx, PTT22-HBs, PTT22-pre S2, PTT22-pre S1, PTT22-HBc, PTT22-HBe, and PTT22-HBp) or control plasmids(PTT22-vector) were transfected into Hep G2 cells. MK886(PPARα) and GW9662(PPARγ) inhibitors were used to explore the effects of HBV on α-1,2-mannosidase expression after the PPARα and PPARγ pathways were blocked.RESULTS We showed that the expression of α-1,2-mannosidases was higher in stably transfected HBV cells than in controls. The expression levels of α-1,2-mannosidase were higher in AD38 cells than those in ND10 cells, which were in turn greater than those in G2.2.15 cells, and positively correlated with the expression of HBsA gin all the cell lines. Levels of α-1,2-mannosidase in nontumorous liver tissues of HBV-related HCC patients were also higher than in the tissues from non-HBVrelated HCC patients. Moreover, transfecting Hep G2 cells with a component of the HBV viral envelope also increased the expression of α-1,2-mannosidases. However, this envelope protein component could not induce MAN1C1 expression in the presence of a PPARα inhibitor, MK886. We also found that MK886 did not affect the expression of MAN1C1 in AD38 cells without tetracycline in the culture medium. This phenomenon was not observed in the case of GW9662.CONCLUSION Our results indicate that HBV increases the expression of α-mannosidases both in vitro and in vivo via activation of the PPARα pathway by its envelope protein.

Effect of Oxytropis glabra DC. Poisoning on α-Mannosidase(AMA) Expression in Mice Brain Tissue

The effect of Oxytropis glabra DC. on α-mannosidase( AMA) expression in mice brain tissue was explored to reveal the toxicity mechanism of O. glabra. Forty mice were randomly divided into four groups,namely control group,experimental group I,experimental group II and experimental group III. The mice in three experimental groups were fed with O. glabra at the doses of 1,5 and 10 g per kilogram weight,respectively. After challenge for 63 d,mice brains were collected to detect changes in distribution and expression of AMA in different brain regions. The results showed that O. glabra poisoning led to declined AMA mRNA expression in mice brain tissue,but the mice in experimental group I had no significant difference with those in control group( P > 0. 05). The AMA mRNA expression in cerebellum,cerebrum and thalamus of mice in experimental groups II and III were significantly lower than that in control group( P < 0. 05),but the AMA mRNA expression in hippocampus and brainstem in three experimental groups had no significant difference with that in control group( P > 0. 05). AMA had very weak expression in hippocampus and brainstem,but it had expressions in other regions,and the expression was positively correlated with the number of neurons and granulosa cells. The results showed that different doses of O. glabra reduced AMA mRNA expression in mice brain tissue,while cerebellum,cerebrum and thalamus were the main target function areas.

Changes in Activities of Three Enzymes Degrading Galactomannan During and Following Rice Seed Germination

To investigate the relationships among β-mannanase, β-mannosidase and a-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA3 on them were surveyed. The activities of β-mannosidase and a-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA3 could promote the activities of β-mannanase and a-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase.

Trimming of N-Glycans by the Golgi-Localized α-1,2-Mannosidases, MNS1 and MNS2, Is Crucial for Maintaining RSW2 Protein Abundance during Salt Stress in Arabidopsis

Asparagine （Asn/N）-Iinked glycans are important for protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotes. The maturation of glycoproteins involves the trimming of mannosyl residues by mannosidases and addition of other sugar molecules to three-branched N-glycans in the Golgi. However, the biological importance of Golgi-mediated mannose trimming is not fully understood. Here, we show that abolishment of two functionally redundant mannosidases, MNS1 and MNS2, responsible for α-1,2-mannose trimming on the A and C branches of plant N-glycans lead to severe root growth inhibition under salt stress conditions in Arabidopsis. In contrast, mutants with defects in the biosynthesis of the oligosaccharide precursor displayed enhanced salt tolerance in the absence of mannose trimming. However, mutation in EBS3, which is required for the formation of the branched N-glycan precursor, suppressed the salt-sensitive phenotype of mnsl mns2 double mutant. Interestingly, we observed that cellulose biosynthesis was compromised in mnsl mns2 roots under high salinity. Consistently, abundance of a membrane anchored endo-13-1,4-endoglucanase （RSW2/KOR） that plays a key role in cellulose biosynthesis and its mutant variant rsw2-1 were modulated by α-1,2-mannose trimming under salt stress. Overexpression of RSW2 could partially rescue the salt-sensitive phenotype of mnsl mns2. Taken together, these results suggest that MNS1/2-mediated mannose trimming of N-glycans is crucial in modulating glycoprotein abundance to withstand salt stress in plants.

Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 a-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 α-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa's staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa's staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 α-mannosidase. The transduction did not affect the expression of Fas molecule on cells.

Directional screening and identification of potential cytotoxic components from Achnatherum inebrians by a combination of surface palsmon resonance and chromatography

Objective:To establish a method for directional screening of the cytotoxic components from the medicinal herb of Achnatherum inebrians by a combination of surface plasmon resonance(SPR)biosensor and chromatographic isolation technology.Methods:Under the guidance of bioactive assessment based on binding abilities between objects and the a-Mannosidase(a-Man)target,the active components from different solvents extracts,different polar extraction parts and fractions were screened orderly and directionally using SPR.Components with a high binding ability to a-Man can be precisely oriented in a narrower fractions range and are easy to isolate.Three human cancer cells were used to evaluate the cytotoxic activity of component with the highest affinity to a-Man.Results:Eight compounds were isolated and identificated from A.inebrians for the first time.Deoxyvasicinone possessed the highest affinity to a-Man among them.Moreover,deoxyvasicinone showed good effects on inhibited proliferation of human hepatoma cells HepG2(IC_(50)=5.7 μmol/L),human breast cancer cells MCF7(IC_(50)=7.21 μmol/L)and human lung cancer cells HCC827(IC_(50)=0.75 μmol/L),respectively.In particular,its inhibitory effect on HCC827 was stronger than the positive drug gefitinib(IC_(50)=1.65 μmol/L).Conclusion:A comprehensive strategy of directional screening potential cytotoxic components from herb based on biomolecular interaction and chromatography was established.Deoxyvasicinone as an effective anti-cancer component was initially isolated from A.inebrians.It is expected that this screening strategy could provide new perspectives for rapid screening and identification of active components from natural plants with the complex matrix.

Inhibition of metastasis to lung of a human nasopharyngeal carcinoma cell line CNE-2L2 transfected with pRc/CMV-antisense 6A8 cDNA in nude mice

The growth of CNE-2L2 cell, a cloned line of human nasopharyngeal carcinoma with a high potentiality of metastasis to lung was inhibited to a certain extent after transfection with a recombinant antisense expression vector of a cDNA encoding a human a-mannosidase (pRc/CMV-antisense 6A8 cDNA) (the Genbank accession number of 6A8 cDNA is U37248) in comparison with that of the cell transfected with the Mock and of the wild cell. Two months after a subcutaneous inoculation of CNE-2L2 cell into the axilla of nude mice metastatic lesions in the lung were observed in 9/10 mice (90%) with grade III in 8 mice and grade II in one mouse in the wild cell group, in 6/8 mice (75%) with grade III in one mouse, grade II in 2 mice and grade I in 3 mice in the Mock-transfection group, in only 3/10 mice (30%) with all grade I in pRc/CMV-antisense 6A8 cDNA-transfection group.