Unusual N-glycosylation of a recombinant human erythropoietin expressed in a human lymphoblastoid cell line does not alter its biological properties.
Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycosylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser-126) and involved in the regulation of the level of red blood cells. Today, only one recombinant human Epo (rHuEpo), produced in CHO cell line, is extensively used in therapy to cure severe anemia. The structure of the glycan chains of this rHuEpo slightly differ of those of the urinary human Epo (uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a human lymphoblastoid cell line, named RPMI 1788. In order to fully characterize the Epo-RPMI, structural characterizations of the protein skeleton as well as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of some N-glycan chains, as mainly determined by ESI-MS, revealed some unusual characteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a sialyl Le(x)motif, and 13% contain more than three LacNAc repeats (up to five per molecule). Despite these unusual structural characteristics, the data concerning the in vitro and in vivo biological activities were not impaired when compared to Epo-CHO and uHuEpo.
Cointe
D
D
Laboratoire de Glycobiologie Structurale et Fonctionnelle, Unité Mixte de Recherche du CNRS No 8576, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France.
Béliard
R
R
Jorieux
S
S
Leroy
Y
Y
Glacet
A
A
Verbert
A
A
Bourel
D
D
Chirat
F
F
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D005591:Chemical Fractionation; D006224:Cricetinae; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006020:Glycopeptides / Q000032:analysis / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis; D010599:Pharmacokinetics; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen; D013329:Structure-Activity Relationship
D006020:Glycopeptides; D009005:Monosaccharides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid