Polypeptide and carbohydrate structure of recombinant human interleukin-6 produced in Chinese hamster ovary cells

• Published in: Journal of biochemistry (Tokyo) Vol. 115; no. 2; p. 345
• Main Authors: Orita, T, Oh-eda, M, Hasegawa, M, Kuboniwa, H, Esaki, K, Ochi, N
• Format: Journal Article
• Language: English
• Published: England 01-02-1994
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Animals; Carbohydrate Conformation; Carbohydrate Sequence; CHO Cells; Cricetinae; Cricetulus; DNA, Complementary - chemistry; DNA, Complementary - genetics; Electrophoresis, Polyacrylamide Gel; Glycosylation; Humans; Interleukin-6 - chemistry; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins - chemistry; Restriction Mapping; Substrate Specificity; Transfection
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a124340

A glycosylated form of human interleukin 6 (hIL-6) has been produced in Chinese hamster ovary (CHO) cells transfected with a cDNA clone for human IL-6. Recombinant hIL-6 was purified from a culture supernatant of the transfected CHO cells, and used for structural characterization. The complete amino acid sequence, composed of 185 amino acid residues, was determined and is identical to that predicted from the cDNA sequence. However, a recombinant hIL-6 species lacking two amino acid residues (Ala-Pro) from the N-terminus was also found. Two disulfide bonds are formed, between Cys45 and Cys51 and between Cys74 and Cys84. Recombinant hIL-6 carries one O-linked carbohydrate chain, and Thr139 is fully O-glycosylated. A portion of recombinant hIL-6 protein carries one N-linked sialooligosaccharide chain, and the N-glycosylation occurs at Asn46. The structure of the N-linked sugar chains was estimated by a combination of sugar mapping and glycosidase digestion. The major structure of the N-linked sugar chain predicted was of a fucosylated biantennary or triantennary complex type. Fucosylated triantennary sugar chains with one or two N-acetyllactosaminyl repeats were also found. The structure of the O-linked sugar chain was determined by 500 mHz 1H-NMR to be NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6) Gal-NAcol.