Improvement in the heterogeneous N-termini and the defective N-glycosylation of human interleukin-6 by genetic engineering.
Recombinant human interleukin-6 (IL-6), expressed in Chinese hamster ovary cells, has heterogeneous N-termini of Ala1 and Val3, as does naturally occurring IL-6. This heterogeneity is thought to be caused by difficulty in cleavage of the signal sequence. To obtain homogeneous IL-6, Pro at -1 was exchanged for Ala by site-directed mutagenesis. Alternatively, the signal sequence was replaced with that of human granulocyte-colony-stimulating factor. In both cases, the IL-6 designed to start with Ala1 was still heterogeneous, while the IL-6 designed to start with Val3 showed a homogeneous N-terminus. It is suggested that the heterogeneity of the N-terminus is caused not only by the signal sequence, but also by the succeeding sequences of the mature protein. Only a portion of recombinant human IL-6 is N-glycosylated. Asn46, being exchanged for Gln by site-directed mutagenesis, was confirmed to be partially N-glycosylated. The defective N-glycosylation was assumed to be caused by interference or tension from a disulfide bond near the N-glycosylation site. To verify this hypothesis, the Cys45 and Cys51 forming the disulfide bond were exchanged for Ser. The N-glycosylated species became predominant upon this substitution, suggesting that formation of the disulfide bond is a cause of the defective N-glycosylation.
Hasegawa
M
M
Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd, Shizuoka, Japan.
Orita
T
T
Kojima
T
T
Tomonoh
K
K
Hirata
Y
Y
Ochi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA; D006031:Glycosylation; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D015850:Interleukin-6; D011994:Recombinant Proteins; D004247:DNA