Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2.
We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and the structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2.
Vita
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Unité Biochimie des Proteines, Sanofi Elf Bio-Recherches, Labège, France.
Magazin
M
M
Marchese
E
E
Lupker
J
J
Ferrara
P
P
D000116:Acetylgalactosamine / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D006224:Cricetinae; D005260:Female; D006031:Glycosylation; D006801:Humans; D007376:Interleukin-2 / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009439:Neuraminidase; D010053:Ovary; D010446:Peptide Fragments; D011994:Recombinant Proteins / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014316:Tritium; D001616:beta-Galactosidase
D002241:Carbohydrates; D007376:Interleukin-2; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D014316:Tritium; D009439:Neuraminidase; D001616:beta-Galactosidase; D000116:Acetylgalactosamine