Unexpected mucin-type O-glycosylation and host-specific N-glycosylation of human recombinant interleukin-17A expressed in a human kidney cell line.
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.
Pfizer Worldwide Research, Groton, CT 06340, USA. kieran.f.geoghegan@pfizer.com
Kieran F
KF
Geoghegan
Xi
X
Song
Lise R
LR
Hoth
Xidong
X
Feng
Suman
S
Shanker
Amira
A
Quazi
Deborah P
DP
Luxenberg
Jill F
JF
Wright
Matthew C
MC
Griffor
D000595:Amino Acid Sequence; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D019476:Insect Proteins / Q000096:biosynthesis / Q000737:chemistry; D020381:Interleukin-17 / Q000096:biosynthesis* / Q000737:chemistry; D008555:Melitten / Q000096:biosynthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry
C508717:IL17A protein, human; D019476:Insect Proteins; D020381:Interleukin-17; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D008555:Melitten