Hayes G R, Williams A, Costello C E, Enns C A, Lucas J J
Department of Biochemistry and Molecular Biology, SUNY Health Science Center at Syracuse 13210, USA.
Glycobiology. 1995 Mar;5(2):227-32. doi: 10.1093/glycob/5.2.227.
The human transferrin receptor (TfR) contains three N-linked oligosaccharides and glycosylation is required for the proper folding and function of the molecule. Earlier studies demonstrated that the oligosaccharide at Asn-727 is vital for the production of fully active TfR. The oligosaccharide(s) present at this site have been analysed using a combination of site-directed mutagenesis and chemical analysis. Wild-type TfR and mutants containing only the Asn-727 site or missing all three sites were transfected into mouse 3T3 cells and receptors were analysed by endo-N-acetylglucosaminidase H (Endo-H) digestion, SDS-PAGE and immunoblotting. These studies suggested that the Asn-727 site contains high-mannose or Endo-H-sensitive hybrid oligosaccharides. Glycosylation of Asn-727 found in the TfR purified from human placentae was analysed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and mass spectrometry following tryptic digestion, peptide purification via reverse-phase high-performance liquid chromatography (RP-HPLC) and peptide sequencing. HPAE-PAD showed the presence of a series of high-mannose oligosaccharides. Mass spectrometry confirmed these observations, but also showed the presence of an 80 Da anionic moiety on a fraction of the oligosaccharides.
人转铁蛋白受体（TfR）含有三个N - 连接寡糖，糖基化对于该分子的正确折叠和功能是必需的。早期研究表明，Asn - 727处的寡糖对于产生完全活性的TfR至关重要。已使用定点诱变和化学分析相结合的方法分析了该位点存在的寡糖。将野生型TfR和仅含有Asn - 727位点或缺失所有三个位点的突变体转染到小鼠3T3细胞中，并通过内切N - 乙酰葡糖胺糖苷酶H（Endo - H）消化、SDS - PAGE和免疫印迹分析受体。这些研究表明，Asn - 727位点含有高甘露糖或Endo - H敏感的杂合寡糖。对从人胎盘中纯化的TfR中发现的Asn - 727的糖基化进行了分析，方法是在胰蛋白酶消化后，通过反相高效液相色谱（RP - HPLC）进行肽纯化和肽测序，然后采用带脉冲安培检测的高pH阴离子交换色谱法（HPAE - PAD）和质谱法。HPAE - PAD显示存在一系列高甘露糖寡糖。质谱法证实了这些观察结果，但也显示在一部分寡糖上存在一个80 Da的阴离子部分。
