Evidence of hydrophobic domains in human respiratory mucins. Effect of sodium chloride on hydrophobic binding properties.
Evidence of hydrophobic domains in human respiratory mucins. Effect of sodium chloride on hydrophobic binding properties.
复制标题
人类呼吸道粘蛋白中疏水域的证据。
DOI：
10.1021/bi00476a030
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Sachdev,GP
中科院分区：
文献类型：
--
作者：
Shankar,V;Naziruddin,B;ReyesdelaRocha,S;Sachdev,GP
摘要：用荧光探针技术研究了纯化的人呼吸道粘蛋白的疏水结合特性。根据我们实验室早期建立的方案，从囊性纤维化（CF）和哮喘患者的气管支气管分泌物以及正常肺的个体中纯化粘蛋白。将纯化的粘蛋白进行还原-烷基化和链霉蛋白酶消化，以研究这些处理对粘蛋白疏水性质的影响。此外，还研究了增加NaCl浓度对天然粘蛋白和还原烷基化粘蛋白的疏水性质的影响。天然粘蛋白显示出大量低亲和力（A！D~ 10-5 M）的疏水配体Mns-Phe的结合位点，并且具有40至50个结合位点/mg粘蛋白。在6 M盐酸胍存在下，用二硫苏糖醇还原粘蛋白，然后用碘乙酰胺烷基化，明显引起粘蛋白分子构象的显著变化，表现为探针的高亲和力（AID~ 10~ 6 M）和低亲和力（KD~ 10 ~ 5 M）结合位点的出现以及探针结合位点数目的增加。链霉蛋白酶消化的天然和还原烷基化粘蛋白几乎完全消除了结合的荧光探针粘蛋白，表明结合位点上的nonglycosylated，链霉蛋白酶敏感的粘蛋白分子的部分。增加NaCl浓度（0.03-1.0 M）没有明显改变天然粘蛋白诱导的Mns-Phe荧光，而还原烷基化粘蛋白诱导的Mns-Phe荧光逐渐增加。结果表明，进一步展开的减少烷基化的粘蛋白在增加的NaCl浓度暴露额外的疏水域。此外，与还原-烷基化非CF粘蛋白相比，还原-烷基化CF粘蛋白中探针结合位点的数量增加更大，表明CF和非CF粘蛋白的三维结构可能存在差异。因此，本研究为人呼吸道粘蛋白中存在疏水结构域提供了证据，这些性质，至少部分地，可能有助于通过非共价相互作用形成粘蛋白的聚合结构。
Revised Manuscript Received March 23, 1990 abstract: Hydrophobic binding properties of purified human respiratory mucins were studied by the fluorescence probe technique using mansylphenylalanine (Mns-Phe) as the fluorescent probe. Mucins were purified from tracheobronchial secretions of cysticfibrosis (CF) and asthmatic patients, as well as from individuals with normal lungs, according to a protocol earlier established in our laboratory. Purified mucins were subjected to reduction-alkylation and Pronase digestion to study the effects of these treatments on the hydrophobic properties of the mucins. In addition, the effects of increased NaCl concentration on the hydrophobic properties of native and reduced-alkylated mucins were also investigated. Native mucins showed evidence of a large number of low-affinity (A! D~ 10-5 M) binding sites for the hydrophobic ligand Mns-Phe and had between 40 and 50 binding sites/mg of mucin. Reduction of mucin using dithiothreitol in thepresence of 6 M guanidine hydrochloride and subsequent alkylation with iodoacetamide apparently caused marked conformational changes in the mucin molecules as revealed by the presence of both high-affinity (AID~ 10~ 6 M) and low-affinity (KD~ 10" 5 M) binding sites for the probe and an increase in the number of probe binding sites. Pronase digestion of the native and reduced-alkylated mucins almost completely eliminated binding of the fluorescentprobe to the mucins, showing that the binding sites are on the nonglycosylated, Pronase-sensitive portion of the mucin molecules. Increasing NaCl concentrations (0.03-1.0 M) did not appreciably alter the native mucin-induced Mns-Phe fluorescence, while that of the reduced-alkylated mucin-induced Mns-Phe fluorescence was progressively increased. The results indicate that further unfolding of reduced-alkylated mucins at increased NaCl concentrations exposes additional hydrophobic domains. Also, the greater increase in the number of probe binding sites in the reduced-alkylated CF mucin as compared to reduced-alkylated non-CF mucins suggests possible differences in the three-dimensional structure of CF and non-CF mucins. Thus, this study provided evidence for the presence of hydrophobicdomains in human respiratory mucins, andthese properties, at least in part, may contribute to the polymeric structure of mucins through noncovalent interactions.
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