Unexpected mucin-type O-glycosylation and host-specific N-glycosylation of human recombinant interleukin-17A expressed in a human kidney cell line.

Abstract The T helper cell-derived cytokine interleukin-17A (IL-17A) is a variably glycosylated disulfide-linked homodimer of 34–38 kDa. Its polypeptide monomer contains one canonical N -glycosylation site at Asn68, and human recombinant IL-17A was partly N -glycosylated when expressed in human kidney (HEK293) cells as a fusion protein with a melittin signal sequence and an N-terminal hexahistidine tag. Orbitrap mass analyses of the tryptic N -glycopeptide 63–69 indicated that the N -glycosylation was of the GalNAc-terminated type characteristic of cultured kidney cells. The mass spectrum of IL-17A monomer also included peaks shifted by +948 Da from the respective masses of unglycosylated and N -glycosylated polypeptides. These were caused by unpredicted partial O -glycosylation of Thr26 with the mucin-like structure -GalNAc(-NeuNAc)-Gal-NeuNAc. Identical O -glycosylation occurred in commercially sourced recombinant IL-17A also expressed in HEK293 cells but with a different N-terminal sequence. Therefore, the kidney host cell line not only imposed its characteristic pattern of N -glycosylation on recombinant IL-17A but additionally created an O -glycosylation not known to be present in the T cell-derived cytokine. Mammalian host cell lines for recombinant protein expression generally impose their characteristic patterns of N -glycosylation on the product, but this work exemplifies how a host may also unpredictably O -glycosylate a protein that is probably not normally O -glycosylated.