Laflamme Brooke A, Avila Frank W, Michalski Kevin, Wolfner Mariana F
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853.
Genetics. 2014 Apr;196(4):1117-29. doi: 10.1534/genetics.113.160101. Epub 2014 Feb 10.
Females and males of sexually reproducing animals must cooperate at the molecular and cellular level for fertilization to succeed, even though some aspects of reproductive molecular biology appear to involve antagonistic interactions. We previously reported the existence of a proteolytic cascade in Drosophila melanogaster seminal fluid that is initiated in the male and ends in the female. This proteolytic cascade, which processes at least two seminal fluid proteins (Sfps), is a useful model for understanding the regulation of Sfp activities, including proteolysis cascades in mammals. Here, we investigated the activation mechanism of the downstream protease in the cascade, the astacin-family metalloprotease Seminal metalloprotease-1 (Semp1, CG11864), focusing on the relative contribution of the male and female to its activation. We identified a naturally occurring semp1 null mutation within the Drosophila Genetic Reference Panel. By expressing mutant forms of Semp1 in males homozygous for the null mutation, we discovered that cleavage is required for the complete activation of Semp1, and we defined at least two sites that are essential for this activational cleavage. These amino acid residues suggest a two-step mechanism for Semp1 activation, involving the action of at least two male-derived proteases. Although the cascade's substrates potentially influence both fertility and sperm competition within the mated female, the role of female factors in the activation or activity of Semp1 is unknown. We show here that Semp1 can undergo its activational cleavage in male ejaculates, without female contributions, but that cleavage of Semp1's substrates does not proceed to completion in ejaculates, indicating an essential role for female factors in Semp1's full activity. In addition, we find that expression of Semp1 in virgin females demonstrates that females can activate this protease on their own, resulting in activity that is complete but substantially delayed.
有性生殖动物的雌性和雄性必须在分子和细胞水平上进行协作，以使受精成功，尽管生殖分子生物学的某些方面似乎涉及对抗性相互作用。我们之前报道过，在黑腹果蝇的精液中存在一种蛋白水解级联反应，该反应始于雄性，结束于雌性。这种蛋白水解级联反应可处理至少两种精液蛋白（Sfps），是理解Sfp活性调控的有用模型，包括哺乳动物中的蛋白水解级联反应。在此，我们研究了该级联反应中下游蛋白酶——虾红素家族金属蛋白酶精液金属蛋白酶-1（Semp1，CG11864）的激活机制，重点关注雄性和雌性对其激活的相对贡献。我们在果蝇遗传参考面板中鉴定出一个天然存在的semp1无效突变。通过在纯合无效突变的雄性中表达Semp1的突变形式，我们发现切割是Semp1完全激活所必需的，并且我们确定了至少两个对这种激活切割至关重要的位点。这些氨基酸残基提示了Semp1激活的两步机制，涉及至少两种雄性来源蛋白酶的作用。尽管该级联反应的底物可能会影响交配雌性体内的生育力和精子竞争，但雌性因素在Semp1激活或活性中的作用尚不清楚。我们在此表明，Semp1可以在雄性射精中进行激活切割，无需雌性的参与，但Semp1底物的切割在射精中并未完成，这表明雌性因素在Semp1的完全活性中起着至关重要的作用。此外，我们发现Semp1在未交配雌性中的表达表明雌性可以自行激活这种蛋白酶，从而产生完整但明显延迟的活性。
