Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine.
Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine.
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DOI：
10.1016/s0021-9258(18)99126-x
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发表时间：
1991-06
期刊：
影响因子：
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通讯作者：
S. Bjoern;Donald C. Foster;Lars Thim;F. Wiberg;M. Christensen;Y. Komiyama;A. Pedersen;Walter Kisiel
中科院分区：
文献类型：
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作者：
S. Bjoern;Donald C. Foster;Lars Thim;F. Wiberg;M. Christensen;Y. Komiyama;A. Pedersen;Walter Kisiel
因子VII是一种多结构域、维生素K依赖性血浆糖蛋白，参与凝血的外源性途径。早期的研究证明了在人血浆因子VII中O-糖苷连接到丝氨酸52的新二糖（Xyl-Glc）或三糖（Xyl 2-Glc）（Nishimura，H.，Kawabata，S.，Kisiel，W.，Hase，S.，池中，T.，Shimonishi，Y.，和Iwanaga，S.（1989）J.Biol.Chem.264，20320-20325）。在本研究中，分离人血浆和重组因子VII并进行酶促片段化。分离包含每种因子VII制剂的第一表皮生长因子样结构域的残基48-62的肽用于比较分析。使用氨基酸测序，碳水化合物和氨基酸组成分析和质谱分析的组合策略，检测到由葡萄糖，葡萄糖-木糖或葡萄糖-（木糖）2组成的三种不同的聚糖结构O-糖苷连接到血浆和重组因子VII中的丝氨酸52。在血浆因子VII中观察到大约等量的三种聚糖结构，而在重组因子VII中葡萄糖和葡萄糖-（木糖）2结构占主导地位。除了在丝氨酸52处观察到的O-连接聚糖结构外，在人血浆和重组因子VII中发现单个岩藻糖在丝氨酸60处共价连接。碳水化合物和质谱分析表明，丝氨酸60的岩藻糖基化几乎是定量的。使用[14 C]岩藻糖的代谢标记研究证实了丝氨酸60处存在O-连接岩藻糖。为了评估丝氨酸52处的碳水化合物部分是否有助于因子VII的生物活性，我们构建了重组因子VII的位点特异性突变体，其中丝氨酸52已被丙氨酸残基取代。在凝血试验中，突变型因子VIIa显示出野生型因子VIIa约60%的凝血活性。突变体因子VIIa的酰胺分解活性与重组野生型因子VIIa所观察到的是不可区分的。此外，突变体因子VIIa与人膀胱癌细胞系（J82）表面上纯化的再脂化组织因子脱辅基蛋白或组织因子复合激活因子X或因子IX的能力与野生型因子VIIa所观察到的几乎相同。这些结果表明，O-糖苷键连接到丝氨酸52的碳水化合物部分似乎不参与因子VIIa与组织因子的相互作用，或与组织因子形成复合物后其对因子X或因子IX的蛋白水解活性的表达。（400字处截断摘要）
Factor VII is a multidomain, vitamin K-dependent plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. Earlier studies demonstrated a novel disaccharide (Xyl-Glc) or trisaccharide (Xyl2-Glc) O-glycosidically linked to serine 52 in human plasma factor VII (Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Shimonishi, Y., and Iwanaga, S. (1989) J. Biol. Chem. 264, 20320-20325). In the present study, human plasma and recombinant factor VII were isolated and subjected to enzymatic fragmentation. Peptides comprising residues 48-62 of the first epidermal growth factor-like domain of each factor VII preparation were isolated for comparative analysis. Using a combined strategy of amino acid sequencing, carbohydrate and amino acid composition analysis, and mass spectrometry, three different glycan structures consisting of either glucose, glucose-xylose, or glucose-(xylose)2 were detected O-glycosidically linked to serine 52 in plasma and recombinant factor VII. Approximately equal amounts of the three glycan structures were observed in plasma factor VII, whereas in recombinant factor VII the glucose and the glucose-(xylose)2 structures predominated. In addition to the O-linked glycan structures observed at serine 52, a single fucose was found to be covalently linked at serine 60 in both human plasma and recombinant factor VII. Carbohydrate and mass spectrometry analyses indicated that the fucosylation of serine 60 was virtually quantitative. Metabolic labeling studies using [14C]fucose confirmed the presence of O-linked fucose at serine 60. In order to assess whether the carbohydrate moiety at serine 52 contributes to the biological activity of factor VII, we have constructed a site-specific mutant of recombinant factor VII in which serine 52 has been replaced with an alanine residue. Mutant factor VIIa exhibited approximately 60% of the coagulant activity of wild-type factor VIIa in a clotting assay. The amidolytic activity of mutant factor VIIa was indistinguishable from that observed for recombinant wild-type factor VIIa. In addition, the ability of mutant factor VIIa in complex with either purified relipidated tissue factor apoprotein or tissue factor on the surface of a human bladder carcinoma cell line (J82) to activate either factor X or factor IX was virtually identical to that observed for wild-type factor VIIa. These results indicate that the carbohydrate moiety O-glycosidically linked to serine 52 does not appear to be involved either in the interaction of factor VIIa with tissue factor, or the expression of its proteolytic activity toward factor X or factor IX following complex formation with tissue factor.(ABSTRACT TRUNCATED AT 400 WORDS)