Edge A S, Spiro R G
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts.
J Biol Chem. 1987 Nov 25;262(33):16135-41.
Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.
通过凝胶过滤、薄层色谱和阴离子交换色谱对经碱性硼氢化物处理从胎球蛋白释放的 O 连接碳水化合物单元进行分析，结果表明该糖蛋白中除了先前描述的四糖和三糖外，还存在一种含酸性葡糖胺的六糖。基于外切糖苷酶消化、高碘酸盐氧化、甲基化分析以及肼 - 亚硝酸断裂法，确定该六糖的结构为 NeuAcα2----3Galβ1----3[NeuAcα2----3Galβ1----4GlcNAcβ1----6]GalNAc。对还原的脱唾液酸六糖进行后一操作时，产生了 Gal----2 - 脱氧半乳糖醇和 Gal----脱水甘露糖，结果表明它们分别源自 Gal----N - 乙酰半乳糖胺醇和 Gal----GlcNAc 序列。用 [14C]甲胺对 Gal----脱水甘露糖二糖进行还原性胺化，从而确定其连接方式为 1----4。虽然肺炎双球菌内切α - D - N - 乙酰半乳糖胺酶作用于脱唾液酸胎球蛋白会释放出不含唾液酸的四糖和三糖（Galβ1----3GalNAc），但该酶不会切割脱唾液酸六糖（Galβ1----3[Galβ1----4GlcNAcβ1----6]GalNAc）的肽连接。基于半乳糖胺醇分析，确定每个胎球蛋白分子中 O 连接的六糖、四糖和三糖的数量分别为 0.2, 0.7 和 2.1。胎球蛋白中不存在含 O 连接 N - 乙酰葡糖胺的四糖或五糖，这表明该糖的连接是一个限速步骤；此外，六糖的有限出现可能表明向 Galβ1----3GalNAc 添加唾液酸以形成 NeuAcα2----3Gal 连接会阻止 GlcNAc 转移酶作用于 GalNAc 残基形成分支点。
