title: Recombinant human lecithin-cholesterol acyltransferase Fc fusion: analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region.
abstract: Recombinant human lecithin-cholesterol acyltransferase Fc fusion (huLCAT-Fc) is a chimeric protein produced by fusing human Fc to the C-terminus of the human enzyme via a linker sequence. The huLCAT-Fc homodimer contains five N-linked glycosylation sites per monomer. The heterogeneity and site-specific distribution of the various glycans were examined using enzymatic digestion and LC-MS/MS, followed by automatic processing. Almost all of the N-linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N-linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N-linked site exclusively contain typical asialobiantennary structures. HuLCAT-Fc was also confirmed to have mucin-type glycans attached at T407 and S409 . When LCAT-Fc fusions were constructed using a G-S-G-G-G-G linker, an unexpected +632 Da xylose-based glycosaminoglycan (GAG) tetrasaccharide core of Xyl-Gal-Gal-GlcA was attached to S418 . Several minor intermediate species including Xyl, Xyl-Gal, Xyl-Gal-Gal, and a phosphorylated GAG core were also present. The mucin-type O-linked glycans can be effectively released by sialidase and O-glycanase; however, the GAG could only be removed and localized using chemical alkaline β-elimination and targeted LC-MS/MS. E416 (the C-terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O-xylosyltransferase. HuLCAT-Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPPE416 GS418 GGGGDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.
authors.affiliation: Biologics Optimization, Therapeutic Discovery, Amgen Inc., Thousand Oaks, California, 91320.
authors.forename: Chris
authors.initials: C
authors.lastname: Spahr
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Kim
authors.forename: Sihong
authors.initials: S
authors.lastname: Deng
authors.forename: Paul
authors.initials: P
authors.lastname: Kodama
authors.forename: Zhen
authors.initials: Z
authors.lastname: Xia
authors.forename: Jay
authors.initials: J
authors.lastname: Tang
authors.forename: Richard
authors.initials: R
authors.lastname: Zhang
authors.forename: Sophia
authors.initials: S
authors.lastname: Siu
authors.forename: Noi
authors.initials: N
authors.lastname: Nuanmanee
authors.forename: Bram
authors.initials: B
authors.lastname: Estes
authors.forename: Jennitte
authors.initials: J
authors.lastname: Stevens
authors.forename: Mingyue
authors.initials: M
authors.lastname: Zhou
authors.forename: Hsieng S
authors.initials: HS
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry* / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D006020:Glycopeptides; D007141:Immunoglobulin Fc Fragments; D009844:Oligosaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase
keywords: N- and O-linked glycans; recombinant human LCAT-Fc; site-specific N-linked glycan profile; xylose-based glycosaminoglycan