Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis
Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis
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DOI：
10.1074/jbc.m601453200
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发表时间：
2006-05-12
影响因子：
4.8
通讯作者：
Oka, S
中科院分区：
文献类型：
--
作者：
Kizuka, Y;Matsui, T;Oka, S
主要在神经系统中表达的HNK-1碳水化合物被认为参与细胞迁移、识别、粘附和突触可塑性。人自然杀伤细胞-1（HNK-1）碳水化合物具有独特的结构，由硫酸化三糖（HSO（3）（-）3GlcA β 1-3Gal-1-4GlcNAc-）组成，并由两种葡萄糖醛酸转移酶（GlcAT-P或GlcAT-S）之一和磺基转移酶（HNK-1ST）依次生物合成。考虑到迄今为止在神经系统中确定的几乎所有HNK-1碳水化合物结构都是硫酸化的，我们假设GlcAT-P或GlcAT-S与HNK-1ST功能性相关，这导致HNK-1碳水化合物的有效连续生物合成。在这项研究中，我们证明了GlcAT-P和GlcAT-S与HNK-1ST在中国仓鼠卵巢细胞中的瞬时表达系统中免疫共沉淀。免疫荧光染色显示，这些酶主要是共定位在高尔基体。为了确定哪个结构域参与这种相互作用，我们制备了GlcAT-P、GlcAT-S和HNK-1ST的C-末端催化结构域，然后用纯化的酶进行下拉测定。结果，我们获得的证据表明，相互催化结构域的GlcAT-P或GlcAT-S和HNK-1ST是重要的，足以形成酶复合物。在体外试验系统中，GlcAT-P或GlcAT-S存在时，HNK-1ST的活性比不存在时增加约2倍。这些结果表明，该酶复合物的功能与HNK-1碳水化合物的有效连续生物合成有关。
HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO(3)(-)3GlcA beta 1-3Gal-1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C- terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT- P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.