Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells

Biologicals. 2004 Jun;32(2):70-7. doi: 10.1016/j.biologicals.2004.04.001.

Abstract

Follistatin (FS), a glycoprotein, plays an important role in cell growth and differentiation through the neutralization of the biological activities of activins. In this study, we analyzed the glycosylation of recombinant human FS (rhFS) produced in Chinese hamster ovary cells. The results of SDS-PAGE and MALDI-TOF MS revealed the presence of both non-glycosylated and glycosylated forms. FS contains two potential N-glycosylation sites, Asn95 and Asn259. Using mass spectrometric peptide/glycopeptide mapping and precursor-ion scanning, we found that both N-glycosylation sites were partially glycosylated. Monosaccharide composition analyses suggested the linkages of fucosylated bi- and triantennary complex-type oligosaccharides on rhFS. This finding was supported by mass spectrometric oligosaccharide profiling, in which the m/z values and elution times of some of the oligosaccharides from rhFS were in good agreement with those of standard oligosaccharides. Site-specific glycosylation was deduced on the basis of the mass spectra of the glycopeptides. It was suggested that biantennary oligosaccharides are major oligosaccharides located at both Asn95 and Asn259, whereas the triantennary structures are present mainly at Asn95.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Asparagine / chemistry
  • Binding Sites
  • CHO Cells
  • Carbohydrates
  • Cell Line
  • Chromatography, Liquid
  • Cricetinae
  • Electrophoresis, Polyacrylamide Gel
  • Follistatin / chemistry*
  • Glycopeptides / chemistry
  • Glycosylation
  • Humans
  • Isoelectric Focusing
  • Mass Spectrometry
  • Molecular Sequence Data
  • Monosaccharides / chemistry
  • Oligosaccharides / chemistry
  • Peptides / chemistry
  • Recombinant Proteins / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Time Factors

Substances

  • Carbohydrates
  • Follistatin
  • Glycopeptides
  • Monosaccharides
  • Oligosaccharides
  • Peptides
  • Recombinant Proteins
  • Asparagine