Production of α2,6-sialylated IgG1 in CHO cells

The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.     

Na-butyrate increases the production and α2,6-sialylation of recombinant interferon-γ expressed by α2,6- sialyltransferase engineered CHO cells

A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme 2,6-sialyltransferase (2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by 2,6-engineered cells contained 68% of the total sialic acids in the 2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the 2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the 2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.     

Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ

The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for 2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat 2,6-ST was expressed in a recombinant CHO cell line making interferon-, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon- being linked in the 2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of 2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN- productivity or other aspects of IFN- glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.Abbreviations AT-III antithrombin-III - CHO Chinese hamster ovary - dhfr dihydrofolate reductase - EPO erythropoietin - IFN- human interferon- - NEO neomycin - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - ST sialyltransferase - tPA tissue plasminogen activator     

Regulation of RNA polymerase II activity in α-amanitin-resistant CHO hybrid cells

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to α-amanitin with mutant cells containing RNA polymerase II activity resistant to α-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing α-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to α-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the α-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975).A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in α-amanitin exhibited a 2–3 fold increase in the activity of the α-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the α-amanitin-inactivated polymerase II of wild-type sensitivity by ³H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of ³H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.     

The effect of α2,6-linked sialic acid on anti-IgM antibody-induced apoptosis in Ramos cells

Apoptosis in B cells is induced through the B cell antigen receptor (BCR) and affects the sialic acid recognition molecules on B cells. We investigated the effects of ₁-acid glycoprotein (AGP), which mainly contains 2,6-linked sialic acid, on anti-IgM antibody (Ab)-induced apoptosis in Ramos cells, which are derived from Burkitt's lymphoma. When Ramos cells were incubated with anti-IgM-Ab in plates coated with AGP, neuraminidase-digested AGP (asAGP) or 2,3-sialylated AGP (2,3AGP), apoptosis was suppressed only in those coated with AGP. We also studied the effects of CD22, which is expressed on the surface of mature B cells and binds to sugar chains containing 2,6-linked sialic acid, with anti-CD22 monoclonal antibody (mAb). Anti-CD22mAb enhanced anti-IgM Ab-induced apoptosis in Ramos cells. These contradictory results suggested that the recognition molecules for 2,6-linked sialic acid on AGP, which inhibits B-cell apoptosis, is distinct from CD22, or that different binding domains of CD22 between 2,6-linked sialic acid and anti-CD22 mAb exert opposite functions of suppression or enhancement to anti-IgM Ab-induced B cells.     

Immunohistochemical localization of α1-acid-glycoprotein in human liver parenchymal cells

Summary ₁-acid glycoprotein, a major human serum glycoprotein was detected and localized in human liver parenchymal cells of a biopsy specimen. A heavy metal salt containing fixative was required to retain sufficient antigen determinants of ₁-acid glycoprotein in order to visualize this protein by the peroxidase-anti-peroxidase unlabeled antibody enzyme method.     

Mechanisms of α-fetoprotein synthesis in hepatoma cells

Summary Immunoreaction of -fetoprotein (AFP) was detected not only in well-differentiated hepatocellular carcinoma but also in hepatocytes forming foci in livers with hyperplastic nodules during 3-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in hepatoma cells was in the rough endoplasmic reticulum, perinuclear space and well-developed Golgi apparatus around the nucleus. In livers with hyperplastic nodules it was also in some parts of the smooth endoplasmic reticulum and Golgi regions in hepatocytes in the vicinity of submembranous areas or bile canaliculi. These findings suggest that the Golgi apparatus in hepatoma cells acts mainly as an organelle for glycosylation of AFP and that the Golgi complexes in the hepatocytes in livers with hyperplastic nodules are organelles for secretion of AFP.Combined light microscopic immunoperoxidase study and autoradiography with ³H-thymidine revealed a higher cumulative labeling index in AFP-positive hepatoma cells than in non-tumorous areas. Combined electron microscopic immunoperoxidase study and autoradiography showed that hepatoma cells with AFP immunoreactivity only in the rough endoplasmic reticulum had a significantly higher labeling index than did cells with AFP immunoreactivity in both rough endoplasmic reticulum and Golgi apparatus. These findings suggest that AFP is synthesized in hepatoma cells before or during the stage of their DNA synthesis and is then transported to the Golgi apparatus.     

