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Sialic Acid Content (sialic + acid_content)
Selected AbstractsEffect of sialic acid content on glycoprotein pI analyzed by two-dimensional electrophoresisELECTROPHORESIS, Issue 17 2010Sílvia Barrabés Abstract 2-DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2-DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2-DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2-DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2-DE pattern of three serum glycoproteins (haptoglobin, ,1-antitrypsin and ribonuclease 1), which N -glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule. [source] Increased sialylation of polymeric ,-IgA1 in patients with IgA nephropathyJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2002Joseph C.K. Leung Abstract The mechanism of mesangial IgA deposition is poorly understood in IgA nephropathy (IgAN). Abnormal glycosylation of carbohydrate moieties in the hinge region of the IgA molecule has recently attracted much attention. In this report, we studied galactosylation and sialylation profiles in ,- and ,-IgA1 from patients with IgAN. Total serum IgA1 was isolated from patients with IgAN or healthy controls by jacalin-affinity chromatography. Six fractions of molecular weight (MW) 50,1,000 kDa were separated by fast protein liquid chromatography (FPLC). Four lectin-binding assays were used to study the sialylation and the presence of terminal galactose or N-acetylgalactosamine (GalNAc) in the O-linked carbohydrate moieties of ,- or ,-IgA1. Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA) lectin recognize ,(2,3)- and ,(2,6)-linked sialic acid, respectively. Peanut agglutinin (PNA) and Helix aspersa (HA) lectin recognize terminal galactose and GalNAc, respectively. Reduced HA was demonstrated in macromolecular , or ,-IgA1 (300,825 kDa) isolated from patients with IgAN (P < 0.05 compared with healthy controls). Lambda- but not ,-IgA1 from patients with IgAN bound less to PNA (P < 0.05). The ,(2,3)-linked sialic acid content in ,- but not ,-IgA1 of MW 150,610 kDa from patients was higher than that of controls (P < 0.005). The ,(2,6)-linked sialic acid content in ,-IgA1 (300,825 kDa) and ,-IgA1 (150,610 kDa) from patients was also higher than that of controls. This unusual glycosylation and sialylation pattern of the ,-IgA1 may have important implications for the pathogenesis of IgAN, as both the masking effect of sialic acid on galactose and the reduced galactosylation will hinder the clearance of macromolecular ,-IgA1 by asialoglycoprotein receptor of hepatocytes. The negative charge from sialic acid may also favor mesangial deposition of macromolecular ,-IgA1 in IgAN. J. Clin. Lab. Anal. 16:11,19, 2002. © 2002 Wiley-Liss, Inc. [source] Relationships between Na+, K+ -ATPase, sialic acid content and fluidity in rainbow trout density-separated erythrocytesJOURNAL OF FISH BIOLOGY, Issue 2 2002E. Salvolini A correlation was found between membrane fluidity, sialic acid (SA) content and Na+, K+ -ATPase activity in the rainbow trout Oncorhynchus mykiss erythrocyte membrane and this suggests that alterations in erythrocyte Na+, K+ -ATPase activity might be caused by modified membrane fluidity through modified SA content. [source] Sialylation enhancement of CTLA4-Ig fusion protein in Chinese hamster ovary cells by dexamethasoneBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Ying Jing Abstract The importance of glycoprotein sialic acid levels is well known, as increased levels have been shown to increase in vivo serum half-life profiles. Here we demonstrate for the first time that dexamethasone (DEX) was capable of improving the sialylation of a CTLA4-Ig fusion protein produced by Chinese hamster ovary (CHO) cells. DEX was shown to enhance the intracellular addition of sialic acid by sialyltransferases as well as reduce extracellular removal of sialic acid by sialidase cleavage. We illustrated that DEX addition resulted in increased expression of the glycosyltransferases ,2,3-sialyltransferase (,2,3-ST) and ,1,4-galactosyltransferase (,1,4-GT) in CHO cells. Based upon our previous results showing DEX addition increased culture cell viability, we confirmed here that cultures treated with DEX also resulted in decreased sialidase activity. Addition of the glucocorticoid receptor (GR) antagonist mifepristone (RU-486) was capable of blocking the increase in sialylation by DEX which further supports that DEX affected sialylation as well as provides evidence that the sialylation enhancement effects of DEX on recombinant CHO cells occurred through the GR. Finally, the effects of DEX on increasing sialylation were then confirmed in 5-L controlled bioreactors. Addition of 1,µM DEX to the bioreactors on day 2 resulted in harvests with average increases of 16.2% for total sialic acid content and 15.8% in the protein fraction with N-linked sialylation. DEX was found to be a simple and effective method for increasing sialylation of this CTLA4-Ig fusion protein expressed in CHO cells. Biotechnol. Bioeng. 2010;107: 488,496. © 2010 Wiley Periodicals, Inc. [source] Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2010Min Zhang Abstract Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFN,) were treated using short interfering RNA (siRNA) and short-hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFN, was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology. Biotechnol. Bioeng. 2010;105: 1094,1105. © 2009 Wiley Periodicals, Inc. [source] Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture systemBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Christopher K. Crowell Abstract The manufacture of secreted proteins is complicated by the need for both high levels of expression and appropriate processing of the nascent polypeptide. For glycoproteins, such as erythropoietin (EPO), posttranslational processing involves the addition of oligosaccharide chains. We initially noted that a subset of the amino acids present in the cell culture media had become depleted by cellular metabolism during the last harvest cycle in our batch fed system and hypothesized that by supplementing these nutrients we would improve EPO yields. By increasing the concentration of these amino acids we increased recombinant human erythropoietin (rHuEPO) biosynthesis in the last harvest cycle as expected but, surprisingly, we also observed a large increase in the amount of rHuEPO with a relatively low sialic acid content. To understand the nature of this process we isolated and characterized the lower sialylated rHuEPO pool. Decreased sialylation correlated with an increase in N-linked carbohydrates missing terminal galactose moieties, suggesting that ,-1,4-galactosyltransferase may be rate limiting in our system. To test this hypothesis we supplemented our cultures with varying concentrations of manganese (Mn2+), a cofactor for ,-1,4-galactosyltransferase. Consistent with our hypothesis we found that Mn2+ addition improved galactosylation and greatly reduced the amount of rHuEPO in the lower sialylated fraction. Additionally, we found that Mn2+ addition increased carbohydrate site occupancy and narrowed carbohydrate branching to bi-antennary structures in these lower sialylated pools. Surprisingly Mn2+ only had this effect late in the culture process. These data indicate that the addition of Mn2+ has complex effects on stressed batch fed cultures. Biotechnol. Bioeng. 2007;96: 538,549. © 2006 Wiley Periodicals, Inc. [source] RNA interference of sialidase improves glycoprotein sialic acid content consistencyBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Frederyk A. Ngantung Abstract An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN-, produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-,, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process. © 2006 Wiley Periodicals, Inc. 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