Importance of N-glycosylation positioning for secretion and folding of ovalbumin

Ito, Kazunari; Ishimaru, Takayuki; Kimura, Fukiko; Matsudomi, Naotoshi

doi:10.1016/j.bbrc.2007.07.066

Cited by 21 publications

(20 citation statements)

References 31 publications

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“…Except for N13S all the produced HRP glyco-variants showed catalytic activity; however, replacement of Asn by Gln never turned out to be the most suitable mutation at any of the eight N-glycosylation sites. Since we did not measure any detectable extracellular protein content for N13S either, we speculate that this mutation caused problems in protein folding and/or secretion, a phenomenon described before (Zhu et al 1998; Ito, Ishimaru, et al 2007; Ito, Seri, et al 2007; Zou et al 2013). Interestingly, we observed significant differences in enzyme activity and stability depending on the introduced mutation (Table I), and identified three mutations which had been described before, namely at positions N13 and N268 (Asad, Khajeh, et al 2011) and N255 (Lin et al 1999), respectively.…”

Section: Resultsmentioning

confidence: 70%

“…Surprisingly, when we calculated the average-specific methanol uptake rate during the consecutive 1% (v/v) methanol pulses ( q s average MeOH ), we observed striking differences between all the strains. One can speculate that the reason for the altered strain physiology lies in the produced HRP glyco-variant itself, since it is known that N-glycosylation can influence protein folding and protein production and thus might affect cell physiology (Zhu et al 1998; Ito, Ishimaru, et al 2007; Ito, Seri, et al 2007; Zou et al 2013). However, as shown in Table II, the amount of total extracellular protein for each strain at the end of the dynamic batch cultivation was basically the same, indicating that the single mutations did not cause significant problems in protein folding or secretion.…”

Section: Resultsmentioning

confidence: 99%

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Glyco-variant library of the versatile enzyme horseradish peroxidase

et al. 2014

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When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library.

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Section: Resultsmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Glyco-variant library of the versatile enzyme horseradish peroxidase

et al. 2014

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“…To circumvent this, we expressed wild type and N -linked glycan mutated forms of NS1 as a transgene using a double subgenomic SINV. Rather than generate N→A mutations that abolish the N -linked glycan site (Crabtree, Kinney, and Miller, 2005; Muylaert et al, 1996; Pryor et al, 1998; Pryor and Wright, 1994; Tajima, Takasaki, and Kurane, 2008), we engineered, like several other groups, the more minimal N→Q to retain polarity and size of the amino acid side chain (Ito et al, 2007a; Ito et al, 2007b; Kayser et al, 2010; Zhou and Tsai, 2009). Using the double subgenomic expression system, we showed as expected, (a) wild type or mutant NS1 had no differential effect on SINV replication and (b) the total intracellular levels of wild type and mutant NS1 were comparably produced.…”

Section: Discussionmentioning

confidence: 99%

N-linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement

et al. 2011

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Dengue virus (DENV) NS1 is a versatile non-structural glycoprotein that is secreted as a hexamer, binds to the cell surface of infected and uninfected cells, and has immune evasive functions. DENV NS1 displays two conserved N-linked glycans at N130 and N207. In this study, we examined the role of these two N-linked glycans on NS1 secretion, stability, and function. Because some groups have reported reduced yields of infectious DENV when N130 and N207 are changed, we analyzed glycosylation-deficient NS1 phenotypes using a transgenic expression system. We show that the N-linked glycan at position 130 is required for stabilization of the secreted hexamer whereas the N-linked glycan at residue 207 facilitates secretion and extracellular protein stability. Moreover, NS1 mutants lacking an N-linked glycan at N130 did not interact efficiently with complement components C1s and C4. In summary, our results elucidate the contribution of N-linked glycosylation to the function of DENV NS1.

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“…The mutation of these N-glycosylation sites (Asn to glutamine [Q]: N292Q and N292/311Q) followed by expression in Pichia pastoris revealed that the secretion and expression of the proteins were greatly reduced compared with wild-type OVA. 17 Furthermore, they found that the N-glycan at N292 of OVA was essential for its secretion and folding in P. pastoris cells.…”

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confidence: 99%

Enhanced expression of heterologous proteins in yeast cells via the modification ofN-glycosylation sites

Han

2015

Bioengineered

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Importance of N-glycosylation positioning for secretion and folding of ovalbumin

Cited by 21 publications

References 31 publications

Glyco-variant library of the versatile enzyme horseradish peroxidase

Glyco-variant library of the versatile enzyme horseradish peroxidase

N-linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement

Enhanced expression of heterologous proteins in yeast cells via the modification ofN-glycosylation sites

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