El Golli Nargès, Issertial Odile, Rosa Jean-Philippe, Briquet-Laugier Véronique
Laboratory of Hemostasis and Thrombosis, Cardiovascular Research Center Inserm Lariboisière, U689-E6 INSERM, IFR139, Université Paris 7, Hôpital Lariboisière, 2 rue Ambroise Paré, 75010 Paris, France.
J Biol Chem. 2005 Aug 26;280(34):30329-35. doi: 10.1074/jbc.M503847200. Epub 2005 Jun 17.
Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed alpha-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory alpha-granule chemokine, and demonstrated that PF4 alpha-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41-50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 +/- 2% to 17 +/- 3%, p < or = 0.0001). Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in alpha-granule targeting was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNG-green fluorescent protein chimera exhibited an 82.5 +/- 1.8% colocalization with the alpha-granule proteins von Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human alpha-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same alpha-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to alpha-granule targeting of chemokines and possibly of other alpha-granule proteins.
血小板通过从其储存颗粒（称为α-颗粒）分泌分泌蛋白，在血管损伤部位实现止血。我们最近分析了血小板因子4（PF4）的颗粒靶向，PF4是一种分泌性α-颗粒趋化因子，并证明PF4在α-颗粒中的储存依赖于PF4成熟序列中的决定因素。为了确定这些决定因素，通过逐步缺失和定点诱变产生了与荧光报告蛋白绿色荧光蛋白融合的PF4突变体。然后将它们转染到AtT20细胞中，并使用激光扫描共聚焦显微镜通过与含促肾上腺皮质激素的颗粒共定位来评估颗粒靶向。该策略确定氨基酸41-50（LIATLKNGRK）序列对PF4颗粒靶向和/或储存最为关键；从PF4中删除该序列会导致颗粒储存显著减少（从81±2%降至17±3%，p≤0.0001）。对LIATLKNGRK进行丙氨酸扫描诱变，将靶向基序缩小到LKNG。在血小板生成素诱导的人巨核细胞Dami细胞中证实了LKNG在α-颗粒靶向中的直接作用，其中LKNG-绿色荧光蛋白嵌合体与α-颗粒蛋白血管性血友病因子和P-选择素的共定位率为82.5±1.8%。LKNG在趋化因子家族中保守性较差。然而，人α-颗粒趋化因子Nap-2（中性粒细胞激活肽）和RANTES（活化正常T细胞表达和分泌调控因子）与PF4的三维比对显示，LKNG是一个表面暴露的亲水性转角/环，与具有相似特征的Nap-2（LKDG）和RANTES（TRKN）肽匹配。此外，Nap-2和RANTES肽表现出与LKNG相同的α-颗粒靶向效率。因此，我们推测PF4 LKNG的三维和物理化学特征与趋化因子以及可能其他α-颗粒蛋白的α-颗粒靶向普遍相关。
