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 :
- Human tissue factor pathway inhibitor is a protease inhibitor with three tandem Kunitz-type inhibitory domains
- The recombinant protein (r- hTFPI ) was produced using Chinese hamster ovary cells, and its polypeptide and carbohydrate chain structures were analyzed
- 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
- Among three potential N-glycosylation sites , both Asn117 and Asn167 were fully N-glycosylated but Asn228 was not
- Thr175 was also fully O-glycosylated, but Ser174 was partially O-glycosylated
- 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)
- 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
- 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
- Fucosylated tri- and tetraantennary carbohydrate chains with one or two N-acetyllactosaminyl repeats were also found (30% of carbohydrate chains determined)
- 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
- These results indicated that the region is occupied by a cluster of three bulky and acidic carbohydrate chains
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. inhibitor, , tissue factor pathway inhibitor, carbohydrate structure, -
- 10. Asn167, , -, one N-linked carbohydrate chain, Asn167
- 10. Ser174, , -, two O-linked carbohydrate chains, Ser174 and Thr175
- 10. Thr175, , -, two O-linked carbohydrate chains, Ser174 and Thr175
- 10. glycosylated, , -, -, region
- 11. occupied, , -, -, region
- 4. N-glycosylated, , -, -, Asn117 and Asn167
- 4. N-glycosylation, , -, -, sites
- 5. O-glycosylated, , -, -, Ser174
- 5. O-glycosylated, , -, -, Thr175
- 8. structures, , hTFPI, structures, -
Output(Part-Of) (sent_index,
protein,
site):
- 0. tissue factor pathway inhibitor, sequence
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 10. tissue factor pathway inhibitor, one N-linked carbohydrate chain, Asn167
- 10. tissue factor pathway inhibitor, two O-linked carbohydrate chains, Ser174 and Thr175
- 4. tissue factor pathway inhibitor, -, Asn117 and Asn167
- 5. tissue factor pathway inhibitor, -, Ser174
- 5. tissue factor pathway inhibitor, -, Thr175