Hansen L K, O'Leary J J, Skubitz A P, Furcht L T, McCarthy J B
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 5545, USA.
Biochim Biophys Acta. 1995 Sep 27;1252(1):135-45. doi: 10.1016/0167-4838(95)00113-9.
This study was undertaken to characterize the potential heparin affinity of an amino-acid sequence within the 70 kDa heat-shock family of proteins (HSPs) that shares homology with a heparin-binding sequence present in the carboxy-terminus of fibronectin (FN), defined by the synthetic peptide, FN-C/H-II (KNNQKSEPLIGRKKT). To first define the heparin binding sequence within FN-C/H-II, solid phase binding assays were performed using overlapping, short (7 amino acids) synthetic peptides corresponding to the amino-acid sequence within FN-C/H-II. Only the sequence LIGRKKT bound [3H] heparin, and the LIGRKKT peptide blocked heparin binding to intact fibronectin by 47% (+/- 0.4, p < 0.001). A computer-generated homology search revealed that two members of the 70 kDa HSP family, HSP70 and HSC70, contain the sequences LIGRK and LIGRR, respectively. Examination of heparin binding using affinity chromatography indicated that while native HSC70 binds heparin, native HSP70 does not. Treatment of the heparin-unbound fraction with heat or urea led to enhanced HSP70 binding to heparin affinity columns. Soluble LIGRKKT peptide or anti-FN-C/H-II IgG also significantly inhibited heparin binding to HSC70 that had been purified by heparin affinity chromatography. Finally, Western blot analysis of HSC70 purified by heparin affinity chromatography demonstrated that polyclonal anti-FN-C/H-II IgG could recognize HSC70. These data demonstrate that LIGRK or LIGRR represent a a common heparin binding motif in fibronectin, HSP70, and HSC70, and are consistent with a proposed role for heparin or similar polyanionic structures in the function of the 70 kDa heat-shock proteins.
本研究旨在表征70 kDa热休克蛋白家族（HSPs）中一个氨基酸序列的潜在肝素亲和力，该序列与纤连蛋白（FN）羧基末端存在的肝素结合序列具有同源性，由合成肽FN-C/H-II（KNNQKSEPLIGRKKT）定义。为了首先确定FN-C/H-II中的肝素结合序列，使用与FN-C/H-II内氨基酸序列相对应的重叠短（7个氨基酸）合成肽进行了固相结合测定。只有序列LIGRKKT结合[3H]肝素，并且LIGRKKT肽使肝素与完整纤连蛋白的结合阻断了47%（±0.4，p<0.001）。计算机生成的同源性搜索显示，70 kDa HSP家族的两个成员HSP70和HSC70分别包含序列LIGRK和LIGRR。使用亲和色谱法检测肝素结合表明，天然HSC70结合肝素，而天然HSP70不结合。用热或尿素处理未结合肝素的部分导致HSP70与肝素亲和柱的结合增强。可溶性LIGRKKT肽或抗FN-C/H-II IgG也显著抑制肝素与通过肝素亲和色谱法纯化的HSC70的结合。最后，对通过肝素亲和色谱法纯化的HSC70进行的蛋白质印迹分析表明，多克隆抗FN-C/H-II IgG可以识别HSC70。这些数据表明，LIGRK或LIGRR代表纤连蛋白、HSP70和HSC70中常见的肝素结合基序，并且与肝素或类似多阴离子结构在70 kDa热休克蛋白功能中的拟议作用一致。
