ABSTRACT
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 alpha2,6-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 N-glycans 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.
Subject(s)
Erythropoietin/chemistry , Erythropoietin/metabolism , Galactosyltransferases/chemistry , Milk Proteins/chemistry , Milk Proteins/metabolism , Milk/chemistry , Milk/metabolism , Animals , Erythropoietin/genetics , Female , Galactosyltransferases/metabolism , Goats , Humans , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.