Fung D C, Somerville M, Richardson P S, Sheehan J K
Department of Physiology, St. George's Hospital Medical School, London, United Kingdom.
Am J Respir Cell Mol Biol. 1995 Mar;12(3):296-306. doi: 10.1165/ajrcmb.12.3.7873196.
This paper describes low-density mucus glycoconjugates released from feline trachea by dirhamnolipid (DRL), a toxin from Pseudomonas aeruginosa. Mucus glycoconjugates in feline tracheas were radiolabeled in vivo with 3H-proline and 14C-glucose. Control mucus and that released by 200 micrograms/ml DRL were dissolved in guanidine hydrochloride buffer (GuHCl) and chromatographed on Sepharose CL-2B. Molecules eluting in the void volume (V0) of the column were isolated by isopycnic density gradient centrifugation in CsCl/GuHCl. All samples gave peaks of radiolabeled and periodic acid/Schiff (PAS)-reactive material at rho = approximately 1.50 and approximately 1.60 g/ml, but DRL-stimulated samples contained low-density material (rho < 1.32 g/ml), also PAS-reactive and radiolabeled. Control secretions incubated with DRL in vitro did not form low-density material. In Triton X-100 (1% vol/vol), a nonionic detergent, low-density material behaved as smaller molecules, running in the partially included volume (Vi) of the column of Sepharose CL-2B, but still in the V0 of Sephacryl S-300. Incubation with chondroitinase ABC, heparinase II and III, and keratanase failed to change its elution profile on S-300, evidence against glycosaminoglycans; but proteolysis with trypsin or proteinase K gave two peaks, peptide fragments near the totally included volume of the column and glycopeptides in V0. The V0 glycopeptides banded between 1.50 and 1.55 g/ml in a CsCl gradient and eluted as a single peak in the Vi of Sephacryl S-400, suggesting a distinct homogeneous glycopeptide, smaller than those from normal mucins. The main 14C-labeled sugars in this glycopeptide were fucose, glucosamine, galactosamine, and galactose, consistent with a mucin. Thus, DRL releases stable but noncovalent complexes containing one or more distinct mucinlike glycoconjugates, probably combined with lipids and peptides. We discuss their possible relevance to airway diseases, including cystic fibrosis.
本文描述了由铜绿假单胞菌产生的毒素鼠李糖脂（DRL）从猫气管中释放出的低密度黏液糖缀合物。猫气管中的黏液糖缀合物在体内用³H - 脯氨酸和¹⁴C - 葡萄糖进行放射性标记。对照黏液以及由200微克/毫升DRL释放出的黏液溶解在盐酸胍缓冲液（GuHCl）中，并在琼脂糖CL - 2B上进行层析。通过在CsCl/GuHCl中进行等密度密度梯度离心分离在柱的空体积（V₀）中洗脱的分子。所有样品在ρ = 约1.50和约1.60克/毫升处给出放射性标记和过碘酸/席夫（PAS）反应性物质的峰，但DRL刺激的样品含有低密度物质（ρ < 1.32克/毫升），同样具有PAS反应性且被放射性标记。在体外与DRL一起孵育的对照分泌物未形成低密度物质。在非离子洗涤剂Triton X - 100（1%体积/体积）中，低密度物质表现为较小的分子，在琼脂糖CL - 2B柱的部分包含体积（Vi）中移动，但仍在Sephacryl S - 300的V₀中。用软骨素酶ABC、肝素酶II和III以及角蛋白酶孵育未能改变其在S - 300上的洗脱图谱，这排除了糖胺聚糖的可能性；但用胰蛋白酶或蛋白酶K进行蛋白水解产生了两个峰，在柱的完全包含体积附近的肽片段和V₀中的糖肽。V₀糖肽在CsCl梯度中在1.50至1.55克/毫升之间形成条带，并在Sephacryl S - 400的Vi中作为单峰洗脱，表明是一种独特的均匀糖肽，比正常黏蛋白中的糖肽小。该糖肽中主要的¹⁴C标记糖是岩藻糖、葡糖胺、半乳糖胺和半乳糖，与黏蛋白一致。因此，DRL释放出含有一种或多种不同的黏蛋白样糖缀合物的稳定但非共价复合物，可能与脂质和肽结合。我们讨论了它们与包括囊性纤维化在内的气道疾病的可能相关性。