Production of albumin and α-fetoprotein in primary culture of fetal human liver cells on collagenous substrata in the presence of hydrocortisone

Summary When primary cultures of fetal human liver cells established on type I collagen gels were compared to sister cultures developed on tissue culture plastic, the cells in contact with type I collagen secreted albumin at a higher rate than those without contact. The albumin secretion was dependent on the presence of hydrocortisone (HC) in the medium. Also, α-fetoprotein (AFP), of which the level decreased gradually and became undetectable after 6 d regardless of the presence or absence of HC in the cells cultured on plastic, was maintained for longer periods of time by plating the cells on type I collagen gels in the presence of HC. Different secretion rates of albumin and AFP were observed after Day 13 and Day 16, respectively, between cells maintained on type I collagen gels and those on film plastic. The cells secreted larger amounts of both albumin and AFP in plates coated with type IV or I collagens than with fibronectin after Day 10. The cells cultured on type I collagen gels were cuboidal in shape, whereas those on plastic were flattened in cultures with HC. These data indicate that the secretion of human albumin and AFP is facilitated by synergies between HC and collagenous substrata.     

Growth and interferon-γ production in batch culture of CHO cells

The relationship between growth and interferon- (IFN-) production in the recombinant cell line CHO 320 was studied by varying the foetal calf serum (FCS) concentration. The specific growth rate varied with the initial FCS concentration in a manner which could be well fitted by the Monod model. TheK _s and _max values were found to be 0.771% (v/v) serum and 0.031 h^–1 respectively. The average specific IFN- production rates during the exponential phase increased with increasing FCS concentration. A good correlation between specific production rate and specific growth rate was found in all phases of the culture except the lag phase and it was clearly demonstrated that IFN- production was growth associated. Specific glucose and glutamine utilisation rates were inversely related to specific growth rates.     

Production of α-amylase fromHalobacterium halobium

Maximum production of extracellular -amylase activity inHalobacterium halobium was at 40°C in a medium containing 25% (w/v) NaCl, 1% (w/v) soluble starch and 1% (w/v) peptone, in presence of 0.1mm ZnSO₄ after 5 days in shaking cultures. The amylase had optimal activity at pH 6.5 in the presence of 1 to 3% (w/v) NaCl at 53°C.S. Patel, N. Jain and D. Madamwar are with the Post Graduate Department of Biosciences, Sadar Patel University, Vallabh Vidyanagar-388120, India.     

Anti-inflammatory IgG production requires functional P1 promoter in β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1) gene

The anti-inflammatory properties associated with intravenous immunoglobulin therapy require the sialic acid modification of the N-glycan of the Fc domain of IgG. Sialylation of the Fc fragment is mediated by β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1), acting on the Gal(β4)GlcNAc terminal structure of the biantennary N-glycans on the Fc domain. However, little is known regarding the in vivo regulation of Fc sialylation and its role in the progression of inflammatory processes. Here, we report that decreased Fc sialylation of circulatory IgG accompanies the acute phase response elicited by turpentine exposure or upon acute exposure to either nontypeable Haemophilus influenzae or ovalbumin. However, Fc sialylation was increased 3-fold from the base line upon transition to chronic inflammation by repeated exposure to challenge. The P1 promoter of the ST6Gal-1 gene is critical for Fc sialylation, but P1 does not drive ST6Gal-1 expression in B cells. The Siat1ΔP1 mouse, with a dysfunctional P1 promoter, was unable to produce sialylated Fc in the systemic circulation, despite the presence of Gal(β4)GlcNAc termini on the Fc glycans. The major contribution of P1 action is to synthesize ST6Gal-1 enzymes that are deposited into the systemic circulation. The data strongly indicate that this pool of extracellular ST6Gal-1 in the blood impacts the sialylation of IgG Fc and that defective Fc sialylation is likely a major contributing mechanism for the proinflammatory tendencies previously noted in Siat1ΔP1 animals.     

