与已知蛋白质缺乏可检测同源性的预测开放阅读框(ORF)称为ORFans。尽管它们在宏基因组中普遍存在,ORFans编码真正蛋白质的程度,它们可以被注释的程度,以及它们的功能贡献,仍然不清楚。为了深入了解这些问题,我们应用敏感的远程同源性检测方法对土壤中的ORFans进行功能分析,海洋,和人类肠道宏基因组集合。ORFans被确认,聚集到序列家族中,并通过与已知结构的蛋白质的概况比较进行注释。我们发现,相当数量的宏基因组ORFans(738484,121,15.3%的73,896)表现出与结构表征蛋白的显着远程同源性,为ORFan功能分析提供了一种方法。检测到的远程同源性的程度远远超过人工蛋白质家族获得的程度(1.4%)。正如真正的基因所预期的那样,ORFans的预测功能与其基因邻居的功能显著相似(p<0.001)。与通过标准同源性搜索预测的功能谱相比,ORFans显示出生物学上有趣的差异。许多富含ORFan的功能是病毒相关的,并且倾向于反映与极端序列多样性相关的生物过程。每个环境还拥有大量独特的ORFan家族和功能,包括一些已知发挥重要作用的社区,如肠道微生物多糖消化。最后,ORFans是寻找新的感兴趣的酶的宝贵资源,正如我们通过鉴定数百种新型ORFan金属蛋白酶所证明的那样,尽管与已知蛋白质普遍缺乏相似性,但它们都具有特征催化基序。我们的ORFan功能预测是发现新蛋白质家族和探索蛋白质序列空间边界的宝贵资源。所有远程同源性预测可在http://doxey获得。uwaterloo.CA/ORFans。
Predicted open reading frames (ORFs) that lack detectable homology to known proteins are termed ORFans. Despite their prevalence in metagenomes, the extent to which ORFans encode real proteins, the degree to which they can be annotated, and their functional contributions, remain unclear. To gain insights into these questions, we applied sensitive remote-homology detection methods to functionally analyze ORFans from soil, marine, and human gut metagenome collections. ORFans were identified, clustered into sequence families, and annotated through profile-profile comparison to proteins of known structure. We found that a considerable number of metagenomic ORFans (73,896 of 484,121, 15.3%) exhibit significant remote homology to structurally characterized proteins, providing a means for
ORFan functional profiling. The extent of detected remote homology far exceeds that obtained for artificial protein families (1.4%). As expected for real genes, the predicted functions of ORFans are significantly similar to the functions of their gene neighbors (p < 0.001). Compared to the functional profiles predicted through standard homology searches, ORFans show biologically intriguing differences. Many
ORFan-enriched functions are virus-related and tend to reflect biological processes associated with extreme sequence diversity. Each environment also possesses a large number of unique
ORFan families and functions, including some known to play important community roles such as gut microbial polysaccharide digestion. Lastly, ORFans are a valuable resource for finding novel enzymes of interest, as we demonstrate through the identification of hundreds of novel
ORFan metalloproteases that all possess a signature catalytic motif despite a general lack of similarity to known proteins. Our
ORFan functional predictions are a valuable resource for discovering novel protein families and exploring the boundaries of protein sequence space. All remote homology predictions are available at http://doxey.uwaterloo.ca/ORFans.