Peptide, Disulfide, and Glycosylation Mapping of Recombinant Human Thrombopoietin from Ser1 to Arg246
- 1 January 1996
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 35 (47), 14849-14861
- https://doi.org/10.1021/bi961075b
Abstract
Thrombopoietin (TPO) is a hematopoietic factor involved in the regulation of megakaryocytopoiesis. Full length recombinant human TPO (332 residues) has been expressed in BHK cells and purified to homogeneity using conventional means. 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). A modification of the ramped orifice method of Carr and co-workers [Carr et al. (1993) Protein Sci. 2, 183−196] is employed, providing additional information for assignment of the LC−ESMS chromatograms. With the modification, b- and y-series peptide ions are produced via front-end CID which confirms the mass-based assignments. 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. Two unique disulfides are formed between the four cysteines in the cytokine domain of TPO: Cys7−Cys151 and Cys29−Cys85. The glycosylation map indicates the position, occupancy, and structures of the N- and O-glycans in TPO 1−246. 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. The N-glycans are of the complex type with the core-fucosylated disialylated biantennary and trisialylated triantennary structures predominating. The O-glycans are of the mucin type with the monosialylated and disialylated GalGalNAc-S/T structures predominating. 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).Keywords
This publication has 21 references indexed in Scilit:
- Structural determination of two N‐linked glycans isolated from recombinant human lactoferrin expressed in BHK cellsFEBS Letters, 1995
- The regulation of megakaryocytopoiesisBlood Reviews, 1995
- Structural Analysis of the Sialylated N- and O-Linked Carbohydrate Chains of Recombinant Human Erythropoietin Expressed in Chinese Hamster Ovary Cells. Sialylation Patterns and Branch Location of Dimeric N-acetyllactosamine UnitsEuropean Journal of Biochemistry, 1995
- The Major N-Linked Carbohydrate Chains from Human Urokinase. The Occurrence of 4-O-sulfated, (alpha2-6)-sialylated or (alpha1-3)-fucosylated N-acetylgalactosamine(beta1-4)-N-acetylglucosamine ElementsEuropean Journal of Biochemistry, 1995
- Comparison of N‐Linked Oligosaccharides of Recombinant Human Tissue Kallikrein Produced by Chinese Hamster Ovary Cells on Microcarrier Beads and in Serum‐Free Suspension CultureBiotechnology Progress, 1994
- Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK‐21 cellsEuropean Journal of Biochemistry, 1993
- Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cellsEuropean Journal of Biochemistry, 1993
- Human interleukin 4: The solution structure of a four-helix bundle proteinJournal of Molecular Biology, 1992
- Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factorJournal of Molecular Biology, 1992
- Correlation of glycosylation forms with position in amino acid sequenceThe Journal of cell biology, 1983