Molecules that mediate reproductive interactions are some of the most rapidly evolving traits. Researchers have often suggested that this is due to coevolution at key physiological interfaces. However, very few of these interfaces are well understood at the functional level. One such interface is the digestion of the spermatophore in Lepidoptera. Female Lepidoptera have a specialized reproductive organ called the bursa copulatrix that receives and processes the male spermatophore, a complex proteinaceous ejaculate. In the cabbage white butterfly, Pieris rapae, the bursa secretes a mixture of proteases hypothesized to digest the spermatophore. However, these proteases remain biochemically uncharacterized. Using a zymogram approach, we identified six proteases in bursal extracts at sufficiently high concentrations to characterize their in vitro activity. We assessed the modes of action of these bursal enzymes by quantifying their activity following exposure to diagnostic protease inhibitors. A serine protease-specific inhibitor failed to reduce bursal protease digestion of casein. However, a cysteine protease-specific inhibitor did decrease the activity of some proteases. To explore the possible molecular mechanisms responsible for these responses, we created protease homology models. The models mirrored the results of our in vitro experiments, indicating that protease homology models may offer insight into underlying functional mechanisms. Whether the observed bursal protease resistance to known inhibitors is important in the context of spermatophore digestion remains to be tested. However, our results suggest the exciting possibility that bursal protease specificity may have evolved in response to interactions with various proteins and inhibitors present within the female tract during the reproductive process.

中文翻译：

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从功能和进化的角度表征蝴蝶中女性生殖蛋白酶。

介导生殖相互作用的分子是一些发展最快的特征。研究人员经常提出，这是由于关键生理界面的共同进化所致。但是，在功能级别上很少能很好地理解这些接口。一种这样的界面是鳞翅目中精细胞的消化。雌性鳞翅目有一个专门的生殖器官，称为法氏囊（Bursa copulatrix），它接受并处理雄性精子，这是一种复杂的蛋白质性射精。在白菜白蝴蝶菜青虫（Pieris rapae）中，法氏囊分泌一种被认为可消化精子的蛋白酶混合物。但是，这些蛋白酶在生化上仍未表征。使用酶谱图方法 我们在法氏囊提取物中鉴定了六种蛋白酶，其浓度足够高以表征其体外活性。我们通过定量暴露于诊断性蛋白酶抑制剂后的活性，评估了这些法氏囊酶的作用方式。丝氨酸蛋白酶特异性抑制剂未能降低酪蛋白的法氏囊蛋白酶消化。但是，半胱氨酸蛋白酶特异性抑制剂确实降低了某些蛋白酶的活性。为了探索负责这些反应的可能的分子机制，我们创建了蛋白酶同源性模型。该模型反映了我们体外实验的结果，表明蛋白酶同源性模型可以提供对潜在功能机制的了解。观察到的法氏囊蛋白酶对已知抑制剂的耐药性在精子消化中是否重要，尚待检验。然而，我们的结果表明，在生殖过程中，法氏囊蛋白酶特异性可能响应于与雌性道内存在的各种蛋白质和抑制剂的相互作用而进化，这一令人振奋的可能性。