Mucin-type proteins produced in the Trichoplusia ni and Spodoptera frugiperda insect cell lines carry novel O-glycans with phosphocholine and sulfate substitutions

作者：Gaunitz Stefan^*; Jin Chunsheng; Nilsson Anki; Liu Jining; Karlsson Niclas G; Holgersson Jan

来源：Glycobiology, 2013, 23(7): 778-796.

DOI：10.1093/glycob/cwt015

• insect cells
• mass spectrometry
• O-glycosylation
• phosphocholine
• sulfate

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 IgG(2b) (PSGL-1/mIgG(2b)) 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/mIgG(2b) 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/mIG(2b) 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 (%26lt; 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.

• 出版日期2013-7