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

Autor: Geoghegan KF; Pfizer Worldwide Research, Groton, CT 06340, USA. kieran.f.geoghegan@pfizer.com, Song X, Hoth LR, Feng X, Shanker S, Quazi A, Luxenberg DP, Wright JF, Griffor MC
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2013 Jan; Vol. 87 (1), pp. 27-34. Date of Electronic Publication: 2012 Oct 13.
DOI: 10.1016/j.pep.2012.09.013
Abstrakt: 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.
(Copyright © 2012 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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