Fuzita Felipe J, Pinkse Martijn W H, Verhaert Peter D E M, Lopes Adriana R
Laboratory of Biochemistry and Biophysics, Instituto Butantan, São Paulo, Brazil; Biotechnology Program, University of São Paulo, São Paulo, Brazil.
Laboratory of Analytical Biotechnology & Innovative Peptide Biology, Delft University of Technology, Delft, The Netherlands.
Insect Biochem Mol Biol. 2015 May;60:47-58. doi: 10.1016/j.ibmb.2015.03.005. Epub 2015 Mar 25.
Cysteine cathepsins are widely spread on living organisms associated to protein degradation in lysosomes, but some groups of Arthropoda (Heteroptera, Coleoptera, Crustacea and Acari) present these enzymes related to digestion of the meal proteins. Although spiders combine a mechanism of extra-oral with intracellular digestion, the sporadic studies on this subject were mainly concerned with the digestive fluid (DF) analysis. Thus, a more complete scenario of the digestive process in spiders is still lacking in the literature. In this paper we describe the identification and characterization of cysteine cathepsins in the midgut diverticula (MD) and DF of the spider Nephilengys cruentata by using enzymological assays. Furthermore, qualitative and quantitative data from transcriptomic followed by proteomic experiments were used together with biochemical assays for results interpretation. Five cathepsins L, one cathepsin F and one cathepsin B were identified by mass spectrometry, with cathepsins L1 (NcCTSL1) and 2 (NcCTSL2) as the most abundant enzymes. The native cysteine cathepsins presented acidic characteristics such as pH optima of 5.5, pH stability in acidic range and zymogen conversion to the mature form after in vitro acidification. NcCTSL1 seems to be a lysosomal enzyme with its recombinant form displaying acidic characteristics as the native ones and being inhibited by pepstatin. Evolutionarily, arachnid cathepsin L may have acquired different roles but its use for digestion is a common feature to studied taxa. Now a more elucidative picture of the digestive process in spiders can be depicted, with trypsins and astacins acting extra-orally under alkaline conditions whereas cysteine cathepsins will act in an acidic environment, likely in the digestive vacuoles or lysosome-like vesicles.
半胱氨酸组织蛋白酶广泛存在于与溶酶体中蛋白质降解相关的生物体中，但节肢动物的一些类群（异翅目、鞘翅目、甲壳纲和蜱螨目）中存在这些与食物蛋白质消化相关的酶。尽管蜘蛛结合了口外消化和细胞内消化机制，但关于这一主题的零星研究主要集中在消化液（DF）分析上。因此，文献中仍缺乏关于蜘蛛消化过程的更完整描述。在本文中，我们通过酶学分析描述了蜘蛛横纹金蛛中肠盲囊（MD）和DF中半胱氨酸组织蛋白酶的鉴定和特性。此外，转录组学随后进行蛋白质组学实验的定性和定量数据与生化分析一起用于结果解释。通过质谱鉴定出五种组织蛋白酶L、一种组织蛋白酶F和一种组织蛋白酶B，其中组织蛋白酶L1（NcCTSL1）和2（NcCTSL2）是最丰富的酶。天然半胱氨酸组织蛋白酶具有酸性特征，如最适pH为5.5，在酸性范围内pH稳定，体外酸化后酶原转化为成熟形式。NcCTSL1似乎是一种溶酶体酶，其重组形式表现出与天然酶相同的酸性特征，并被胃蛋白酶抑制剂抑制。在进化上，蛛形纲组织蛋白酶L可能具有不同的作用，但用于消化是所研究类群的共同特征。现在可以描绘出蜘蛛消化过程的更清晰图景，胰蛋白酶和虾红素在碱性条件下在口外起作用，而半胱氨酸组织蛋白酶将在酸性环境中起作用，可能在消化液泡或类溶酶体小泡中。
