Hogenkamp David G, Arakane Yasuyuki, Kramer Karl J, Muthukrishnan Subbaratnam, Beeman Richard W
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Insect Biochem Mol Biol. 2008 Apr;38(4):478-89. doi: 10.1016/j.ibmb.2007.08.002. Epub 2007 Aug 17.
Enzymes belonging to the beta-N-acetylhexosaminidase family cleave chitin oligosaccharides produced by the action of chitinases on chitin into the constituent N-acetylglucosamine monomer. Four genes encoding putative chitooligosaccharidolytic beta-N-acetylhexosaminidases (hereafter referred to as N-acetylglucosaminidases (NAGs)) in the red flour beetle, Tribolium castaneum, namely TcNAG1, TcFDL, TcNAG2, and TcNAG3, and three other related hexosaminidases were identified by searching the recently completed genome [Tribolium Genome Sequencing Consortium, 2007. The first genome sequence of a beetle, Tribolium castaneum, a model for insect development and pest biology. Nature, submitted for publication]. Full-length cDNAs for all four NAGs were cloned and sequenced, and the exon-intron organization of the corresponding genes was determined. Analyses of their developmental expression patterns indicated that, although all four of the NAGs are transcribed during most developmental stages, each gene had a distinct spatial and temporal expression pattern. TcNAG1 transcripts are the most abundant, particularly at the late pupal stage, while TcNAG3 transcripts are the least abundant, even at their peak levels in the late larval stages. The function of each NAG during different developmental stages was assessed by observations of lethal phenotypes after gene-specific double-stranded RNA (dsRNA)-mediated transcript depletion as verified by real-time PCR. TcNAG1 dsRNA was most effective in interrupting all three types of molts: larval-larval, larval-pupal, and pupal-adult. Treated insects died after failing to completely shed their old cuticles. Knockdown of transcripts for the other three NAG genes resulted in phenotypes similar to those of TcNAG1 dsRNA-treated insects, but the effects were somewhat variable and less severe. Sequence comparisons with other enzymatically characterized insect homologs suggested that TcFDL, unlike the other NAGs, may have a role in N-glycan processing in addition to its apparent role in cuticular chitin turnover. These results support the hypothesis that TcNAGs participate in chitin turnover and/or N-glycan processing during insect development and that each NAG fulfills an essential and distinct function.
属于β-N-乙酰己糖胺酶家族的酶可将几丁质酶作用于几丁质产生的几丁质寡糖切割成组成成分N-乙酰葡糖胺单体。通过搜索最近完成的基因组 [Tribolium Genome Sequencing Consortium, 2007. 一种甲虫赤拟谷盗的首个基因组序列，昆虫发育和害虫生物学的模型。《自然》，已提交发表]，在赤拟谷盗中鉴定出四个编码假定的几丁质寡糖分解性β-N-乙酰己糖胺酶（以下简称N-乙酰葡糖胺酶（NAGs））的基因，即TcNAG1、TcFDL、TcNAG2和TcNAG3，以及其他三个相关的己糖胺酶。克隆并测序了所有四个NAGs的全长cDNA，并确定了相应基因的外显子-内含子结构。对它们发育表达模式的分析表明，虽然所有四个NAGs在大多数发育阶段都有转录，但每个基因都有独特的时空表达模式。TcNAG1转录本最为丰富，尤其是在蛹后期，而TcNAG3转录本最为稀少，即使在幼虫后期达到峰值水平时也是如此。通过实时PCR验证基因特异性双链RNA（dsRNA）介导的转录本缺失后，观察致死表型来评估每个NAG在不同发育阶段的功能。TcNAG1 dsRNA在中断所有三种类型的蜕皮方面最为有效：幼虫-幼虫、幼虫-蛹和蛹-成虫。处理后的昆虫在未能完全蜕去旧表皮后死亡。敲低其他三个NAG基因的转录本导致的表型与TcNAG1 dsRNA处理的昆虫相似，但效果有所不同且不太严重。与其他已进行酶学表征的昆虫同源物的序列比较表明，与其他NAGs不同，TcFDL除了在表皮几丁质周转中具有明显作用外，可能还在N-聚糖加工中发挥作用。这些结果支持了这样的假设，即TcNAGs在昆虫发育过程中参与几丁质周转和/或N-聚糖加工，并且每个NAG都履行着重要且独特的功能。
