Sodium butyrate alters erythropoietin glycosylation via multiple mechanisms

Crowell, Christopher K.; Qin, Qiang; Grampp, Gustavo; Radcliffe, Richard A.; Rogers, Gary N.; Scheinman, Robert I.

doi:10.1002/bit.21539

Cited by 23 publications

(13 citation statements)

References 39 publications

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“…The changes here were more consistent if SB altered the levels or activity of the CMAH enzyme producing CMP-Neu5Gc or increased production of CMP-Neu5Ac. Crowell et al (2008) reported increased levels of CMPNeu5Ac with SB treatment of HT-1080 cells expressing EPO. A change in the ratio of CMP-Neu5Ac and CMP-Neu5Gc could potentially change Neu5Gc expression.…”

Section: Discussionmentioning

confidence: 96%

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Borys

Dalal

Abu‐Absi

et al. 2010

Biotech & Bioengineering

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CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well-known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50-62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early-temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO(2) with this product showed that the Neu5Gc levels were 46% lower at high pCO(2) conditions (140 mmHg) compared to moderate pCO(2) levels (20-80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics.

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Section: Discussionmentioning

confidence: 96%

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Borys

Dalal

Abu‐Absi

et al. 2010

Biotech & Bioengineering

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“…Furthermore, the addition of sodium butyrate and peptone into the culture media containing the transfected cells can have positive effect on the expression of target protein. However, long-term consequences of using such components in culture media remain controversial and unknown (18, 19). Results in this report demonstrate that a high level expression of Tf-fusion proteins can be achieved by the insertion of a helical peptide linker between the two protein domains.…”

Section: Discussionmentioning

confidence: 99%

Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins

2008

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Purpose To demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains. Methods Tf-based fusion proteins were designed to contain oligonucleotides encoding a helical linker inserted between the protein domains. Plasmid constructs were transfected into HEK293 cells and the secreted fusion proteins were purified from conditioned serum free media. Expression was assessed using both SDS-PAGE and Western Blot using anti-hGH, G-CSF, or Tf antibodies; protein bands were analyzed using Quantity One software. The function of fusion proteins consisting of human growth hormone (hGH) and Tf was evaluated in Nb2 cell proliferation assays. Results The fusion proteins containing a helical linker, hGH-(H4)2-Tf and Tf-(H4)2-hGH, were expressed 1.7- and 2.4-fold higher, respectively, with a 2-fold lower ED50 than the hGH-Tf fusion protein without a helical linker. The Tf-(H4)2-G-CSF fusion protein exhibited a greater expression with an 11.2-fold increase compared with Tf-G-CSF fusion protein. Conclusions The helical linker introduced in Tf-fusion proteins resulted in a high-level of expression with improved in vitro bioactivity. This approach provides a simple method to increase poor expression of other fusion proteins.

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“…Butyrate is a well-known inhibitor of histone deacetylase (HDAC) and was recently reported to promote protein glycosylation [24,25]. Selective modulators of histone acetylation and protein glycosylation were used to elucidate the mechanism of action of butyrate.…”

Section: Mechanism Of Butyrate-induced Nod2 Expressionmentioning

confidence: 99%

Butyrate mediates nucleotide‐binding and oligomerisation domain (NOD) 2‐dependent mucosal immune responses against peptidoglycan

Leung

Lam

et al. 2009

Eur J Immunol

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The interaction between digestive tract microbiological flora and food has an important influence on human health. Butyrate is produced during the fermentation of dietary fibres by intestinal bacteria and plays an important role in the regulation of mucosal immunity. In this report, we studied the impact of butyrate on the defence mechanism against the bacterial membrane component peptidoglycan (PGN). Butyrate was found to enhance PGN-mediated IL-8 and GRO-a production. The expression of these chemokines required the activation of NF-jB and was dependent on the concentrations of butyrate and PGN. Butyrate was found to up-regulate nucleotide-binding and oligomerisation domain (NOD) 2, but not NOD1 or TLR2. NOD2 up-regulation was mediated by an increase in histone acetylation in the Nod2 promoter region, leading to enhanced PGN-induced IL-8 and GRO-a secretion. Knockdown of NOD2 and TLR2 by siRNA significantly reduced PGNmediated chemokine production, suggesting that both NOD2 and TLR2 are required for maximal response. Our findings provide a better understanding of the mechanism by which butyrate regulates mucosal immunity for normal intestinal function. Based on the results of this study, we infer that dietary fibres can impact inflammatory bowel diseases.Key words: Butyrate . Intestinal immunity . Nucleotide-binding and oligomerization domains (NOD) .Peptidoglycan . TLR IntroductionIntestinal epithelial cells (IEC) function as a physical barrier against foreign substances in the intestinal lumen, such as food and pathogenic and non-pathogenic microorganisms. These cells are also involved in the host immune defence. Cytokines or chemokines are produced by IEC in response to potentially harmful substances, which are detected by a battery of pathogenrecognition receptors. These receptors are involved in the regulation of physiological and pathophysiological processes of the intestinal mucosa, including immunity and inflammation, via activation of MAPK and the key transcription factor, NF-kB [1][2][3][4]. The first widely studied family of receptors was the TLR family. Eleven mammalian TLR have been identified, of which TLR1-6 and TLR9 are expressed in IEC [3][4][5]. Another group of pathogenrecognition receptors, termed nucleotide-binding and oligomerisation domain (NOD) proteins, provide a second line of innate immune defence against bacterial infection. This family consists of more than 20 members and is located primarily in the cytosol. While NOD1 is constitutively expressed in IEC, the level of NOD2 expression is very low [1,2]. The bi-directional interaction between digestive tract microbiological flora and food has an important influence on human health, particularly on the immune response [6,7]. Butyrate is There is an increasing body of evidence demonstrating the importance of butyrate in maintaining intestinal mucosal immunity [9][10][11]. Butyrate has been shown to switch the transcriptional pattern of chemokine production through promotion of histone acetylation [12]. Several studies have also suggested...

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Sodium butyrate alters erythropoietin glycosylation via multiple mechanisms

Cited by 23 publications

References 39 publications

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins

Butyrate mediates nucleotide‐binding and oligomerisation domain (NOD) 2‐dependent mucosal immune responses against peptidoglycan

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