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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 et al. (Sun,) studied this question.