Department of Genetics and Evolution, Laboratory of Molecular Biology, Federal University of São Carlos, 13565-905 São Carlos, Brazil.
Insect Biochem Mol Biol. 2012 Jan;42(1):58-69. doi: 10.1016/j.ibmb.2011.10.008. Epub 2011 Nov 12.
A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37°C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K(m)=37.53 mMS(-1)), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K(i)=0.196 nM), indicating that this protease is a potential target for pest control.
从 Sphenophorus levis 幼虫 cDNA 文库中分离出一种编码消化组织蛋白酶 L 的 cDNA，命名为 Sl-CathL，这是一种在中肠中负责蛋白水解的最丰富的 EST（10.49%）。该开放阅读框长 972bp，编码一个类似于其他鞘翅目昆虫中肠组织蛋白酶 L 样酶的前酶原。重组 Sl-CathL 在毕赤酵母中表达，分子量约为 42kDa。该重组蛋白在低 pH 值下被催化激活，成熟酶的分子量为 39kDa，在 37°C 和 pH6.0 时表现出热不稳定性和最大活性。免疫细胞化学分析显示 Sl-CathL 在中肠上皮细胞中产生，并从含有该酶的囊泡中分泌到肠腔中，这证实了该酶在昆虫幼虫消化过程中发挥了重要作用。通过生物周期的 RT-PCR 表达谱分析表明，Sl-CathL 主要在幼虫阶段产生，在 30 日龄幼虫时表达量最高。在这个阶段，该酶的表达量比蛹期高出 1250 倍，而在蛹期检测到的表达量最低。该酶也在成虫期产生，但丰度较低，这表明成虫消化系统中存在不同的酶。组织特异性分析显示，Sl-CathL mRNA 的合成主要发生在幼虫中肠，这与免疫定位分析中检测到的消化酶功能相一致。使用不同的底物（Z-Leu-Arg-AMC、Z-Arg-Arg-AMC 和 Z-Phe-Arg-AMC）计算纯化重组酶的催化效率，rSl-CathL 对 Z-Leu-Arg-AMC 的水解偏好性（k(cat)/K(m)=37.53mMS(-1)），与其他昆虫组织蛋白酶 L 样酶相似。使用四种重组甘蔗cystatins 进行 rSl-CathL 活性抑制测定。rSl-CathL 被重组cystatin CaneCPI-4 强烈抑制（K(i)=0.196nM），表明该蛋白酶是害虫防治的潜在靶标。
