Semino C E, Specht C A, Raimondi A, Robbins P W
Massachusetts Institute of Technology, Center for Cancer Research, Cambridge 02139-4307, USA.
Proc Natl Acad Sci U S A. 1996 May 14;93(10):4548-53. doi: 10.1073/pnas.93.10.4548.
The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.
非洲爪蟾发育基因DG42在胚胎发育早期,即囊胚中期和神经胚形成阶段之间表达。推导的非洲爪蟾DG42蛋白序列与根瘤菌Nod C、链球菌Has A和真菌几丁质合酶具有相似性。此前,我们发现体外转录/翻译系统中产生的DG42蛋白催化了一系列几丁质寡糖的合成。在此我们表明,非洲爪蟾和斑马鱼早期胚胎的细胞提取物也能合成几丁质寡糖。还克隆并测序了与斑马鱼和小鼠DG42同源的cDNA片段。基于Northern印迹和Western印迹分析,这些同源物的表达与非洲爪蟾中所描述的相似。非洲爪蟾抗DG42抗体在斑马鱼胚胎提取物中识别出一种63 kDa的蛋白,其发育表达模式与之前描述的非洲爪蟾相似。提取物中发现的几丁质寡糖合酶活性被特异性DG42抗体灭活;在测试条件下,透明质酸(HA)的合成不受影响。其他实验表明,在质粒控制下,DG42在小鼠3T3细胞中的表达产生了几丁质寡糖合酶活性,而HA合酶水平没有增加。其他研究者在哺乳动物细胞培养系统中发现DG42可刺激HA合成,我们的结果与他们的结果之间可能存在的关系,将由其他地方发表的结构分析提供,这些分析表明几丁质寡糖存在于HA链的还原端。由于在至少一种脊椎动物系统中,透明质酸的形成可被纯几丁质酶抑制,几丁质寡糖似乎有可能作为透明质酸合成的引物。