Malapert M, Pellissier B, Borgese F
Laboratoire Jean Maetz, ERS 1253, Département de Biologie Cellulaire et Moléculaire CEA, Villefranche-sur-mer, France.
Eur J Biochem. 1998 Oct 1;257(1):228-35. doi: 10.1046/j.1432-1327.1998.2570228.x.
The Na+/H+ exchanger (NHE) is a plasma membrane transport protein found in a wide range of biological systems. NHE is involved in various functions including pH homeostasis, volume regulation, cell proliferation and transcellular Na+ absorption. This study reports immunodetection results obtained with antibodies generated against the C-terminus of the NHE of trout red blood cells, betaNHE. Immunoblotting of cell membrane preparation reveals that betaNHE is a protein with an apparent molecular mass of 95 kDa. Moreover enzymatic glycosidase treatment demonstrates that the antiporter is an N-glycosylated but not O-glycosylated protein. The primary structure of betaNHE contains three putative N-glycosylation consensus sites (N-X-S/T) at Asn49, Asn338 and Asn378. Expression of betaNHE in PS120 fibroblasts, a cell line which lacks an endogenous Na+/H+ exchange, allows to determine the precise sites of glycosylation. The construction of a site-directed mutated betaNHE antiporter, lacking the first predicted motif, shows that betaNHE possesses an unique glycosylation site located on the first extracellular loop of the exchanger (Asn49). Expression of this deglycosylated antiporter shows that deglycosylation of the protein modifies neither the pH(i) dependency of the antiporter nor its hormonal stimulation.
钠氢交换体（NHE）是一种存在于多种生物系统中的质膜转运蛋白。NHE参与多种功能，包括pH稳态、体积调节、细胞增殖和跨细胞钠吸收。本研究报告了用针对虹鳟红细胞NHE（βNHE）C末端产生的抗体获得的免疫检测结果。细胞膜制剂的免疫印迹显示，βNHE是一种表观分子量为95 kDa的蛋白质。此外，酶糖苷酶处理表明，该反向转运蛋白是一种N-糖基化而非O-糖基化的蛋白质。βNHE的一级结构在Asn49、Asn338和Asn378处含有三个假定的N-糖基化共有序列（N-X-S/T）。在缺乏内源性钠氢交换的PS120成纤维细胞中表达βNHE，可以确定糖基化的精确位点。构建一个缺乏第一个预测基序的定点突变βNHE反向转运蛋白，表明βNHE在该转运蛋白的第一个细胞外环（Asn49）上有一个独特的糖基化位点。这种去糖基化反向转运蛋白的表达表明，该蛋白的去糖基化既不改变反向转运蛋白对细胞内pH（i）的依赖性，也不改变其激素刺激作用。
