Thornton D J, Sheehan J K, Lindgren H, Carlstedt I
Department of Biochemistry and Molecular Biology, University of Manchester, U.K.
Biochem J. 1991 Jun 15;276 ( Pt 3)(Pt 3):667-75. doi: 10.1042/bj2760667.
Mucus glycoproteins (mucins) were isolated from sputum of patients with cystic fibrosis (CF) after separation into sol and gel phases. The mucus gel was solubilized with gentle stirring in 6 M-guanidinium chloride supplemented with proteinase inhibitors, and purification of mucins was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. Density-gradient centrifugation also revealed a heterogeneity of the macromolecules, the pattern of which varied between individuals, and mucins from the gel phase was pooled as 'heavy' and 'light' fractions. Gel chromatography on Sepharose CL-2B showed that the heavy fraction contained a larger proportion of smaller species than the 'light' fraction and that the gel phase mucins were much larger than those from the sol. An apparently homogeneous high-Mr mucin population from one individual contained approx. 70% (w/w) carbohydrate, the major sugars being N-acetylglucosamine (17.8%), N-acetylgalactosamine (6.7%), galactose (20.7%), fucose (13.2%) and sialic acid (11.4%). These mucins had an S020.w of 47 S, and an Mr of 15 x 10(6) -20 x 10(6), and rate-zonal centrifugation revealed a polydisperse size distribution [range (5-30) x 10(6)] with a weight-average Mr of 17 x 10(6). The whole mucins were visualized with electron microscopy as linear and apparently flexible threads, disperse in size. Reduction produced subunits which were included on Sepharose CL-2B, and subsequent trypsin digestion yielded high-Mr glycopeptides which were further retarded. The size distributions and fragmentation patterns of mucin from two other CF patients were the same, as studied by gel chromatography, rate-zonal centrifugation and electron microscopy. We conclude that CF mucins are heterogeneous in both size and buoyant density and that the various populations, though differing in buoyant density, share the same architecture and macromolecular properties and are, in this respect, similar to mucins from normal respiratory secretions [Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt (1990) Biochem. J. 265, 179-186] and human cervical mucus [Carlstedt & Sheehan (1989) SEB Symp. XLIII 289-316].
黏液糖蛋白（黏蛋白）从囊性纤维化（CF）患者的痰液中分离出来，先将痰液分为溶胶相和凝胶相。在添加蛋白酶抑制剂的6M - 氯化胍中轻轻搅拌使黏液凝胶溶解，随后通过在CsCl/氯化胍中进行等密度梯度离心来纯化黏蛋白。密度梯度离心还揭示了大分子的异质性，其模式在个体之间有所不同，凝胶相的黏蛋白被收集为“重”和“轻”组分。在Sepharose CL - 2B上进行凝胶过滤显示，重组分比“轻”组分含有更大比例的较小分子，并且凝胶相黏蛋白比溶胶相的黏蛋白大得多。来自一个个体的明显均一的高Mr黏蛋白群体含有约70%（w/w）的碳水化合物，主要糖类为N - 乙酰葡糖胺（17.8%）、N - 乙酰半乳糖胺（6.7%）、半乳糖（20.7%）、岩藻糖（13.2%）和唾液酸（11.4%）。这些黏蛋白的S020.w为47 S，Mr为15×10(6) - 20×10(6)，速率区带离心显示其具有多分散的大小分布[范围（5 - 30）×10(6)]，重均Mr为17×10(6)。通过电子显微镜观察，完整的黏蛋白呈现为线性且明显柔韧的细丝，大小不一。还原反应产生的亚基可被Sepharose CL - 2B保留，随后用胰蛋白酶消化产生更高Mr的糖肽，其进一步被滞留。通过凝胶过滤、速率区带离心和电子显微镜研究发现，另外两名CF患者的黏蛋白的大小分布和片段化模式相同。我们得出结论，CF黏蛋白在大小和浮力密度方面均具有异质性，并且各种群体虽然浮力密度不同，但具有相同的结构和大分子性质，在这方面，它们与正常呼吸道分泌物中的黏蛋白[Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt (1990) Biochem. J. 265, 179 - 186]以及人宫颈黏液中的黏蛋白[Carlstedt & Sheehan (1989) SEB Symp. XLIII 289 - 316]相似。
