Brito Catarina, Escrevente Cristina, Reis Celso A, Lee Virginia M-Y, Trojanowski John Q, Costa Júlia
Instituto de Tecnologia Química e Biológica, Oeiras, Portugal.
J Neurosci Res. 2007 May 1;85(6):1260-70. doi: 10.1002/jnr.21230.
The expression of the fucosylated carbohydrate Lewis(x) (Le(x)) determinant (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R) has been found in glycoproteins, proteoglycans, and glycolipids from the nervous system. Evidence suggests its association with cell-cell recognition, neurite outgrowth, and neuronal migration during central nervous system development. In the present work, we detected increased levels of Le(x) in differentiated human NT2N neurons cultured in vitro. To identify which fucosyltransferase (FUT) synthesized the Le(x) in NT2N neurons, RT-PCR, FUT substrate specificity and Western blot analysis were carried out. Strong activity toward acceptors Galbeta4GlcNAc-O-R and Fucalpha2Galbeta4GlcNAc-O-R [R = -(CH(2))(3)NHCO(CH(2))(5)NH-biotin], together with strong FUT9 detection by Western blot and presence of transcripts showed that FUT9 was the enzyme associated with Le(x) biosynthesis in NT2N neurons. Le(x) was detected at the plasma membrane of NT2N neurons, in lysosomes marked with lysosomal-associated membrane protein 1 (LAMP-1), and it was found for the first time to colocalize with the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) that defines the TI-VAMP exocytic compartment that is involved in neurite outgrowth. Furthermore, incubation with anti-Le(x) monoclonal antibody L5 led to impaired adhesion of NT2N neurons to the surface matrix and inhibited neurite initiation. In conclusion, FUT9 and its product Le(x) are detected specifically in human NT2N neurons and our results indicate that they underlie cell differentiation, cell adhesion, and initiation of neurite outgrowth in those neurons.
岩藻糖基化碳水化合物路易斯x（Le(x)）决定簇（Gal(β1-4)[Fuc(α1-3)]GlcNAc-R）已在神经系统的糖蛋白、蛋白聚糖和糖脂中被发现。有证据表明其在中枢神经系统发育过程中与细胞间识别、神经突生长和神经元迁移有关。在本研究中，我们检测到体外培养的分化人NT2N神经元中Le(x)水平升高。为了确定哪种岩藻糖基转移酶（FUT）在NT2N神经元中合成Le(x)，我们进行了逆转录聚合酶链反应（RT-PCR）、FUT底物特异性分析和蛋白质印迹分析。对受体Galβ4GlcNAc-O-R和Fucα2Galβ4GlcNAc-O-R [R = -(CH(2))(3)NHCO(CH(2))(5)NH-生物素]具有强活性，同时蛋白质印迹法检测到强FUT9信号以及转录本的存在，表明FUT9是与NT2N神经元中Le(x)生物合成相关的酶。在NT2N神经元的质膜、用溶酶体相关膜蛋白1（LAMP-1）标记的溶酶体中检测到Le(x)，并且首次发现它与破伤风神经毒素不敏感的囊泡相关膜蛋白（TI-VAMP）共定位，TI-VAMP定义了参与神经突生长的TI-VAMP胞吐区室。此外，用抗Le(x)单克隆抗体L5孵育导致NT2N神经元与表面基质的粘附受损，并抑制神经突起始。总之，在人NT2N神经元中特异性检测到FUT9及其产物Le(x)，我们的结果表明它们是这些神经元中细胞分化、细胞粘附和神经突生长起始的基础。
