Kojima T, Katsumi A, Yamazaki T, Muramatsu T, Nagasaka T, Ohsumi K, Saito H
First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan.
J Biol Chem. 1996 Mar 8;271(10):5914-20. doi: 10.1074/jbc.271.10.5914.
Ryudocan, a heparan sulfate proteoglycan, was isolated from human endothelium-like EAhy926 cells by a combination of ion-exchange and immunoaffinity chromatography. Purified human ryudocan has biochemical properties similar to those of rat ryudocan isolated from microvascular endothelial cells. Human ryudocan contains only heparan sulfate (HS) glycosaminoglycan chains along with a core protein with an apparent molecular mass of 30 kDa. We evaluated the interactions between purified human ryudocan and several extracellular ligands by using a solid-phase binding assay. We found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibit significant ryudocan binding. Heparitinase (but not chondroitin ABC lyase) treatment destroyed the ability of ryudocan binding to bFGF, MK, and TFPI. Heparin and HS, but not chondroitin sulfate, inhibited such ryudocan binding. Thus, the HS chains of ryudocan appear to be responsible for its binding to bFGF, MK, and TFPI. The apparent dissociation constants for purified ryudocan were as follows: bFGF, 0.50 nM; MK, 0.30 nM; and TFPI, 0.74 nM. Immunohistochemical analysis revealed that ryudocan was expressed in fibrous connective tissues, peripheral nerve tissues, and placental trophoblasts. These findings suggest that ryudocan may possess multiple biological functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via HS-binding effectors in the cellular microenvironment.
硫酸乙酰肝素蛋白聚糖Ryudocan是通过离子交换和免疫亲和层析相结合的方法从人内皮样EAhy926细胞中分离出来的。纯化后的人Ryudocan具有与从微血管内皮细胞中分离出的大鼠Ryudocan相似的生化特性。人Ryudocan仅包含硫酸乙酰肝素（HS）糖胺聚糖链以及一个表观分子量为30 kDa的核心蛋白。我们通过固相结合试验评估了纯化后的人Ryudocan与几种细胞外配体之间的相互作用。我们发现碱性成纤维细胞生长因子（bFGF）、中期因子（MK）和组织因子途径抑制剂（TFPI）与Ryudocan有显著的结合。肝素酶（而非软骨素ABC裂解酶）处理破坏了Ryudocan与bFGF、MK和TFPI结合的能力。肝素和HS可抑制这种Ryudocan结合，而硫酸软骨素则不能。因此，Ryudocan的HS链似乎负责其与bFGF、MK和TFPI的结合。纯化后的Ryudocan的表观解离常数如下：bFGF为0.50 nM；MK为0.30 nM；TFPI为0.74 nM。免疫组织化学分析显示，Ryudocan在纤维结缔组织、外周神经组织和胎盘滋养层细胞中表达。这些发现表明，Ryudocan可能通过细胞微环境中的HS结合效应物具有多种生物学功能，如bFGF调节、促进神经突生长和抗凝作用。
