Valnickova Zuzana, Christensen Trine, Skottrup Peter, Thøgersen Ida B, Højrup Peter, Enghild Jan J
Center for Insoluble Protein Structure (inSPIN) at the Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wied's Vej 10C, 8000 Aarhus C, Denmark.
Biochemistry. 2006 Feb 7;45(5):1525-35. doi: 10.1021/bi051956v.
Thrombin-activable fibrinolysis inhibitor (TAFI) is distinct from pancreatic procarboxypeptidase B in several ways. The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes. During this study, we observed that (i) the isoelectric point (pI) of TAFI shifts dramatically from pH 5 toward pH 8 upon activation and (ii) TAFIa is significantly less soluble than TAFI. The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity. The analyses revealed that all five potential N-glycosylation sites were utilized including Asn22, Asn51, Asn63, Asn86 (located in the activation peptide), and Asn219 (located in the catalytic domain). Asn219 was also found in an unglycosylated variant. Four of the glycans, Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous. In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver. Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation. TAFI contains eight cysteine residues, of which two, Cys69 and Cys383, are not involved in disulfides and contain free sulfhydryl groups. The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257. This pattern is homologous to pancreatic procarboxypeptidase B, and it is therefore unlikely that permutations in the cysteine connectivity are responsible for the enzymatic instability. LC-MS/MS analyses covering more than 90% of the TAFI amino acid sequence revealed no additional modifications. When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications. However, after activation, TAFIa loses 80% of the attached glycans, generating a large shift in pI and a propensity to precipitate. These changes are likely to significantly affect the properties of TAFIa as compared to TAFI.
凝血酶激活的纤溶抑制物（TAFI）在多个方面与胰腺羧肽酶原B不同。TAFIa的酶活性不稳定，半衰期为几分钟。在本研究中，我们观察到：（i）TAFI的等电点（pI）在激活后从pH 5显著向pH 8偏移；（ii）TAFIa的溶解度明显低于TAFI。通过表征所有翻译后修饰，包括连接的聚糖和二硫键连接，研究了这些观察结果的结构基础。分析表明，所有五个潜在的N-糖基化位点都被利用，包括Asn22、Asn51、Asn63、Asn86（位于激活肽中）和Asn219（位于催化结构域中）。Asn219也存在未糖基化的变体。其中四个聚糖，Asn51、Asn63、Asn86和Asn219表现出微观异质性，而连接到Asn22的聚糖似乎是均匀的。此外，检测到连接到三甘露糖核心的平分型GlcNAc，这表明其来源不是肝脏。单糖组成和LC-MS/MS分析未提供O-糖基化的证据。TAFI含有八个半胱氨酸残基，其中两个，Cys69和Cys383，不参与二硫键形成，含有游离巯基。其余六个半胱氨酸形成二硫键，包括Cys156-Cys169、Cys228-Cys252和Cys243-Cys257。这种模式与胰腺羧肽酶原B同源，因此二硫键连接的排列不太可能是酶不稳定性的原因。覆盖TAFI氨基酸序列90%以上的LC-MS/MS分析未发现其他修饰。综合这些结果表明，TAFIa固有的不稳定性不是由翻译后修饰引起的。然而，激活后，TAFIa失去80%的连接聚糖，导致pI大幅偏移并倾向于沉淀。与TAFI相比，这些变化可能会显著影响TAFIa的性质。
