Melikian H E, McDonald J K, Gu H, Rudnick G, Moore K R, Blakely R D
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322.
J Biol Chem. 1994 Apr 22;269(16):12290-7.
Antibodies have been raised against synthetic peptides derived from the predicted primary sequence of the human cocaine- and antidepressant-sensitive norepinephrine (NE) transporter (NET). One antibody (N430), raised and purified against a putative intracellular human norepinephrine transporter (hNET) epitope, detects hNET expression in a stably transfected cell line (LLC-NET) by indirect immunofluorescence only in the presence of detergent, while no immunoreactivity is observed in either the parental cells (LLC-PK1) or in LLC-NET cells incubated with preimmune sera or peptide absorbed antibody. N430 immunoblots of LLC-NET cell extracts reveal two major immunoreactive hNET species in these cells, migrating at 80 and 54 kDa, respectively. Pulse-chase N430 immunoprecipitation studies confirm that the 54-kDa species is a transient, glycosylated intermediate of a longer lived, more highly glycosylated protein with an apparent M(r) of 80,000. In contrast, a 54-kDa species is the primary hNET product in vaccinia virus T7-infected HeLa cells, transiently transfected with hNET cDNA. PNGase F digestion of extracts prepared from LLC-NET- and hNET-transfected HeLa cells convert all immunoreactive species to a 46-kDa form, equivalent to that observed following incubation of whole cells with the glycosylation inhibitor tunicamycin. As transiently transfected HeLa and stable LLC-NET cells exhibit a pharmacologically similar NE transport activity, it appears likely that the additional glycosylation evident in the stable line does not contribute significantly to antagonist sensitivity. On the other hand, NE transport and antagonist ([125I]RTI-55) binding assays on whole LLC-NET cells treated with tunicamycin reveal a pronounced reduction in NE transport activity and hNET membrane density paralleled by an inability of NET proteins to replenish the higher M(r) hNET pool. These findings suggest an obligate role for N-linked glycosylation in hNET biosynthetic maturation, stability, and functional expression. In summary, N430 antibody is a useful tool for the visualization and characterization of hNET gene products and has permitted the first direct evaluation of biosynthetic steps leading to functional catecholamine transporter expression.
已经制备了针对源自人可卡因和抗抑郁药敏感去甲肾上腺素（NE）转运体（NET）预测一级序列的合成肽的抗体。一种抗体（N430）是针对假定的细胞内人去甲肾上腺素转运体（hNET）表位制备并纯化的，仅在存在去污剂的情况下通过间接免疫荧光检测稳定转染细胞系（LLC-NET）中的hNET表达，而在亲本细胞（LLC-PK1）或用免疫前血清或肽吸收抗体孵育的LLC-NET细胞中均未观察到免疫反应性。LLC-NET细胞提取物的N430免疫印迹显示这些细胞中有两种主要的免疫反应性hNET蛋白，迁移率分别为80 kDa和54 kDa。脉冲追踪N430免疫沉淀研究证实，54 kDa的蛋白是一种寿命更长、糖基化程度更高的蛋白（表观分子量为80,000）的瞬时糖基化中间体。相比之下，54 kDa的蛋白是痘苗病毒T7感染的HeLa细胞中瞬时转染hNET cDNA后的主要hNET产物。对LLC-NET和hNET转染的HeLa细胞提取物进行PNGase F消化后，所有免疫反应性蛋白均转化为46 kDa的形式，这与用糖基化抑制剂衣霉素孵育全细胞后观察到的情况相同。由于瞬时转染的HeLa细胞和稳定的LLC-NET细胞表现出药理学上相似的NE转运活性，因此稳定细胞系中明显的额外糖基化似乎对拮抗剂敏感性没有显著贡献。另一方面，对用衣霉素处理的全LLC-NET细胞进行的NE转运和拮抗剂（[125I]RTI-55）结合测定显示，NE转运活性和hNET膜密度显著降低，同时NET蛋白无法补充较高分子量的hNET池。这些发现表明N-连接糖基化在hNET生物合成成熟、稳定性和功能表达中起重要作用。总之，N430抗体是用于可视化和表征hNET基因产物的有用工具，并首次允许对导致功能性儿茶酚胺转运体表达的生物合成步骤进行直接评估。
