N-glycosylation microheterogeneity and site occupancy of an Asn-X-Cys sequon in plasma-derived and recombinant protein C

Proteomics. 2009 May;9(9):2555-67. doi: 10.1002/pmic.200800775.


Human protein C (hPC) is glycosylated at three Asn-X-Ser/Thr and one atypical Asn-X-Cys sequons. We have characterized the micro- and macro-heterogeneity of plasma-derived hPC and compared the glycosylation features with recombinant protein C (tg-PC) produced in a transgenic pig bioreactor from two animals having approximately tenfold different expression levels. The N-glycans of hPC are complex di- and tri-sialylated structures, and we measured 78% site occupancy at Asn-329 (the Asn-X-Cys sequon). The N-glycans of tg-PC are complex sialylated structures, but less branched and partially sialylated. The porcine mammary epithelial cells glycosylate the Asn-X-Cys sequon with a similar efficiency as human hepatocytes even at these high expression levels, and site occupancy at this sequon was not affected by expression level. A distinct bias for particular structures was present at each of the four glycosylation sites for both hPC and tg-PC. Interestingly, glycans with GalNAc in the antennae were predominant at the Asn-329 site. The N-glycan structures found for tg-PC are very similar to those reported for a recombinant Factor IX produced in transgenic pig milk, and similar to the endogenous milk protein lactoferrin, which may indicate that N-glycan processing in the porcine mammary epithelial cells is more uniform than in other tissues.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Asparagine / chemistry
  • Asparagine / metabolism
  • Bioreactors
  • Chromatography, High Pressure Liquid
  • Cysteine / chemistry
  • Cysteine / metabolism
  • Epithelium / chemistry
  • Glycopeptides / chemistry*
  • Glycopeptides / genetics
  • Glycopeptides / metabolism
  • Glycosylation
  • Humans
  • Mammary Glands, Animal / cytology
  • Mass Spectrometry
  • N-Acetylneuraminic Acid / chemistry
  • Plasma / chemistry
  • Protein C / chemistry*
  • Protein C / genetics
  • Protein C / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Swine


  • Glycopeptides
  • Protein C
  • Recombinant Proteins
  • Asparagine
  • N-Acetylneuraminic Acid
  • Cysteine