title: Mucin-type proteins produced in the Trichoplusia ni and Spodoptera frugiperda insect cell lines carry novel O-glycans with phosphocholine and sulfate substitutions.
abstract: The O-glycans of a recombinant mucin-type protein expressed in insect cell lines derived from Trichoplusia ni (Hi-5) and Spodoptera frugiperda (Sf9) were characterized. The P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b) fusion protein carrying 106 potential O-glycosylation sites and 6 potential N-glycosylation sites was expressed and purified from the Hi-5 and Sf9 cell culture medium using affinity chromatography and gel filtration. Liquid chromatography mass spectrometry (LC-MS) of O-glycans released from PSGL-1/mIgG2b revealed a large repertoire of structurally diverse glycans, which is in contrast to previous reports of only simple glycans. O-Glycans containing hexuronic acid (HexA, here glucuronic acid and galacturonic acid) were found to be prevalent. Also sulfate (Hi-5 and Sf9) and phosphocholine (PC; Sf9) O-glycan substitutions were detected. Western blotting confirmed the presence of O-linked PC on PSGL-1/mIG2b produced in Sf9 cells. To our knowledge, this is the first structural characterization of PC-substituted O-glycans in any species. The MS analyses revealed that Sf9 oligosaccharides consisted of short oligosaccharides (<6 residues) low in hexose (Hex) and with terminating N-acetylhexosamine (HexNAc) units, whereas Hi-5 produced a family of large O-glycans with (HexNAc-HexA-Hex) repeats and sulfate substitution on terminal residues. In both cell lines, the core N-acetylgalactosamine was preferentially non-branched, but small amounts of O-glycan cores with single fucose or hexose branches were found.
authors: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital at Huddinge, SE-141 86 Huddinge, Sweden. stefan.gaunitz@ki.se
authors: Stefan
authors: S
authors: Gaunitz
authors: Chunsheng
authors: C
authors: Jin
authors: Anki
authors: A
authors: Nilsson
authors: Jining
authors: J
authors: Liu
authors: Niclas G
authors: NG
authors: Karlsson
authors: Jan
authors: J
authors: Holgersson
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006031:Glycosylation; D006603:Hexuronic Acids / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009036:Moths / Q000737:chemistry; D009077:Mucins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010767:Phosphorylcholine / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry*
chemical_list: D006603:Hexuronic Acids; D008562:Membrane Glycoproteins; D009077:Mucins; C084882:P-selectin ligand protein; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D013431:Sulfates; D010767:Phosphorylcholine; D000116:Acetylgalactosamine
keywords: O-glycosylation; insect cells; mass spectrometry; phosphocholine; sulfate