Nakahara Y, Miyata T, Hamuro T, Funatsu A, Miyagi M, Tsunasawa S, Kato H
Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan.
Biochemistry. 1996 May 21;35(20):6450-9. doi: 10.1021/bi9524880.
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.
人组织因子途径抑制物是一种具有三个串联Kunitz型抑制结构域的蛋白酶抑制剂。使用中国仓鼠卵巢细胞生产重组蛋白（r-hTFPI），并对其多肽和糖链结构进行分析。蛋白酶消化后，使用蛋白质测序仪确定了由276个残基组成的完整氨基酸序列，该序列与从cDNA序列预测的序列相同。在三个潜在的N-糖基化位点中，Asn117和Asn167均被完全N-糖基化，但Asn228未被糖基化。Thr175也被完全O-糖基化，但Ser174部分被O-糖基化。对十一肽OG-11（残基Leu 170至Leu180）的碳水化合物组成和质谱分析表明，两条O-连接的糖链由具有0至3摩尔N-乙酰神经氨酸的1型核心结构（Gal-GalNAc-Ser/Thr）组成。在碳水化合物残基的还原端用2-氨基吡啶标记，并用唾液酸酶去除非还原端唾液酸后，通过二维碳水化合物图谱结合顺序糖苷酶消化分析N-连接的糖链。r-hTFPI中的所有N-连接结构均为复杂型糖链，在还原端的GlcNAc上连接有一个岩藻糖残基，由双天线、三天线和四天线糖链以1.9:1.3:1.0的比例组成。还发现了具有一个或两个N-乙酰乳糖胺重复序列的岩藻糖基化三天线和四天线糖链（占确定的糖链的30%）。因此，由外显子7编码的Kunitz结构域2和3之间的区域被Ser174和Thr175处的两条O-连接糖链和Asn167处一条N-连接糖链高度糖基化。这些结果表明，该区域被一簇三个庞大的酸性糖链占据。
