Mehrotra R, Thornton D J, Sheehan J K
Wellcome Trust Centre for Cell-Matrix Research, Division of Biochemistry, 2.205 School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
Biochem J. 1998 Sep 1;334 ( Pt 2)(Pt 2):415-22. doi: 10.1042/bj3340415.
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.
唾液中含有两类主要的黏蛋白（MG1和MG2）；这些糖蛋白中较小的一种（MG2）的多肽已被确定为MUC7基因的产物。在本研究中，我们设计了一种快速的两步法，该方法能够回收这种基本上不含其他成分且数量充足的糖蛋白，以便进行物理和自身相互作用研究。将未处理的唾液溶解于4M氯化胍中，然后进行琼脂糖CL-4B柱层析。富含MG2的组分被回收，其中不含较大的MG1糖蛋白以及较小的蛋白质/糖蛋白（分子量小于100kDa）。最终通过在Mono Q上进行阴离子交换层析对MG2糖蛋白进行纯化。在用[14C]乙酸酐对分子进行放射性标记后，通过SDS/PAGE评估制备物的纯度。肽图谱分析、N端测序和氨基酸分析证实黏蛋白的多肽为MUC7基因产物。通过电子显微镜检查分离出的分子，其呈现为长度为30 - 120nm的短而柔韧的蠕虫状结构。分布是不均匀的，包含一个主要成分，其数均长度和重均长度分别为52nm和55nm，以及一个次要成分，其数均长度和重均长度分别为94nm和98nm。我们认为这两种不同大小的群体分别代表黏蛋白的单体和二聚体形式。在0.2M NaCl中进行的凝胶层析表明存在单体、二聚体和四聚体；该制备物的平均分子量为192kDa。然而，在4M氯化胍中分子量为158kDa，还原后的黏蛋白制备物的分子量测定为类似的155kDa。这些结果表明黏蛋白可能通过蛋白质介导的相互作用进行自身缔合。基于这些结果，提出了一个MUC7黏蛋白自身缔合的模型，这可能对其生物学功能很重要。
