Veiga Ana B G, Ribeiro José M C, Francischetti Ivo M B, Xu Xueqing, Guimarães Jorge A, Andersen John F
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, Maryland, United States of America; Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre RS, Brazil.
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, Maryland, United States of America.
PLoS One. 2014 Jun 27;9(6):e95424. doi: 10.1371/journal.pone.0095424. eCollection 2014.
The bilin-binding proteins (BBP) from lepidopteran insects are members of the lipocalin family of proteins and play a special role in pigmentation through the binding of biliverdin IXγ. Lopap, a BBP-like protein from the venom of the toxic caterpillar Lonomia obliqua has been reported to act as a serine protease that activates the coagulation proenzyme prothrombin. Here we show that BBPLo, a variant of lopap from the same organism binds biliverdin IXγ, forming a complex that is spectrally identical with previously described BBP proteins. Although BBPLo is nearly identical in sequence to lopap, no prothrombinase activity was detected in our recombinant preparations using reconstituted systems containing coagulation factors Xa and Va, as well as anionic phospholipids. In addition to biliverdin, BBPLo was found to form a 1:1 complex with heme prompting us to examine whether the unusual biliverdin IXγ ligand of BBPs forms as a result of oxidation of bound heme in situ rather than by a conventional heme oxygenase. Using ascorbate or a NADPH(+)-ferredoxin reductase-ferredoxin system as a source of reducing equivalents, spectral changes are seen that suggest an initial reduction of heme to the Fe(II) state and formation of an oxyferrous complex. The complex then disappears and a product identified as a 5-coordinate carbonyl complex of verdoheme, an intermediate in the biosynthesis of biliverdin, is formed. However, further reaction to form biliverdin was not observed, making it unlikely that biliverdin IXγ is formed by this pathway.
鳞翅目昆虫的胆绿素结合蛋白（BBP）是脂质运载蛋白家族的成员，通过与胆绿素IXγ结合在色素沉着中发挥特殊作用。据报道，来自有毒毛虫斜纹枯叶蛾毒液中的一种类BBP蛋白Lopap可作为激活凝血酶原酶原的丝氨酸蛋白酶。在此我们表明，来自同一生物体的Lopap变体BBPLo与胆绿素IXγ结合，形成一种在光谱上与先前描述的BBP蛋白相同的复合物。尽管BBPLo在序列上与Lopap几乎相同，但在我们使用含有凝血因子Xa和Va以及阴离子磷脂的重组系统的重组制剂中未检测到凝血酶原酶活性。除了胆绿素外，还发现BBPLo与血红素形成1:1复合物，这促使我们研究BBP异常的胆绿素IXγ配体是否是由于结合的血红素原位氧化而非传统的血红素加氧酶形成的。使用抗坏血酸或NADPH（+） - 铁氧化还原蛋白还原酶 - 铁氧化还原蛋白系统作为还原当量的来源，可以看到光谱变化，这表明血红素最初还原为Fe（II）状态并形成氧合亚铁复合物。然后该复合物消失，形成一种被鉴定为胆绿素生物合成中间体的胆血红素5配位羰基复合物的产物。然而，未观察到进一步反应形成胆绿素，因此胆绿素IXγ不太可能通过该途径形成。
