Mannion B A, Kolesnikova T V, Lin S H, Wang S, Thompson N L, Hemler M E
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1998 Dec 11;273(50):33127-9. doi: 10.1074/jbc.273.50.33127.
The 80/40-kDa CD98 protein complex was purified using an anti-CD98 heavy chain monoclonal antibody coupled to Sepharose beads. Eluted proteins were subjected to preparative SDS-polyacrylamide gel electrophoresis, and protein corresponding to the 40-kDa CD98 light chain was excised. Following proteolysis with trypsin, a peptide fragment was sequenced by mass spectrometry. The nine residues obtained were identical to established C-terminal sequences of the human E16 and rat TA1 proteins, suggesting that TA1/E16 protein is the CD98 light chain. Consistent with this, anti-TA1/E16 antibodies specifically immunoblotted the approximately 35-40-kDa light chain present upon immunoprecipitation of the human CD98 complex. Furthermore, anti-CD98 heavy chain antibody specifically co-immunoprecipitated hemagglutinin-tagged light chain from cells transfected with hemagglutinin-tagged E16 cDNA. In conclusion, the CD98 light chain is identical to the TA1/E16 protein, based on partial amino acid sequence identity, antibody cross-reactivity, genetic reconstitution evidence, similar molecular size, and comparable cell distribution.
使用与琼脂糖珠偶联的抗CD98重链单克隆抗体纯化80/40-kDa CD98蛋白复合物。将洗脱的蛋白进行制备性SDS-聚丙烯酰胺凝胶电泳，切下对应于40-kDa CD98轻链的蛋白条带。用胰蛋白酶进行蛋白水解后，通过质谱对一个肽片段进行测序。获得的九个残基与已确定的人E16和大鼠TA1蛋白的C末端序列相同，表明TA1/E16蛋白是CD98轻链。与此一致的是，抗TA1/E16抗体特异性免疫印迹人CD98复合物免疫沉淀时出现的约35-40-kDa轻链。此外，抗CD98重链抗体从用血凝素标记的E16 cDNA转染的细胞中特异性共免疫沉淀血凝素标记的轻链。总之，基于部分氨基酸序列同一性、抗体交叉反应性、基因重组证据、相似的分子大小和可比的细胞分布，CD98轻链与TA1/E16蛋白相同。