Role of γδ T cells in α-galactosylceramide-mediated immunity

Attempts to harness mouse type I NKT cells in different therapeutic settings including cancer, infection, and autoimmunity have proven fruitful using the CD1d-binding glycolipid α-galactosylceramide (α-GalCer). In these different models, the effects of α-GalCer mainly relied on the establishment of a type I NKT cell-dependent immune cascade involving dendritic cell, NK cell, B cell, or conventional CD4(+) and CD8(+) T cell activation/regulation as well as immunomodulatory cytokine production. In this study, we showed that γδ T cells, another population of innate-like T lymphocytes, displayed a phenotype of activated cells (cytokine production and cytotoxic properties) and were required to achieve an optimal α-GalCer-induced immune response. Using gene-targeted mice and recombinant cytokines, a critical need for IL-12 and IL-18 has been shown in the α-GalCer-induced IFN-γ production by γδ T cells. Moreover, this cytokine production occurred downstream of type I NKT cell response, suggesting their bystander effect on γδ T cells. In line with this, γδ T cells failed to directly recognize the CD1d/α-GalCer complex. We also provided evidence that γδ T cells increase their cytotoxic properties after α-GalCer injection, resulting in an increase in killing of tumor cell targets. Moreover, using cancer models, we demonstrated that γδ T cells were required for an optimal α-GalCer-mediated anti-tumor activity. Finally, we reported that immunization of wild-type mice with α-GalCer enhanced the adaptive immune response elicited by OVA, and this effect was strongly mediated by γδ T cells. We conclude that γδ T cells amplify the innate and acquired response to α-GalCer, with possibly important outcomes for the therapeutic effects of this compound.     

N-glycan analysis of human α1-antitrypsin produced in Chinese hamster ovary cells

Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1?-?6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2?-?3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2?-?6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.     

Production of α-Glucosidase and Its Role in Regulation of Amylase Production by Bacillus circulons F-2

Bacillus circulans F-2 requires a special carbon source or cultural conditions for amylase production. The α-glucosidase production of this bacterium was studied in various cultural conditions with measured glucose concèntrations. High amylase production was always accompanied by low α-glucosidase production and the absence of glucose in culture broth. Usually higher α-glucosidase production was observed in cultural conditions where little amylase was produced. In the presence of 1-deoxynojirimycin, an inhibitor of α-glucosidase activity, the bacterium produced significant amounts of amylase even in conditions giving high α-glucosidase production. It was concluded that the special requirement of this bacterium to produce amylase is effected by its high sensitivity to glucose repression and by the production of α-glucosidase which leads to the formation of glucose. Production of α-glucosidase was, like that of amylase, induced by maltooligosaccharides and repressed by glucose, but both its induction and repression are less sensitive to glucose than those of amylase.     

Activity of α-galactosidase in immobilized cells of Amsonia tabernaemontana Walt

Cell suspension culture Amsonia tabernaemontana Walt. were permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest α-galactosidase activity was at pH 5.3 and temperature 70 °C. The hydrolysis of substrate was linear for 3 h reaching 70 - 75 % conversion. The cells characterized by high enzyme activity and stability in long-term storage showed convenient physico-mechanical properties (physical protection from shear forces and easy separation of product from biocatalysts). This revised version was published online in July 2006 with corrections to the Cover Date.     

IFN-λ1 in CHO cells: its expression and biological activity

Background

Many studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor.

Results

A full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities. The anti-viral activity of rhIFN-λ1 was determined as 10⁶ IU/mg using the vesicular stomatitis virus (WISH-VSV) assay system. The anti-proliferation activity of rhIFN-λ1 was measured using the MTS method and the growth inhibition ratio was 57% higher than that for recombinant human IFN-α2b (rhIFN-α2b) when the rhIFN-λ1 concentration was 1000 IU/ml. rhIFN-λ1 had lower natural killer cell cytotoxicity than rhIFN-α2b.

Conclusion

The Flp-In-CHO system is suitable for stably expressing rhIFN-λ1 that possesses the predicted anti-viral, anti-proliferation and natural killer cell cytotoxicity-promoting activities.

     

Production of α-linolenic acid in Yarrowia lipolytica using low-temperature fermentation

α-Linolenic acid (ALA) is an essential ω-3 fatty with reported health benefits. However, this molecule is naturally found in plants such as flaxseed and canola which currently limits production. Here, we demonstrate the potential to sustainably produce ALA using the oleaginous yeast Yarrowia lipolytica. Through the use of a recently identified Δ12–15 desaturase (Rk Δ12–15), we were able to enable production in Y. lipolytica. When combined with a previously engineered lipid-overproducing strain with high precursor availability, further improvements of ALA production were achieved. Finally, the cultivation of this strain at lower temperatures significantly increased ALA content, with cells fermented at 20 °C accumulating nearly 30% ALA of the total lipids in this cell. This low-temperature fermentation represents improved ALA titer up to 3.2-fold compared to standard growth conditions. Scale-up into a fed-batch bioreactor produced ALA at 1.4 g/L, representing the highest published titer of this ω-3 fatty acid in a yeast host.