Hokke C H, Damm J B, Penninkhof B, Aitken R J, Kamerling J P, Vliegenthart J F
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Eur J Biochem. 1994 Apr 1;221(1):491-512. doi: 10.1111/j.1432-1033.1994.tb18762.x.
The N-linked carbohydrate chains of porcine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and subsequently separated from the O-glycoprotein by gel-permeation chromatography on Bio-Gel P-100. The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment. Fractionation of the extremely heterogeneous mixture of O-linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of 32 O-glycans were determined by one- and two-dimensional 1H-NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Gal beta 1-4GlcNAc beta 1-3Gal beta 1-3GalNAc-ol, Gal beta 1-4(6SO4-)GlcNAc, and alpha 2-3-linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n = 0 to > 6: [Neu5Gc/Ac alpha2-3]0-1[Gal beta 1-4(6SO4-)GlcNAc beta 1-3]nGal beta 1-4GlcNAc beta 1-3Gal Beta 1-3GalNAc-ol. In addition, smaller compounds were identified in which the Gal beta 1-3GalNAc-ol core is substituted by Neu5Gc/Ac alpha 2-6-linked to GalNAc-ol and/or Neu5Gc/Ac alpha 2-3-linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6-O-sulfated GlcNAc residues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAc beta 1-3GalNAc-ol core structure. Analysis of the endo-beta-galactosidase digests of pools of N- and O-glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N-acetyllactosamine) chains. In the case of the N-linked carbohydrate chains, multiple branching of the core structures occurs, resulting in an even larger heterogeneity than observed for the O-linked carbohydrate chains.
用肽 - N4 -（N - 乙酰 - β - 葡糖胺基）天冬酰胺酶F消化猪透明带糖蛋白的N - 连接碳水化合物链，随后通过在Bio - Gel P - 100上的凝胶渗透色谱法将其与O - 糖蛋白分离。通过碱性硼氢化钠处理从O - 糖蛋白中释放O - 连接的碳水化合物链。通过包括在Bio - Gel P - 4和P - 6上的凝胶渗透色谱法、在Mono Q上的阴离子交换快速蛋白质液相色谱法以及在CarboPac PA - 1上的高pH阴离子交换色谱法的色谱技术组合，实现了O - 连接低聚糖糖醇极其不均一混合物的分级分离。通过一维和二维1H - NMR光谱法测定了32种O - 聚糖的一级结构。分析的化合物的主要部分包含结构元件Galβ1 - 4GlcNAcβ1 - 3Galβ1 - 3GalNAc - ol、Galβ1 - 4(6SO4 - )GlcNAc和α2 - 3连接的Neu5Gc或Neu5Ac的组合。这一系列化合物具有以下结构，其中n = 0至> 6：[Neu5Gc/Acα2 - 3]0 - 1[Galβ1 - 4(6SO4 - )GlcNAcβ1 - 3]nGalβ1 - 4GlcNAcβ1 - 3Galβ1 - 3GalNAc - ol。此外，鉴定出了较小的化合物，其中Galβ1 - 3GalNAc - ol核心被与GalNAc - olα2 - 6连接和/或与Galα2 - 3连接的Neu5Gc/Ac取代。此外，还获得了其中6 - O - 硫酸化GlcNAc残基的分布与上述一般结构不同的低聚糖，并且一小部分低聚糖具有GlcNAcβ1 - 3GalNAc - ol核心结构。对N - 和O - 聚糖池的内切β - 半乳糖苷酶消化物的分析表明，这两种类型的低聚糖在质量上含有相似的聚（N - 乙酰乳糖胺）链。在N - 连接碳水化合物链的情况下，核心结构会发生多个分支，导致比O - 连接碳水化合物链观察到的更大的不均一性。
