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

2013; Oxford University Press; Volume: 23; Issue: 7 Linguagem: Inglês

10.1093/glycob/cwt015

1460-2423

Stefan Gaunitz, Chunsheng Jin, Anki Nilsson, Jining Liu, Niclas G. Karlsson, Jan Holgersson,

Invertebrate Immune Response Mechanisms

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.