Chen Rui, Jin Ge, McIntyre Thomas M
From the Departments of Cellular and Molecular Medicine and.
the Department of Biological Sciences, Case Western Reserve University School of Dental Medicine, Cleveland, Ohio 44106.
J Biol Chem. 2017 Jun 16;292(24):10112-10122. doi: 10.1074/jbc.M116.771642. Epub 2017 Apr 28.
Platelets are the sole source of EGF in circulation, yet how EGF is stored or released from stimulated cells is undefined. In fact, we found platelets did not store EGF, synthesized as a single 6-kDa domain in pro-EGF, but rather expressed intact pro-EGF precursor on granular and plasma membranes. Activated platelets released high-molecular-weight (HMW)-EGF, produced by a single cleavage between the EGF and the transmembrane domains of pro-EGF. We synthesized a fluorogenic peptide encompassing residues surrounding the putative sessile arginyl residue and found stimulated platelets released soluble activity that cleaved this pro-EGF peptide. High throughput screening identified chymostatins, bacterial peptides with a central cyclic arginyl structure, as inhibitors of this activity. In contrast, the matrix metalloproteinase/TACE (tumor necrosis factor-α-converting enzyme) inhibitor GM6001 was ineffective. Stimulated platelets released the soluble protease ADAMDEC1, recombinant ADAMDEC1 hydrolyzed pro-EGF, and this activity was inhibited by chymostatin and not GM6001. Biotinylating platelet surface proteins showed ADAMDEC1 hydrolyzed surface pro-EGF to HMW-EGF that stimulated HeLa EGF receptor (EGFR) reporter cells and EGFR-dependent tumor cell migration. This proteolysis was inhibited by chymostatin and not GM6001. Metabolizing pro-EGF Arg to citrulline with recombinant polypeptide arginine deiminase 4 (PAD4) abolished ADAMDEC1-catalyzed pro-EGF peptidolysis, while pretreating intact platelets with PAD4 suppressed ADAMDEC1-, thrombin-, or collagen-induced release of HMW-EGF. We conclude that activated platelets release ADAMDEC1, which hydrolyzes pro-EGF to soluble HMW-EGF, that HMW-EGF is active, that proteolytic cleavage of pro-EGF first occurs at the C-terminal arginyl residue of the EGF domain, and that proteolysis is the regulated and rate-limiting step in generating soluble EGF bioactivity from activated platelets.
血小板是循环中表皮生长因子（EGF）的唯一来源，但EGF如何在受刺激细胞中储存或释放尚不清楚。事实上，我们发现血小板并不储存作为前体EGF中单一6 kDa结构域合成的EGF，而是在颗粒膜和质膜上表达完整的前体EGF前体。活化的血小板释放出高分子量（HMW）-EGF，它是由前体EGF的EGF结构域和跨膜结构域之间的单次切割产生的。我们合成了一种包含假定的固定精氨酰残基周围残基的荧光肽，发现受刺激的血小板释放出可切割该前体EGF肽的可溶性活性物质。高通量筛选确定抑糜酶素（具有中心环状精氨酰结构的细菌肽）是这种活性的抑制剂。相比之下，基质金属蛋白酶/TACE（肿瘤坏死因子-α转化酶）抑制剂GM6001无效。受刺激的血小板释放出可溶性蛋白酶ADAMDEC1，重组ADAMDEC1水解前体EGF，并且这种活性被抑糜酶素抑制而不被GM6001抑制。对血小板表面蛋白进行生物素化显示，ADAMDEC1将表面前体EGF水解为HMW-EGF，后者刺激了HeLa表皮生长因子受体（EGFR）报告细胞以及EGFR依赖性肿瘤细胞迁移。这种蛋白水解被抑糜酶素抑制而不被GM6001抑制。用重组多肽精氨酸脱亚氨酶4（PAD4）将前体EGF中的精氨酸代谢为瓜氨酸消除了ADAMDEC1催化的前体EGF肽水解作用，而用PAD4预处理完整的血小板则抑制了ADAMDEC1、凝血酶或胶原诱导的HMW-EGF释放。我们得出结论，活化的血小板释放ADAMDEC1，其将前体EGF水解为可溶性HMW-EGF，HMW-EGF具有活性，前体EGF的蛋白水解切割首先发生在EGF结构域的C末端精氨酰残基处，并且蛋白水解是从活化血小板产生可溶性EGF生物活性的调节和限速步骤。
