Berg D T, Burck P J, Berg D H, Grinnell B W
Cardiovascular Research Department, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285-0434.
Blood. 1993 Mar 1;81(5):1312-22.
Native tissue plasminogen activator (ntPA) has a variable glycosylation site on its kringle-2 domain. We have examined the effects of kringle glycosylation on functional properties by studying the simplified tPA molecule, tPA-6. tPA-6 is composed of kringle-2 and the serine protease domains and, like ntPA, cells expressing tPA-6 process it into two glycoforms: the monoglycosylated tPA-6-primary (tPA-6P, type II) with N-linked glycosylation at Asn-448 in the serine protease domain and diglycosylated tPA-6-variant (tPA-6V, type I) with glycosylation at Asn-448 and at Asn-184 in kringle-2. When the two glycoforms were separated, we found that purified tPA-6V had reduced fibrin-stimulated plasminogenolytic activity toward Glu-plasminogen when compared to purified tPA-6P. However, in the presence of fibrin, tPA-6V unexpectedly exhibited a sixfold increase in selectivity toward Lys-plasminogen. In addition, tPA-6V was less susceptible than tPA-6P to plasmin-mediated conversion to the two-chain form. By site-directed mutagenesis of tPA-6, we eliminated variable glycosylation at Asn-184 and engineered a new glycosylation signal at a remnant site in the kringle. This derivative, designated tPA-6D, was secreted with complete kringle glycosylation. Like the naturally occurring tPA-6V, tPA-6D had lower rates of fibrin-stimulated Glu-plasminogen activation, increased specificity toward Lys-plasminogen, and greater resistance to plasmin digestion. Although the activity of tPA-6D could be stimulated by fibrin, its activity was not stimulated significantly by fibrinogen, and in human plasma the rate of fibrinogen depletion was reduced threefold. Although fibrin binding to kringle-2 of tPA-6D was slightly improved, there was a substantial increase in the dissociation constant (kd) for lysine binding, demonstrating a lack of correlation between these ligand-binding sites. Overall, our data demonstrate the marked effect of kringle glycosylation on functional properties. In addition, we have generated a derivative with properties that potentially improve clot specificity and single-chain half-life and reduce the potential for plasminogen activation in the plasma.
天然组织型纤溶酶原激活剂（ntPA）在其kringle-2结构域上有一个可变糖基化位点。我们通过研究简化的tPA分子tPA-6，来考察kringle糖基化对功能特性的影响。tPA-6由kringle-2和丝氨酸蛋白酶结构域组成，与ntPA一样，表达tPA-6的细胞将其加工成两种糖型：单糖基化的tPA-6-初级型（tPA-6P，II型），其在丝氨酸蛋白酶结构域的Asn-448处有N-连接糖基化；双糖基化的tPA-6-变体型（tPA-6V，I型），其在kringle-2的Asn-448和Asn-184处有糖基化。当分离出这两种糖型时，我们发现，与纯化的tPA-6P相比，纯化的tPA-6V对纤维蛋白刺激的向Glu-纤溶酶原的纤溶酶原溶解活性降低。然而，在有纤维蛋白存在的情况下，tPA-6V对Lys-纤溶酶原的选择性意外地增加了六倍。此外，tPA-6V比tPA-6P更不易被纤溶酶介导转化为双链形式。通过对tPA-6进行定点诱变，我们消除了Asn-184处的可变糖基化，并在kringle的一个残留位点设计了一个新的糖基化信号。这种衍生物称为tPA-6D，以完全的kringle糖基化形式分泌。与天然存在的tPA-6V一样，tPA-6D的纤维蛋白刺激的Glu-纤溶酶原激活率较低，对Lys-纤溶酶原的特异性增加，对纤溶酶消化的抵抗力更强。虽然tPA-6D的活性可被纤维蛋白刺激，但它的活性不会被纤维蛋白原显著刺激，并且在人血浆中纤维蛋白原消耗率降低了三倍。虽然纤维蛋白与tPA-6D的kringle-2的结合略有改善，但赖氨酸结合的解离常数（kd）大幅增加，表明这些配体结合位点之间缺乏相关性。总体而言，我们的数据证明了kringle糖基化对功能特性的显著影响。此外，我们生成了一种衍生物，其特性可能改善凝块特异性和单链半衰期，并降低血浆中纤溶酶原激活的可能性。
