Edge A S, Spiro R G
J Biol Chem. 1985 Dec 5;260(28):15332-8.
The distribution along the polypeptide of the carbohydrate units of two major calf thyroid cell surface glycoproteins, GP-1 and GP-3, was obtained from a study of their glycopeptides obtained after Pronase digestion. The GP-3 molecule (Mr = 20,000) yielded two large glycopeptides (Mr = 9,500 and 7,000) in equimolar amounts which each consisted of one N-linked (Mr = 5,400) and several small O-linked oligosaccharides accounting for a total of nine carbohydrate attachment sites in a 27-amino acid residue segment of the peptide chain. The Pronase treatment of GP-1 (Mr = 100,000) revealed the presence of a large protease-resistant fragment (Mr = 50,000) which contained 34 carbohydrate units (eight N-linked and 26 O-linked) in a segment of 105 amino acids. In addition to these densely glycosylated peptides (one glycosylation site/3 amino acid residues), small glycopeptides with polymannose saccharide units were found in the digests of both proteins. The occurrence of repeating N-acetyllactosamine sequences in the N-linked carbohydrate units of GP-1 and GP-3 was suggested by the composition and size of the oligosaccharides released by hydrazinolysis and was demonstrated by endo-beta-galactosidase treatment. The cleavage products from digestion with this enzyme were identified as NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----3Gal, Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal, Gal beta 1----4GlcNAc beta 1----3Gal, and GlcNAc beta 1----3Gal with the tetrasaccharides constituting the predominant species. The terminal alpha-D-Gal residues accounted for the binding of GP-1 and GP-3 glycopeptides to Bandeiraea simplicifolia I-agarose; concanavalin A-Sepharose affinity chromatography indicated that most of the N-linked carbohydrate units of both glycoproteins contained more than two branches. Difference in the branching on the poly-N-acetyllactosamine sequences of GP-1 and GP-3 was suggested by the finding that only the latter glycoprotein, as well as its glycopeptides, reacted with anti-blood group I antibodies; neither glycoprotein demonstrated blood group i antigenicity. Examination of cultured thyroid follicular cells revealed that both I and i determinants were present at the cell surface.
通过对两种主要的小牛甲状腺细胞表面糖蛋白GP - 1和GP - 3经链霉蛋白酶消化后得到的糖肽进行研究，获得了碳水化合物单元沿多肽链的分布情况。GP - 3分子（Mr = 20,000）产生了两个等摩尔量的大糖肽（Mr = 9,500和7,000），每个糖肽由一个N - 连接（Mr = 5,400）和几个小的O - 连接寡糖组成，在肽链的一个27个氨基酸残基片段中共有九个碳水化合物连接位点。对GP - 1（Mr = 100,000）进行链霉蛋白酶处理后发现存在一个大的抗蛋白酶片段（Mr = 50,000），该片段在105个氨基酸的片段中含有34个碳水化合物单元（八个N - 连接和26个O - 连接）。除了这些高度糖基化的肽（一个糖基化位点/3个氨基酸残基）外，在两种蛋白质的消化物中还发现了带有多甘露糖聚糖单元的小糖肽。通过肼解释放的寡糖的组成和大小表明，GP - 1和GP - 3的N - 连接碳水化合物单元中存在重复的N - 乙酰乳糖胺序列，并通过内切β - 半乳糖苷酶处理得到证实。用该酶消化产生的裂解产物被鉴定为NeuAcα2----6Galβ1----4GlcNAcβ1----3Gal、Galα1----3Galβ1----4GlcNAcβ1----3Gal、Galβ1----4GlcNAcβ1----3Gal和GlcNAcβ1----3Gal，其中四糖是主要成分。末端α - D - Gal残基导致GP - 1和GP - 3糖肽与单叶豆凝集素I - 琼脂糖结合；伴刀豆球蛋白A - 琼脂糖亲和层析表明，两种糖蛋白的大多数N - 连接碳水化合物单元含有两个以上的分支。GP - 1和GP - 3的多聚N - 乙酰乳糖胺序列分支的差异通过以下发现表明：只有后一种糖蛋白及其糖肽与抗血型I抗体反应；两种糖蛋白均未表现出血型i抗原性。对培养的甲状腺滤泡细胞的检查表明，I和i决定簇均存在于细胞表面。
