Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells

Toledo, Jorge R.; Sánchez, Oliberto; Seguí, Raquel Montesino; García, Yaiza Fernández; Rodríguez, María Pilar; Cremata, José A.

doi:10.1016/j.bbagen.2005.06.007

Cited by 18 publications

(11 citation statements)

References 33 publications

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“…A target protein could be obtained from milk by employing the recombinant adenovirus directly transfected into the mammary gland. However, as suggested in previous reports [15,16], the successful expression of a recombinant protein using this approach relies on many factors, including the physiological and biological properties of the recombinant protein itself, the promoter driving the recombinant protein expression in vivo, and the animals used to express the recombinant protein.…”

Section: Introductionmentioning

confidence: 97%

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Expression of Recombinant Human Nerve Growth Factor Beta in Milk of Goats by Recombinant Replication-Defective Adenovirus

Feng

et al. 2008

Appl Biochem Biotechnol

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For producing a large quantity of human nerve growth factor beta (hNGF-β), the recombinant adenovirus containing an expression cassette of hNGF-β complementary DNA gene was constructed and then instilled directly into goats' mammary glands. The recombinant hNGF-β was detected in the milk. The maximal expression level of recombinant hNGF-β in the milk reached 196.8 mg/l. The biological activity of the recombinant hNGF-β, compared with the commercial hNGF-β, was tested using PC12 cells. The results demonstrate that instilling recombinant adenovirus directly into the mammary gland of goat is an efficient approach to producing a large quantity of hNGF-β.

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Section: Introductionmentioning

confidence: 97%

“…In recent years, a new approach for expressing exogenous genes in secretory mammary epithelial cells has been proven to be the most effective versatile alternative [14][15][16][17][18]. A target protein could be obtained from milk by employing the recombinant adenovirus directly transfected into the mammary gland.…”

Section: Introductionmentioning

confidence: 99%

Expression of Recombinant Human Nerve Growth Factor Beta in Milk of Goats by Recombinant Replication-Defective Adenovirus

Feng

et al. 2008

Appl Biochem Biotechnol

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“…All together, the approach presented here is fully compatible with the current standard in proteomics analysis with the added value of addressing the most important PTM in a relatively rapid, straight-forward, and reliable manner. No doubt that this will contribute to a more comprehensive analysis in several research areas including clinical environments, drug quality control, study of specific cell-type glycosylation patterns [42], or even antidoping control analysis.…”

mentioning

confidence: 99%

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

Llop¹,

Gallego²,

Belalcazar³

et al. 2007

Proteomics

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The structure, function, and physico-chemical properties of many proteins are determined by PTM, being glycosylation the most complex. This study describes how a combination of typical proteomics methods (2-DE) combines with glycomics strategies (HPLC, MALDI-TOF-MS, exoglycosidases sequencing) to yield comprehensive data about single spot-microheterogeneity, providing meaningful information for the detection of disease markers, pharmaceutical industry, antidoping control, etc. Recombinant erythropoietin and its hyperglycosylated analogue darbepoetin-a were chosen as showcases because of their relevance in these fields and the analytical challenge they represent. The combined approach yielded good results in terms of sample complexity (mixture glycoforms), reproducibility, sensitivity (,25 pmoles of glycoprotein/spot), and identification of the underlying protein. Heterogeneity was present in all spots but with a clear tendency; spots proximal to the anode contained the highest amount of tetra-antennary tetrasialylated glycans, whereas the opposite occurred for spots proximal to the cathode with the majority of the structures being undersialylated. Spot microheterogeneity proved a consequence of the multiple glycosylation sites as they contributed directly to the number of possibilities to account for a discrete charge in a single spot. The interest of this combined glycoproteomics method resides in the efficiency for detecting and quantifying subtle dissimilarities originated from altered ratios of identical glycans including N-acetyl-lactosamine repeats, acetylation, or antigenic epitopes, that do not significantly contribute to the electrophoretic mobility, but affect the glycan microheterogeneity and the potential underlying related functionality.

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“…Recently, our group has expressed hEPO in the milk of mice and goats (Capra hircus) by adenoviral transduction of the mammary secretory epithelial cells [19,20]. An adenoviral vector, carrying the hEPO cDNA, allowed the expression of the recombinant glycoprotein at levels over 2 g/L in goat milk.…”

mentioning

confidence: 99%

Monosialylated biantennary N-glycoforms containing GalNAc–GlcNAc antennae predominate when human EPO is expressed in goat milk

Montesino

Toledo

Sánchez

et al. 2008

Archives of Biochemistry and Biophysics

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Recently, our group reported the expression of recombinant human erythropoietin in goat milk (rhEPO-milk) as well as in the mammary epithelial cell line GMGE (EPO-GMGE) by cell culture using the adenoviral transduction system. N-Glycosylation characterization of rhEPO-milk by Normal-Phase HPLC profiling of the fluorophore, 4-aminobenzoic acid-labeled enzymatically released N-glycan pool from rhEPO-goat milk, combined with MALDI, ESI-MS and LC/MS, revealed that low branched, core-fucosylated, N-glycans predominate. The labeled N-glycans were separated into neutral and charged fractions by anion exchange chromatography and the charged N-glycans were found to be mostly a2,6-monosialylated with Neu5Ac or Neu5Gc in a ratio of 1:1. Unlike the N-glycans from rhEPO produced in CHO cells, where the glycans are multiantennary highly sialylated, core-fucosylated oligosaccahrides, or even in the goat mammary gland epithelial cell line cultured in vitro in which multiantennary, core-and outer-arm fucosylated, monosialylated Nglycans are the most abundant species, a large proportion of the N-glycans from rhEPO-milk were monosialylated, biantennary, antennae mostly terminating with the more unusual GalNAc-GlcNAc motive and without outer-arm fucosylation. These findings, emphasizing the difference in the N-glycan repertoire between the rhEPO-milk and EPO-GMGE, are consistent with the principle that glycosylation is cell-type dependent and that the cell environment is crucial as well.

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Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells

Cited by 18 publications

References 33 publications

Expression of Recombinant Human Nerve Growth Factor Beta in Milk of Goats by Recombinant Replication-Defective Adenovirus

Expression of Recombinant Human Nerve Growth Factor Beta in Milk of Goats by Recombinant Replication-Defective Adenovirus

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

Monosialylated biantennary N-glycoforms containing GalNAc–GlcNAc antennae predominate when human EPO is expressed in goat milk

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