Monosialylated biantennary N-glycoforms containing GalNAc-GlcNAc antennae predominate when human EPO is expressed in goat milk.
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.
Montesino
R
R
Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba.
Toledo
J R
JR
Sánchez
O
O
Sánchez
A
A
Harvey
D J
DJ
Royle
L
L
Dwek
R A
RA
Rudd
P M
PM
Gerwig
G J
GJ
Kamerling
J P
JP
Cremata
J A
JA
D000818:Animals; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005700:Galactosyltransferases / Q000737:chemistry* / Q000378:metabolism; D006041:Goats; D006801:Humans; D008892:Milk / Q000737:chemistry* / Q000378:metabolism*; D008894:Milk Proteins / Q000737:chemistry* / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D008894:Milk Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D004921:Erythropoietin; D005700:Galactosyltransferases