Beaufort Nathalie, Leduc Dominique, Eguchi Hiroshi, Mengele Karin, Hellmann Daniela, Masegi Tsukio, Kamimura Takashi, Yasuoka Susumu, Fend Falko, Chignard Michel, Pidard Dominique
Inserm, E0336, Paris, France.
Am J Physiol Lung Cell Mol Physiol. 2007 May;292(5):L1263-72. doi: 10.1152/ajplung.00191.2006. Epub 2007 Jan 19.
The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase-type plasminogen activator receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein that plays a crucial role in innate immunity and inflammation by supporting cell migration and matrix degradation, with structure and biological properties that can be regulated via limited endoproteolysis. With the use of immunoblotting, flow immunocytometry, and ELISA analyses applied to a recombinant uPAR protein and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range of 10-500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into the truncated (D2D3) species, with cleavage occurring in the D1 to D2 linker sequence after arginine residues at position 83 and 89. Using immunohistochemistry, we found that both HAT and uPAR were expressed in the human bronchial epithelium. Moreover, transient cotransfection in epithelial cells showed that membrane coexpression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways.
人气道类胰蛋白酶（HAT）是一种与呼吸道上皮相关的II型跨膜丝氨酸蛋白酶，在气道炎症性疾病期间，它也作为一种细胞外酶在肺液中被检测到。我们评估了它影响尿激酶型纤溶酶原激活物受体（uPAR）的能力，uPAR是一种膜糖脂锚定的三结构域（D1D2D3）糖蛋白，通过支持细胞迁移和基质降解在先天免疫和炎症中起关键作用，其结构和生物学特性可通过有限的内切蛋白水解作用进行调节。通过对重组uPAR蛋白以及表达uPAR的单核细胞和人支气管上皮细胞进行免疫印迹、流式免疫细胞术和ELISA分析，结果表明，uPAR暴露于10 - 500 nM范围内的可溶性HAT会导致全长（D1D2D3）被蛋白水解加工成截短的（D2D3）形式，切割发生在第83和89位精氨酸残基后的D1至D2连接序列中。通过免疫组织化学，我们发现HAT和uPAR在人支气管上皮中均有表达。此外，上皮细胞中的瞬时共转染表明，两个蛋白的膜共表达会导致D1结构域的组成性和广泛脱落，这种情况发生在纳摩尔范围内的膜相关HAT浓度下。由于发现截短的受体无法结合uPAR的两种主要配体，即黏附性基质蛋白玻连蛋白和丝氨酸蛋白酶尿激酶，因此，HAT对uPAR的蛋白水解调节似乎可能会调节气道炎症反应期间的细胞黏附与运动以及组织重塑。
