PMID: 8639592

 

    Legend: Sugar

Title : Amino acid sequence and carbohydrate structure of a recombinant human tissue factor pathway inhibitor expressed in Chinese hamster ovary cells: one N-and two O-linked carbohydrate chains are located between Kunitz domains 2 and 3 and one N-linked carbohydrate chain is in Kunitz domain 2

Abstract :
  1. Human tissue factor pathway inhibitor is a protease inhibitor with three tandem Kunitz-type inhibitory domains
  2. The recombinant protein (r- hTFPI ) was produced using Chinese hamster ovary cells, and its polypeptide and carbohydrate chain structures were analyzed
  3. The complete amino acid sequence , composed of 276 residues, was determined using a protein sequencer after protease digestion and it was identical to that predicted from the cDNA sequence
  4. Among three potential N-glycosylation sites , both Asn117 and Asn167 were fully N-glycosylated but Asn228 was not
  5. Thr175 was also fully O-glycosylated, but Ser174 was partially O-glycosylated
  6. Carbohydrate composition and mass spectrometric analyses of the undecapeptide OG-11 ( residues Leu 170approximately Leu180 ) showed that two O-linked carbohydrate chains consisted of a type-1 core structure (Gal-GalNAc-Ser/Thr) with 0-3 mol of N-acetylneuraminic acid(s)
  7. The N-linked carbohydrate chains were analyzed by two-dimensional carbohydrate mapping combined with sequential glycosidase digestion, after the reducing-ends of carbohydrate residues were tagged with 2-aminopyridine and non-reducing-end sialic acids were removed with sialidase
  8. All the N-linked structures in r- hTFPI were complex-type carbohydrate chains with one fucose residue attached to the reducing-end GlcNAc and consisted of bi-, tri-, and tetraantennary carbohydrate chains in the ratio 1.9:1.3:1.0
  9. Fucosylated tri- and tetraantennary carbohydrate chains with one or two N-acetyllactosaminyl repeats were also found (30% of carbohydrate chains determined)
  10. Thus, the region between Kunitz domains 2 and 3 encoded by exon 7 was highly glycosylated by two O-linked carbohydrate chains at Ser174 and Thr175 and one N-linked carbohydrate chain at Asn167
  11. These results indicated that the region is occupied by a cluster of three bulky and acidic carbohydrate chains