Stability and Refolding of Prophenol Oxidase Protein with 2-Propanol in Drosophila melanogaster

Phenol oxidase in Drosophila melanogaster occurs as folded phase precursors designated as prophenol oxidase A1 and A3, and prophenol oxidase is activated with alcohol, especially 2-propanol, within a few minutes as unfolded-phase in vitro. To clarify a common effect of alcohols on proteins and peptides, the extract containing prophenol oxidase protein was prepared. Phenol oxidase activity activated with 2-propanol has been maintained stable at least 24 hours remains as it is. Protein of prophenol oxidase was not denatured opposite hypnoses known as the instability of protein with alcohol. Activated prophenol oxidase with 2-propanol remain enzyme activity with no aggregation, stable, renaturation, and the refolding phenomena occurred around the active phase within the catalytic active center of prophenol oxidase protein in Drosophila melanogaster. This study is important to induce the wide range applications of the effect in many fields for rational drag design.

Activation of Prophenol Oxidase PHOX-S with Limited Proteolysis in Drosophila Melanogaster

Prophenol oxidase isoform A1 was isolated from Drosophila melanogaster （The sequence has been deposited in GenBank data base under accession number AB557586） PHOX-S strain, and its characteristics and activation mechanism were determined. The NH2-terminal region of PHOX-S A1 was determined to be comprised of 15 amino acids with the following sequence MTNMKMKMKAMMR. Comparison of an alignment in the known prophenol oxidase protein sequences from Drosophila melanogaster strains showed high homology in the copper-binding sequences at the Cu （A） site of the active center. Limited proteolysis takes place between Arg-50 and Val-51. Therefore, it is concluded that prophenol oxidase PHOX-S protein was evolved at the upstream, but no evolved at the central site in Drosophila melanogaster.

家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase,PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。