Rawitch A B, Pollock G, Yang S X, Taurog A
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66103.
Arch Biochem Biophys. 1992 Sep;297(2):321-7. doi: 10.1016/0003-9861(92)90679-q.
Highly purified, trypsin/detergent-solubilized thyroid peroxidase (TPO), prepared from pig thyroid tissue, was subjected to reduction and alkylation followed by trypsin digestion. The resulting peptides were fractionated using HPLC. Corresponding carbohydrate positive regions from three separate HPLC experiments were pooled and further chromatography was carried out to yield purified peptide suitable for sequence analysis and complete carbohydrate composition analysis. Four of the five putative sites for N-linked glycosylation were found to carry oligosaccharide units in which mannose and glucosamine were the sole or predominant sugars. Three of the four glycosylations occur at asparagine residues which are likely to be at beta turns or bends. The fifth putative glycosylation site could not be confirmed and may either be poorly glycosylated or escape glycosylation. All of the confirmed glycosylated sites occur in the N-terminal third of the TPO polypeptide chain, in the portion of the molecule believed to be extracellular. The isolation of at least two chromatographic forms of glycopeptide derived from each of the confirmed sites suggests microheterogeneity in the structure of the oligosaccharide units of thyroid peroxidase similar to that observed in many other glycoproteins.
从猪甲状腺组织中制备的高度纯化的、经胰蛋白酶/去污剂溶解的甲状腺过氧化物酶（TPO），先进行还原和烷基化处理，然后用胰蛋白酶消化。所得肽段通过高效液相色谱（HPLC）进行分离。将来自三个独立HPLC实验的相应碳水化合物阳性区域合并，进一步进行色谱分离，以得到适合序列分析和完整碳水化合物组成分析的纯化肽段。五个假定的N-连接糖基化位点中有四个被发现带有寡糖单元，其中甘露糖和氨基葡萄糖是唯一或主要的糖类。四个糖基化中有三个发生在可能处于β转角或弯曲处的天冬酰胺残基上。第五个假定的糖基化位点无法得到证实，可能糖基化程度较低或未发生糖基化。所有已确认的糖基化位点都位于TPO多肽链的N端三分之一处，即在分子中被认为是细胞外的部分。从每个已确认位点衍生出至少两种色谱形式的糖肽，这表明甲状腺过氧化物酶寡糖单元的结构存在微不均一性，类似于在许多其他糖蛋白中观察到的情况。
