Purification, cloning, and characterization of a profibrinolytic plasminogen-binding protein, TIP49a
Purification, cloning, and characterization of a profibrinolytic plasminogen-binding protein, TIP49a
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DOI：
10.1074/jbc.m004919200
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发表时间：
2001-01-05
影响因子：
4.8
通讯作者：
Miles, LA
中科院分区：
文献类型：
--
作者：
Hawley, SB;Tamura, T;Miles, LA
负责增强细胞表面依赖性纤溶酶原活化的纤溶酶原受体暴露细胞表面上的COOH-末端赖氨酸，并且对羧肽酶B（CpB）的蛋白水解敏感。我们用CpB处理U937细胞，然后将膜组分进行二维凝胶电泳，然后用I-125-纤溶酶原进行配体印迹。一个54 kDa的蛋白质失去了能力，结合I-125-纤溶酶原处理后的完整细胞，并通过二维凝胶电泳纯化，然后通过质谱测序。获得了两个独立的氨基酸序列，并且与人和大鼠TIP 49 a中所含的序列相同。该54 kDa蛋白的cDNA与人TIP 49 a序列相匹配，并编码一个COOH末端赖氨酸，这与对CpB的易感性一致。针对大鼠TIP 49 a的抗体识别U937细胞膜的二维Western印迹上的纤溶酶原结合蛋白。人I-125-Glu-纤溶酶原与TIP 49 a蛋白特异性结合，结合被α-氨基己酸抑制。检测到一类单一的结合位点，并确定K-d为0.57 +/- 0.14 μ M。与BSA对照相比，TIP 49 a增强纤溶酶原活化8倍，这相当于纤溶酶处理的纤维蛋白原介导的增强。这些结果表明，TIP 49 a是一个以前未被识别的纤溶酶原结合蛋白的U937细胞表面。
The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel electrophoresis followed by ligand blotting with I-125-plasminogen. A 54-kDa protein lost the ability to bind I-125-plasminogen after treatment of intact cells and was purified by two-dimensional gel electrophoresis and then sequenced by mass spectrometry. Two separate amino acid sequences were obtained and were identical to sequences contained within human and rat TIP49a. The cDNA for the 54-kDa protein matched the human TIP49a sequence, and encoded a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodies against rat TIP49a recognized the plasminogen-binding protein on two-dimensional Western blots of U937 cell membranes. Human I-125-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by epsilon -aminocaproic acid. A single class of binding sites was detected, and a K-d of 0.57 +/- 0.14 muM was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhancement mediated by plasmin-treated fibrinogen. These results suggest that TIP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface.