West C M, Mao J, van der Wel H, Erdos G W, Zhang Y
Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville 32610-0235, USA.
Microbiology (Reading). 1996 Aug;142 ( Pt 8):2227-43. doi: 10.1099/13500872-142-8-2227.
Highly purified spore coats of Dictyostelium discoideum each contained about 5 x 10(6) protein molecules as determined by amino acid composition analysis. By two-dimensional gel electrophoresis the coats were found to contain nine major-abundance and numerous minor protein species, most of which were highly enriched relative to the adjacent interspore matrix. Protein was nearly quantitatively eluted by denaturants and 2-mercaptoethanol, showing that it was not irreversibly cross-linked. Because a reducing agent is required together with denaturants to elute most proteins if their free thiol groups have been prealkylated, it was concluded that the D. discoideum spore coat proteins are disulfide cross-linked into the matrix. One major coat protein, SP75, was partially sequenced and found to be encoded by the previously identified DP87 gene; this finding was supported by additional physical, genetic, biochemical and microscopic evidence. The five major proteins for which genes have been cloned were associated with the outer layer of the coat. In coats missing one or more of four of these proteins as a result of gene disruption, there were physical changes but, with one exception, the other major coat proteins appeared to be incorporated normally. Sequence analysis showed that these five outer layer coat proteins are homologous and consist of alternating sequence motifs related to epithelial mucin repeats, basic proline repeats found in salivary acidic proline-rich proteins, the NH2-terminal subdomain of epidermal growth factor modules and other cysteine repeats. Based on these and other observations, outer layer coat proteins are predicted to organize indeterminately to form a cell surface microenvironment supportive of cellulose morphogenesis during spore coat formation.
通过氨基酸组成分析确定,高度纯化的盘基网柄菌孢子壁每个含有约5×10⁶个蛋白质分子。通过二维凝胶电泳发现,孢子壁含有9种主要丰度的蛋白质和众多次要蛋白质种类,其中大多数相对于相邻的孢子间基质高度富集。蛋白质几乎能被变性剂和2-巯基乙醇定量洗脱,表明其并非不可逆交联。由于如果蛋白质的游离巯基已被预烷基化,洗脱大多数蛋白质时需要还原剂与变性剂一起使用,因此得出结论,盘基网柄菌孢子壁蛋白质通过二硫键交联到基质中。对一种主要的孢子壁蛋白质SP75进行了部分测序,发现它由先前鉴定的DP87基因编码;这一发现得到了其他物理、遗传、生化和显微镜证据的支持。已克隆出基因的5种主要蛋白质与孢子壁的外层相关。在因基因破坏而缺失其中4种蛋白质中的一种或多种的孢子壁中,存在物理变化,但除一个例外,其他主要的孢子壁蛋白质似乎正常掺入。序列分析表明,这5种外层孢子壁蛋白质是同源的,由与上皮粘蛋白重复序列、唾液富含酸性脯氨酸蛋白质中发现的碱性脯氨酸重复序列、表皮生长因子模块的NH₂末端亚结构域以及其他半胱氨酸重复序列相关的交替序列基序组成。基于这些及其他观察结果,预计外层孢子壁蛋白质会无规组织形成细胞表面微环境,在孢子壁形成过程中支持纤维素形态发生。