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

Glycobiology. 2013 Jul;23(7):778-96. doi: 10.1093/glycob/cwt015. Epub 2013 Mar 5.


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.

Keywords: O-glycosylation; insect cells; mass spectrometry; phosphocholine; sulfate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylgalactosamine / chemistry
  • Acetylgalactosamine / metabolism
  • Animals
  • Cell Line
  • Glycosylation
  • Hexuronic Acids / chemistry
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Mice
  • Moths / chemistry
  • Mucins / chemistry*
  • Mucins / genetics
  • Mucins / metabolism
  • Phosphorylcholine / chemistry*
  • Polysaccharides / chemistry*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Sf9 Cells
  • Spodoptera / chemistry
  • Sulfates / chemistry*


  • Hexuronic Acids
  • Membrane Glycoproteins
  • Mucins
  • P-selectin ligand protein
  • Polysaccharides
  • Recombinant Fusion Proteins
  • Sulfates
  • Phosphorylcholine
  • Acetylgalactosamine