Institute for Clinical Chemistry, University of Cologne, D-50924 Cologne, Germany.
Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center (BMC), Lund University, SE-221 00 Lund, Sweden.
J Biol Chem. 2018 Jan 5;293(1):203-214. doi: 10.1074/jbc.M117.818930. Epub 2017 Nov 16.
C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins. Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator (tPA) thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown. Here, we recombinantly expressed murine and human full-length CLEC3As as well as truncated forms of CLEC3A in HEK-293 Epstein-Barr nuclear antigen (EBNA) cells. We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy. We found that CLEC3A mainly occurs as a monomer, but also forms dimers and trimers, potentially via a coiled-coil α-helix. We also noted that CLEC3A can be modified with chondroitin/dermatan sulfate side chains and tends to oligomerize to form higher aggregates. We show that CLEC3A is present in resting, proliferating, and hypertrophic growth-plate cartilage and assembles into an extended extracellular network in cultures of rat chondrosarcoma cells. Further, we found that CLEC3A specifically binds to plasminogen and enhances tPA-mediated plasminogen activation. In summary, we have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation.
C 型凝集素结构域家族 3 成员 A（CLEC3A）是一种特征不明确的蛋白，属于 C 型凝集素超家族。其最接近的同源物四连接蛋白结合纤溶酶原的kringle 4 结构域，并增强其与组织纤溶酶原激活物（tPA）的结合，从而增强纤溶酶的产生，但 CLEC3A 是否有助于纤溶酶原的激活尚不清楚。在这里，我们在 HEK-293 Epstein-Barr 核抗原（EBNA）细胞中重组表达了鼠和人全长 CLEC3A 以及 CLEC3A 的截断形式。我们通过 SDS-PAGE 和免疫印迹、糖分析、基质辅助激光解吸电离飞行时间质谱、尺寸排阻色谱、圆二色光谱和电子显微镜分析了重组 CLEC3A 的结构；比较了重组蛋白与软骨中提取的 CLEC3A 的性质；并通过免疫组织化学和免疫荧光显微镜研究了其组织分布和细胞外组装。我们发现 CLEC3A 主要以单体形式存在，但也可以形成二聚体和三聚体，可能通过卷曲螺旋α-螺旋。我们还注意到 CLEC3A 可以被软骨素/硫酸皮肤素侧链修饰，并倾向于寡聚形成更高的聚集体。我们表明 CLEC3A 存在于静止、增殖和肥大的生长板软骨中，并在大鼠软骨肉瘤细胞的培养中组装成扩展的细胞外网络。此外，我们发现 CLEC3A 特异性结合纤溶酶原并增强 tPA 介导的纤溶酶原激活。总之，我们确定了软骨特异性凝集素 CLEC3A 的结构、组织分布和分子功能，并表明 CLEC3A 结合纤溶酶原并参与 tPA 介导的纤溶酶原激活。
