Bisecting GlcNAc mediates the binding of annexin V to Hsp47
Bisecting GlcNAc mediates the binding of annexin V to Hsp47
复制标题
DOI：
10.1093/glycob/cwj005
复制
发表时间：
2005-11-01
期刊：
影响因子：
4.3
通讯作者：
Taniguchi, N
中科院分区：
文献类型：
--
作者：
Gao, CX;Miyoshi, E;Taniguchi, N
通过UDP-N-乙酰葡糖胺：β-D-甘露糖苷β-1，4-N-乙酰葡糖胺转移酶III（GnT-III）催化形成的二等分N-乙酰葡糖胺（GlcNAc）结构负责多种生物学功能。我们以前已经表明，膜联蛋白V，钙/磷脂结合膜联蛋白家族的蛋白质的成员，具有对二等分GIcNAe结构的结合活性。在这项研究中，我们报告了在大鼠肝癌细胞系M31中寻找膜联蛋白的潜在靶糖蛋白。使用谷胱甘肽S-转移酶（GST）-膜联蛋白V固定化Sepharose 4 B亲和柱捕获相互作用的蛋白质产生的GnT-III转染的M31细胞，我们分离到一个47 kDa的蛋白质。经N-末端序列分析鉴定为Hsp 47。免疫沉淀实验表明，膜联蛋白V与热休克蛋白47相互作用。通过用N-糖苷酶F处理或用含有二等分GlcNAc的糖链预孵育来废除膜联蛋白V和HSP 47的关联，这表明二等分GlcNAc在相互作用中起重要作用。从GnT-III转染的M31细胞中纯化的Hsp 47的寡糖分析显示具有二等分GlcNAc结构，如通过红细胞凝集植物血凝素（E-4-PHA）和基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱（MS）分析检测到的。表面等离子体共振分析表明，膜联蛋白V被绑定到Hsp 47，轴承平分GlcNAc的Kd为5.5 μ M，而没有观察到显着的结合的情况下，Hsp 47没有平分GlcNAc。此外，免疫荧光显微镜显示共定位的膜联蛋白V，热休克蛋白47，和一个平分的GlcNAc糖链周围的高尔基体。总之，这些结果表明膜联蛋白V与Hsp 47的结合是由二等分的GlcNAc寡糖结构介导的，并且Hsp 47是膜联蛋白V的细胞内配体糖蛋白。
The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GIcNAe structure. In this study, we reported on a search for potential target glycoproteins for annexin in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E-4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a K-d of 5.5 mu M, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.