Isolation and physical characterization of the MUC7 (MG2) mucin from saliva: evidence for self-association.
Isolation and physical characterization of the MUC7 (MG2) mucin from saliva: evidence for self-association.
复制标题
唾液中 MUC7 (MG2) 粘蛋白的分离和物理特征：自关联的证据。
作者：
Ravi Mehrotra;David J. Thornton;J K Sheehan
唾液含有两个主要的粘蛋白家族（MG 1和MG 2）;这些糖蛋白中较小的糖蛋白（MG 2）的多肽已被指定为MUC 7基因的产物。在这项研究中，我们设计了一个快速的两步程序，回收这种糖蛋白基本上不含其他成分，并在足够的数量，使物理和自我相互作用的研究。将原始唾液溶解在4 M氯化胍中，然后进行琼脂糖CL-4 B层析。回收的富含MG 2的部分不含较大的MG 1糖蛋白和较小的蛋白质/糖蛋白（分子量小于100 kDa）。MG 2糖蛋白最后通过Mono Q上的阴离子交换色谱法纯化。用[14 C]乙酸酐放射性标记分子后，通过SDS/PAGE评估制备物的纯度。肽图、N-末端测序和氨基酸分析证实粘蛋白多肽为MUC 7基因产物。通过电子显微镜检查分离的分子，并显示为长度为30-120 nm的短柔性蠕虫状结构。分布是不均匀的，包含一个主要成分的数均和重量平均长度分别为52和55 nm和一个次要成分的数均和重量平均长度分别为94和98 nm。我们建议，这两个不同大小的人口代表粘蛋白的单体和二聚体物种。在0.2 M NaCl中进行的凝胶色谱表明存在单体、二聚体和四聚体;制备物的平均分子量为192 kDa。然而，在4 M氯化胍中，分子量为158 kDa，还原后的粘蛋白制剂测定到类似的分子量（155 kDa）。这些结果表明，粘蛋白可能通过蛋白质介导的相互作用自我关联。结果的基础上，提出了一个模型的MUC 7粘蛋白，这可能是重要的，其生物学功能的自关联。
Saliva contains two major families of mucins (MG1 and MG2); the polypeptide of the smaller of these glycoproteins (MG2) has been assigned as the product of the MUC7 gene. In this study we have devised a rapid two-step procedure that recovers this glycoprotein essentially free of other components and in sufficient quantity to enable physical and self-interaction studies. Raw saliva was solubilized in 4 M guanidinium chloride and thereafter subjected to Sepharose CL-4B chromatography. The MG2-rich fraction was recovered free from the larger MG1 glycoproteins and also smaller proteins/glycoproteins (molecular mass less than 100 kDa). MG2 glycoproteins were finally purified by anion-exchange chromatography on Mono Q. The purity of the preparation was assessed by SDS/PAGE after radiolabelling of the molecules with [14C]acetic anhydride. Peptide mapping, N-terminal sequencing and amino acid analysis verified the polypeptide of the mucins as the MUC7 gene product. The isolated molecules were examined by electron microscopy and appeared as short flexible worm-like structures 30-120 nm in length. The distribution was heterogeneous, containing a major component with number-average and weight-average lengths of 52 and 55 nm respectively and a minor component with number-average and weight-average lengths of 94 and 98 nm respectively. We propose that the two differently sized populations represent monomeric and dimeric species of the mucins. Gel chromatography performed in 0.2 M NaCl indicated the presence of monomers, dimers and tetramers; an average molecular mass for the preparation was 192 kDa. However, in 4 M guanidinium chloride the molecular mass was 158 kDa and a similar molecular mass (155 kDa) was determined for the mucin preparation after reduction. These results suggest that the mucins might self-associate via a protein-mediated interaction. On the basis of the results a model is proposed for the self-association of the MUC7 mucin, which might be important for its biological function.