Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor
Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor
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DOI：
10.1021/bi0600695
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发表时间：
2006-05-30
期刊：
影响因子：
2.9
通讯作者：
Mason, Anne B.
中科院分区：
文献类型：
--
作者：
Byrne, Shaina L.;Leverence, Rachael;Mason, Anne B.
本文报道了用幼仓鼠肾细胞（BHK）生产转铁蛋白受体可溶性部分（sTFR）的方法，并首次研究了糖基化对sTFR生物学功能的影响。sTFR（残基121-760）具有三个N-连接的糖基化位点（Asn 251、Asn 317和Asn 727）。虽然完全糖基化的sTFR分泌到组织培养基中（类似于40 mg/L），但不能产生非糖基化的sTFR，表明碳水化合物对受体的折叠、稳定性和/或分泌至关重要。其中在位置251和727处的糖基化被消除的突变体（N251 D和N727 D）被良好地表达，而N317 D突变体的产生较差。电喷雾电离质谱分析证实了sTFR的二聚化以及每个突变体中单个位点不存在碳水化合物。糖基化对二铁人转铁蛋白结合的影响（Fe-2 hTF），一种真正的单铁hTF，在C叶中含有铁（命名为Fe-C hTF），和突变体（命名为Mut-Fe-C hTF，铁释放速率慢30倍）通过表面等离子体共振测定;对于Fe-C hTF和Mut-Fe-C hTF与sTFR N317 D突变体的结合，注意到小的（类似于20%）但一致的差异。在pH 5.6下，从与sTFR和sTFR突变体复合的Fe-C hTF和Mut-Fe-C hTF释放铁的速率揭示了仅N317 D突变体具有显著效果。位置317处的碳水化合物靠近之前显示与hTF相互作用的TFR区域。
Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium (similar to 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe-2 hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe-C hTF), and a mutant (designated Mut-Fe-C hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small (similar to 20%) but consistent difference is noted for the binding of Fe-C hTF and the Mut-Fe-C hTF to the sTFR N317D mutant. The rate of iron release from Fe-C hTF and Mut-Fe-C hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.