PMID: 8942648

 

    Legend: Sugar

Title : Peptide , disulfide, and glycosylation mapping of recombinant human thrombopoietin from ser1 to Arg246

Abstract :
  1. Thrombopoietin ( TPO ) is a hematopoietic factor involved in the regulation of megakaryocytopoiesis
  2. Full length recombinant human TPO (332 residues ) has been expressed in BHK cells and purified to homogeneity using conventional means
  3. Peptide , disulfide, and glycosylation mapping of human TPO from residues 1 to 246 has been carried out using liquid chromatography-electrospray mass spectrometry (LC-ESMS)
  4. A modification of the ramped orifice method of Carr and co-workers [ Carr et al. ( 1993 ) Protein Sci
  5. 2, 183-196] is employed, providing additional information for assignment of the LC-ESMS chromatograms
  6. With the modification, b- and y-series peptide ions are produced via front-end CID which confirms the mass-based assignments
  7. The results of our analysis of TPO indicate that the amino acid sequence of TPO 1-246 is as expected from the transfected cDNA with complete cleavage of the signal peptide
  8. Two unique disulfides are formed between the four cysteines in the cytokine domain of TPO : Cys7-Cys151 and Cys29-Cys85
  9. The glycosylation map indicates the position, occupancy, and structures of the N- and O-glycans in TPO 1-246
  10. In addition, site specific structural characterization of the PNGase F-liberated N-glycans has been performed following purification by high-pH anionic exchange chromatography with pulsed amperometric detection (HPAEC-PAD); the results corroborate the LC-ESMS data
  11. The N-glycans are of the complex type with the core-fucosylated disialylated biantennary and trisialylated triantennary structures predominating
  12. The O-glycans are of the mucin type with the monosialylated and disialylated GalGalNAc-S/T structures predominating
  13. Furthermore, we propose that the C-terminal domain of TPO be further divided into two domains on the basis of sequence homology among the cloned sequences and glycosylation/structural features: an N-glycan domain (154-246) and an O-glycan domain (247-332)