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.
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.
Nakahara
Y
Y
Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan.
Miyata
T
T
Hamuro
T
T
Funatsu
A
A
Miyagi
M
M
Tsunasawa
S
S
Kato
H
H
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D002241:Carbohydrates; D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor