Magdolen V, Rettenberger P, Koppitz M, Goretzki L, Kessler H, Weidle U H, König B, Graeff H, Schmitt M, Wilhelm O
Frauenklinik der Technischen Universität München, Germany.
Eur J Biochem. 1996 May 1;237(3):743-51. doi: 10.1111/j.1432-1033.1996.0743p.x.
The amino-terminal fragment of human uPA (ATF; amino acids 1-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His6 tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR. The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine. Lys23, Tyr24, Phe25, IIe28, and Trp30 were important determinants for uPAR binding. A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATF. Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPAR-binding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thr18 by alanine or Asn32 by serine had hardly any effect, replacement of Asn22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR. Deletion of either Val20, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants. Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity. The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated uPA/uPAR interaction.
人尿激酶型纤溶酶原激活物（uPA）的氨基末端片段（ATF；氨基酸1 - 135），其包含尿激酶型纤溶酶原激活物受体（uPAR，CD87）的结合位点，在酿酒酵母中表达。通过在氨基末端框内插入His6序列对重组酵母ATF进行修饰和延伸，然后通过镍螯合亲和层析从总蛋白提取物中纯化，结果表明其具有功能活性，因为它能有效地与uPA竞争结合细胞表面相关的uPAR。ATF表达质粒用作构建一系列定点突变体的模板，以确定那些对与uPAR结合很重要的氨基酸。除了一个突变体（Ser26缺失）外，所有突变的ATF蛋白均以稳定形式表达（约20 - 30 ng/mg总蛋白），并且通过使用固定在微量滴定板上的重组uPAR的uPA - 配体结合试验来测试每个突变体的结合能力。uPA结合区域环B的11个氨基酸（氨基酸20 - 30）中的每一个都分别被丙氨酸取代。Lys23、Tyr24、Phe25、Ile28和Trp30是uPAR结合的重要决定因素。还对化学合成的跨越ATF氨基酸14 - 32的线性肽进行了系统的丙氨酸扫描。获得了与酵母ATF突变体类似的结果。在另一组实验中，改变了uPA的uPAR结合区域中仅在人和狒狒之间保守而在其他物种中不保守的那些氨基酸：用丙氨酸取代Thr18或用丝氨酸取代Asn32几乎没有任何影响，而用酪氨酸取代Asn22和用精氨酸取代Trp30（这两个位置在其他哺乳动物中严格保守）导致ATF变体无法与人uPAR相互作用。分别缺失Val20、Ser21、Lys23、His29或Val20加Ser21也产生了无反应性的ATF突变体。最后，将ATF中的Lys23替换为某些氨基酸：用丙氨酸、组氨酸或谷氨酰胺取代Lys23产生了具有中等uPAR结合活性的ATF变体，而引入带负电荷的氨基酸（用谷氨酸替换Lys23）则完全消除了uPAR结合活性。关于ATF突变体和uPA衍生肽的结果可能为确定uPA与其受体物理相互作用的性质提供必要线索，并可能有助于开发uPA衍生的肽类似物作为潜在的治疗剂来阻断肿瘤细胞相关的uPA/uPAR相互作用。
