Bachman E S, McClay D R
Developmental Cell and Molecular Biology Group, Duke University, Durham, NC 27708, USA.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6808-13. doi: 10.1073/pnas.93.13.6808.
We report the molecular cloning of the first beta-1,3 glucanase from animal tissue. Three peptide sequences were obtained from beta-1,3 glucanase that had been purified from eggs of the sea urchin Strongylocentrotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences. The full-length cDNA shows a predicted enzyme structure of 499 aa with a hydrophobic signal sequence. A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue. A polyclonal antibody to the native 68-kDa enzyme recognizes a single band during early embryogenesis that reappears in the adult gut, and recognizes a 57-kDa fusion protein made from a full-length cDNA clone for beta-1,3 glucanase. The identity of this molecule as beta-1,3 glucanase is confirmed by sequence homology, by the presence of all three peptide sequences in the deduced amino acid sequence, and by the recognition of the bacterial fusion protein by the antibody directed against the native enzyme. Data base searches show significant homology at the amino acid level to beta-1,3 glucanases from two species of bacteria and a clotting factor from the horseshoe crab. The homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four divergent species. These four species were also found to have two homologous regions in common with more distantly related plant, fungal, and bacterial proteins. A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes. In particular, there is an especially deep split within genes taken from the bacterial genus Bacillus.
我们报道了从动物组织中首次克隆出β-1,3葡聚糖酶的分子克隆过程。从紫海胆（Strongylocentrotus purpuratus）卵中纯化得到的β-1,3葡聚糖酶获得了三个肽序列，并使用从这些肽序列推导而来的寡核苷酸通过PCR克隆了该基因。全长cDNA显示预测的酶结构为499个氨基酸，带有一个疏水信号序列。在卵、早期胚胎发育过程以及成年肠道组织中存在一条3.2 kb的信使RNA。针对天然68 kDa酶的多克隆抗体在早期胚胎发育过程中识别出一条单一的条带，该条带在成年肠道中再次出现，并且识别由β-1,3葡聚糖酶的全长cDNA克隆制备的57 kDa融合蛋白。通过序列同源性、推导的氨基酸序列中所有三个肽序列的存在以及针对天然酶的抗体对细菌融合蛋白的识别，证实了该分子为β-1,3葡聚糖酶。数据库搜索显示，在氨基酸水平上与两种细菌的β-1,3葡聚糖酶以及鲎的一种凝血因子具有显著同源性。与细菌的同源性集中在一个304个氨基酸的区域，在这四个不同物种之间有七个分散的高度同源区域。还发现这四个物种与亲缘关系更远的植物、真菌和细菌蛋白有两个共同的同源区域。基于这些区域的全局系统发育强烈表明，葡聚糖酶是一个非常古老的基因家族。特别是，来自芽孢杆菌属的基因之间存在一个特别深的分化。
