Spiro R G, Bhoyroo V D
J Biol Chem. 1984 Aug 10;259(15):9858-66.
Treatment of thyroglobulins from several mammalian sources (calf, sheep, pig, dog, rat, rabbit, guinea pig, and man) with alpha-galactosidase demonstrated a species-dependent occurrence of terminal alpha-D-galactosyl residues which ranged from 11 mol/mol of protein (23% of total galactose) in calf to a complete absence in man. The presence of the alpha-D-galactosyl groups resulted in a partial binding of the thyroglobulins (greater than 70% in calf and sheep) to Bandeiraea simplicifolia I-agarose, and this lectin-thyroglobulin interaction could be quantitated by a solid-phase assay utilizing 125I-labeled B. simplicifolia I. Sequential glycosidase digestions of calf thyroglobulin glycopeptides containing the complex carbohydrate unit (unit B) and characterization of oligosaccharide obtained by partial acid hydrolysis indicated that the alpha-D-galactosyl residues are located on oligosaccharide branches with an alpha-D-Gal-(1----3)-beta-D-Gal-(1----4)-D-GlcNAc sequence. While mild acid treatment of calf thyroglobulin glycopeptides yielded a disaccharide, alpha-D-Gal-(1----3)-D-Gal, and a trisaccharide, alpha-D-Gal-(1----3)-beta-D-Gal-(1----4)-D-GlcNAc, which could be resolved by B. simplicifolia I-agarose or thin-layer chromatography, similar hydrolysis of the human unit B-containing glycopeptides did not produce such components. A study of various glycopeptides indicated that the alpha-D-galactosyl residues are unevenly distributed among the multiple complex carbohydrate units of calf thyroglobulin and are preferentially located in units with a relatively low sialic acid content. During affinity chromatography on B. simplicifolia I-agarose, glycopeptides with multiple alpha-D-galactosyl groups bound more firmly to the lectin than those which contained only a single residue. In contrast to the alpha-D-galactosyl residues, beta-linked galactose of calf thyroglobulin was primarily bound in penultimate locations being susceptible to enzymatic release only after prior removal of capping sialyl and alpha-D-galactosyl groups. The isolation of N-acetyllactosamine and a beta-D-Gal----beta-D-GlcNAc----D-Man trisaccharide from partial acid hydrolysates helped to position the beta-D-galactosyl residues in the oligosaccharide branches of the complex carbohydrate units.
用α-半乳糖苷酶处理来自多种哺乳动物来源（小牛、绵羊、猪、狗、大鼠、兔子、豚鼠和人）的甲状腺球蛋白，结果表明末端α-D-半乳糖基残基的出现存在物种依赖性，其含量范围从小牛的11摩尔/摩尔蛋白质（占总半乳糖的23%）到人的完全缺失。α-D-半乳糖基的存在导致甲状腺球蛋白部分结合（小牛和绵羊中大于70%）到单叶豆（Bandeiraea simplicifolia）I-琼脂糖上，并且这种凝集素-甲状腺球蛋白相互作用可以通过使用125I标记的单叶豆I的固相测定法定量。对含有复合碳水化合物单元（单元B）的小牛甲状腺球蛋白糖肽进行连续糖苷酶消化，并对部分酸水解获得的寡糖进行表征，结果表明α-D-半乳糖基残基位于具有α-D-半乳糖-（1→3）-β-D-半乳糖-（1→4）-D-葡萄糖胺序列的寡糖分支上。虽然对小牛甲状腺球蛋白糖肽进行温和酸处理可产生二糖α-D-半乳糖-（1→3）-D-半乳糖和三糖α-D-半乳糖-（1→3）-β-D-半乳糖-（1→4）-D-葡萄糖胺，它们可通过单叶豆I-琼脂糖或薄层色谱法分离，但对含有人单元B的糖肽进行类似水解不会产生此类成分。对各种糖肽的研究表明，α-D-半乳糖基残基在小牛甲状腺球蛋白的多个复合碳水化合物单元中分布不均，并且优先位于唾液酸含量相对较低的单元中。在单叶豆I-琼脂糖上进行亲和色谱时，具有多个α-D-半乳糖基的糖肽比仅含有单个残基的糖肽更牢固地结合到凝集素上。与α-D-半乳糖基残基不同，小牛甲状腺球蛋白的β-连接半乳糖主要结合在倒数第二个位置，只有在预先去除封端的唾液酸基和α-D-半乳糖基后才易受酶释放。从部分酸水解产物中分离出N-乙酰乳糖胺和β-D-半乳糖→β-D-葡萄糖胺→D-甘露糖三糖有助于确定β-D-半乳糖基残基在复合碳水化合物单元寡糖分支中的位置。
