Laboratory of Extracellular Matrix Biochemistry, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Matrix Biol. 2013 Apr 24;32(3-4):188-95. doi: 10.1016/j.matbio.2013.01.005. Epub 2013 Jan 26.
Nephronectin is a basement membrane protein comprising five N-terminal epidermal growth factor (EGF)-like repeats, a central linker segment containing an Arg-Gly-Asp (RGD) motif and a C-terminal meprin-A5 protein-receptor protein tyrosine phosphatase μ (MAM) domain. Nephronectin has been shown to interact with α8β1 integrin through the central linker segment, but its interactions with other molecules remain to be elucidated. Here, we examined the binding of nephronectin to a panel of glycosaminoglycan (GAG) chains. Nephronectin bound strongly to heparin and chondroitin sulfate (CS)-E and moderately to heparan sulfate (HS), but failed to bind to CS-A, CS-C, CS-D, dermatan sulfate and hyaluronic acid. Deletion of the MAM domain severely impaired the binding of nephronectin to heparin but not CS-E, whereas deletion of the EGF-like repeats reduced its binding to CS-E but not heparin, suggesting that nephronectin interacts with CS-E and heparin through the EGF-like repeats and MAM domain, respectively. Consistent with these results, nephronectin bound to agrin and perlecan, which are heparan sulfate proteoglycans (HSPGs) in basement membranes, in HS-dependent manners. Site-directed mutagenesis of the MAM domain revealed that multiple basic amino acid residues in the putative loop regions were involved in the binding of the MAM domain to agrin. The binding of nephronectin to basement membrane HSPGs was further confirmed by in situ nephronectin overlay assays using mouse frozen tissue sections. Taken together, these findings indicate that nephronectin is capable of binding to HSPGs in basement membranes via the MAM domain, and thereby raise the possibility that interactions with basement membrane HSPGs may be involved in the deposition of nephronectin onto basement membranes.
纤连蛋白是一种基底膜蛋白，由五个 N 端表皮生长因子（EGF）样重复序列、一个包含 Arg-Gly-Asp（RGD）基序的中央连接片段和一个 C 端 Meprin-A5 蛋白-受体蛋白酪氨酸磷酸酶 μ（MAM）结构域组成。纤连蛋白已被证明通过中央连接片段与α8β1 整合素相互作用，但它与其他分子的相互作用仍有待阐明。在这里，我们检查了纤连蛋白与一系列糖胺聚糖（GAG）链的结合。纤连蛋白与肝素和硫酸软骨素（CS）-E 结合紧密，与硫酸乙酰肝素（HS）结合中等，但与硫酸软骨素（CS）-A、CS-C、CS-D、硫酸皮肤素和透明质酸不结合。MAM 结构域缺失严重削弱了纤连蛋白与肝素的结合，但不影响 CS-E 的结合，而 EGF 样重复序列缺失则降低了其与 CS-E 的结合，但不影响肝素的结合，这表明纤连蛋白分别通过 EGF 样重复序列和 MAM 结构域与 CS-E 和肝素相互作用。与这些结果一致，纤连蛋白与硫酸乙酰肝素蛋白聚糖（HSPG）在基底膜中的 agrin 和 perlecan 结合，呈 HS 依赖性。MAM 结构域的定点突变显示，假定环区中的多个碱性氨基酸残基参与了 MAM 结构域与 agrin 的结合。纤连蛋白与基底膜 HSPG 的结合通过使用小鼠冷冻组织切片的原位纤连蛋白覆盖测定进一步得到证实。总之，这些发现表明纤连蛋白能够通过 MAM 结构域与基底膜 HSPG 结合，从而提出与基底膜 HSPG 的相互作用可能参与纤连蛋白在基底膜上的沉积的可能性。
