title: O-glycans and O-glycosylation sites of recombinant human GM-CSF derived from suspension-cultured rice cells, and their structural role.
abstract: Recombinant human GM-CSF (rhGM-CSF) from yeast has been clinically applied to immunosuppressed patients. The production of suspension-cultured rice-cell-derived rhGM-CSF (rrhGM-CSF), which has a longer blood clearance time and the same bioactivity as yeast-derived rhGM-CSF, and the analysis of its N-glycans have been reported recently. However, there are no previous reports of the O-glycosylation of rhGM-CSF from plant cells, and so this study investigated O-glycans, O-glycosylation sites, and their structural role in rrhGM-CSF. Monosaccharide analysis revealed the presence of O-glycans comprising arabinose and galactose. Eight O-glycans comprising four arabinose residues with zero to seven galactose residues along with their relative quantities were analyzed. Analysis of pronase-digested glycopeptides indicated that the O-glycans are partially attached to Ser 5, Ser 7, Ser 9, or Thr 10 residues, and glycan heterogeneity was confirmed at each site. Pro-to-hydroxyproline conversions occurred at Pro 2, Pro 6, and Pro 8 residues. The preparation of deglycosylated rrhGM-CSFs revealed that deglycosylation greatly affects their α-helix structures. These findings indicate that O-glycans of rrhGM-CSF are essential for maintaining its structural stability and result in an extended in vivo half-life, but without affecting its biological function. This is the first report on the O-glycosylation of rhGM-CSF derived from plant cells.
authors.affiliation: Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, South Korea.
authors.forename: Jihye
authors.initials: J
authors.lastname: Kim
authors.affiliation: Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, South Korea.
authors.forename: Heajin
authors.initials: H
authors.lastname: Park
authors.affiliation: Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, South Korea.
authors.forename: Byung Tae
authors.initials: BT
authors.lastname: Park
authors.affiliation: Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, South Korea.
authors.forename: Hye Seong
authors.initials: HS
authors.lastname: Hwang
authors.affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712, South Korea.
authors.forename: Jae Il
authors.initials: JI
authors.lastname: Kim
authors.affiliation: Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, South Korea.
authors.forename: Dae Kyong
authors.initials: DK
authors.lastname: Kim
authors.affiliation: Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, South Korea. Electronic address: hahyung@cau.ac.kr.
authors.forename: Ha Hyung
authors.initials: HH
authors.lastname: Kim
mesh_terms: D001089:Arabinose / Q000737:chemistry; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005690:Galactose / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D009005:Monosaccharides / Q000737:chemistry; D012275:Oryza / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry*; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006020:Glycopeptides; D009005:Monosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D001089:Arabinose; D005690:Galactose
keywords: Human GM-CSF; In vivo half-life; O-glycosylation; Rice cell; Structural stability